Understanding the interactions of different substrates with wild-type and mutant acylaminoacyl peptidase using molecular dynamics simulations.

PubMed

Zhu, Jingxuan; Wang, Yan; Li, Xin; Han, Weiwei; Zhao, Li

2017-12-20

Acylaminoacylpeptidase (AAP) belongs to peptidase protein family, which can degrade amyloid β-peptide forms in the brains of patients, and hence leads to Alzheimer's disease. And so, AAP is considered to be a novel target in the design of drugs against Alzheimer's disease. In this investigation, six molecular dynamics simulations were used to find that the interaction between the wild-type and R526V AAP with two different substrates (p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide). Our results were as follows: firstly, Ac-Leu-p-nitroanilide bound to R526V AAP to form a more disordered loop (residues 552-562) in the α/β-hydrolase fold like of AAP, which caused an open and inactive AAP domain form, secondly, binding p-nitrophenylcaprylate and Ac-Leu-p-nitroanilide to AAP can decrease the flexibility of residues 225-250, 260-270, and 425-450, in which the ordered secondary structures may contain the suitable geometrical structure and so it is useful to serine attack. Our theoretical results showed that the binding of the two substrates can induce specific conformational changes responsible for the diverse AAP catalytic specificity. These theoretical substrate-induced structural diversities can help explain the abilities of AAPs to recognize and hydrolyze extremely different substrates.

Volcanic ash supports a diverse bacterial community in a marine mesocosm.

PubMed

Witt, V; Ayris, P M; Damby, D E; Cimarelli, C; Kueppers, U; Dingwell, D B; Wörheide, G

2017-05-01

Shallow-water coral reef ecosystems, particularly those already impaired by anthropogenic pressures, may be highly sensitive to disturbances from natural catastrophic events, such as volcanic eruptions. Explosive volcanic eruptions expel large quantities of silicate ash particles into the atmosphere, which can disperse across millions of square kilometres and deposit into coral reef ecosystems. Following heavy ash deposition, mass mortality of reef biota is expected, but little is known about the recovery of post-burial reef ecosystems. Reef regeneration depends partly upon the capacity of the ash deposit to be colonised by waterborne bacterial communities and may be influenced to an unknown extent by the physiochemical properties of the ash substrate itself. To determine the potential for volcanic ash to support pioneer bacterial colonisation, we exposed five well-characterised volcanic and coral reef substrates to a marine aquarium under low light conditions for 3 months: volcanic ash, synthetic volcanic glass, carbonate reef sand, calcite sand and quartz sand. Multivariate statistical analysis of Automated Ribosomal Intergenic Spacer Analysis (ARISA) fingerprinting data demonstrates clear segregation of volcanic substrates from the quartz and coral reef substrates over 3 months of bacterial colonisation. Overall bacterial diversity showed shared and substrate-specific bacterial communities; however, the volcanic ash substrate supported the most diverse bacterial community. These data suggest a significant influence of substrate properties (composition, granulometry and colour) on bacterial settlement. Our findings provide first insights into physicochemical controls on pioneer bacterial colonisation of volcanic ash and highlight the potential for volcanic ash deposits to support bacterial diversity in the aftermath of reef burial, on timescales that could permit cascading effects on larval settlement. © 2017 The Authors. Geobiology Published by John Wiley & Sons Ltd.

Insights into the Specificity of Lysine Acetyltransferases

DOE PAGES

Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

2014-11-07

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

PubMed Central

Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

2017-01-01

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

PEGylated substrates of NSP4 protease: A tool to study protease specificity

NASA Astrophysics Data System (ADS)

Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

2016-03-01

Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Activity and Phylogenetic Diversity of Bacterial Cells with High and Low Nucleic Acid Content and Electron Transport System Activity in an Upwelling Ecosystem

PubMed Central

Longnecker, K.; Sherr, B. F.; Sherr, E. B.

2005-01-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea. PMID:16332746

Activity and phylogenetic diversity of bacterial cells with high and low nucleic acid content and electron transport system activity in an upwelling ecosystem.

PubMed

Longnecker, K; Sherr, B F; Sherr, E B

2005-12-01

We evaluated whether bacteria with higher cell-specific nucleic acid content (HNA) or an active electron transport system, i.e., positive for reduction of 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), were responsible for the bulk of bacterioplankton metabolic activity. We also examined whether the phylogenetic diversity of HNA and CTC-positive cells differed from the diversity of Bacteria with low nucleic acid content (LNA). Bacterial assemblages were sampled both in eutrophic shelf waters and in mesotrophic offshore waters in the Oregon coastal upwelling region. Cytometrically sorted HNA, LNA, and CTC-positive cells were assayed for their cell-specific [3H]leucine incorporation rates. Phylogenetic diversity in sorted non-radioactively labeled samples was assayed using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes. Cell-specific rates of leucine incorporation of HNA and CTC-positive cells were on average only slightly greater than the cell-specific rates of LNA cells. HNA cells accounted for most bacterioplankton substrate incorporation due to high abundances, while the low abundances of CTC-positive cells resulted in only a small contribution by these cells to total bacterial activity. The proportion of the total bacterial leucine incorporation attributable to LNA cells was higher in offshore regions than in shelf waters. Sequence data obtained from DGGE bands showed broadly similar phylogenetic diversity across HNA, LNA, and CTC-positive cells, with between-sample and between-region variability in the distribution of phylotypes. Our results suggest that LNA bacteria are not substantially different from HNA bacteria in either cell-specific rates of substrate incorporation or phylogenetic composition and that they can be significant contributors to bacterial metabolism in the sea.

A conserved amino acid residue critical for product and substrate specificity in plant triterpene synthases

PubMed Central

Salmon, Melissa; Thimmappa, Ramesha B.; Minto, Robert E.; Melton, Rachel E.; O’Maille, Paul E.; Hemmings, Andrew M.; Osbourn, Anne

2016-01-01

Triterpenes are structurally complex plant natural products with numerous medicinal applications. They are synthesized through an origami-like process that involves cyclization of the linear 30 carbon precursor 2,3-oxidosqualene into different triterpene scaffolds. Here, through a forward genetic screen in planta, we identify a conserved amino acid residue that determines product specificity in triterpene synthases from diverse plant species. Mutation of this residue results in a major change in triterpene cyclization, with production of tetracyclic rather than pentacyclic products. The mutated enzymes also use the more highly oxygenated substrate dioxidosqualene in preference to 2,3-oxidosqualene when expressed in yeast. Our discoveries provide new insights into triterpene cyclization, revealing hidden functional diversity within triterpene synthases. They further open up opportunities to engineer novel oxygenated triterpene scaffolds by manipulating the precursor supply. PMID:27412861

Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1

PubMed Central

Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin

2016-01-01

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50–60 °C and pH 5.5–6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates. PMID:27271847

Functional diversity of family 3 β-glucosidases from thermophilic cellulolytic fungus Humicola insolens Y1.

PubMed

Xia, Wei; Bai, Yingguo; Cui, Ying; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Zhang, Wei; Luo, Huiying; Zhan, Xiuan; Yao, Bin

2016-06-08

The fungus Humicola insolens is one of the most powerful decomposers of crystalline cellulose. However, studies on the β-glucosidases from this fungus remain insufficient, especially on glycosyl hydrolase family 3 enzymes. In the present study, we analyzed the functional diversity of three distant family 3 β-glucosidases from Humicola insolens strain Y1, which belonged to different evolutionary clades, by heterogeneous expression in Pichia pastoris strain GS115. The recombinant enzymes shared similar enzymatic properties including thermophilic and neutral optima (50-60 °C and pH 5.5-6.0) and high glucose tolerance, but differed in substrate specificities and kinetics. HiBgl3B was solely active towards aryl β-glucosides while HiBgl3A and HiBgl3C showed broad substrate specificities including both disaccharides and aryl β-glucosides. Of the three enzymes, HiBgl3C exhibited the highest specific activity (158.8 U/mg on pNPG and 56.4 U/mg on cellobiose) and catalytic efficiency and had the capacity to promote cellulose degradation. Substitutions of three key residues Ile48, Ile278 and Thr484 of HiBgl3B to the corresponding residues of HiBgl3A conferred the enzyme activity towards sophorose, and vice versa. This study reveals the functional diversity of GH3 β-glucosidases as well as the key residues in recognizing +1 subsite of different substrates.

Guiding Empirical and Theoretical Explorations of Organic Matter Decay By Synthesizing Temperature Responses of Enzyme Kinetics, Microbes, and Isotope Fluxes

NASA Astrophysics Data System (ADS)

Billings, S. A.; Ballantyne, F.; Lehmeier, C.; Min, K.

2014-12-01

Soil organic matter (SOM) transformation rates generally increase with temperature, but whether this is realized depends on soil-specific features. To develop predictive models applicable to all soils, we must understand two key, ubiquitous features of SOM transformation: the temperature sensitivity of myriad enzyme-substrate combinations and temperature responses of microbial physiology and metabolism, in isolation from soil-specific conditions. Predicting temperature responses of production of CO2 vs. biomass is also difficult due to soil-specific features: we cannot know the identity of active microbes nor the substrates they employ. We highlight how recent empirical advances describing SOM decay can help develop theoretical tools relevant across diverse spatial and temporal scales. At a molecular level, temperature effects on purified enzyme kinetics reveal distinct temperature sensitivities of decay of diverse SOM substrates. Such data help quantify the influence of microbial adaptations and edaphic conditions on decay, have permitted computation of the relative availability of carbon (C) and nitrogen (N) liberated upon decay, and can be used with recent theoretical advances to predict changes in mass specific respiration rates as microbes maintain biomass C:N with changing temperature. Enhancing system complexity, we can subject microbes to temperature changes while controlling growth rate and without altering substrate availability or identity of the active population, permitting calculation of variables typically inferred in soils: microbial C use efficiency (CUE) and isotopic discrimination during C transformations. Quantified declines in CUE with rising temperature are critical for constraining model CUE estimates, and known changes in δ13C of respired CO2 with temperature is useful for interpreting δ13C-CO2 at diverse scales. We suggest empirical studies important for advancing knowledge of how microbes respond to temperature, and ideas for theoretical work to enhance the relevance of such work to the world's soils.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

PubMed

Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

2016-03-01

In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

Stand Structure and Substrate Diversity as Two Major Drivers for Bryophyte Distribution in a Temperate Montane Ecosystem

PubMed Central

Chen, Yun; Niu, Shuai; Li, Peikun; Jia, Hongru; Wang, Hailiang; Ye, Yongzhong; Yuan, Zhiliang

2017-01-01

Elucidating the major drivers of bryophyte distribution is the first step to protecting bryophyte diversity. Topography, forest, substrates (ground, tree trunks, roots, rocks, and rotten wood), and spatial factor, which factors are the major drivers of bryophyte distribution? In this study, 53 plots were set in 400 m2 along the elevation gradient in Xiaoqinling, China. All bryophytes in the plots were collected and identified. Regression analysis was used to examine the relationship between bryophyte and substrate diversity. We compared the patterns of overall bryophyte diversity and diversity of bryophytes found on the ground, tree, and rock along elevational gradients. Canonical correspondence analysis was applied to relate species composition to selected environmental variables. The importance of topography, forest, substrates, and spatial factors was determined by variance partitioning. A total of 1378 bryophyte specimens were collected, and 240 species were identified. Bryophyte diversity was closely related to substrate diversity. The overall bryophyte diversity significantly increased with elevation; however, the response varied among ground, tree, and rock bryophytes. Tree diversity and herb layer were considered important environmental factors in determining bryophyte distribution. Species abundance was best explained by stand structure (17%), and species diversity was best explained by stand structure (35%) and substrate (40%). Results directly indicated that substrate diversity can improve bryophyte species diversity. The effects of micro-habitat formed by stand structure and substrate diversity were higher than those of spatial processes and topography factors on bryophyte distribution. This study proved that the determinant factors influencing bryophyte diversity reflect the trends in recent forest management, providing a real opportunity to improve forest biodiversity conservation. PMID:28603535

Stand Structure and Substrate Diversity as Two Major Drivers for Bryophyte Distribution in a Temperate Montane Ecosystem.

PubMed

Chen, Yun; Niu, Shuai; Li, Peikun; Jia, Hongru; Wang, Hailiang; Ye, Yongzhong; Yuan, Zhiliang

2017-01-01

Elucidating the major drivers of bryophyte distribution is the first step to protecting bryophyte diversity. Topography, forest, substrates (ground, tree trunks, roots, rocks, and rotten wood), and spatial factor, which factors are the major drivers of bryophyte distribution? In this study, 53 plots were set in 400 m 2 along the elevation gradient in Xiaoqinling, China. All bryophytes in the plots were collected and identified. Regression analysis was used to examine the relationship between bryophyte and substrate diversity. We compared the patterns of overall bryophyte diversity and diversity of bryophytes found on the ground, tree, and rock along elevational gradients. Canonical correspondence analysis was applied to relate species composition to selected environmental variables. The importance of topography, forest, substrates, and spatial factors was determined by variance partitioning. A total of 1378 bryophyte specimens were collected, and 240 species were identified. Bryophyte diversity was closely related to substrate diversity. The overall bryophyte diversity significantly increased with elevation; however, the response varied among ground, tree, and rock bryophytes. Tree diversity and herb layer were considered important environmental factors in determining bryophyte distribution. Species abundance was best explained by stand structure (17%), and species diversity was best explained by stand structure (35%) and substrate (40%). Results directly indicated that substrate diversity can improve bryophyte species diversity. The effects of micro-habitat formed by stand structure and substrate diversity were higher than those of spatial processes and topography factors on bryophyte distribution. This study proved that the determinant factors influencing bryophyte diversity reflect the trends in recent forest management, providing a real opportunity to improve forest biodiversity conservation.

Structure and Functional Diversity of GCN5-Related N-Acetyltransferases (GNAT)

PubMed Central

Salah Ud-Din, Abu Iftiaf Md; Tikhomirova, Alexandra; Roujeinikova, Anna

2016-01-01

General control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT) catalyze the transfer of an acyl moiety from acyl coenzyme A (acyl-CoA) to a diverse group of substrates and are widely distributed in all domains of life. This review of the currently available data acquired on GNAT enzymes by a combination of structural, mutagenesis and kinetic methods summarizes the key similarities and differences between several distinctly different families within the GNAT superfamily, with an emphasis on the mechanistic insights obtained from the analysis of the complexes with substrates or inhibitors. It discusses the structural basis for the common acetyltransferase mechanism, outlines the factors important for the substrate recognition, and describes the mechanism of action of inhibitors of these enzymes. It is anticipated that understanding of the structural basis behind the reaction and substrate specificity of the enzymes from this superfamily can be exploited in the development of novel therapeutics to treat human diseases and combat emerging multidrug-resistant microbial infections. PMID:27367672

AGCVIII Kinases: at the crossroads of cellular signaling

USDA-ARS?s Scientific Manuscript database

AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

Patterns and Variation in Benthic Biodiversity in a Large Marine Ecosystem.

PubMed

Piacenza, Susan E; Barner, Allison K; Benkwitt, Cassandra E; Boersma, Kate S; Cerny-Chipman, Elizabeth B; Ingeman, Kurt E; Kindinger, Tye L; Lee, Jonathan D; Lindsley, Amy J; Reimer, Jessica N; Rowe, Jennifer C; Shen, Chenchen; Thompson, Kevin A; Thurman, Lindsey L; Heppell, Selina S

2015-01-01

While there is a persistent inverse relationship between latitude and species diversity across many taxa and ecosystems, deviations from this norm offer an opportunity to understand the conditions that contribute to large-scale diversity patterns. Marine systems, in particular, provide such an opportunity, as marine diversity does not always follow a strict latitudinal gradient, perhaps because several hypothesized drivers of the latitudinal diversity gradient are uncorrelated in marine systems. We used a large scale public monitoring dataset collected over an eight year period to examine benthic marine faunal biodiversity patterns for the continental shelf (55-183 m depth) and slope habitats (184-1280 m depth) off the US West Coast (47°20'N-32°40'N). We specifically asked whether marine biodiversity followed a strict latitudinal gradient, and if these latitudinal patterns varied across depth, in different benthic substrates, and over ecological time scales. Further, we subdivided our study area into three smaller regions to test whether coast-wide patterns of biodiversity held at regional scales, where local oceanographic processes tend to influence community structure and function. Overall, we found complex patterns of biodiversity on both the coast-wide and regional scales that differed by taxonomic group. Importantly, marine biodiversity was not always highest at low latitudes. We found that latitude, depth, substrate, and year were all important descriptors of fish and invertebrate diversity. Invertebrate richness and taxonomic diversity were highest at high latitudes and in deeper waters. Fish richness also increased with latitude, but exhibited a hump-shaped relationship with depth, increasing with depth up to the continental shelf break, ~200 m depth, and then decreasing in deeper waters. We found relationships between fish taxonomic and functional diversity and latitude, depth, substrate, and time at the regional scale, but not at the coast-wide scale, suggesting that coast-wide patterns can obscure important correlates at smaller scales. Our study provides insight into complex diversity patterns of the deep water soft substrate benthic ecosystems off the US West Coast.

Diversity and mineral substrate preference in endolithic microbial communities from marine intertidal outcrops (Isla de Mona, Puerto Rico)

NASA Astrophysics Data System (ADS)

Couradeau, Estelle; Roush, Daniel; Guida, Brandon Scott; Garcia-Pichel, Ferran

2017-01-01

Endolithic microbial communities are prominent features of intertidal marine habitats, where they colonize a variety of substrates, contributing to their erosion. Almost 2 centuries worth of naturalistic studies focused on a few true-boring (euendolithic) phototrophs, but substrate preference has received little attention. The Isla de Mona (Puerto Rico) intertidal zone offers a unique setting to investigate substrate specificity of endolithic communities since various phosphate rock, limestone and dolostone outcrops occur there. High-throughput 16S rDNA genetic sampling, enhanced by targeted cultivation, revealed that, while euendolithic cyanobacteria were dominant operational taxonomic units (OTUs), the communities were invariably of high diversity, well beyond that reported in traditional studies and implying an unexpected metabolic complexity potentially contributed by secondary colonizers. While the overall community composition did not show differences traceable to the nature of the mineral substrate, we detected specialization among particular euendolithic cyanobacterial clades towards the type of substrate they excavate but only at the OTU phylogenetic level, implying that close relatives have specialized recurrently into particular substrates. The cationic mineral component was determinant in this preference, suggesting the existence in nature of alternatives to the boring mechanism described in culture that is based exclusively on transcellular calcium transport.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

Atypical profiles and modulations of heme-enzymes catalyzed outcomes by low amounts of diverse additives suggest diffusible radicals' obligatory involvement in such redox reactions.

PubMed

Manoj, Kelath Murali; Parashar, Abhinav; Venkatachalam, Avanthika; Goyal, Sahil; Satyalipsu; Singh, Preeti Gunjan; Gade, Sudeep K; Periyasami, Kalaiselvi; Jacob, Reeba Susan; Sardar, Debosmita; Singh, Shanikant; Kumar, Rajan; Gideon, Daniel A

2016-06-01

Peroxidations mediated by heme-enzymes have been traditionally studied under a single-site (heme distal pocket), non-sequential (ping-pong), two-substrates binding scheme of Michaelis-Menten paradigm. We had reported unusual modulations of peroxidase and P450 reaction outcomes and explained it invoking diffusible reactive species [Manoj, 2006; Manoj et al., 2010; Andrew et al., 2011, Parashar et al., 2014 & Venkatachalam et al., 2016]. A systematic investigation of specific product formation rates was undertaken to probe the hypothesis that involvement of diffusible reactive species could explain undefined substrate specificities and maverick modulations (sponsored by additives) of heme-enzymes. When the rate of specific product formation was studied as a function of reactants' concentration or environmental conditions, we noted marked deviations from normal profiles. We report that heme-enzyme mediated peroxidations of various substrates are inhibited (or activated) by sub-equivalent concentrations of diverse redox-active additives and this is owing to multiple redox equilibriums in the milieu. At low enzyme and peroxide concentrations, the enzyme is seen to recycle via a one-electron (oxidase) cycle, which does not require the substrate to access the heme centre. Schemes are provided that explain the complex mechanistic cycle, kinetics & stoichiometry. It is not obligatory for an inhibitor or substrate to interact with the heme centre for influencing overall catalysis. Roles of diffusible reactive species explain catalytic outcomes at low enzyme and reactant concentrations. The current work highlights the scope/importance of redox enzyme reactions that could occur "out of the active site" in biological or in situ systems. Copyright © 2016 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Genetic Control and Evolution of Anthocyanin Methylation1[W

PubMed Central

Provenzano, Sofia; Spelt, Cornelis; Hosokawa, Satoko; Nakamura, Noriko; Brugliera, Filippa; Demelis, Linda; Geerke, Daan P.; Schubert, Andrea; Tanaka, Yoshikazu; Quattrocchio, Francesca; Koes, Ronald

2014-01-01

Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins’ structural diversity. PMID:24830298

Is the molecular diversity of marine dissolved organic matter already imprinted in the exometabolome of single strains?

NASA Astrophysics Data System (ADS)

Noriega-Ortega, B. E.; Wienhausen, G.; Dittmar, T.; Simon, M.; Niggemann, J.

2016-02-01

Dissolved organic matter (DOM) in the ocean, the marine geometabolome, is an extremely complex mixture composed of a wide variety of compounds. The molecular chemodiversity affects the function and turnover rate of DOM in the ocean. We hypothesize that the active microbial community essentially contributes to the complexity of the DOM pool through uptake and excretion of compounds. We tested this hypothesis in culture experiments with fully-sequenced strains of the Roseobacter clade. Bacteria of the Roseobacter clade are among the most abundant microbial players in the ocean. We studied the exometabolome of two representatives of the Roseobacter clade, Phaeobacter inhibens DSM 17395 and Dinoroseobacter shibae. The organisms were grown separately in cultures on defined single model substrates (acetate, succinate, glutamate, glucose). We used a non-targeted analytical approach via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to characterize the exometabolome at the molecular level, complemented by compound-specific analyses of free and combined amino acids and carbohydrates. The exometabolome composition varied between the tested strains, which released a different suite of compounds depending on the growth phase as well as on growth conditions (substrate). Both organisms exhibited a core exometabolome with compounds released when growing on either substrate and at all growth phases, and a variable exometabolome specific for different substrates and growth phases. However, only a small fraction of the exometabolites detected by FT-ICR-MS could be directly linked to the genome or transcriptome. We interpret these findings as evidence for the excretion of molecularly highly-diverse metabolic waste, whose composition is dependent on the metabolic state and genetic repertoire of the organisms. The molecular diversity of compounds excreted by a single strain is extraordinary and is likely the reason for the molecular diversity of natural DOM in the ocean.

Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

PubMed

Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

2004-03-01

Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

Structural features, substrate specificity, kinetic properties of insect α-amylase and specificity of plant α-amylase inhibitors.

PubMed

Kaur, Rimaljeet; Kaur, Narinder; Gupta, Anil Kumar

2014-11-01

α-Amylase is an important digestive enzyme required for the optimal growth and development of insects. Several insect α-amylases had been purified and their physical and chemical properties were characterized. Insect α-amylases of different orders display variability in structure, properties and substrate specificity. Such diverse properties of amylases could be due to different feeding habits and gut environment of insects. In this review, structural features and properties of several insect α-amylases were compared. This could be helpful in exploring the diversity in characteristics of α-amylase between the members of the same class (insecta). Properties like pH optima are reflected in enzyme structural features. In plants, α-amylase inhibitors (α-AIs) occur as part of natural defense mechanisms against pests by interfering in their digestion process and thus could also provide access to new pest management strategies. AIs are quite specific in their action; therefore, these could be employed according to their effectiveness against target amylases. Potential of transgenics with α-AIs has also been discussed for insect resistance and controlling infestation. The differences in structural features of insect α-amylases provided reasons for their efficient functioning at different pH and the specificity towards various substrates. Various proteinaceous and non-proteinaceous inhibitors discussed could be helpful in controlling pest infestation. In depth detailed studies are required on proteinaceous α-AI-α-amylase interaction at different pH's as well as the insect proteinase action on these inhibitors before selecting the α-AI for making transgenics resistant to particular insect. Copyright © 2014 Elsevier Inc. All rights reserved.

Patterns and Variation in Benthic Biodiversity in a Large Marine Ecosystem

PubMed Central

Lee, Jonathan D.

2015-01-01

While there is a persistent inverse relationship between latitude and species diversity across many taxa and ecosystems, deviations from this norm offer an opportunity to understand the conditions that contribute to large-scale diversity patterns. Marine systems, in particular, provide such an opportunity, as marine diversity does not always follow a strict latitudinal gradient, perhaps because several hypothesized drivers of the latitudinal diversity gradient are uncorrelated in marine systems. We used a large scale public monitoring dataset collected over an eight year period to examine benthic marine faunal biodiversity patterns for the continental shelf (55–183 m depth) and slope habitats (184–1280 m depth) off the US West Coast (47°20′N—32°40′N). We specifically asked whether marine biodiversity followed a strict latitudinal gradient, and if these latitudinal patterns varied across depth, in different benthic substrates, and over ecological time scales. Further, we subdivided our study area into three smaller regions to test whether coast-wide patterns of biodiversity held at regional scales, where local oceanographic processes tend to influence community structure and function. Overall, we found complex patterns of biodiversity on both the coast-wide and regional scales that differed by taxonomic group. Importantly, marine biodiversity was not always highest at low latitudes. We found that latitude, depth, substrate, and year were all important descriptors of fish and invertebrate diversity. Invertebrate richness and taxonomic diversity were highest at high latitudes and in deeper waters. Fish richness also increased with latitude, but exhibited a hump-shaped relationship with depth, increasing with depth up to the continental shelf break, ~200 m depth, and then decreasing in deeper waters. We found relationships between fish taxonomic and functional diversity and latitude, depth, substrate, and time at the regional scale, but not at the coast-wide scale, suggesting that coast-wide patterns can obscure important correlates at smaller scales. Our study provides insight into complex diversity patterns of the deep water soft substrate benthic ecosystems off the US West Coast. PMID:26308521

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

PubMed Central

Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

2014-01-01

ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

Genetic Control and Evolution of Anthocyanin Methylation.

PubMed

Provenzano, Sofia; Spelt, Cornelis; Hosokawa, Satoko; Nakamura, Noriko; Brugliera, Filippa; Demelis, Linda; Geerke, Daan P; Schubert, Andrea; Tanaka, Yoshikazu; Quattrocchio, Francesca; Koes, Ronald

2014-07-01

Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins' structural diversity. © 2014 American Society of Plant Biologists. All Rights Reserved.

Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease.

PubMed

Rampello, Anthony J; Glynn, Steven E

2017-03-24

The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

PubMed

Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof

2011-01-01

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

A perspective on the structural studies of inner membrane electrochemical potential-driven transporters.

PubMed

Lemieux, M Joanne

2008-09-01

Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists.

The active site of O-GlcNAc transferase imposes constraints on substrate sequence

PubMed Central

Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.

2016-01-01

O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE PAGES

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

2015-03-09

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

PubMed Central

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

2015-01-01

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

Terminal Olefin Profiles and Phylogenetic Analyses of Olefin Synthases of Diverse Cyanobacterial Species.

PubMed

Zhu, Tao; Scalvenzi, Thibault; Sassoon, Nathalie; Lu, Xuefeng; Gugger, Muriel

2018-07-01

Cyanobacteria can synthesize alkanes and alkenes, which are considered to be infrastructure-compatible biofuels. In terms of physiological function, cyanobacterial hydrocarbons are thought to be essential for membrane flexibility for cell division, size, and growth. The genetic basis for the biosynthesis of terminal olefins (1-alkenes) is a modular type I polyketide synthase (PKS) termed olefin synthase (Ols). The modular architectures of Ols and structural characteristics of alkenes have been investigated only in a few species of the small percentage (approximately 10%) of cyanobacteria that harbor putative Ols pathways. In this study, investigations of the domains, modular architectures, and phylogenies of Ols in 28 cyanobacterial strains suggested distinctive pathway evolution. Structural feature analyses revealed 1-alkenes with three carbon chain lengths (C 15 , C 17 , and C 19 ). In addition, the total cellular fatty acid profile revealed the diversity of the carbon chain lengths, while the fatty acid feeding assay indicated substrate carbon chain length specificity of cyanobacterial Ols enzymes. Finally, in silico analyses suggested that the N terminus of the modular Ols enzyme exhibited characteristics typical of a fatty acyl-adenylate ligase (FAAL), suggesting a mechanism of fatty acid activation via the formation of acyl-adenylates. Our results shed new light on the diversity of cyanobacterial terminal olefins and a mechanism for substrate activation in the biosynthesis of these olefins. IMPORTANCE Cyanobacterial terminal olefins are hydrocarbons with promising applications as advanced biofuels. Despite the basic understanding of the genetic basis of olefin biosynthesis, the structural diversity and phylogeny of the key modular olefin synthase (Ols) have been poorly explored. An overview of the chemical structural traits of terminal olefins in cyanobacteria is provided in this study. In addition, we demonstrated by in vivo fatty acid feeding assays that cyanobacterial Ols enzymes might exhibit substrate carbon chain length specificity. Furthermore, by performing bioinformatic analyses, we observed that the substrate activation domain of Ols exhibited features typical of a fatty acyl-adenylate ligase (FAAL), which activates fatty acids by converting them to fatty acyl-adenylates. Our results provide further insight into the chemical structures of terminal olefins and further elucidate the mechanism of substrate activation for terminal olefin biosynthesis in cyanobacteria. Copyright © 2018 American Society for Microbiology.

Structure of the Antibiotic Resistance Factor Spectinomycin Phosphotransferase from Legionella pneumophila

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fong, D.; Lemke, C; Huang, J

2010-01-01

Aminoglycoside phosphotransferases (APHs) constitute a diverse group of enzymes that are often the underlying cause of aminoglycoside resistance in the clinical setting. Several APHs have been extensively characterized, including the elucidation of the three-dimensional structure of two APH(3{prime}) isozymes and an APH(2{double_prime}) enzyme. Although many APHs are plasmid-encoded and are capable of inactivating numerous 2-deoxystreptmaine aminoglycosides with multiple regiospecificity, APH(9)-Ia, isolated from Legionella pneumophila, is an unusual enzyme among the APH family for its chromosomal origin and its specificity for a single non-2-deoxystreptamine aminoglycoside substrate, spectinomycin. We describe here the crystal structures of APH(9)-Ia in its apo form, its binarymore » complex with the nucleotide, AMP, and its ternary complex bound with ADP and spectinomycin. The structures reveal that APH(9)-Ia adopts the bilobal protein kinase-fold, analogous to the APH(3{prime}) and APH(2{double_prime}) enzymes. However, APH(9)-Ia differs significantly from the other two types of APH enzymes in its substrate binding area and that it undergoes a conformation change upon ligand binding. Moreover, kinetic assay experiments indicate that APH(9)-Ia has stringent substrate specificity as it is unable to phosphorylate substrates of choline kinase or methylthioribose kinase despite high structural resemblance. The crystal structures of APH(9)-Ia demonstrate and expand our understanding of the diversity of the APH family, which in turn will facilitate the development of new antibiotics and inhibitors.« less

Annexins - scaffolds modulating PKC localization and signaling.

PubMed

Hoque, Monira; Rentero, Carles; Cairns, Rose; Tebar, Francesc; Enrich, Carlos; Grewal, Thomas

2014-06-01

Spatial and temporal organization of signal transduction is critical to link different extracellular stimuli with distinct cellular responses. A classical example of hormones and growth factors creating functional diversity is illustrated by the multiple signaling pathways activated by the protein kinase C (PKC) family of serine/threonine protein kinases. The molecular requirements for diacylglycerol (DAG) and calcium (Ca(2+)) to promote PKC membrane translocation, the hallmark of PKC activation, have been clarified. However, the underlying mechanisms that establish selectivity of individual PKC family members to facilitate differential substrate phosphorylation and varied signal output are still not fully understood. It is now well believed that the coordinated control and functional diversity of PKC signaling involves the formation of PKC isozyme-specific protein complexes in certain subcellular sites. In particular, interaction of PKC isozymes with compartment and signal-organizing scaffolds, including receptors for activated C-kinase (RACKs), A-kinase-anchoring proteins (AKAPs), 14-3-3, heat shock proteins (HSP), and importins target PKC isozymes to specific cellular locations, thereby delivering PKC isozymes into close proximity of their substrates. In addition, several annexins (Anx), including AnxA1, A2, A5 and A6, display specific and distinct abilities to interact and promote membrane targeting of different PKC isozymes. Together with the ability of annexins to create specific membrane microenvironments, this is likely to enable PKCs to phosphorylate certain substrates and regulate their downstream effector pathways in specific cellular sites. This review aims to summarize the capacity of annexins to modulate the localization and activity of PKC family members and participate in the spatiotemporal regulation of PKC signaling in health and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Marine Mesocosm Bacterial Colonisation of Volcanic Ash

NASA Astrophysics Data System (ADS)

Witt, V.; Cimarelli, C.; Ayris, P. M.; Kueppers, U.; Erpenbeck, D.; Dingwell, D. B.; Woerheide, G.

2014-12-01

Explosive volcanic eruptions regularly eject large quantities of ash particles into the atmosphere, which can be deposited via fallout into oceanic environments. Such fallout has the potential to alter pH, light and nutrient availability at local or regional scales. Shallow-water coral reef ecosystems - "rainforests of the sea" - are highly sensitive to disturbances, such as ocean acidification, sedimentation and eutrophication. Therefore, ash deposition may lead to burial and mortality of such reefs. Coral reef ecosystem resilience may depend on pioneer bacterial colonisation of the ash layer, supporting subsequent establishment of the micro- and ultimately the macro-community. However, it is currently unknown which bacteria are involved in pioneer colonisation. We hypothesize that physico-chemical properties (i.e., morphology, chemistry, mineralogy) of the ash may dictate bacterial colonisation. We have tested the effect of substrate properties on bacterial diversity and abundance colonising five substrates: i) quartz sand ii) crystalline ash from the Sakurajima volcano (Japan) iii) volcanic glass iv) carbonate reef sand and v) calcite sand of similar grain size - by incubation in a controlled marine mesocosm (coral reef aquarium) under low light conditions for three months. Bacterial communities were screened every month by Automated Ribosomal Intergenic Spacer Analysis of the 16S-23S rRNA Internal Transcribed Spacer region. Multivariate statistics revealed discrete groupings of bacterial communities on substrates of volcanic origin (ash and glass) and reef origin (three sands). Analysis Of Similarity supports significantly different communities associated with all substrates (p=0.0001), only quartz did not differ from both carbonate and calcite sands. The ash substrate exhibited the most diverse bacterial community and carried the most substrate-specific bacterial operational taxonomic units. Our findings suggest that bacterial diversity and community composition during colonisation of volcanic ash in a coral reef-like environment is controlled by the physico-chemical composition of the substrate. Knowledge on pioneer bacterial colonisation may increase our understanding on the resilience of coral reefs to natural "catastrophes", such as volcanic ash fallout.

Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation

PubMed Central

Chen, Chun-I; Keusch, Jeremy J; Klein, Dominique; Hess, Daniel; Hofsteenge, Jan; Gut, Heinz

2012-01-01

Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the enzyme protein O-fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function. PMID:22588082

Tree species diversity affects decomposition through modified micro-environmental conditions across European forests.

PubMed

Joly, François-Xavier; Milcu, Alexandru; Scherer-Lorenzen, Michael; Jean, Loreline-Katia; Bussotti, Filippo; Dawud, Seid Muhie; Müller, Sandra; Pollastrini, Martina; Raulund-Rasmussen, Karsten; Vesterdal, Lars; Hättenschwiler, Stephan

2017-05-01

Different tree species influence litter decomposition directly through species-specific litter traits, and indirectly through distinct modifications of the local decomposition environment. Whether these indirect effects on decomposition are influenced by tree species diversity is presently not clear. We addressed this question by studying the decomposition of two common substrates, cellulose paper and wood sticks, in a total of 209 forest stands of varying tree species diversity across six major forest types at the scale of Europe. Tree species richness showed a weak but positive correlation with the decomposition of cellulose but not with that of wood. Surprisingly, macroclimate had only a minor effect on cellulose decomposition and no effect on wood decomposition despite the wide range in climatic conditions among sites from Mediterranean to boreal forests. Instead, forest canopy density and stand-specific litter traits affected the decomposition of both substrates, with a particularly clear negative effect of the proportion of evergreen tree litter. Our study suggests that species richness and composition of tree canopies modify decomposition indirectly through changes in microenvironmental conditions. These canopy-induced differences in the local decomposition environment control decomposition to a greater extent than continental-scale differences in macroclimatic conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

PubMed

Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

2015-07-24

The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.

PubMed

Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz

2009-08-25

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.

Tree species, tree genotypes and tree genotypic diversity levels affect microbe-mediated soil ecosystem functions in a subtropical forest.

PubMed

Purahong, Witoon; Durka, Walter; Fischer, Markus; Dommert, Sven; Schöps, Ricardo; Buscot, François; Wubet, Tesfaye

2016-11-18

Tree species identity and tree genotypes contribute to the shaping of soil microbial communities. However, knowledge about how these two factors influence soil ecosystem functions is still lacking. Furthermore, in forest ecosystems tree genotypes co-occur and interact with each other, thus the effects of tree genotypic diversity on soil ecosystem functions merit attention. Here we investigated the effects of tree species, tree genotypes and genotypic diversity levels, alongside soil physicochemical properties, on the overall and specific soil enzyme activity patterns. Our results indicate that tree species identity, tree genotypes and genotypic diversity level have significant influences on overall and specific soil enzyme activity patterns. These three factors influence soil enzyme patterns partly through effects on soil physicochemical properties and substrate quality. Variance partitioning showed that tree species identity, genotypic diversity level, pH and water content all together explained ~30% variations in the overall patterns of soil enzymes. However, we also found that the responses of soil ecosystem functions to tree genotypes and genotypic diversity are complex, being dependent on tree species identity and controlled by multiple factors. Our study highlights the important of inter- and intra-specific variations in tree species in shaping soil ecosystem functions in a subtropical forest.

Tree species, tree genotypes and tree genotypic diversity levels affect microbe-mediated soil ecosystem functions in a subtropical forest

PubMed Central

Purahong, Witoon; Durka, Walter; Fischer, Markus; Dommert, Sven; Schöps, Ricardo; Buscot, François; Wubet, Tesfaye

2016-01-01

Tree species identity and tree genotypes contribute to the shaping of soil microbial communities. However, knowledge about how these two factors influence soil ecosystem functions is still lacking. Furthermore, in forest ecosystems tree genotypes co-occur and interact with each other, thus the effects of tree genotypic diversity on soil ecosystem functions merit attention. Here we investigated the effects of tree species, tree genotypes and genotypic diversity levels, alongside soil physicochemical properties, on the overall and specific soil enzyme activity patterns. Our results indicate that tree species identity, tree genotypes and genotypic diversity level have significant influences on overall and specific soil enzyme activity patterns. These three factors influence soil enzyme patterns partly through effects on soil physicochemical properties and substrate quality. Variance partitioning showed that tree species identity, genotypic diversity level, pH and water content all together explained ~30% variations in the overall patterns of soil enzymes. However, we also found that the responses of soil ecosystem functions to tree genotypes and genotypic diversity are complex, being dependent on tree species identity and controlled by multiple factors. Our study highlights the important of inter- and intra-specific variations in tree species in shaping soil ecosystem functions in a subtropical forest. PMID:27857198

Comparative interactions of withanolides and sterols with two members of sterol glycosyltransferases from Withania somnifera.

PubMed

Pandey, Vibha; Dhar, Yogeshwar Vikram; Gupta, Parul; Bag, Sumit K; Atri, Neelam; Asif, Mehar Hasan; Trivedi, Prabodh Kumar; Misra, Pratibha

2015-04-16

Sterol glycosyltransferases (SGTs) are ubiquitous but one of the most diverse group of enzymes of glycosyltransferases family. Members of this family modulate physical and chemical properties of secondary plant products important for various physiological processes. The role of SGTs has been demonstrated in the biosynthesis of pharmaceutically important molecules of medicinal plants like Withania somnifera. Analysis suggested conserved behaviour and high similarity in active sites of WsSGTs with other plant GTs. Substrate specificity of WsSGTs were analysed through docking performance of WsSGTs with different substrates (sterols and withanolides). Best docking results of WsSGTL1 in the form of stable enzyme-substrate complex having lowest binding energies were obtained with brassicasterol, transandrosteron and WsSGTL4 with solasodine, stigmasterol and 24-methylene cholesterol. This study reveals topological characters and conserved nature of two SGTs from W. somnifera (WsSGTs) i.e. WsSGTL1 and WsSGTL4. However, besides being ubiquitous in nature and with broad substrate specificity, difference between WsSGTL1 and WsSGTL4 is briefly described by difference in stability (binding energy) of enzyme-substrate complexes through comparative docking.

Phylogenetically conserved resource partitioning in the coastal microbial loop

DOE PAGES

Bryson, Samuel; Li, Zhou; Chavez, Francisco; ...

2017-08-11

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populationsmore » did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.« less

Phylogenetically conserved resource partitioning in the coastal microbial loop

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bryson, Samuel; Li, Zhou; Chavez, Francisco

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populationsmore » did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone.« less

Phylogenetically conserved resource partitioning in the coastal microbial loop

PubMed Central

Bryson, Samuel; Li, Zhou; Chavez, Francisco; Weber, Peter K; Pett-Ridge, Jennifer; Hettich, Robert L; Pan, Chongle; Mayali, Xavier; Mueller, Ryan S

2017-01-01

Resource availability influences marine microbial community structure, suggesting that population-specific resource partitioning defines discrete niches. Identifying how resources are partitioned among populations, thereby characterizing functional guilds within the communities, remains a challenge for microbial ecologists. We used proteomic stable isotope probing (SIP) and NanoSIMS analysis of phylogenetic microarrays (Chip-SIP) along with 16S rRNA gene amplicon and metagenomic sequencing to characterize the assimilation of six 13C-labeled common metabolic substrates and changes in the microbial community structure within surface water collected from Monterey Bay, CA. Both sequencing approaches indicated distinct substrate-specific community shifts. However, observed changes in relative abundance for individual populations did not correlate well with directly measured substrate assimilation. The complementary SIP techniques identified assimilation of all six substrates by diverse taxa, but also revealed differential assimilation of substrates into protein and ribonucleotide biomass between taxa. Substrate assimilation trends indicated significantly conserved resource partitioning among populations within the Flavobacteriia, Alphaproteobacteria and Gammaproteobacteria classes, suggesting that functional guilds within marine microbial communities are phylogenetically cohesive. However, populations within these classes exhibited heterogeneity in biosynthetic activity, which distinguished high-activity copiotrophs from low-activity oligotrophs. These results indicate distinct growth responses between populations that is not apparent by genome sequencing alone. PMID:28800138

Exometabolite niche partitioning among sympatric soil bacteria

DOE PAGES

Baran, Richard; Brodie, Eoin L.; Mayberry-Lewis, Jazmine; ...

2015-09-22

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13-26% of available metabolites,more » with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. In conclusion, these results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity.« less

Exometabolite niche partitioning among sympatric soil bacteria

PubMed Central

Baran, Richard; Brodie, Eoin L.; Mayberry-Lewis, Jazmine; Hummel, Eric; Da Rocha, Ulisses Nunes; Chakraborty, Romy; Bowen, Benjamin P.; Karaoz, Ulas; Cadillo-Quiroz, Hinsby; Garcia-Pichel, Ferran; Northen, Trent R.

2015-01-01

Soils are arguably the most microbially diverse ecosystems. Physicochemical properties have been associated with the maintenance of this diversity. Yet, the role of microbial substrate specialization is largely unexplored since substrate utilization studies have focused on simple substrates, not the complex mixtures representative of the soil environment. Here we examine the exometabolite composition of desert biological soil crusts (biocrusts) and the substrate preferences of seven biocrust isolates. The biocrust's main primary producer releases a diverse array of metabolites, and isolates of physically associated taxa use unique subsets of the complex metabolite pool. Individual isolates use only 13−26% of available metabolites, with only 2 out of 470 used by all and 40% not used by any. An extension of this approach to a mesophilic soil environment also reveals high levels of microbial substrate specialization. These results suggest that exometabolite niche partitioning may be an important factor in the maintenance of microbial diversity. PMID:26392107

Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge.

PubMed

Zaiat, M; Vieira, L G; Foresti, E

1997-01-20

This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (r(obs)) under a diversity of external mass transfer resistance conditions. The r(obs) curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters r(max), the maximum specific substrate utilization rate, and K(s), the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30 degrees C resulted in intrinsic r(max) and K(s), respectively, of 0.330 mg chemical oxygen demand (COD) . mg(-1) volatile suspended solids (VSS) . h(-1) and 72 mg COD . L(-1). In comparison with the values found in the literature, intrinsic r(max) is significantly high and intrinsic K(s) is relatively low. (c) 1997 John Wiley & Sons, Inc.

Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

PubMed Central

Poreba, M; Kasperkiewicz, P; Snipas, S J; Fasci, D; Salvesen, G S; Drag, M

2014-01-01

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases – the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time. PMID:24832467

Fabricating nanowire devices on diverse substrates by simple transfer-printing methods.

PubMed

Lee, Chi Hwan; Kim, Dong Rip; Zheng, Xiaolin

2010-06-01

The fabrication of nanowire (NW) devices on diverse substrates is necessary for applications such as flexible electronics, conformable sensors, and transparent solar cells. Although NWs have been fabricated on plastic and glass by lithographic methods, the choice of device substrates is severely limited by the lithographic process temperature and substrate properties. Here we report three new transfer-printing methods for fabricating NW devices on diverse substrates including polydimethylsiloxane, Petri dishes, Kapton tapes, thermal release tapes, and many types of adhesive tapes. These transfer-printing methods rely on the differences in adhesion to transfer NWs, metal films, and devices from weakly adhesive donor substrates to more strongly adhesive receiver substrates. Electrical characterization of fabricated NW devices shows that reliable ohmic contacts are formed between NWs and electrodes. Moreover, we demonstrated that Si NW devices fabricated by the transfer-printing methods are robust piezoresistive stress sensors and temperature sensors with reliable performance.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate

PubMed Central

Malyavantham, Kishore S.; Suresh, Lakshmanan

2018-01-01

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern. PMID:29364249

"Phylogenetic and evolutionary analysis of functional divergence among Gamma glutamyl transpeptidase (GGT) subfamilies".

PubMed

Verma, Ved Vrat; Gupta, Rani; Goel, Manisha

2015-09-14

γ-glutamyltranspeptidase (GGT) is a bi-substrate enzyme conserved in all three domains of life. It catalyzes the cleavage and transfer of γ-glutamyl moiety of glutathione to either water (hydrolysis) or substrates like peptides (transpeptidation). GGTs exhibit great variability in their enzyme kinetics although the mechanism of catalysis is conserved. Recently, GGT has been shown to be a virulence factor in microbes like Helicobacter pylori and Bacillus anthracis. In mammalian cells also, GGT inhibition prior to chemotherapy has been shown to sensitize tumors to the therapy. Therefore, lately both bacterial and eukaryotic GGTs have emerged as potential drug targets, but the efforts directed towards finding suitable inhibitors have not yielded any significant results yet. We propose that delineating the residues responsible for the functional diversity associated with these proteins could help in design of species/clade specific inhibitors. In the present study, we have carried out phylogenetic analysis on a set of 47 GGT-like proteins to address the functional diversity. These proteins segregate into various subfamilies, forming separate clades on the tree. Sequence conservation and motif prediction studies show that even though most of the highly conserved residues have been characterized biochemically in previous studies, a significant number of novel putative sites and motifs are discovered that vary in a clade specific manner. Many of the putative sites predicted during the functional divergence type I and type II analysis, lie close to the known catalytic residues and line the walls of the substrate binding cavity, reinforcing their role in modulating the substrate specificity, catalytic rates and stability of this protein. The study offers interesting insights into the evolution of GGT-like proteins in pathogenic vs. non-pathogenic bacteria, archaea and eukaryotes. Our analysis delineates residues that are highly specific to each GGT subfamily. We propose that these sites not only explain the differences in stability and catalytic variability of various GGTs but can also aid in design of specific inhibitors against particular GGTs. Thus, apart from the commonly used in-silico inhibitor screening approaches, evolutionary analysis identifying the functional divergence hotspots in GGT proteins could augment the structure based drug design approaches.

Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays)

PubMed Central

2005-01-01

Unlike animals which accumulate glutathione (γ-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or γ-glutamyl-L-cysteinyl-β-alanine)], wheat (hydroxymethylglutathione or γ-glutamyl-L-cysteinyl-serine) and maize (γ-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into γ-Glu-Cys. The soya bean enzyme primarily incorporated β-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and α-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor. PMID:16008521

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion.

PubMed

Bibo-Verdugo, Betsaida; O'Donoghue, Anthony J; Rojo-Arreola, Liliana; Craik, Charles S; García-Carreño, Fernando

2016-04-01

Crustaceans are a diverse group, distributed in widely variable environmental conditions for which they show an equally extensive range of biochemical adaptations. Some digestive enzymes have been studied by purification/characterization approaches. However, global analysis is crucial to understand how digestive enzymes interplay. Here, we present the first proteomic analysis of the digestive fluid from a crustacean (Homarus americanus) and identify glycosidases and peptidases as the most abundant classes of hydrolytic enzymes. The digestion pathway of complex carbohydrates was predicted by comparing the lobster enzymes to similar enzymes from other crustaceans. A novel and unbiased substrate profiling approach was used to uncover the global proteolytic specificity of gastric juice and determine the contribution of cysteine and aspartic acid peptidases. These enzymes were separated by gel electrophoresis and their individual substrate specificities uncovered from the resulting gel bands. This new technique is called zymoMSP. Each cysteine peptidase cleaves a set of unique peptide bonds and the S2 pocket determines their substrate specificity. Finally, affinity chromatography was used to enrich for a digestive cathepsin D1 to compare its substrate specificity and cold-adapted enzymatic properties to mammalian enzymes. We conclude that the H. americanus digestive peptidases may have useful therapeutic applications, due to their cold-adaptation properties and ability to hydrolyze collagen.

A single amino acid limits the substrate specificity of Thermus thermophilus uridine-cytidine kinase to cytidine.

PubMed

Tomoike, Fumiaki; Nakagawa, Noriko; Kuramitsu, Seiki; Masui, Ryoji

2011-05-31

The salvage pathways of nucleotide biosynthesis are more diverse and are less well understood as compared with de novo pathways. Uridine-cytidine kinase (UCK) is the rate-limiting enzyme in the pyrimidine-nucleotide salvage pathway. In this study, we have characterized a UCK homologue of Thermus thermophilus HB8 (ttCK) biochemically and structurally. Unlike other UCKs, ttCK had substrate specificity toward only cytidine and showed no inhibition by UTP, suggesting uridine does not bind to ttCK as substrate. Structural analysis revealed that the histidine residue located near the functional group at position 4 of cytidine or uridine in most UCKs is substituted with tyrosine, Tyr93, in ttCK. Replacement of Tyr93 by histidine or glutamine endowed ttCK with phosphorylation activity toward uridine. These results suggested that a single amino acid residue, Tyr93, gives cytidine-limited specificity to ttCK. However, replacement of Tyr93 by Phe or Leu did not change the substrate specificity of ttCK. Therefore, we conclude that a residue at this position is essential for the recognition of uridine by UCK. In addition, thymidine phosphorylase from T. thermophilus HB8 was equally active with thymidine and uridine, which indicates that this protein is the sole enzyme metabolizing uridine in T. Thermophilus HB8. On the basis of these results, we discuss the pyrimidine-salvage pathway in T. thermophilus HB8.

Characterization and Engineering of the Adenylation Domain of a NRPS-Like Protein: A Potential Biocatalyst for Aldehyde Generation

PubMed Central

2015-01-01

The adenylation (A) domain acts as the first “gate-keeper” to ensure the activation and thioesterification of the correct monomer to nonribosomal peptide synthetases (NRPSs). Our understanding of the specificity-conferring code and our ability to engineer A domains are critical for increasing the chemical diversity of nonribosomal peptides (NRPs). We recently discovered a novel NRPS-like protein (ATEG_03630) that can activate 5-methyl orsellinic acid (5-MOA) and reduce it to 2,4-dihydroxy-5,6-dimethyl benzaldehyde. A NRPS-like protein is much smaller than multidomain NRPSs, but it still represents the thioesterification half-reaction, which is otherwise missed from a stand-alone A domain. Therefore, a NRPS-like protein may serve as a better model system for A domain engineering. Here, we characterize the substrate specificity of ATEG_03630 and conclude that the hydrogen-bond donor at the 4-position is crucial for substrate recognition. Next, we show that the substrate specificity of ATEG_03630 can be engineered toward our target substrate anthranilate via bioinformatics analysis and mutagenesis. The resultant mutant H358A increased its activity toward anthranilate by 10.9-fold, which led to a 26-fold improvement in specificity. Finally, we demonstrate one-pot chemoenzymatic synthesis of 4-hydroxybenzaldoxime from 4-hydroxybenzoic acid with high yield. PMID:24804152

Soil functional diversity analysis of a bauxite-mined restoration chronosequence.

PubMed

Lewis, Dawn E; White, John R; Wafula, Denis; Athar, Rana; Dickerson, Tamar; Williams, Henry N; Chauhan, Ashvini

2010-05-01

Soil microorganisms are sensitive to environmental perturbations such that changes in microbial community structure and function can provide early signs of anthropogenic disturbances and even predict restoration success. We evaluated the bacterial functional diversity of un-mined and three chronosequence sites at various stages of rehabilitation (0, 10, and 20 years old) located in the Mocho Mountains of Jamaica. Samples were collected during the dry and wet seasons and analyzed for metal concentrations, microbial biomass carbon, bacterial numbers, and functional responses of soil microbiota using community-level physiological profile (CLPP) assays. Regardless of the season, un-mined soils consisted of higher microbial biomass and numbers than any of the rehabilitated sites. Additionally, the number and rate of substrates utilized and substrate evenness (the distribution of color development between the substrates) were significantly greater in the un-mined soils with carbohydrates being preferentially utilized than amino acids, polymers, carboxylic acids, and esters. To some extent, functional responses varied with the seasons but the least physiological activity was shown by the site rehabilitated in 1987 indicating long-term perturbation to this ecosystem. Small subunit ribosomal DNA (SSUrDNA)-denaturing gradient-gel electrophoresis analyses on the microbiota collected from the most preferred CLPP substrates followed by taxonomic analyses showed Proteobacteria, specifically the gamma-proteobacteria, as the most functionally active phyla, indicating a propensity of this phyla to out-compete other groups under the prevailing conditions. Additionally, multivariate statistical analyses, Shannon's diversity, and evenness indices, principal component analysis, biplot and un-weighted-pair-group method with arithmetic averages dendrograms further confirmed that un-mined sites were distinctly different from the rehabilitated soils.

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

PubMed

Maeda, Hiroshi; Sakai, Daisuke; Kobayashi, Takuji; Morita, Hiroto; Okamoto, Ayako; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei

2016-06-01

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.

PubMed

Wenig, Katja; Chatwell, Lorenz; von Pawel-Rammingen, Ulrich; Björck, Lars; Huber, Robert; Sondermann, Peter

2004-12-14

Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.

PrenDB, a Substrate Prediction Database to Enable Biocatalytic Use of Prenyltransferases.

PubMed

Gunera, Jakub; Kindinger, Florian; Li, Shu-Ming; Kolb, Peter

2017-03-10

Prenyltransferases of the dimethylallyltryptophan synthase (DMATS) superfamily catalyze the attachment of prenyl or prenyl-like moieties to diverse acceptor compounds. These acceptor molecules are generally aromatic in nature and mostly indole or indole-like. Their catalytic transformation represents a major skeletal diversification step in the biosynthesis of secondary metabolites, including the indole alkaloids. DMATS enzymes thus contribute significantly to the biological and pharmacological diversity of small molecule metabolites. Understanding the substrate specificity of these enzymes could create opportunities for their biocatalytic use in preparing complex synthetic scaffolds. However, there has been no framework to achieve this in a rational way. Here, we report a chemoinformatic pipeline to enable prenyltransferase substrate prediction. We systematically catalogued 32 unique prenyltransferases and 167 unique substrates to create possible reaction matrices and compiled these data into a browsable database named PrenDB. We then used a newly developed algorithm based on molecular fragmentation to automatically extract reactive chemical epitopes. The analysis of the collected data sheds light on the thus far explored substrate space of DMATS enzymes. To assess the predictive performance of our virtual reaction extraction tool, 38 potential substrates were tested as prenyl acceptors in assays with three prenyltransferases, and we were able to detect turnover in >55% of the cases. The database, PrenDB (www.kolblab.org/prendb.php), enables the prediction of potential substrates for chemoenzymatic synthesis through substructure similarity and virtual chemical transformation techniques. It aims at making prenyltransferases and their highly regio- and stereoselective reactions accessible to the research community for integration in synthetic work flows. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Consecutive Spray Printing Strategy to Construct and Integrate Diverse Supercapacitors on Various Substrates.

PubMed

Wang, Xinyu; Lu, Qiongqiong; Chen, Chen; Han, Mo; Wang, Qingrong; Li, Haixia; Niu, Zhiqiang; Chen, Jun

2017-08-30

The rapid development of printable electronic devices with flexible and wearable characteristics requires supercapacitor devices to be printable, light, thin, integrated macro- and micro-devices with flexibility. Herein, we developed a consecutive spray printing strategy to controllably construct and integrate diverse supercapacitors on various substrates. In such a strategy, all supercapacitor components are fully printable, and their thicknesses and shapes are well controlled. As a result, supercapacitors obtained by this strategy achieve diverse structures and shapes. In addition, different nanocarbon and pseudocapacitive materials are applicable for the fabrication of these diverse supercapacitors. Furthermore, the diverse supercapacitors can be readily constructed on various objects with planar, curved, or even rough surfaces (e.g., plastic film, glass, cloth, and paper). More importantly, the consecutive spray printing process can integrate several supercapacitors together in the perpendicular and parallel directions of one substrate by designing the structure of electrodes and separators. This enlightens the construction and integration of fully printable supercapacitors with diverse configurations to be compatible with fully printable electronics on various substrates.

The hydrolysis and biogas production of complex cellulosic substrates using three anaerobic biomass sources.

PubMed

Keating, C; Cysneiros, D; Mahony, T; O'Flaherty, V

2013-01-01

In this study, the ability of various sludges to digest a diverse range of cellulose and cellulose-derived substrates was assessed at different temperatures to elucidate the factors affecting hydrolysis. For this purpose, the biogas production was monitored and the specific biogas activity (SBA) of the sludges was employed to compare the performance of three anaerobic sludges on the degradation of a variety of complex cellulose sources, across a range of temperatures. The sludge with the highest performance on complex substrates was derived from a full-scale bioreactor treating sewage at 37 °C. Hydrolysis was the rate-limiting step during the degradation of complex substrates. No activity was recorded for the synthetic cellulose compound carboxymethylcellulose (CMC) using any of the sludges tested. Increased temperature led to an increase in hydrolysis rates and thus SBA values. The non-granular nature of the mesophilic sludge played a positive role in the hydrolysis of solid substrates, while the granular sludges proved more effective on the degradation of soluble compounds.

Structural Diversity in the Dandelion (Taraxacum officinale) Polyphenol Oxidase Family Results in Different Responses to Model Substrates

PubMed Central

Dirks-Hofmeister, Mareike E.; Singh, Ratna; Leufken, Christine M.; Inlow, Jennifer K.; Moerschbacher, Bruno M.

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure–function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a “selector” for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates. PMID:24918587

Structural diversity in the dandelion (Taraxacum officinale) polyphenol oxidase family results in different responses to model substrates.

PubMed

Dirks-Hofmeister, Mareike E; Singh, Ratna; Leufken, Christine M; Inlow, Jennifer K; Moerschbacher, Bruno M

2014-01-01

Polyphenol oxidases (PPOs) are ubiquitous type-3 copper enzymes that catalyze the oxygen-dependent conversion of o-diphenols to the corresponding quinones. In most plants, PPOs are present as multiple isoenzymes that probably serve distinct functions, although the precise relationship between sequence, structure and function has not been addressed in detail. We therefore compared the characteristics and activities of recombinant dandelion PPOs to gain insight into the structure-function relationships within the plant PPO family. Phylogenetic analysis resolved the 11 isoenzymes of dandelion into two evolutionary groups. More detailed in silico and in vitro analyses of four representative PPOs covering both phylogenetic groups were performed. Molecular modeling and docking predicted differences in enzyme-substrate interactions, providing a structure-based explanation for grouping. One amino acid side chain positioned at the entrance to the active site (position HB2+1) potentially acts as a "selector" for substrate binding. In vitro activity measurements with the recombinant, purified enzymes also revealed group-specific differences in kinetic parameters when the selected PPOs were presented with five model substrates. The combination of our enzyme kinetic measurements and the in silico docking studies therefore indicate that the physiological functions of individual PPOs might be defined by their specific interactions with different natural substrates.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates.

PubMed

Khadempour, Lily; Burnum-Johnson, Kristin E; Baker, Erin S; Nicora, Carrie D; Webb-Robertson, Bobbie-Jo M; White, Richard A; Monroe, Matthew E; Huang, Eric L; Smith, Richard D; Currie, Cameron R

2016-11-01

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants' fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats or a mixture of all three. Across all treatments, our analysis identified and quantified 1766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of subcolonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores. © 2016 John Wiley & Sons Ltd.

The fungal cultivar of leaf-cutter ants produces specific enzymes in response to different plant substrates

PubMed Central

Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.; Nicora, Carrie D.; Webb-Robertson, Bobbie-Jo M.; White, Richard A.; Monroe, Matthew E.; Huang, Eric L.; Smith, Richard D.; Currie, Cameron R.

2016-01-01

Herbivores use symbiotic microbes to help derive energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, cultivating their mutualistic fungus Leucoagaricus gongylophorus on plant biomass that workers forage from a diverse collection of plant species. Here, we investigate the metabolic flexibility of the ants’ fungal cultivar for utilizing different plant biomass. Using feeding experiments and a novel approach in metaproteomics, we examine the enzymatic response of L. gongylophorus to leaves, flowers, oats, or a mixture of all three. Across all treatments, our analysis identified and quantified 1,766 different fungal proteins, including 161 putative biomass-degrading enzymes. We found significant differences in the protein profiles in the fungus gardens of sub-colonies fed different plant substrates. When provided with leaves or flowers, which contain the majority of their energy as recalcitrant plant polymers, the fungus gardens produced more proteins predicted to break down cellulose: endoglucanase, exoglucanase, and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, while the mixed substrate treatment closely resembled the treatment with oats alone. This indicates that when provided a mixture of plant substrates, fungus gardens preferentially break down the simpler, more digestible substrates. This flexible, substrate-specific enzymatic response of the fungal cultivar allows leaf-cutter ants to derive energy from a wide range of substrates, which likely contributes to their ability to be dominant generalist herbivores. PMID:27696597

Functions of Calcium-Dependent Protein Kinases in Plant Innate Immunity

PubMed Central

Gao, Xiquan; Cox, Kevin L.; He, Ping

2014-01-01

An increase of cytosolic Ca2+ is generated by diverse physiological stimuli and stresses, including pathogen attack. Plants have evolved two branches of the immune system to defend against pathogen infections. The primary innate immune response is triggered by the detection of evolutionarily conserved pathogen-associated molecular pattern (PAMP), which is called PAMP-triggered immunity (PTI). The second branch of plant innate immunity is triggered by the recognition of specific pathogen effector proteins and known as effector-triggered immunity (ETI). Calcium (Ca2+) signaling is essential in both plant PTI and ETI responses. Calcium-dependent protein kinases (CDPKs) have emerged as important Ca2+ sensor proteins in transducing differential Ca2+ signatures, triggered by PAMPs or effectors and activating complex downstream responses. CDPKs directly transmit calcium signals by calcium binding to the elongation factor (EF)-hand domain at the C-terminus and substrate phosphorylation by the catalytic kinase domain at the N-terminus. Emerging evidence suggests that specific and overlapping CDPKs phosphorylate distinct substrates in PTI and ETI to regulate diverse plant immune responses, including production of reactive oxygen species, transcriptional reprogramming of immune genes, and the hypersensitive response. PMID:27135498

Microbial diversity arising from thermodynamic constraints

PubMed Central

Großkopf, Tobias; Soyer, Orkun S

2016-01-01

The microbial world displays an immense taxonomic diversity. This diversity is manifested also in a multitude of metabolic pathways that can utilise different substrates and produce different products. Here, we propose that these observations directly link to thermodynamic constraints that inherently arise from the metabolic basis of microbial growth. We show that thermodynamic constraints can enable coexistence of microbes that utilise the same substrate but produce different end products. We find that this thermodynamics-driven emergence of diversity is most relevant for metabolic conversions with low free energy as seen for example under anaerobic conditions, where population dynamics is governed by thermodynamic effects rather than kinetic factors such as substrate uptake rates. These findings provide a general understanding of the microbial diversity based on the first principles of thermodynamics. As such they provide a thermodynamics-based framework for explaining the observed microbial diversity in different natural and synthetic environments. PMID:27035705

Direct growth of cerium oxide nanorods on diverse substrates for superhydrophobicity and corrosion resistance

NASA Astrophysics Data System (ADS)

Cho, Young Jun; Jang, Hanmin; Lee, Kwan-Soo; Kim, Dong Rip

2015-06-01

Superhydrophobic surfaces with anti-corrosion properties have attracted great interest in many industrial fields, particularly to enhance the thermal performance of offshore applications such as heat exchangers, pipelines, power plants, and platform structures. Nanostructures with hydrophobic materials have been widely utilized to realize superhydrophobicity of surfaces, and cerium oxide has been highlighted due to its good corrosion resistive and intrinsically hydrophobic properties. However, few studies of direct growth of cerium oxide nanostructures on diverse substrates have been reported. Herein we report a facile hydrothermal method to directly grow cerium oxide nanorods on diverse substrates, such as aluminum alloy, stainless steel, titanium, and silicon. Diverse substrates with cerium oxide nanorods exhibited superhydrophobicity with no hydrophobic modifiers on their surfaces, and showed good corrosion resistive properties in corrosive medium. We believe our method could pave the way for realization of scalable and sustainable corrosion resistive superhydrophobic surfaces in many industrial fields.

Peptide interfaces with graphene: an emerging intersection of analytical chemistry, theory, and materials.

PubMed

Russell, Shane R; Claridge, Shelley A

2016-04-01

Because noncovalent interface functionalization is frequently required in graphene-based devices, biomolecular self-assembly has begun to emerge as a route for controlling substrate electronic structure or binding specificity for soluble analytes. The remarkable diversity of structures that arise in biological self-assembly hints at the possibility of equally diverse and well-controlled surface chemistry at graphene interfaces. However, predicting and analyzing adsorbed monolayer structures at such interfaces raises substantial experimental and theoretical challenges. In contrast with the relatively well-developed monolayer chemistry and characterization methods applied at coinage metal surfaces, monolayers on graphene are both less robust and more structurally complex, levying more stringent requirements on characterization techniques. Theory presents opportunities to understand early binding events that lay the groundwork for full monolayer structure. However, predicting interactions between complex biomolecules, solvent, and substrate is necessitating a suite of new force fields and algorithms to assess likely binding configurations, solvent effects, and modulations to substrate electronic properties. This article briefly discusses emerging analytical and theoretical methods used to develop a rigorous chemical understanding of the self-assembly of peptide-graphene interfaces and prospects for future advances in the field.

Synergetic effect of pH and biochemical components on bacterial diversity during mesophilic anaerobic fermentation of biomass-origin waste.

PubMed

Lü, F; Shao, L M; Bru, V; Godon, J J; He, P J

2009-02-01

To investigate the synergetic effect of pH and biochemical components on bacterial community structure during mesophilic anaerobic degradation of solid wastes with different origins, and under acidic or neutral conditions. The bacterial community in 16 samples of solid wastes with different biochemical compositions and origins was evaluated during mesophilic anaerobic degradation at acidic and neutral pH. Denaturing gradient gel electrophoresis (DGGE) and single-strand conformation polymorphism (SSCP) were used to compare the communities. Multivariate analysis of the DGGE and SSCP results revealed that most of the dominant microbes were dependent on the content of easily degradable carbohydrates in the samples. Furthermore, the dominant microbes were divided into two types, those that preferred an acid environment and those that preferred a neutral environment. A shift in pH was found to change their preference for medium substrates. Although most of the substrates with similar origin and biochemical composition had similar microbial diversity during fermentation, some microbes were found only in substrates with specific origins. For example, two microbes were only found in substrate that contained lignocellulose and animal protein without starch. These microbes were related to micro-organisms that are found in swine manure, as well as in other intestinal or oral niches. In addition, the distribution of fermentation products was less sensitive to the changes in pH and biochemical components than the microbial community. Bacterial diversity during anaerobic degradation of organic wastes was affected by both pH and biochemical components; however, pH exerted a greater effect. The results of this study reveal that control of pH may be an effective method to produce a stable bacterial community and relatively similar product distribution during anaerobic digestion of waste, regardless of variation in the waste feedstocks.

Diversity and Evolution of Bacterial Twin Arginine Translocase Protein, TatC, Reveals a Protein Secretion System That Is Evolving to Fit Its Environmental Niche

PubMed Central

Simone, Domenico; Bay, Denice C.; Leach, Thorin; Turner, Raymond J.

2013-01-01

Background The twin-arginine translocation (Tat) protein export system enables the transport of fully folded proteins across a membrane. This system is composed of two integral membrane proteins belonging to TatA and TatC protein families and in some systems a third component, TatB, a homolog of TatA. TatC participates in substrate protein recognition through its interaction with a twin arginine leader peptide sequence. Methodology/Principal Findings The aim of this study was to explore TatC diversity, evolution and sequence conservation in bacteria to identify how TatC is evolving and diversifying in various bacterial phyla. Surveying bacterial genomes revealed that 77% of all species possess one or more tatC loci and half of these classes possessed only tatC and tatA genes. Phylogenetic analysis of diverse TatC homologues showed that they were primarily inherited but identified a small subset of taxonomically unrelated bacteria that exhibited evidence supporting lateral gene transfer within an ecological niche. Examination of bacilli tatCd/tatCy isoform operons identified a number of known and potentially new Tat substrate genes based on their frequent association to tatC loci. Evolutionary analysis of these Bacilli isoforms determined that TatCy was the progenitor of TatCd. A bacterial TatC consensus sequence was determined and highlighted conserved and variable regions within a three dimensional model of the Escherichia coli TatC protein. Comparative analysis between the TatC consensus sequence and Bacilli TatCd/y isoform consensus sequences revealed unique sites that may contribute to isoform substrate specificity or make TatA specific contacts. Synonymous to non-synonymous nucleotide substitution analyses of bacterial tatC homologues determined that tatC sequence variation differs dramatically between various classes and suggests TatC specialization in these species. Conclusions/Significance TatC proteins appear to be diversifying within particular bacterial classes and its specialization may be driven by the substrates it transports and the environment of its host. PMID:24236045

Dynamics of Preferential Substrate Recognition in HIV-1 Protease: Redefining the Substrate Envelope

PubMed Central

Özen, Ayşegül; Haliloğlu, Türkan; Schiffer, Celia A.

2011-01-01

HIV-1 protease (PR) permits viral maturation by processing the Gag and Gag-Pro-Pol polyproteins. Though HIV-1 PR inhibitors (PIs) are used in combination antiviral therapy, the emergence of drug resistance has limited their efficacy. The rapid evolution of HIV-1 necessitates the consideration of drug resistance in novel drug-design strategies. Drug-resistant HIV-1 PR variants, while no longer efficiently inhibited, continue to efficiently hydrolyze the natural viral substrates. Though highly diverse in sequence, the HIV-1 PR substrates bind in a conserved three-dimensional shape we defined as the “substrate envelope”. We previously showed that resistance mutations arise where PIs protrude beyond the substrate envelope, as these regions are crucial for drug binding but not for substrate recognition. Here, we extend this model by considering the role of protein dynamics in the interaction of HIV-1 PR with its substrates. Seven molecular dynamics simulations of PR-substrate complexes were performed to estimate the conformational flexibility of substrates in their complexes. Interdependency of the substrate-protease interactions may compensate for the variations in cleavage-site sequences, and explain how a diverse set of sequences can be recognized as substrates by the same enzyme. This diversity may be essential for regulating sequential processing of substrates. We also define a dynamic substrate envelope as a more accurate representation of PR-substrate interactions. This dynamic substrate envelope, described by a probability distribution function, is a powerful tool for drug design efforts targeting ensembles of resistant HIV-1 PR variants with the aim of developing drugs that are less susceptible to resistance. PMID:21762811

The contributions of forest structure and substrate to bryophyte diversity and abundance in mature coniferous forests of the Pacific Northwest

Treesearch

Shelley A. Evans; Charles B. Halpern; Donald McKenzie

2012-01-01

Many aspects of forest structure are thought to contribute to the presence, abundance, and diversity of forest-floor bryophytes. To what extent easily measured characteristics of local environment (overstory structure or substrate availability) explain patterns of abundance and diversity remains unclear in most forest ecosystems. We explore these relationships in four...

Initial nitrogen enrichment conditions determines variations in nitrogen substrate utilization by heterotrophic bacterial isolates.

PubMed

Ghosh, Suchismita; Ayayee, Paul A; Valverde-Barrantes, Oscar J; Blackwood, Christopher B; Royer, Todd V; Leff, Laura G

2017-04-04

The nitrogen (N) cycle consists of complex microbe-mediated transformations driven by a variety of factors, including diversity and concentrations of N compounds. In this study, we examined taxonomic diversity and N substrate utilization by heterotrophic bacteria isolated from streams under complex and simple N-enrichment conditions. Diversity estimates differed among isolates from the enrichments, but no significant composition were detected. Substrate utilization and substrate range of bacterial assemblages differed within and among enrichments types, and not simply between simple and complex N-enrichments. N substrate use patterns differed between isolates from some complex and simple N-enrichments while others were unexpectedly similar. Taxonomic composition of isolates did not differ among enrichments and was unrelated to N use suggesting strong functional redundancy. Ultimately, our results imply that the available N pool influences physiology and selects for bacteria with various abilities that are unrelated to their taxonomic affiliation.

The role of macrobiota in structuring microbial communities along rocky shores

DOE PAGES

Pfister, Catherine A.; Gilbert, Jack A.; Gibbons, Sean M.

2014-10-16

Rocky shore microbial diversity presents an excellent system to test for microbial habitat specificity or generality, enabling us to decipher how common macrobiota shape microbial community structure. At two coastal locations in the northeast Pacific Ocean, we show that microbial composition was significantly different between inert surfaces, the biogenic surfaces that included rocky shore animals and an alga, and the water column plankton. While all sampled entities had a core of common OTUs, rare OTUs drove differences among biotic and abiotic substrates. For the mussel Mytilus californianus, the shell surface harbored greater alpha diversity compared to internal tissues of themore » gill and siphon. Strikingly, a 7-year experimental removal of this mussel from tidepools did not significantly alter the microbial community structure of microbes associated with inert surfaces when compared with unmanipulated tidepools. However, bacterial taxa associated with nitrate reduction had greater relative abundance with mussels present, suggesting an impact of increased animal-derived nitrogen on a subset of microbial metabolism. Because the presence of mussels did not affect the structure and diversity of the microbial community on adjacent inert substrates, microbes in this rocky shore environment may be predominantly affected through direct physical association with macrobiota.« less

The role of macrobiota in structuring microbial communities along rocky shores

PubMed Central

Gilbert, Jack A.; Gibbons, Sean M.

2014-01-01

Rocky shore microbial diversity presents an excellent system to test for microbial habitat specificity or generality, enabling us to decipher how common macrobiota shape microbial community structure. At two coastal locations in the northeast Pacific Ocean, we show that microbial composition was significantly different between inert surfaces, the biogenic surfaces that included rocky shore animals and an alga, and the water column plankton. While all sampled entities had a core of common OTUs, rare OTUs drove differences among biotic and abiotic substrates. For the mussel Mytilus californianus, the shell surface harbored greater alpha diversity compared to internal tissues of the gill and siphon. Strikingly, a 7-year experimental removal of this mussel from tidepools did not significantly alter the microbial community structure of microbes associated with inert surfaces when compared with unmanipulated tidepools. However, bacterial taxa associated with nitrate reduction had greater relative abundance with mussels present, suggesting an impact of increased animal-derived nitrogen on a subset of microbial metabolism. Because the presence of mussels did not affect the structure and diversity of the microbial community on adjacent inert substrates, microbes in this rocky shore environment may be predominantly affected through direct physical association with macrobiota. PMID:25337459

Microbial community changes as a possible factor controlling carbon sequestration in subsoil

NASA Astrophysics Data System (ADS)

Strücker, Juliane; Jörgensen, Rainer Georg

2015-04-01

In order to gain more knowledge regarding the microbial community and their influence on carbon sequestration in subsoil two depth profiles with different soil organic carbon (SOC) concentrations were sampled. The SOC concentrations developed naturally due to deposition and erosion. This experiment offers the opportunity to investigate to which extend natural SOC availability or other subsoil specific conditions influence the composition and the functional diversity of the microbial community and in return if there is any evidence how the microbial community composition affects carbon sequestration under these conditions. Soil samples were taken at four different depths on two neighbouring arable sites; one Kolluvisol with high SOC concentrations (8-12 g/kg) throughout the profile and one Luvisol with low SOC concentrations (3-4 g/kg) below 30 cm depth. The multi substrate induced respiration (MSIR) method was used to identify shifts in the functional diversity of the microbial community along the depth profiles. Amino sugars Muramic Acid and Glucosamine were measured as indicators for bacterial and fungal residues and ergosterol was determined as marker for saprotrophic fungi. The results of the discriminant analysis of the respiration values obtained from the 17 substrates used in the MSIR show that the substrate use in subsoil is different from the substrate use in topsoil. The amino sugar analysis and the ratio of ergosterol to microbial biomass C indicate that the fungal dominance of the microbial community decreases with depth. The results from this study support previous findings, which also observed decreasing fungal dominance with depth. Furthermore the MSIR approach shows clearly that not only the composition of the microbial community but also their substrate use changes with depth. Thus, a different microbial community with altered substrate requirements could be an important reason for enhanced carbon sequestration in subsoil. The fact that the MSIR was also able to differentiate between the two sites proves the assumption that resources are an important factor controlling the functional diversity of the microbial community, as abiotic factors are very similar for the two profiles, but the sites show a different depth gradient for SOC.

A novel esterase gene cloned from a metagenomic library from neritic sediments of the South China Sea

PubMed Central

2011-01-01

Background Marine microbes are a large and diverse group, which are exposed to a wide variety of pressure, temperature, salinity, nutrient availability and other environmental conditions. They provide a huge potential source of novel enzymes with unique properties that may be useful in industry and biotechnology. To explore the lipolytic genetic resources in the South China Sea, 23 sediment samples were collected in the depth < 100 m marine areas. Results A metagenomic library of South China Sea sediments assemblage in plasmid vector containing about 194 Mb of community DNA was prepared. Screening of a part of the unamplified library resulted in isolation of 15 unique lipolytic clones with the ability to hydrolyze tributyrin. A positive recombinant clone (pNLE1), containing a novel esterase (Est_p1), was successfully expressed in E. coli and purified. In a series of assays, Est_p1 displayed maximal activity at pH 8.57, 40°C, with ρ-Nitrophenyl butyrate (C4) as substrate. Compared to other metagenomic esterases, Est_p1 played a notable role in specificity for substrate C4 (kcat/Km value 11,500 S-1m M-1) and showed no inhibited by phenylmethylsulfonyl fluoride, suggested that the substrate binding pocket was suitable for substrate C4 and the serine active-site residue was buried at the bottom of substrate binding pocket which sheltered by a lid structure. Conclusions Esterase, which specificity towards short chain fatty acids, especially butanoic acid, is commercially available as potent flavoring tools. According the outstanding activity and specificity for substrate C4, Est_p1 has potential application in flavor industries requiring hydrolysis of short chain esters. PMID:22067554

Evolution of Substrate Specificity within a Diverse Family of [beta/alpha]-Barrel-fold Basic Amino Acid Decarboxylases X-ray Structure Determination of Enzymes with Specificity for L-Arginine and Carboxynorspermidine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Xiaoyi; Lee, Jeongmi; Michael, Anthony J.

2010-08-26

Pyridoxal 5{prime}-phosphate (PLP)-dependent basic amino acid decarboxylases from the {beta}/{alpha}-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-{angstrom} resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the {beta}/{alpha}-barrel domain of one subunit and the {beta}-barrel of the other. ADC contains bothmore » a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the {beta}-barrel domain we refer to as the 'specificity helix.' Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu{sup 314}, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5{prime}-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.« less

Cys-X scanning for expansion of active-site residues and modulation of catalytic functions in a glutathione transferase.

PubMed

Norrgård, Malena A; Hellman, Ulf; Mannervik, Bengt

2011-05-13

We propose Cys-X scanning as a semisynthetic approach to engineer the functional properties of recombinant proteins. As in the case of Ala scanning, key residues in the primary structure are identified, and one of them is replaced by Cys via site-directed mutagenesis. The thiol of the residue introduced is subsequently modified by alternative chemical reagents to yield diverse Cys-X mutants of the protein. This chemical approach is orthogonal to Ala or Cys scanning and allows the expansion of the repertoire of amino acid side chains far beyond those present in natural proteins. In its present application, we have introduced Cys-X residues in human glutathione transferase (GST) M2-2, replacing Met-212 in the substrate-binding site. To achieve selectivity of the modifications, the Cys residues in the wild-type enzyme were replaced by Ala. A suite of simple substitutions resulted in a set of homologous Met derivatives ranging from normethionine to S-heptyl-cysteine. The chemical modifications were validated by HPLC and mass spectrometry. The derivatized mutant enzymes were assayed with alternative GST substrates representing diverse chemical reactions: aromatic substitution, epoxide opening, transnitrosylation, and addition to an ortho-quinone. The Cys substitutions had different effects on the alternative substrates and differentially enhanced or suppressed catalytic activities depending on both the Cys-X substitution and the substrate assayed. As a consequence, the enzyme specificity profile could be changed among the alternative substrates. The procedure lends itself to large-scale production of Cys-X modified protein variants.

The molecular response of the white-rot fungus Dichomitus squalens to wood and non-woody biomass as examined by transcriptome and exoproteome analyses.

PubMed

Rytioja, Johanna; Hildén, Kristiina; Di Falco, Marcos; Zhou, Miaomiao; Aguilar-Pontes, Maria Victoria; Sietiö, Outi-Maaria; Tsang, Adrian; de Vries, Ronald P; Mäkelä, Miia R

2017-03-01

The ability to obtain carbon and energy is a major requirement to exist in any environment. For several ascomycete fungi, (post-)genomic analyses have shown that species that occupy a large variety of habitats possess a diverse enzymatic machinery, while species with a specific habitat have a more focused enzyme repertoire that is well-adapted to the prevailing substrate. White-rot basidiomycete fungi also live in a specific habitat, as they are found exclusively in wood. In this study, we evaluated how well the enzymatic machinery of the white-rot fungus Dichomitus squalens is tailored to degrade its natural wood substrate. The transcriptome and exoproteome of D. squalens were analyzed after cultivation on two natural substrates, aspen and spruce wood, and two non-woody substrates, wheat bran and cotton seed hulls. D. squalens produced ligninolytic enzymes mainly at the early time point of the wood cultures, indicating the need to degrade lignin to get access to wood polysaccharides. Surprisingly, the response of the fungus to the non-woody polysaccharides was nearly as good a match to the substrate composition as observed for the wood polysaccharides. This indicates that D. squalens has preserved its ability to efficiently degrade plant biomass types not present in its natural habitat. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

PubMed

Lohman, Jeremy R; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N; Shen, Ben

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

PubMed Central

Lohman, Jeremy R.; Ma, Ming; Osipiuk, Jerzy; Nocek, Boguslaw; Kim, Youngchang; Chang, Changsoo; Cuff, Marianne; Mack, Jamey; Bigelow, Lance; Li, Hui; Endres, Michael; Babnigg, Gyorgy; Joachimiak, Andrzej; Phillips, George N.; Shen, Ben

2015-01-01

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs. PMID:26420866

Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE

PubMed Central

Wang, Jianmin; Liu, Yingli; Tang, Lijuan; Qi, Sufen; Mi, Yingjun; Liu, Dianwu; Tian, Qingbao

2017-01-01

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spec troscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13. PMID:28498477

Temperature, inocula and substrate: Contrasting electroactive consortia, diversity and performance in microbial fuel cells.

PubMed

Heidrich, E S; Dolfing, J; Wade, M J; Sloan, W T; Quince, C; Curtis, T P

2018-02-01

The factors that affect microbial community assembly and its effects on the performance of bioelectrochemical systems are poorly understood. Sixteen microbial fuel cell (MFC) reactors were set up to test the importance of inoculum, temperature and substrate: Arctic soil versus wastewater as inoculum; warm (26.5°C) versus cold (7.5°C) temperature; and acetate versus wastewater as substrate. Substrate was the dominant factor in determining performance and diversity: unexpectedly the simple electrogenic substrate delivered a higher diversity than a complex wastewater. Furthermore, in acetate fed reactors, diversity did not correlate with performance, yet in wastewater fed ones it did, with greater diversity sustaining higher power densities and coulombic efficiencies. Temperature had only a minor effect on power density, (Q 10 : 2 and 1.2 for acetate and wastewater respectively): this is surprising given the well-known temperature sensitivity of anaerobic bioreactors. Reactors were able to operate at low temperature with real wastewater without the need for specialised inocula; it is speculated that MFC biofilms may have a self-heating effect. Importantly, the warm acetate fed reactors in this study did not act as direct model for cold wastewater fed systems. Application of this technology will encompass use of real wastewater at ambient temperatures. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Quantitative isotope incorporation reveals substrate partitioning in a coastal microbial community.

PubMed

Mayali, Xavier; Weber, Peter K

2018-05-01

To quantitatively link microbial identity with biogeochemical function, we carried out 14 simultaneous stable isotope probing experiments with organic and inorganic C and N substrates to measure the isotope incorporation by over one hundred co-occurring eukaryotic and prokaryotic populations in a coastal community. We found that nitrate was the most commonly incorporated substrate, and that light-driven carbon fixation was carried out by some bacterial taxa from the Flavobacteriales and OM60 (NOR5) clade, in addition to photoautotrophic phytoplankton. We found that organisms that incorporated starch, maltose, glucose, lactose and bicarbonate were phylogenetically clustered, suggesting that specific bacterial lineages specialized in the incorporation of these substrates. The data further revealed that coastal microorganisms spanned a range of resource utilization strategies from generalists to specialists and demonstrated a high level of substrate partitioning, with two thirds of taxa exhibiting unique substrate incorporation patterns and the remaining third shared by no more than three OTUs each. Specialists exhibited more extreme incorporation levels (high or low), whereas generalists displayed more intermediate activity levels. These results shed valuable insights into the bottom-up ecological strategies enabling the persistence of high microbial diversity in aquatic ecosystems.

Solid state fermentation (SSF): diversity of applications to valorize waste and biomass.

PubMed

Lizardi-Jiménez, M A; Hernández-Martínez, R

2017-05-01

Solid state fermentation is currently used in a range of applications including classical applications, such as enzyme or antibiotic production, recently developed products, such as bioactive compounds and organic acids, new trends regarding bioethanol and biodiesel as sources of alternative energy, and biosurfactant molecules with environmental purposes of valorising unexploited biomass. This work summarizes the diversity of applications of solid state fermentation to valorize biomass regarding alternative energy and environmental purposes. The success of applying solid state fermentation to a specific process is affected by the nature of specific microorganisms and substrates. An exhaustive number of microorganisms able to grow in a solid matrix are presented, including fungus such as Aspergillus or Penicillum for antibiotics, Rhizopus for bioactive compounds, Mortierella for biodiesel to bacteria, Bacillus for biosurfactant production, or yeast for bioethanol.

Hypersusceptibility to substrate analogs conferred by mutations in human immunodeficiency virus type 1 reverse transcriptase.

PubMed

Smith, Robert A; Anderson, Donovan J; Preston, Bradley D

2006-07-01

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) contains four structural motifs (A, B, C, and D) that are conserved in polymerases from diverse organisms. Motif B interacts with the incoming nucleotide, the template strand, and key active-site residues from other motifs, suggesting that motif B is an important determinant of substrate specificity. To examine the functional role of this region, we performed "random scanning mutagenesis" of 11 motif B residues and screened replication-competent mutants for altered substrate analog sensitivity in culture. Single amino acid replacements throughout the targeted region conferred resistance to lamivudine and/or hypersusceptibility to zidovudine (AZT). Substitutions at residue Q151 increased the sensitivity of HIV-1 to multiple nucleoside analogs, and a subset of these Q151 variants was also hypersusceptible to the pyrophosphate analog phosphonoformic acid (PFA). Other AZT-hypersusceptible mutants were resistant to PFA and are therefore phenotypically similar to PFA-resistant variants selected in vitro and in infected patients. Collectively, these data show that specific amino acid replacements in motif B confer broad-spectrum hypersusceptibility to substrate analog inhibitors. Our results suggest that motif B influences RT-deoxynucleoside triphosphate interactions at multiple steps in the catalytic cycle of polymerization.

Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation.

PubMed

Brandt, Martin; Szewczuk, Lawrence M; Zhang, Hong; Hong, Xuan; McCormick, Patricia M; Lewis, Tia S; Graham, Taylor I; Hung, Sunny T; Harper-Jones, Amber D; Kerrigan, John J; Wang, Da-Yuan; Dul, Edward; Hou, Wangfang; Ho, Thau F; Meek, Thomas D; Cheung, Mui H; Johanson, Kyung O; Jones, Christopher S; Schwartz, Benjamin; Kumar, Sanjay; Oliff, Allen I; Kirkpatrick, Robert B

2013-06-01

Small ubiquitin-like modifier (SUMO) belongs to the family of ubiquitin-like proteins (Ubls) that can be reversibly conjugated to target-specific lysines on substrate proteins. Although covalently sumoylated products are readily detectible in gel-based assays, there has been little progress toward the development of robust quantitative sumoylation assay formats for the evaluation of large compound libraries. In an effort to identify inhibitors of ubiquitin carrier protein 9 (Ubc9)-dependent sumoylation, a high-throughput fluorescence polarization assay was developed, which allows detection of Lys-1201 sumoylation, corresponding to the major site of functional sumoylation within the transcriptional repressor trichorhino-phalangeal syndrome type I protein (TRPS1). A minimal hexapeptide substrate peptide, TMR-VVK₁₂₀₁TEK, was used in this assay format to afford high-throughput screening of the GlaxoSmithKline diversity compound collection. A total of 728 hits were confirmed but no specific noncovalent inhibitors of Ubc9 dependent trans-sumoylation were found. However, several diaminopyrimidine compounds were identified as inhibitors in the assay with IC₅₀ values of 12.5 μM. These were further characterized to be competent substrates which were subject to sumoylation by SUMO-Ubc9 and which were competitive with the sumoylation of the TRPS1 peptide substrates.

Marine mesocosm bacterial colonisation of volcanic ash

NASA Astrophysics Data System (ADS)

Witt, Verena; Cimarelli, Corrado; Ayris, Paul; Kueppers, Ulrich; Erpenbeck, Dirk; Dingwell, Donald; Woerheide, Gert

2015-04-01

Volcanic eruptions regularly eject large quantities of ash particles into the atmosphere, which can be deposited via fallout into oceanic environments. Such fallout has the potential to alter pH, light and nutrient availability at local scales. Shallow-water coral reef ecosystems - "rainforests of the sea" - are highly sensitive to disturbances, such as ocean acidification, sedimentation and eutrophication. Therefore, wind-delivered volcanic ash may lead to burial and mortality of such reefs. Coral reef ecosystem resilience may depend on pioneer bacterial colonisation of the ash layer, supporting subsequent establishment of the micro- and ultimately the macro-community. However, which bacteria are involved in pioneer colonisation remain unknown. We hypothesize that physico-chemical properties (i.e., morphology, mineralogy) of the ash may dictate bacterial colonisation. The effect of substrate properties on bacterial colonisation was tested by exposing five substrates: i) quartz sand ii) crystalline ash (Sakurajima, Japan) iii) volcanic glass iv) carbonate reef sand and v) calcite sand of similar grain size, in controlled marine coral reef aquaria under low light conditions for six months. Bacterial communities were screened every month by Automated Ribosomal Intergenic Spacer Analysis of the 16S-23S rRNA Internal Transcribed Spacer region. Multivariate statistics revealed discrete groupings of bacterial communities on substrates of volcanic origin (ash and glass) and reef origin (three sands). Analysis of Similarity supported significantly different communities associated with all substrates (p=0.0001), only quartz did not differ from both carbonate and calcite sands. The ash substrate exhibited the most diverse bacterial community with the most substrate-specific bacterial operational taxonomic units. Our findings suggest that bacterial diversity and community composition during colonisation of volcanic ash in a coral reef-like environment is controlled by the physico-chemical composition of the substrate. Knowledge on pioneer bacterial colonisation may increase our understanding on the resilience of coral reefs to natural "catastrophes", such as volcanic ash fallout.

Functional characterization of a novel β-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697 on structurally diverse fructans.

PubMed

Ávila-Fernández, Á; Cuevas-Juárez, E; Rodríguez-Alegría, M E; Olvera, C; López-Munguía, A

2016-07-01

In this study, we describe the isolation of a gene encoding a novel β-fructofuranosidase from Bifidobacterium longum subsp. infantis ATCC 15697, and the characterization of the enzyme, the second one found in this strain, significantly different in primary sequence to the already reported bifidobacterial β-fructofuranosidases. The gene, found through genome-mining was expressed in Escherichia coli C41(DE3). The recombinant enzyme (B.longum_l1) has a molecular weight of 75 kDa, with optimal activity at 50°C, pH 6·0-6·5, and a remarkable stability with a half-life of 75·5 h at 50°C. B.longum_l1 has a wide specificity for fructans, hydrolysing all substrates through an exo-type mechanism, including Oligofructose P95 (β2-1 fructooligosaccharides (FOS), DP 2-8), Raftilose Synergy 1(β2-1 FOS & inulin, DP 2-60), Raftiline HP (inulin, DP 2-60), bacterial inulin (3000 kDa) and levan (8·3 & 3500 kDa), Agave fructans (mixed fructans, DP 3-29) and levan-type FOS (β2-6 FOS, DP 2-8), with the highest relative activity and turnover number found for levan-type FOS. The apparent affinity of the enzyme for levan-type FOS and Oligofructose P95 was found to be 9·2 and 4·6 mmol l(-1) (Km ) with a specific activity of 908 and 725 μmol min(-1)  mg(-1) of protein (k2 ), respectively, more than twice the activity for sucrose. B.longum_l1 is a wide substrate specificity enzyme, which may contribute to the competitiveness and persistence of this strain in the colon. The bifidobacterial β-fructofuranosidase activity was evaluated with a wide variety of substrates including noncommercial fructans, such as levan-type and mixed agave fructans. Its activity on these substrates certainly strengthens their commercial prebiotic character and contributes to the understanding of bifidobacteria stimulation by structurally diverse fructans. © 2016 The Society for Applied Microbiology.

Tilt Grain Boundary Topology Induced by Substrate Topography.

PubMed

Yu, Henry; Gupta, Nitant; Hu, Zhili; Wang, Kai; Srijanto, Bernadeta R; Xiao, Kai; Geohegan, David B; Yakobson, Boris I

2017-09-26

Synthesis of two-dimensional (2D) crystals is a topic of great current interest, since their chemical makeup, electronic, mechanical, catalytic, and optical properties are so diverse. A universal challenge, however, is the generally random formation of defects caused by various growth factors on flat surfaces. Here we show through theoretical analysis and experimental demonstration that nonplanar, curved-topography substrates permit the intentional and controllable creation of topological defects within 2D materials. We augment a common phase-field method by adding a geometric phase to track the crystal misorientation on a curved surface and to detect the formation of grain boundaries, especially when a growing monocrystal "catches its own tail" on a nontrivial topographical feature. It is specifically illustrated by simulated growth of a trigonal symmetry crystal on a conical-planar substrate, to match the experimental synthesis of WS 2 on silicon template, with satisfactory and in some cases remarkable agreement of theory predictions and experimental evidence.

Ubiquitin Ligases: Structure, Function, and Regulation.

PubMed

Zheng, Ning; Shabek, Nitzan

2017-06-20

Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.

Substrate-dependent denitrification of abundant probe-defined denitrifying bacteria in activated sludge.

PubMed

Morgan-Sagastume, Fernando; Nielsen, Jeppe Lund; Nielsen, Per Halkjaer

2008-11-01

The denitrification capacity of different phylogenetic bacterial groups was investigated on addition of different substrates in activated sludge from two nutrient-removal plants. Nitrate/nitrite consumption rates (CRs) were calculated from nitrate and nitrite biosensor, in situ measurements. The nitrate/nitrite CRs depended on the substrate added, and acetate alone or combined with other substrates yielded the highest rates (3-6 mg N gVSS(-1) h(-1)). The nitrate CRs were similar to the nitrite CRs for most substrates tested. The structure of the active denitrifying population was investigated using heterotrophic CO2 microautoradiography (HetCO2-MAR) and FISH. Probe-defined denitrifiers appeared as specialized substrate utilizers despite acetate being preferentially used by most of them. Azoarcus and Accumulibacter abundance in the two different sludges was related to differences in their substrate-specific nitrate/nitrite CRs. Aquaspirillum-related bacteria were the most abundant potential denitrifiers (c. 20% of biovolume); however, Accumulibacter (3-7%) and Azoarcus (2-13%) may have primarily driven denitrification by utilizing pyruvate, ethanol, and acetate. Activated sludge denitrification was potentially conducted by a diverse, versatile population including not only Betaproteobacteria (Aquaspirillum, Thauera, Accumulibacter, and Azoarcus) but also some Alphaproteobacteria and Gammaproteobacteria, as indicated by the assimilation of 14CO2 by these probe-defined groups with a complex substrate mixture as an electron donor and nitrite as an electron acceptor in HetCO2-MAR-FISH tests.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lohman, Jeremy; Ma, Ming; Osipiuk, Jerzy

2015-10-13

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representationmore » of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.« less

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii.

PubMed

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved.

Delineating Substrate Diversity of Disparate Short-Chain Dehydrogenase Reductase from Debaryomyces hansenii

PubMed Central

Ghatak, Arindam; Bharatham, Nagakumar; Shanbhag, Anirudh P.; Datta, Santanu; Venkatraman, Janani

2017-01-01

Short-chain dehydrogenase reductases (SDRs) have been utilized for catalyzing the reduction of many aromatic/aliphatic prochiral ketones to their respective alcohols. However, there is a paucity of data that elucidates their innate biological role and diverse substrate space. In this study, we executed an in-depth biochemical characterization and substrate space mapping (with 278 prochiral ketones) of an unannotated SDR (DHK) from Debaryomyces hansenii and compared it with structurally and functionally characterized SDR Synechococcus elongatus. PCC 7942 FabG to delineate its industrial significance. It was observed that DHK was significantly more efficient than FabG, reducing a diverse set of ketones albeit at higher conversion rates. Comparison of the FabG structure with a homology model of DHK and a docking of substrate to both structures revealed the presence of additional flexible loops near the substrate binding site of DHK. The comparative elasticity of the cofactor and substrate binding site of FabG and DHK was experimentally substantiated using differential scanning fluorimetry. It is postulated that the loop flexibility may account for the superior catalytic efficiency of DHK although the positioning of the catalytic triad is conserved. PMID:28107498

Controls over fungal communities and consequences for nutrient cycling

NASA Astrophysics Data System (ADS)

Treseder, K. K.; Majumder, P.; Bent, E.; Borneman, J.; Allison, S. D.; Hanson, C. A.

2007-12-01

Soils harbor a high diversity of microbes-- as many as 100 species of fungi within a square meter. If different species target different components of litter, a more diverse community of fungi should lead to faster decomposition rates. We examined the hypotheses that variation in substrate use among fungal groups and variation in nitrogen availability are both important controls over the diversity of fungi in an Alaskan boreal forest. Nitrogen availability was considered because microbes are often N-limited, and because humans are altering N availability via anthropogenic N deposition and global warming. We used nucleotide analogs to link fungal groups with their role in decomposition in field samples. Leaf litter collected from the forest floor was supplemented with one of four N-containing compounds. Bromodeoxyuridine (BrdU, a thymidine analog) was also added. After 48 hours incubation, DNA was extracted. Most growing fungi should have assimilated the BrdU into new DNA. Their genetic identity was determined using oligonucleotide fingerprinting of rRNA genes (OFRG). OFRG is an rRNA gene profiling method that sorts genes into taxonomic groups with a high degree of resolution, and has a large capacity for sample processing. Fungal groups that proliferated following the addition of a given compound probably metabolized that compound. We found that fungal taxa varied in their responses to different substrates, indicating that they differed in substrate use. Specifically, community composition of fungi was significantly different among substrate treatments (P < 0.001). In addition, of the 15 dominant taxa, seven displayed significant preferences for one substrate over another. For instance, taxa within the Helotiales preferred glutamate (P = 0.001); Sporidiales, tannin-protein complexes (P = 0.014); Saccharomycetales, arginine (P = 0.042); and Polyporales, arginine and lignocellulose (P = 0.040). In a complementary experiment, we used BrdU labeling to characterize effects of N fertilization on fungal community composition. We observed that N fertilization decreased the richness of fungal taxa by 22%. Helotiales and Saccharomycetales tended to increase under N fertilization, whereas Polyporales did not change significantly. Together, these results indicate that shifts in the community composition of fungi under anthropogenic N deposition could lead to changes in nutrient dynamics.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria.

PubMed

Ross, Avena C; Xu, Ying; Lu, Liang; Kersten, Roland D; Shao, Zongze; Al-Suwailem, Abdulaziz M; Dorrestein, Pieter C; Qian, Pei-Yuan; Moore, Bradley S

2013-01-23

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Biosynthetic Multitasking Facilitates Thalassospiramide Structural Diversity in Marine Bacteria

PubMed Central

Ross, Avena C.; Xu, Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Pei-Yuan; Moore, Bradley S.

2013-01-01

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multi-module skipping and iteration. Preliminary biochemical analysis of the N-terminal NRPS module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N-terminus. PMID:23270364

Recent Advances in the Structural Mechanisms of DNA Glycosylases

PubMed Central

Brooks, Sonja C.; Adhikary, Suraj; Rubinson, Emily H.; Eichman, Brandt F.

2012-01-01

DNA glycosylases safeguard the genome by locating and excising a diverse array of aberrant nucleobases created from oxidation, alkylation, and deamination of DNA. Since the discovery 28 years ago that these enzymes employ a base flipping mechanism to trap their substrates, six different protein architectures have been identified to perform the same basic task. Work over the past several years has unraveled details for how the various DNA glycosylases survey DNA, detect damage within the duplex, select for the correct modification, and catalyze base excision. Here, we provide a broad overview of these latest advances in glycosylase mechanisms gleaned from structural enzymology, highlighting features common to all glycosylases as well as key differences that define their particular substrate specificities. PMID:23076011

Substrate Specificity of Cysteine Proteases Beyond the S2 Pocket: Mutagenesis and Molecular Dynamics Investigation of Fasciola hepatica Cathepsins L

PubMed Central

Corvo, Ileana; Ferraro, Florencia; Merlino, Alicia; Zuberbühler, Kathrin; O'Donoghue, Anthony J.; Pastro, Lucía; Pi-Denis, Natalia; Basika, Tatiana; Roche, Leda; McKerrow, James H.; Craik, Charles S.; Caffrey, Conor R.; Tort, José F.

2018-01-01

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity. PMID:29725596

Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goldfarb, K.C.; Karaoz, U.; Hanson, C.A.

2011-04-18

Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeledmore » DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin-protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial composition.« less

Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

PubMed

Boutard, Magali; Cerisy, Tristan; Nogue, Pierre-Yves; Alberti, Adriana; Weissenbach, Jean; Salanoubat, Marcel; Tolonen, Andrew C

2014-11-01

Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

Partial Reconstruction of Flavonoid and Isoflavonoid Biosynthesis in Yeast Using Soybean Type I and Type II Chalcone Isomerases1[w

PubMed Central

Ralston, Lyle; Subramanian, Senthil; Matsuno, Michiyo; Yu, Oliver

2005-01-01

Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4′-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3β-hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux. PMID:15778463

Substrate specificity characterization for eight putative nudix hydrolases. Evaluation of criteria for substrate identification within the Nudix family.

PubMed

Nguyen, Vi N; Park, Annsea; Xu, Anting; Srouji, John R; Brenner, Steven E; Kirsch, Jack F

2016-12-01

The nearly 50,000 known Nudix proteins have a diverse array of functions, of which the most extensively studied is the catalyzed hydrolysis of aberrant nucleotide triphosphates. The functions of 171 Nudix proteins have been characterized to some degree, although physiological relevance of the assayed activities has not always been conclusively demonstrated. We investigated substrate specificity for eight structurally characterized Nudix proteins, whose functions were unknown. These proteins were screened for hydrolase activity against a 74-compound library of known Nudix enzyme substrates. We found substrates for four enzymes with k cat /K m values >10,000 M -1  s -1 : Q92EH0_LISIN of Listeria innocua serovar 6a against ADP-ribose, Q5LBB1_BACFN of Bacillus fragilis against 5-Me-CTP, and Q0TTC5_CLOP1 and Q0TS82_CLOP1 of Clostridium perfringens against 8-oxo-dATP and 3'-dGTP, respectively. To ascertain whether these identified substrates were physiologically relevant, we surveyed all reported Nudix hydrolytic activities against NTPs. Twenty-two Nudix enzymes are reported to have activity against canonical NTPs. With a single exception, we find that the reported k cat /K m values exhibited against these canonical substrates are well under 10 5 M -1  s -1 . By contrast, several Nudix enzymes show much larger k cat /K m values (in the range of 10 5 to >10 7 M -1  s -1 ) against noncanonical NTPs. We therefore conclude that hydrolytic activities exhibited by these enzymes against canonical NTPs are not likely their physiological function, but rather the result of unavoidable collateral damage occasioned by the enzymes' inability to distinguish completely between similar substrate structures. Proteins 2016; 84:1810-1822. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

PubMed

Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

2017-10-01

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Introducing N-glycans into natural products through a chemoenzymatic approach.

PubMed

Huang, Wei; Ochiai, Hirofumi; Zhang, Xinyu; Wang, Lai-Xi

2008-11-24

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Scent evolution in Chinese roses

PubMed Central

Scalliet, Gabriel; Piola, Florence; Douady, Christophe J.; Réty, Stéphane; Raymond, Olivier; Baudino, Sylvie; Bordji, Karim; Bendahmane, Mohammed; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe

2008-01-01

The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic “tea scent” that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses. PMID:18413608

Remarkable recovery and colonization behaviour of methane oxidizing bacteria in soil after disturbance is controlled by methane source only.

PubMed

Pan, Yao; Abell, Guy C J; Bodelier, Paul L E; Meima-Franke, Marion; Sessitsch, Angela; Bodrossy, Levente

2014-08-01

Little is understood about the relationship between microbial assemblage history, the composition and function of specific functional guilds and the ecosystem functions they provide. To learn more about this relationship we used methane oxidizing bacteria (MOB) as model organisms and performed soil microcosm experiments comprised of identical soil substrates, hosting distinct overall microbial diversities(i.e., full, reduced and zero total microbial and MOB diversities). After inoculation with undisturbed soil, the recovery of MOB activity, MOB diversity and total bacterial diversity were followed over 3 months by methane oxidation potential measurements and analyses targeting pmoA and 16S rRNA genes. Measurement of methane oxidation potential demonstrated different recovery rates across the different treatments. Despite different starting microbial diversities, the recovery and succession of the MOB communities followed a similar pattern across the different treatment microcosms. In this study we found that edaphic parameters were the dominant factor shaping microbial communities over time and that the starting microbial community played only a minor role in shaping MOB microbial community.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and custom bioinformatic analyses to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycetemore » fungi over a three-week time course. We demonstrate that although the fungi produce a similar suite of extracellular enzymes, they exhibit striking differences in the regulation of these enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Paraconiothyrium sporulosum AP3s5-JAC2a and Alternaria alternata SRC1lrK2f employ sequential enzyme secretion patterns concomitant with decreasing resource availability, Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among cellulose-degrading Ascomycetes and enhances our understanding of their role in C turnover in the environment.« less

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi.

PubMed

Zeiner, Carolyn A; Purvine, Samuel O; Zink, Erika M; Paša-Tolić, Ljiljana; Chaput, Dominique L; Wu, Si; Santelli, Cara M; Hansel, Colleen M

2017-09-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein composition of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. This work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment. Copyright © 2017. Published by Elsevier Inc.

Simultaneous saccharification and fermentation of Agave tequilana fructans by Kluyveromyces marxianus yeasts for bioethanol and tequila production.

PubMed

Flores, Jose-Axel; Gschaedler, Anne; Amaya-Delgado, Lorena; Herrera-López, Enrique J; Arellano, Melchor; Arrizon, Javier

2013-10-01

Agave tequilana fructans (ATF) constitute a substrate for bioethanol and tequila industries. As Kluyveromyces marxianus produces specific fructanases for ATF hydrolysis, as well as ethanol, it can perform simultaneous saccharification and fermentation. In this work, fifteen K. marxianus yeasts were evaluated to develop inoculums with fructanase activity on ATF. These inoculums were added to an ATF medium for simultaneous saccharification and fermentation. All the yeasts, showed exo-fructanhydrolase activity with different substrate specificities. The yeast with highest fructanase activity in the inoculums showed the lowest ethanol production level (20 g/l). Five K. marxianus strains were the most suitable for the simultaneous saccharification and fermentation of ATF. The volatile compounds composition was evaluated at the end of fermentation, and a high diversity was observed between yeasts, nevertheless all of them produced high levels of isobutyl alcohol. The simultaneous saccharification and fermentation of ATF with K. marxianus strains has potential for industrial application. Copyright © 2013 Elsevier Ltd. All rights reserved.

Proteomic analysis reveals diverse proline hydroxylation-mediated oxygen-sensing cellular pathways in cancer cells

PubMed Central

Liu, Bing; Gao, Yankun; Ruan, Hai-Bin; Chen, Yue

2016-01-01

Proline hydroxylation is a critical cellular mechanism regulating oxygen-response pathways in tumor initiation and progression. Yet, its substrate diversity and functions remain largely unknown. Here, we report a system-wide analysis to characterize proline hydroxylation substrates in cancer cells using an immunoaffinity-purification assisted proteomics strategy. We identified 562 sites from 272 proteins in HeLa cells. Bioinformatic analysis revealed that proline hydroxylation substrates are significantly enriched with mRNA processing and stress-response cellular pathways with canonical and diverse flanking sequence motifs. Structural analysis indicates a significant enrichment of proline hydroxylation participating in the secondary structure of substrate proteins. Our study identified and validated Brd4, a key transcription factor, as a novel proline hydroxylation substrate. Functional analysis showed that the inhibition of proline hydroxylation pathway significantly reduced the proline hydroxylation abundance on Brd4 and affected Brd4-mediated transcriptional activity as well as cell proliferation in AML leukemia cells. Taken together, our study identified a broad regulatory role of proline hydroxylation in cellular oxygen-sensing pathways and revealed potentially new targets that dynamically respond to hypoxia microenvironment in tumor cells. PMID:27764789

Chemoenzymatic syntheses of prenylated aromatic small molecules using Streptomyces prenyltransferases with relaxed substrate specificities

PubMed Central

Kumano, Takuto; Richard, Stéphane B.; Noel, Joseph P.; Nishiyama, Makoto; Kuzuyama, Tomohisa

2010-01-01

NphB is a soluble prenyltransferase from Streptomyces sp. strain CL190 that attaches a geranyl group to a 1,3,6,8-tetrahydroxynaphthalene-derived polyketide during the biosynthesis of anti-oxidant naphterpin. Here we report multiple chemoenzymatic syntheses of various prenylated compounds from aromatic substrates including flavonoids using two prenyltransferases NphB and SCO7190, a NphB homolog from Streptomyces coelicolor A3(2), as biocatalysts. NphB catalyzes carbon–carbon-based and carbon–oxygen-based geranylation of a diverse collection of hydroxyl-containing aromatic acceptors. Thus, this simple method using the prenyltransferases can be used to explore novel prenylated aromatic compounds with biological activities. Kinetic studies with NphB reveal that the prenylation reaction follows a sequential ordered mechanism. PMID:18682327

The complicated substrates enhance the microbial diversity and zinc leaching efficiency in sphalerite bioleaching system.

PubMed

Xiao, Yunhua; Xu, YongDong; Dong, Weiling; Liang, Yili; Fan, Fenliang; Zhang, Xiaoxia; Zhang, Xian; Niu, Jiaojiao; Ma, Liyuan; She, Siyuan; He, Zhili; Liu, Xueduan; Yin, Huaqun

2015-12-01

This study used an artificial enrichment microbial consortium to examine the effects of different substrate conditions on microbial diversity, composition, and function (e.g., zinc leaching efficiency) through adding pyrite (SP group), chalcopyrite (SC group), or both (SPC group) in sphalerite bioleaching systems. 16S rRNA gene sequencing analysis showed that microbial community structures and compositions dramatically changed with additions of pyrite or chalcopyrite during the sphalerite bioleaching process. Shannon diversity index showed a significantly increase in the SP (1.460), SC (1.476), and SPC (1.341) groups compared with control (sphalerite group, 0.624) on day 30, meanwhile, zinc leaching efficiencies were enhanced by about 13.4, 2.9, and 13.2%, respectively. Also, additions of pyrite or chalcopyrite could increase electric potential (ORP) and the concentrations of Fe3+ and H+, which were the main factors shaping microbial community structures by Mantel test analysis. Linear regression analysis showed that ORP, Fe3+ concentration, and pH were significantly correlated to zinc leaching efficiency and microbial diversity. In addition, we found that leaching efficiency showed a positive and significant relationship with microbial diversity. In conclusion, our results showed that the complicated substrates could significantly enhance microbial diversity and activity of function.

Methods of Antimicrobial Coating of Diverse Materials

NASA Technical Reports Server (NTRS)

Akse, James R.; Holtsnider, John T.; Kliestik, Helen

2011-01-01

Methods of coating diverse substrate materials with antimicrobial agents have been developed. Originally intended to reduce health risks to astronauts posed by pathogenic microorganisms that can grow on surfaces in spacecraft, these methods could also be used on Earth for example, to ensure sterility of surgical inserts and other medical equipment. The methods involve, generally, chemical preparation of substrate surfaces to enable attachment of antimicrobial molecules to the substrate surfaces via covalent bonds. Substrate materials that have been treated successfully include aluminum, glass, a corrosion-resistant nickel alloy, stainless steel, titanium, and poly(tetrafluoroethylene). Antimicrobial agents that have been successfully immobilized include antibiotics, enzymes, bacteriocins, bactericides, and fungicides. A variety of linkage chem istries were employed. Activity of antimicrobial coatings against gram-positive bacteria, gram-negative bacteria, and fungi was demonstrated. Results of investigations indicate that the most suitable combination of antimicrobial agent, substrate, and coating method depends upon the intended application.

The PP1 binding code: a molecular-lego strategy that governs specificity.

PubMed

Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

2013-01-01

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Islam, Kabirul; Chen, Yuling; Wu, Hong

2013-11-18

Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. Withmore » two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.« less

In Silico Prediction of Cytochrome P450-Mediated Biotransformations of Xenobiotics: A Case Study of Epoxidation.

PubMed

Zhang, Jing; Ji, Li; Liu, Weiping

2015-08-17

Predicting the biotransformation of xenobiotics is important in toxicology; however, as more compounds are synthesized than can be investigated experimentally, powerful computational methods are urgently needed to prescreen potentially useful candidates. Cytochrome P450 enzymes (P450s) are the major enzymes involved in xenobiotic metabolism, and many substances are bioactivated by P450s to form active compounds. An example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species of P450 enzymes (an iron-oxo porphyrin cation radical oxidant, compound I) for a diverse set of 36 olefinic substrates with state-of-the-art density functional theory (DFT) methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation. This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational method used in this work, single-point energy calculations with the B3LYP functional including zero-point energy and solvation and dispersion corrections based on B3LYP-optimized geometries, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions.

Inhibition Kinetics and Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase†,‡

PubMed Central

Korman, Tyler Paz; Tan, Yuhong; Wong, Justin; Luo, Rui; Tsai, Shiou-Chuan

2008-01-01

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5–10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio- and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP+ and the inhibitor emodin were solved with the wild type and P94L mutant of actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns. PMID:18205400

Inhibition Kinetics And Emodin Cocrystal Structure of a Type II Polyketide Ketoreductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korman, T.P.; Tan, Y.-H.; Wong, J.

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5-10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio- and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP{sup +} and the inhibitor emodin were solved with the wild type and P94L mutant ofmore » actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bahl, C.; Morisseau, C; Bomberger, J

Cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is a virulence factor secreted by Pseudomonas aeruginosa that reduces the quantity of CFTR in the apical membrane of human airway epithelial cells. Initial sequence analysis suggested that Cif is an epoxide hydrolase (EH), but its sequence violates two strictly conserved EH motifs and also is compatible with other {alpha}/{beta} hydrolase family members with diverse substrate specificities. To investigate the mechanistic basis of Cif activity, we have determined its structure at 1.8-{angstrom} resolution by X-ray crystallography. The catalytic triad consists of residues Asp129, His297, and Glu153, which are conserved across themore » family of EHs. At other positions, sequence deviations from canonical EH active-site motifs are stereochemically conservative. Furthermore, detailed enzymatic analysis confirms that Cif catalyzes the hydrolysis of epoxide compounds, with specific activity against both epibromohydrin and cis-stilbene oxide, but with a relatively narrow range of substrate selectivity. Although closely related to two other classes of {alpha}/{beta} hydrolase in both sequence and structure, Cif does not exhibit activity as either a haloacetate dehalogenase or a haloalkane dehalogenase. A reassessment of the structural and functional consequences of the H269A mutation suggests that Cif's effect on host-cell CFTR expression requires the hydrolysis of an extended endogenous epoxide substrate.« less

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

PubMed

He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

Microbial Life Driving Low-Temperature Basalt Alteration in the Subsurface: Decoupling Abiotic Processes from Biologically-Mediated Rock Alteration

NASA Astrophysics Data System (ADS)

Moore, R.; Lecoeuvre, A.; Stephant, S.; Dupraz, S.; Ranchou-Peyruse, M.; Ranchou-Peyruse, A.; Gérard, E.; Ménez, B.

2017-12-01

Microorganisms are involved with specific rock alteration processes in the deep subsurface. It is a challenge to link any contribution microbial life may have on rock alteration with specific functions or phyla because many alteration features and secondary minerals produced by metabolic processes can also produce abiotically. Here, two flow-through experiments were designed to mimic the circulation of a CO2-rich fluid through crystalline basalt. In order to identify microbially-mediated alteration and be able to link it with specific metabolisms represented in the subsurface, a relatively fresh crystalline basalt substrate was subsampled, sterilized and used as the substrate for both experiments. In one experiment, the substrate was left sterile, and in the other it was inoculated with an enrichment culture derived from the same aquifer as the rock substrate. Initial results show that the inoculum contained Proteobacteria and Firmicutes, which have diverse metabolic potentials. Fluid and rock analyses before, during, and after the experiments show that mineralogy, fluid chemistry, and dissolution processes differ between the sterile and inoculated systems. In the inoculated experiment iron-rich orthopyroxenes were preferentially dissolved while in the sterile system clinopyroxenes and plagioclases both exhibited a higher degree of dissolution. Additionally, the patterns of CO2 consumption and production over the duration of both experiments is different. This suggest that in a low-temperature basalt system with microorganisms CO2 is either consumed to produce biomass, or that carbonates are produced and then subsequently preserved. This suite of results combined with molecular ecology analyses can be used to conclude that in low-temperature basalts microorganisms play an intrinsic role in rock alteration.

Laser-Induced Graphene by Multiple Lasing: Toward Electronics on Cloth, Paper, and Food.

PubMed

Chyan, Yieu; Ye, Ruquan; Li, Yilun; Singh, Swatantra Pratap; Arnusch, Christopher J; Tour, James M

2018-03-27

A simple and facile method for obtaining patterned graphene under ambient conditions on the surface of diverse materials ranging from renewable precursors such as food, cloth, paper, and cardboard to high-performance polymers like Kevlar or even on natural coal would be highly desirable. Here, we report a method of using multiple pulsed-laser scribing to convert a wide range of substrates into laser-induced graphene (LIG). With the increased versatility of the multiple lase process, highly conductive patterns can be achieved on the surface of a diverse number of substrates in ambient atmosphere. The use of a defocus method results in multiple lases in a single pass of the laser, further simplifying the procedure. This method can be implemented without increasing processing times when compared with laser induction of graphene on polyimide (Kapton) substrates as previously reported. In fact, any carbon precursor that can be converted into amorphous carbon can be converted into graphene using this multiple lase method. This may be a generally applicable technique for forming graphene on diverse substrates in applications such as flexible or even biodegradable and edible electronics.

Diversity of metabolic capacities among strains degrading polycyclic aromatic hydrocarbons

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bouchez, M.; Besnaienou, B.; Blanchet, D.

1995-12-31

Strains of Pseudomonas and Rhodococcus genera were isolated for their capacity to use, as a sole carbon and energy source, one of the following polycyclic aromatic hydrocarbons (PAHs): naphthalene (NAP), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), and pyrene (PYR). The range of PAHs supporting growth of these pure strains was usually restricted, but several other hydrocarbons were used by Rhodococcus sp. All strains could grow on simple organic acids. Maximal specific growth rates ({mu}{sub max}) of all strains on their PAH growth substrates were determined by respirometry. No clear relationships between {mu}{sub max} values and the molecular weightmore » or water solubility of PAHs were apparent, but Pseudomonas sp. exhibited the highest {mu}{sub max} values. Carbon balances for PAH biodegradation were established. Differences between strains were observed, but high mineralization rates and low production of soluble metabolites were obtained for all PAHs. Bacterial biomass represented 16% to 35% of the carbon consumed. Strain diversity was also apparent in the interactions observed in the degradation of a mixture of two PAHs by individual strains, which often involved inhibition of PAH substrate degradation, with or without cometabolization of the second PAH.« less

Microbial Breakdown of Organic Carbon in the Diverse Sediments of Guaymas Basin

NASA Astrophysics Data System (ADS)

Hoarfrost, A.; Snider, R.; Arnosti, C.

2015-12-01

Guaymas Basin is characterized by sediments under conditions ranging from hemipelagic to hydrothermal. This wide range in geochemical contexts results in diverse microbial communities that may have varying abilities to access organic matter. We can address these functional differences by comparing enzyme activities initializing the breakdown of organic matter across these sediment types; however, previous direct measurements of the extracellular hydrolysis of complex organic carbon in sediments are sparse. We measured this first step of heterotrophic processing of organic matter in sediments at 5-10cm and 55-60cm depth from a wide range of environmental settings in Guaymas Basin. Sediment sources included sulfidic seeps on the Sonora Margin, hemipelagic ridge flank sediments, and hydrothermically altered Sonora Margin sediments bordering a methane seep site. Hydrolysis of organic substrates varied by depth and by sediment source, but despite high energy potential and organic carbon load in sulfidic sediments, activity was not highest where hydrothermal influence was highest. These results suggest that heterotrophic breakdown of organic carbon in Guaymas Basin sediments may be sensitive to factors including varying composition of organic carbon available in different sediment types, or differences in microbial community capacities to access specific organic substrates.

Biochemical and Structural Analysis of an Eis Family Aminoglycoside Acetyltransferase from Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Green, Keith D.; Biswas, Tapan; Chang, Changsoo

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was alsomore » different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.« less

Linking soil bacterial biodiversity and soil carbon stability.

PubMed

Mau, Rebecca L; Liu, Cindy M; Aziz, Maliha; Schwartz, Egbert; Dijkstra, Paul; Marks, Jane C; Price, Lance B; Keim, Paul; Hungate, Bruce A

2015-06-01

Native soil carbon (C) can be lost in response to fresh C inputs, a phenomenon observed for decades yet still not understood. Using dual-stable isotope probing, we show that changes in the diversity and composition of two functional bacterial groups occur with this 'priming' effect. A single-substrate pulse suppressed native soil C loss and reduced bacterial diversity, whereas repeated substrate pulses stimulated native soil C loss and increased diversity. Increased diversity after repeated C amendments contrasts with resource competition theory, and may be explained by increased predation as evidenced by a decrease in bacterial 16S rRNA gene copies. Our results suggest that biodiversity and composition of the soil microbial community change in concert with its functioning, with consequences for native soil C stability.

Polymorphism of antibiotic-inactivating enzyme driven by ecology expands the environmental resistome.

PubMed

Kim, Dae-Wi; Thawng, Cung Nawl; Choi, Jung-Hye; Lee, Kihyun; Cha, Chang-Jun

2018-01-01

The environmental resistome has been recognized as the origin and reservoir of antibiotic resistance genes and considered to be dynamic and ever expanding. In this study, a targeted gene sequencing approach revealed that the polymorphic diversity of the aminoglycoside-inactivating enzyme AAC(6')-Ib was ecological niche-specific. AAC(6')-Ib-cr, previously known as a clinical variant, was prevalent in various soils and the intestines of chickens and humans, suggesting that this variant might not have arisen from adaptive mutations in the clinic but instead originated from the environment. Furthermore, ecologically dominant polymorphic variants of AAC(6')-Ib were characterized and found to display different substrate specificities for quinolones and aminoglycosides, conferring the altered resistance spectra. Interestingly, a novel variant with the D179Y substitution showed an extended resistance spectrum to the recently developed fluoroquinolone gemifloxacin. Our results suggest that soil and animal microbiomes could be major reservoirs of antibiotic resistance; polymorphic diversity expands the antibiotic resistome in the environment, resulting in the potential emergence of novel resistance.

Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

PubMed Central

Yung, Hong Wa

2011-01-01

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling. PMID:21445305

Multi-year assessment of coastal planktonic fungi reveals environmental drivers of diversity and abundance

PubMed Central

Taylor, Joe D; Cunliffe, Michael

2016-01-01

Mycoplankton have so far been a neglected component of pelagic marine ecosystems, having been poorly studied relative to other plankton groups. Currently, there is a lack of understanding of how mycoplankton diversity changes through time, and the identity of controlling environmental drivers. Using Fungi-specific high-throughput sequencing and quantitative PCR analysis of plankton DNA samples collected over 6 years from the coastal biodiversity time series site Station L4 situated off Plymouth (UK), we have assessed changes in the temporal variability of mycoplankton diversity and abundance in relation to co-occurring environmental variables. Mycoplankton diversity at Station L4 was dominated by Ascomycota, Basidiomycota and Chytridiomycota, with several orders within these phyla frequently abundant and dominant in multiple years. Repeating interannual mycoplankton blooms were linked to potential controlling environmental drivers, including nitrogen availability and temperature. Specific relationships between mycoplankton and other plankton groups were also identified, with seasonal chytrid blooms matching diatom blooms in consecutive years. Mycoplankton α-diversity was greatest during periods of reduced salinity at Station L4, indicative of riverine input to the ecosystem. Mycoplankton abundance also increased during periods of reduced salinity, and when potential substrate availability was increased, including particulate organic matter. This study has identified possible controlling environmental drivers of mycoplankton diversity and abundance in a coastal sea ecosystem, and therefore sheds new light on the biology and ecology of an enigmatic marine plankton group. Mycoplankton have several potential functional roles, including saprotrophs and parasites, that should now be considered within the consensus view of pelagic ecosystem functioning and services. PMID:26943623

An expert-based model for selecting the most suitable substrate material type for antenna circuits

NASA Astrophysics Data System (ADS)

AL-Oqla, Faris M.; Omar, Amjad A.

2015-06-01

Quality and properties of microwave circuits depend on all the circuit components. One of these components is the substrate. The process of substrate material selection is a decision-making problem that involves multicriteria with objectives that are diverse and conflicting. The aim of this work was to select the most suitable substrate material type to be used in antennas in the microwave frequency range that gives best performance and reliability of the substrate. For this purpose, a model was built to ease the decision-making that includes hierarchical alternatives and criteria. The substrate material type options considered were limited to fiberglass-reinforced epoxy laminates (FR4 εr = 4.8), aluminium (III) oxide (alumina εr = 9.6), gallium arsenide III-V compound (GaAs εr = 12.8) and PTFE composites reinforced with glass microfibers (Duroid εr = 2.2-2.3). To assist in building the model and making decisions, the analytical hierarchy process (AHP) was used. The decision-making process revealed that alumina substrate material type was the most suitable choice for the antennas in the microwave frequency range that yields best performance and reliability. In addition, both the size of the circuit and the loss tangent of the substrates were found to be the most contributing subfactors in the antenna circuit specifications criterion. Experimental assessments were conducted utilising The Expert Choice™ software. The judgments were tested and found to be precise, consistent and justifiable, and the marginal inconsistency values were found to be very narrow. A sensitivity analysis was also presented to demonstrate the confidence in the drawn conclusions.

Bovine Milk as a Source of Functional Oligosaccharides for Improving Human Health12

PubMed Central

Zivkovic, Angela M.; Barile, Daniela

2011-01-01

Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk–like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising. PMID:22332060

Bovine milk as a source of functional oligosaccharides for improving human health.

PubMed

Zivkovic, Angela M; Barile, Daniela

2011-05-01

Human milk oligosaccharides are complex sugars that function as selective growth substrates for specific beneficial bacteria in the gastrointestinal system. Bovine milk is a potentially excellent source of commercially viable analogs of these unique molecules. However, bovine milk has a much lower concentration of these oligosaccharides than human milk, and the majority of the molecules are simpler in structure than those found in human milk. Specific structural characteristics of milk-derived oligosaccharides are crucial to their ability to selectively enrich beneficial bacteria while inhibiting or being less than ideal substrates for undesirable and pathogenic bacteria. Thus, if bovine milk products are to provide human milk-like benefits, it is important to identify specific dairy streams that can be processed commercially and cost-effectively and that can yield specific oligosaccharide compositions that will be beneficial as new food ingredients or supplements to improve human health. Whey streams have the potential to be commercially viable sources of complex oligosaccharides that have the structural resemblance and diversity of the bioactive oligosaccharides in human milk. With further refinements to dairy stream processing techniques and functional testing to identify streams that are particularly suitable for enriching beneficial intestinal bacteria, the future of oligosaccharides isolated from dairy streams as a food category with substantiated health claims is promising.

Functional reconstitution of the Mycobacterium tuberculosis long-chain acyl-CoA carboxylase from multiple acyl-CoA subunits.

PubMed

Bazet Lyonnet, Bernardo; Diacovich, Lautaro; Gago, Gabriela; Spina, Lucie; Bardou, Fabienne; Lemassu, Anne; Quémard, Annaïk; Gramajo, Hugo

2017-04-01

Mycobacterium tuberculosis produces a large number of structurally diverse lipids that have been implicated in the pathogenicity, persistence and antibiotic resistance of this organism. Most building blocks involved in the biosynthesis of all these lipids are generated by acyl-CoA carboxylases whose subunit composition and physiological roles have not yet been clearly established. Inconclusive data in the literature refer to the exact protein composition and substrate specificity of the enzyme complex that produces the long-chain α-carboxy-acyl-CoAs, which are substrates involved in the last step of condensation mediated by the polyketide synthase 13 to synthesize mature mycolic acids. Here we have successfully reconstituted the long-chain acyl-CoA carboxylase (LCC) complex from its purified components, the α subunit (AccA3), the ε subunit (AccE5) and the two β subunits (AccD4 and AccD5), and demonstrated that the four subunits are essential for its activity. Furthermore, we also showed by substrate competition experiments and the use of a specific inhibitor that the AccD5 subunit's role in the carboxylation of the long acyl-CoAs, as part of the LCC complex, was structural rather than catalytic. Moreover, AccD5 was also able to carboxylate its natural substrates, acetyl-CoA and propionyl-CoA, in the context of the LCC enzyme complex. Thus, the supercomplex formed by these four subunits has the potential to generate the main substrates, malonyl-CoA, methylmalonyl-CoA and α-carboxy-C 24-26 -CoA, used as condensing units for the biosynthesis of all the lipids present in this pathogen. © 2017 Federation of European Biochemical Societies.

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

Quantitative iTRAQ-based secretome analysis reveals species-specific and temporal shifts in carbon utilization strategies among manganese(II)-oxidizing Ascomycete fungi

DOE PAGES

Zeiner, Carolyn A.; Purvine, Samuel O.; Zink, Erika M.; ...

2017-07-01

Fungi generate a wide range of extracellular hydrolytic and oxidative enzymes and reactive metabolites, collectively known as the secretome, that synergistically drive plant litter decomposition in the environment. While secretome studies of model organisms have greatly expanded our knowledge of these enzymes, few have extended secretome characterization to environmental isolates, particularly filamentous Ascomycetes, or directly compared temporal patterns of enzyme utilization among diverse species. Thus, the mechanisms of carbon (C) degradation by many ubiquitous soil fungi remain poorly understood. Here in this study we use a combination of iTRAQ proteomics and extracellular enzyme activity assays to compare the protein compositionmore » of the secretomes of four manganese(II)-oxidizing Ascomycete fungi over a three-week time course. We demonstrate that the fungi exhibit striking differences in the regulation of extracellular lignocellulose-degrading enzymes among species and over time, revealing species-specific and temporal shifts in C utilization strategies as they degrade the same substrate. Specifically, our findings suggest that Alternaria alternata SRC1lrK2f and Paraconiothyrium sporulosum AP3s5-JAC2a employ sequential enzyme secretion patterns concomitant with decreasing resource availability. Stagonospora sp. SRC1lsM3a preferentially degrades proteinaceous substrate before switching to carbohydrates, and Pyrenochaeta sp. DS3sAY3a utilizes primarily peptidases to aggressively attack carbon sources in a concentrated burst. In conclusion, this work highlights the diversity of operative metabolic strategies among understudied yet ubiquitous cellulose-degrading Ascomycetes, enhancing our understanding of their contribution to C turnover in the environment.« less

Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms

PubMed Central

James, Karen E.; Schneider, Harald; Ansell, Stephen W.; Evers, Margaret; Robba, Lavinia; Uszynski, Grzegorz; Pedersen, Niklas; Newton, Angela E.; Russell, Stephen J.; Vogel, Johannes C.; Kilian, Andrzej

2008-01-01

Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography. PMID:18301759

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernstein, Hans C.; Brislawn, Colin J.; Dana, Karl

Benthic microbial ecosystems are widespread yet knowledge gaps still remain on the relationships between the diversity of species across kingdoms and productivity. Here, we ask two fundamental questions: 1) How does species diversity relate to the rates of primary and heterotrophic productivity? 2) How do diel variations in light-energy inputs influence productivity and microbiome diversity? To answer these questions, microbial mats from a magnesium sulfate hypersaline Lake were used to establish microcosms. Both the number and relatedness between bacterial and eukaryotic taxa in the microbiome were assayed via amplicon based sequencing of 16S and 18S rRNA genes over two dielmore » cycles. These results correlated with biomass productivity obtained from substrate-specific 13C stable isotope incorporation that enabled comparisons between primary and heterotrophic productivity. Both bacterial and eukaryotic species richness and evenness were related only to the rates of 13C labeled glucose and acetate biomass incorporation. Interestingly, measures of these heterotrophic relationships changed from positive and negative correlations depending on carbon derived from glucose and acetate, respectively. Bacterial and eukaryotic diversity of this ecosystem is also controlled, in part, energy constraints imposed by changing irradiance over a diel cycle.« less

Carboxylesterases: General detoxifying enzymes

PubMed Central

Hatfield, M. Jason; Umans, Robyn A.; Hyatt, Janice L.; Edwards, Carol C; Wierdl, Monika; Tsurkan, Lyudmila; Taylor, Michael R.; Potter, Philip M.

2016-01-01

Carboxylesterases (CE) are members of the esterase family of enzymes, and as their name suggests, they are responsible for the hydrolysis of carboxylesters into the corresponding alcohol and carboxylic acid. To date, no endogenous CE substrates have been identified and as such, these proteins are thought to act as a mechanism to detoxify ester-containing xenobiotics. As a consequence, they are expressed in tissues that might be exposed to such agents (lung and gut epithelia, liver, kidney, etc.). CEs demonstrate very broad substrate specificities and can hydrolyze compounds as diverse as cocaine, oseltamivir (Tamiflu), permethrin and irinotecan. In addition, these enzymes are irreversibly inhibited by organophosphates such as Sarin and Tabun. In this overview, we will compare and contrast the two human enzymes that have been characterized, and evaluate the biology of the interaction of these proteins with organophosphates (principally nerve agents). PMID:26892220

The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy.

PubMed

Kwon, Yong Tae; Ciechanover, Aaron

2017-11-01

The conjugation of the 76 amino acid protein ubiquitin to other proteins can alter the metabolic stability or non-proteolytic functions of the substrate. Once attached to a substrate (monoubiquitination), ubiquitin can itself be ubiquitinated on any of its seven lysine (Lys) residues or its N-terminal methionine (Met1). A single ubiquitin polymer may contain mixed linkages and/or two or more branches. In addition, ubiquitin can be conjugated with ubiquitin-like modifiers such as SUMO or small molecules such as phosphate. The diverse ways to assemble ubiquitin chains provide countless means to modulate biological processes. We overview here the complexity of the ubiquitin code, with an emphasis on the emerging role of linkage-specific degradation signals (degrons) in the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system (hereafter autophagy). Copyright © 2017 Elsevier Ltd. All rights reserved.

Primary and heterotrophic productivity relate to multikingdom diversity in a hypersaline mat

DOE PAGES

Bernstein, Hans C.; Brislawn, Colin J.; Dana, Karl; ...

2017-10-17

Benthic microbial ecosystems are widespread yet knowledge gaps still remain on the relationships between the diversity of species across kingdoms and productivity. Here, we ask two fundamental questions: (i) How does species diversity relate to the rates of primary and heterotrophic productivity? (ii) How do diel variations in light-energy inputs influence productivity and microbiome diversity? To answer these questions, microbial mats from a magnesium sulfate hypersaline lake were used to establish microcosms. Both the number and relatedness between bacterial and eukaryotic taxa in the microbiome were assayed via amplicon-based sequencing of 16S and 18S rRNA genes over two diel cycles.more » These results correlated with biomass productivity obtained from substrate-specific 13C stable isotope tracers that enabled comparisons between primary and heterotrophic productivity. Both bacterial and eukaryotic species richness and evenness were related only to the rates of 13C-labeled glucose and acetate biomass incorporation. Interestingly, measures of these heterotrophic relationships changed from positive and negative correlations depending on carbon derived from glucose or acetate, respectively. The bacterial and eukaryotic diversity of this ecosystem is also controlled, in part, from energy constraints imposed by changing irradiance over a diel cycle.« less

Primary and heterotrophic productivity relate to multikingdom diversity in a hypersaline mat

PubMed Central

Brislawn, Colin J.; Dana, Karl; Flores-Wentz, Tobias; Cory, Alexandra B.; Fansler, Sarah J.; Fredrickson, James K.; Moran, James J.

2017-01-01

Abstract Benthic microbial ecosystems are widespread yet knowledge gaps still remain on the relationships between the diversity of species across kingdoms and productivity. Here, we ask two fundamental questions: (i) How does species diversity relate to the rates of primary and heterotrophic productivity? (ii) How do diel variations in light-energy inputs influence productivity and microbiome diversity? To answer these questions, microbial mats from a magnesium sulfate hypersaline lake were used to establish microcosms. Both the number and relatedness between bacterial and eukaryotic taxa in the microbiome were assayed via amplicon-based sequencing of 16S and 18S rRNA genes over two diel cycles. These results correlated with biomass productivity obtained from substrate-specific 13C stable isotope tracers that enabled comparisons between primary and heterotrophic productivity. Both bacterial and eukaryotic species richness and evenness were related only to the rates of 13C-labeled glucose and acetate biomass incorporation. Interestingly, measures of these heterotrophic relationships changed from positive and negative correlations depending on carbon derived from glucose or acetate, respectively. The bacterial and eukaryotic diversity of this ecosystem is also controlled, in part, from energy constraints imposed by changing irradiance over a diel cycle. PMID:29045626

Genetics, Genomics and Evolution of Ergot Alkaloid Diversity

PubMed Central

Young, Carolyn A.; Schardl, Christopher L.; Panaccione, Daniel G.; Florea, Simona; Takach, Johanna E.; Charlton, Nikki D.; Moore, Neil; Webb, Jennifer S.; Jaromczyk, Jolanta

2015-01-01

The ergot alkaloid biosynthesis system has become an excellent model to study evolutionary diversification of specialized (secondary) metabolites. This is a very diverse class of alkaloids with various neurotropic activities, produced by fungi in several orders of the phylum Ascomycota, including plant pathogens and protective plant symbionts in the family Clavicipitaceae. Results of comparative genomics and phylogenomic analyses reveal multiple examples of three evolutionary processes that have generated ergot-alkaloid diversity: gene gains, gene losses, and gene sequence changes that have led to altered substrates or product specificities of the enzymes that they encode (neofunctionalization). The chromosome ends appear to be particularly effective engines for gene gains, losses and rearrangements, but not necessarily for neofunctionalization. Changes in gene expression could lead to accumulation of various pathway intermediates and affect levels of different ergot alkaloids. Genetic alterations associated with interspecific hybrids of Epichloë species suggest that such variation is also selectively favored. The huge structural diversity of ergot alkaloids probably represents adaptations to a wide variety of ecological situations by affecting the biological spectra and mechanisms of defense against herbivores, as evidenced by the diverse pharmacological effects of ergot alkaloids used in medicine. PMID:25875294

Primary and heterotrophic productivity relate to multikingdom diversity in a hypersaline mat

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernstein, Hans C.; Brislawn, Colin J.; Dana, Karl

Benthic microbial ecosystems are widespread yet knowledge gaps still remain on the relationships between the diversity of species across kingdoms and productivity. Here, we ask two fundamental questions: (i) How does species diversity relate to the rates of primary and heterotrophic productivity? (ii) How do diel variations in light-energy inputs influence productivity and microbiome diversity? To answer these questions, microbial mats from a magnesium sulfate hypersaline lake were used to establish microcosms. Both the number and relatedness between bacterial and eukaryotic taxa in the microbiome were assayed via amplicon-based sequencing of 16S and 18S rRNA genes over two diel cycles.more » These results correlated with biomass productivity obtained from substrate-specific 13C stable isotope tracers that enabled comparisons between primary and heterotrophic productivity. Both bacterial and eukaryotic species richness and evenness were related only to the rates of 13C-labeled glucose and acetate biomass incorporation. Interestingly, measures of these heterotrophic relationships changed from positive and negative correlations depending on carbon derived from glucose or acetate, respectively. The bacterial and eukaryotic diversity of this ecosystem is also controlled, in part, from energy constraints imposed by changing irradiance over a diel cycle.« less

Biomechanics of substrate boring by fig wasps.

PubMed

Kundanati, Lakshminath; Gundiah, Namrata

2014-06-01

Female insects of diverse orders bore into substrates to deposit their eggs. Such insects must overcome several biomechanical challenges to successfully oviposit, which include the selection of suitable substrates through which the ovipositor can penetrate without itself fracturing. In many cases, the insect may also need to steer and manipulate the ovipositor within the substrate to deliver eggs at desired locations before rapidly retracting her ovipositor to avoid predation. In the case of female parasitoid ichneumonid wasps, this process is repeated multiple times during her lifetime, thus testing the ability of the ovipositioning apparatus to endure fracture and fatigue. What specific adaptations does the ovipositioning apparatus of a female ichneumonoid wasp possess to withstand these challenges? We addressed this question using a model system composed of parasitoid and pollinator fig wasps. First, we show that parasitoid ovipositor tips have teeth-like structures, preferentially enriched with zinc, unlike the smooth morphology of pollinator ovipositors. We describe sensillae present on the parasitoid ovipositor tip that are likely to aid in the detection of chemical species and mechanical deformations and sample microenvironments within the substrate. Second, using atomic force microscopy, we show that parasitoid tip regions have a higher modulus compared with regions proximal to the abdomen in parasitoid and pollinator ovipositors. Finally, we use videography to film wasps during substrate boring and analyse buckling of the ovipositor to estimate the forces required for substrate boring. Together, these results allow us to describe the biomechanical principles underlying substrate boring in parasitoid ichneumonid wasps. Such studies may be useful for the biomimetic design of surgical tools and in the use of novel mechanisms to bore through hard substrates. © 2014. Published by The Company of Biologists Ltd.

Characterization of type 2 diacylglycerol acyltransferases in Chlamydomonas reinhardtii reveals their distinct substrate specificities and functions in triacylglycerol biosynthesis.

PubMed

Liu, Jin; Han, Danxiang; Yoon, Kangsup; Hu, Qiang; Li, Yantao

2016-04-01

Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Substrate specificity of the violaxanthin de-epoxidase of the primitive green alga Mantoniella squamata (Prasinophyceae).

PubMed

Goss, Reimund

2003-09-01

The substrate specificity of the enzyme violaxanthin de-epoxidase (VDE) of the primitive green alga Mantoniella squamata (Prasinophyceae) was tested in in vitro enzyme assays employing the following xanthophyll mono-epoxides: antheraxanthin (Ax), diadinoxanthin (Ddx), lutein-epoxide (LE), cryptoxanthin-epoxide (CxE), 9- cis neoxanthin (cNx), all- trans neoxanthin (Nx), and xanthophyll di-epoxides: 9- cis violaxanthin (cVx), all- trans violaxanthin (Vx), cryptoxanthin-di-epoxide (CxDE). The data presented in this study show that the VDE of M. squamata not only exhibits a low affinity for the mono-epoxide Ax, as has been reported by R. Frommolt et al. (2001, Planta 213:446-456), but has a reduced substrate affinity for the mono-epoxides Ddx, LE, CxE, and Nx as well. On the other hand, xanthophylls with a second epoxy-group (Vx, CxDE) can be de-epoxidized with a higher efficiency. Such a preference for xanthophyll di-epoxides cannot be observed for the higher-plant VDE, where, in general, no marked differences in the pigment de-epoxidation rates between xanthophyll mono- and di-epoxides are visible. Despite this substantial difference between the VDEs of M. squamata and S. oleracea there are also features common to both enzymes. Neither VDE is able to convert xanthophylls with a 9- cis configuration in the acyclic polyene chain and both rely on substrates in the all- trans configuration. Both enzymes furthermore exhibit a dependence of enzyme activity on the polarity of the substrate. Highly polar (Nx) or non-polar (CxE) xanthophylls are de-epoxidized with greatly reduced rates in comparison to substrates with an intermediate polarity (Vx, Ax, LE, Ddx). This dependence on substrate polarity becomes more obvious when the higher-plant VDE is examined, as the substrate affinity of the VDE of M. squamata is more strongly influenced by the existence or absence of a second epoxy-group. In summary, the data presented in this study underline the fact that different VDEs, although in general catalyzing the same reaction sequence, are functionally diverse.

Performance and bacterial community structure of a 10-years old constructed mangrove wetland.

PubMed

Tian, Tingting; Tam, Nora F Y; Zan, Qijie; Cheung, S G; Shin, Paul K S; Wong, Y S; Zhang, Li; Chen, Zhanghe

2017-11-30

Constructed mangrove wetland has been used for wastewater treatment but its long-term performance has not been reported. One-year monitoring of a 10-years old horizontal subsurface-flow constructed mangrove wetland consisting of three belts, two with mangrove plants and one without, revealed that the system maintained high and stable removal percentages of organic matter and nutrients, and planted belts performed better than unplanted control. Substrates in belts planted with Aegiceras corniculatum or Kandelia obovata had higher abundance of ammonifiers, nitrifiers and denitrifiers but lower total heterotrophic bacteria than unplanted substrate. Denaturing gradient gel electrophoresis showed that microbial diversity in planted substrate was significantly lower than that in unplanted one. The bacteria in substrates, irrespective to belts, were phylogenetically related to Proteobacteria (most dominant), Acidobacteria, Firmicutes, Nitrospirae, Gemmatimonadetes, Chloroflexi and Cyanobacteria. The steady performance of this 10-year old constructed mangrove wetland was affected by the abundance and diversity of bacterial community in substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

C/N Ratio Drives Soil Actinobacterial Cellobiohydrolase Gene Diversity

PubMed Central

Prendergast-Miller, Miranda T.; Poonpatana, Pabhon; Farrell, Mark; Bissett, Andrew; Macdonald, Lynne M.; Toscas, Peter; Richardson, Alan E.; Thrall, Peter H.

2015-01-01

Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle. PMID:25710367

Substrates of Peltigera Lichens as a Potential Source of Cyanobionts.

PubMed

Zúñiga, Catalina; Leiva, Diego; Carú, Margarita; Orlando, Julieta

2017-10-01

Photobiont availability is one of the main factors determining the success of the lichenization process. Although multiple sources of photobionts have been proposed, there is no substantial evidence confirming that the substrates on which lichens grow are one of them. In this work, we obtained cyanobacterial 16S ribosomal RNA gene sequences from the substrates underlying 186 terricolous Peltigera cyanolichens from localities in Southern Chile and maritime Antarctica and compared them with the sequences of the cyanobionts of these lichens, in order to determine if cyanobacteria potentially available for lichenization were present in the substrates. A phylogenetic analysis of the sequences showed that Nostoc phylotypes dominated the cyanobacterial communities of the substrates in all sites. Among them, an overlap was observed between the phylotypes of the lichen cyanobionts and those of the cyanobacteria present in their substrates, suggesting that they could be a possible source of lichen photobionts. Also, in most cases, higher Nostoc diversity was observed in the lichens than in the substrates from each site. A better understanding of cyanobacterial diversity in lichen substrates and their relatives in the lichens would bring insights into mycobiont selection and the distribution patterns of lichens, providing a background for hypothesis testing and theory development for future studies of the lichenization process.

Habitat connectivity and local conditions shape taxonomic and functional diversity of arthropods on green roofs.

PubMed

Braaker, Sonja; Obrist, Martin Karl; Ghazoul, Jaboury; Moretti, Marco

2017-05-01

Increasing development of urban environments creates high pressure on green spaces with potential negative impacts on biodiversity and ecosystem services. There is growing evidence that green roofs - rooftops covered with vegetation - can contribute mitigate the loss of urban green spaces by providing new habitats for numerous arthropod species. Whether green roofs can contribute to enhance taxonomic and functional diversity and increase connectivity across urbanized areas remains, however, largely unknown. Furthermore, only limited information is available on how environmental conditions shape green roof arthropod communities. We investigated the community composition of arthropods (Apidae, Curculionidae, Araneae and Carabidae) on 40 green roofs and 40 green sites at ground level in the city of Zurich, Switzerland. We assessed how the site's environmental variables (such as area, height, vegetation, substrate and connectivity among sites) affect species richness and functional diversity using generalized linear models. We used an extension of co-inertia analysis (RLQ) and fourth-corner analysis to highlight the mechanism underlying community assemblages across taxonomic groups on green roof and ground communities. Species richness was higher at ground-level sites, while no difference in functional diversity was found between green roofs and ground sites. Green roof arthropod diversity increased with higher connectivity and plant species richness, irrespective of substrate depth, height and area of green roofs. The species trait analysis reviewed the mechanisms related to the environmental predictors that shape the species assemblages of the different taxa at ground and roof sites. Our study shows the important contribution of green roofs in maintaining high functional diversity of arthropod communities across different taxonomic groups, despite their lower species richness compared with ground sites. Species communities on green roofs revealed to be characterized by specific trait assemblages. The study also provides details on the environmental conditions that influence arthropod diversity and gives new perspectives on how the design of green roofs can be improved to increase their ecological value. Furthermore, the study highlights the importance of integrating green roofs in planning policies which aim to enhance urban habitat connectivity. © 2017 The Authors. Journal of Animal Ecology © 2017 British Ecological Society.

The Aminolysis Reaction of Streptomyces S9 Aminopeptidase Promotes the Synthesis of Diverse Prolyl Dipeptides▿ †

PubMed Central

Arima, Jiro; Morimoto, Masazumi; Usuki, Hirokazu; Mori, Nobuhiro; Hatanaka, Tadashi

2010-01-01

Prolyl dipeptide synthesis by S9 aminopeptidase from Streptomyces thermocyaneoviolaceus (S9AP-St) has been demonstrated. In the synthesis, S9AP-St preferentially used l-Pro-OBzl as the acyl donor, yielding synthesized dipeptides having an l-Pro-Xaa structure. In addition, S9AP-St showed broad specificity toward the acyl acceptor. Furthermore, S9AP-St produced cyclo (l-Pro-l-His) with a conversion ratio of substrate to cyclo (l-Pro-l-His) higher than 40%. PMID:20418423

Suzuki-Miyaura Cross-Coupling Reactions of Primary Alkyltrifluoroborates with Aryl Chlorides

PubMed Central

Dreher, Spencer D.; Lim, Siang-Ee; Sandrock, Deidre L.; Molander, Gary A.

2009-01-01

Parallel microscale experimentation was used to develop general conditions for the Suzuki-Miyaura cross-coupling of diversely functionalized primary alkyltrifluoroborates with a variety of aryl chlorides. These conditions were found to be amenable to coupling with aryl bromides, iodides, and triflates as well. The conditions that were previously identified through similar techniques to promote the cross-coupling of secondary alkyltrifluoroborates with aryl chlorides were not optimal for the primary alkyltrifluoroborates, thus demonstrating the value of parallel experimentation to develop novel, substrate specific results. PMID:19271726

FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

NASA Astrophysics Data System (ADS)

Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

2013-07-01

Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

Structural Basis for Substrate Specificity in Phosphate Binding (beta/alpha)8-Barrels: D-Allulose 6-Phosphate 3-Epimerase from Escherichia coli K-12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chan,K.; Fedorov, A.; Almo, S.

2008-01-01

Enzymes that share the ({beta}/{alpha})8-barrel fold catalyze a diverse range of reactions. Many utilize phosphorylated substrates and share a conserved C-terminal ({beta}/a)2-quarter barrel subdomain that provides a binding motif for the dianionic phosphate group. We recently reported functional and structural studies of d-ribulose 5-phosphate 3-epimerase (RPE) from Streptococcus pyogenes that catalyzes the equilibration of the pentulose 5-phosphates d-ribulose 5-phosphate and d-xylulose 5-phosphate in the pentose phosphate pathway [J. Akana, A. A. Fedorov, E. Fedorov, W. R. P. Novack, P. C. Babbitt, S. C. Almo, and J. A. Gerlt (2006) Biochemistry 45, 2493-2503]. We now report functional and structural studies ofmore » d-allulose 6-phosphate 3-epimerase (ALSE) from Escherichia coli K-12 that catalyzes the equilibration of the hexulose 6-phosphates d-allulose 6-phosphate and d-fructose 6-phosphate in a catabolic pathway for d-allose. ALSE and RPE prefer their physiological substrates but are promiscuous for each other's substrate. The active sites (RPE complexed with d-xylitol 5-phosphate and ALSE complexed with d-glucitol 6-phosphate) are superimposable (as expected from their 39% sequence identity), with the exception of the phosphate binding motif. The loop following the eighth {beta}-strand in ALSE is one residue longer than the homologous loop in RPE, so the binding site for the hexulose 6-phosphate substrate/product in ALSE is elongated relative to that for the pentulose 5-phosphate substrate/product in RPE. We constructed three single-residue deletion mutants of the loop in ALSE, ?T196, ?S197 and ?G198, to investigate the structural bases for the differing substrate specificities; for each, the promiscuity is altered so that d-ribulose 5-phosphate is the preferred substrate. The changes in kcat/Km are dominated by changes in kcat, suggesting that substrate discrimination results from differential transition state stabilization. In both ALSE and RPE, the phosphate group hydrogen bonds not only with the conserved motif but also with an active site loop following the sixth {beta}-strand, providing a potential structural mechanism for coupling substrate binding with catalysis.« less

Stream channel responses to streamflow diversion on small streams of the Snake River drainage, Idaho

Treesearch

Carolyn C. Bohn; John G. King

2000-01-01

The effects on channels of small, low-head seasonal water diversions in the Snake River drainage were investigated. Channels below small diversions were compared to the channels immediately above the same diversions to determine if differences in flow conveyance, substrate sediment size distribution, or streamside vegetation density were present. Estimates of flow...

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

PubMed

Pons, T; Hernández, L; Batista, F R; Chinea, G

2000-11-01

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family.

Prediction of a common beta-propeller catalytic domain for fructosyltransferases of different origin and substrate specificity.

PubMed Central

Pons, T.; Hernández, L.; Batista, F. R.; Chinea, G.

2000-01-01

The three-dimensional (3D) structure of fructan biosynthetic enzymes is still unknown. Here, we have explored folding similarities between reported microbial and plant enzymes that catalyze transfructosylation reactions. A sequence-structure compatibility search using TOPITS, SDP, 3D-PSSM, and SAM-T98 programs identified a beta-propeller fold with scores above the confidence threshold that indicate a structurally conserved catalytic domain in fructosyltransferases (FTFs) of diverse origin and substrate specificity. The predicted fold appeared related to that of neuraminidase and sialidase, of glycoside hydrolase families 33 and 34, respectively. The most reliable structural model was obtained using the crystal structure of neuraminidase (Protein Data Bank file: 5nn9) as template, and it is consistent with the location of previously identified functional residues of bacterial levansucrases (Batista et al., 1999; Song & Jacques, 1999). The sequence-sequence analysis presented here reinforces the recent inclusion of fungal and plant FTFs into glycoside hydrolase family 32, and suggests a modified sequence pattern H-x (2)-[PTV]-x (4)-[LIVMA]-[NSCAYG]-[DE]-P-[NDSC][GA]3 for this family. PMID:11305239

Glycomacropeptide Sustains Microbiota Diversity and Promotes Specific Taxa in an Artificial Colon Model of Elderly Gut Microbiota.

PubMed

Ntemiri, Alexandra; Chonchúir, Fodhla Ní; O'Callaghan, Tom F; Stanton, Catherine; Ross, R Paul; O'Toole, Paul W

2017-03-01

The potential of milk-derived glycomacropeptide (GMP) and lactose for modulating the human gut microbiota of older people, in whom loss of diversity correlates with inferior health, was investigated. We used an in vitro batch fermentation (artificial colon model) to simulate colonic fermentation processes of two GMP products, i.e., a commercially available GMP concentrate and a semipurified GMP concentrate, and lactose. Faecal samples were collected from healthy and frail older people. Samples were analyzed by Illumina Miseq sequencing of rRNA gene amplicons. The commercial GMP preparation had a positive effect on the growth of Coprococcus and Clostridium cluster XIVb and sustained a higher faecal microbiota diversity compared to control substrates or lactose. Lactose fermentation promoted the growth of Proteobacteria including Escherichia/Shigella. This work provides an in-depth insight on the potential of GMP and lactose for modulating the gut microbiota and contributes more evidence confirming the prebiotic activity of GMP.

Microbial Diversity in Soil, Sand Dune and Rock Substrates of the Thar Monsoon Desert, India.

PubMed

Rao, Subramanya; Chan, Yuki; Bugler-Lacap, Donnabella C; Bhatnagar, Ashish; Bhatnagar, Monica; Pointing, Stephen B

2016-03-01

A culture-independent diversity assessment of archaea, bacteria and fungi in the Thar Desert in India was made. Six locations in Ajmer, Jaisalmer, Jaipur and Jodhupur included semi-arid soils, arid soils, arid sand dunes, plus arid cryptoendolithic substrates. A real-time quantitative PCR approach revealed that bacteria dominated soils and cryptoendoliths, whilst fungi dominated sand dunes. The archaea formed a minor component of all communities. Comparison of rRNA-defined community structure revealed that substrate and climate rather than location were the most parsimonious predictors. Sequence-based identification of 1240 phylotypes revealed that most taxa were common desert microorganisms. Semi-arid soils were dominated by actinobacteria and alpha proteobacteria, arid soils by chloroflexi and alpha proteobacteria, sand dunes by ascomycete fungi and cryptoendoliths by cyanobacteria. Climatic variables that best explained this distribution were mean annual rainfall and maximum annual temperature. Substrate variables that contributed most to observed diversity patterns were conductivity, soluble salts, Ca(2+) and pH. This represents an important addition to the inventory of desert microbiota, novel insight into the abiotic drivers of community assembly, and the first report of biodiversity in a monsoon desert system.

Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

PubMed Central

Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

2015-01-01

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

Plant-microbe interactions driven by exometabolite preferences of rhizosphere bacteria

NASA Astrophysics Data System (ADS)

Zhalnina, K.; Louie, K. B.; Mansoori, N.; Hao, Z.; Gao, J.; Cho, H. J.; Karaoz, U.; Loqué, D.; Bowen, B.; Firestone, M.; Brodie, E.; Northen, T.

2016-12-01

It is known that rhizosphere bacteria can impact important processes during plant development. In `return' plants release substantial quantities of soluble C into the soil surrounding its roots, attracting bacteria and other soil organisms. Given the potential beneficial and detrimental consequences of stimulating high densities of organisms adjacent to newly formed root, regulating the chemical composition of exudates would represent a potential means of plant selection for beneficial microorganisms. If exudate resource composition functions to select specific microorganisms, then one would expect that substrate specialization exists within the rhizosphere microbiome. Here we provide evidence that in the rhizosphere of wild oats (Avena barbata), specific metabolites are exuded that are preferentially used by selected bacteria in rhizosphere and this substrate specialization, together with the changing composition of root exudates, drives the observed successional patterns. To investigate the relationship between exudates and rhizosphere bacteria we first analyzed exudate composition of hydroponically grown plants using LC-MS/MS based metabolomics. We then designed a medium to simulate plant exudates and using this medium we examined the substrate preferences of a diversity of rhizosphere bacterial isolates. We then assessed the ability of soil isolates to consume exudate components by LC-MS/MS based metabolomics. These substrate preferences were then related to genomic features and successional patterns of bacteria in the Avena rhizosphere. The major fraction of plant exudates was found to be composed of amino- and carboxylic acids, sugars, nucleosides, quaternary amines and plant hormones. Amino acids, sugars and nucleosides were consumed by all analyzed isolates. However, isolates that were preferentially stimulated by plant growth, revealed substrate utilization preferences towards aromatic organic acids, while those not responding to growing roots did not utilize these compounds under these conditions. This substrate partitioning among rhizosphere bacteria can be suggested as a potential mechanism for how plants influence the structure of their rhizosphere microbiome and provides a key insight into the mechanisms underlying patterns of ecological succession in soil.

Substrate Scope of O-Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides.

PubMed

Parajuli, Prakash; Pandey, Ramesh Prasad; Nguyen, Thi Huyen Trang; Dhakal, Dipesh; Sohng, Jae Kyung

2018-04-01

Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

Resource availability controls fungal diversity across a plant diversity gradient

USGS Publications Warehouse

Waldrop, M.P.; Zak, D.R.; Blackwood, C.B.; Curtis, C.D.; Tilman, D.

2006-01-01

Despite decades of research, the ecological determinants of microbial diversity remain poorly understood. Here, we test two alternative hypotheses concerning the factors regulating fungal diversity in soil. The first states that higher levels of plant detritus production increase the supply of limiting resources (i.e. organic substrates) thereby increasing fungal diversity. Alternatively, greater plant diversity increases the range of organic substrates entering soil, thereby increasing the number of niches to be filled by a greater array of heterotrophic fungi. These two hypotheses were simultaneously examined in experimental plant communities consisting of one to 16 species that have been maintained for a decade. We used ribosomal intergenic spacer analysis (RISA), in combination with cloning and sequencing, to quantify fungal community composition and diversity within the experimental plant communities. We used soil microbial biomass as a temporally integrated measure of resource supply. Plant diversity was unrelated to fungal diversity, but fungal diversity was a unimodal function of resource supply. Canonical correspondence analysis (CCA) indicated that plant diversity showed a relationship to fungal community composition, although the occurrence of RISA bands and operational taxonomic units (OTUs) did not differ among the treatments. The relationship between fungal diversity and resource availability parallels similar relationships reported for grasslands, tropical forests, coral reefs, and other biotic communities, strongly suggesting that the same underlying mechanisms determine the diversity of organisms at multiple scales. ?? 2006 Blackwell Publishing Ltd/CNRS.

Identification of Arabidopsis MYB56 as a novel substrate for CRL3(BPM) E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2015-02-01

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work, MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies have indicated that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling flowering time in plants. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library.

PubMed

Macdonald, Spencer S; Patel, Ankoor; Larmour, Veronica L C; Morgan-Lang, Connor; Hallam, Steven J; Mark, Brian L; Withers, Stephen G

2018-03-02

Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to N -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural basis for the substrate selectivity of a HAD phosphatase from Thermococcus onnurineus NA1.

PubMed

Ngo, Tri Duc; Van Le, Binh; Subramani, Vinod Kumar; Thi Nguyen, Chi My; Lee, Hyun Sook; Cho, Yona; Kim, Kyeong Kyu; Hwang, Hye-Yeon

2015-05-22

Proteins in the haloalkaloic acid dehalogenase (HAD) superfamily, which is one of the largest enzyme families, is generally composed of a catalytic core domain and a cap domain. Although proteins in this family show broad substrate specificities, the mechanisms of their substrate recognition are not well understood. In this study, we identified a new substrate binding motif of HAD proteins from structural and functional analyses, and propose that this motif might be crucial for interacting with hydrophobic rings of substrates. The crystal structure of TON_0338, one of the 17 putative HAD proteins identified in a hyperthermophilic archaeon, Thermococcus onnurineus NA1, was determined as an apo-form at 2.0 Å resolution. In addition, we determined the crystal structure TON_0338 in complex with Mg(2+) or N-cyclohexyl-2-aminoethanesulfonic acid (CHES) at 1.7 Å resolution. Examination of the apo-form and CHES-bound structures revealed that CHES is sandwiched between Trp58 and Trp61, suggesting that this Trp sandwich might function as a substrate recognition motif. In the phosphatase assay, TON_0338 was shown to have high activity for flavin mononucleotide (FMN), and the docking analysis suggested that the flavin of FMN may interact with Trp58 and Trp61 in a way similar to that observed in the crystal structure. Moreover, the replacement of these tryptophan residues significantly reduced the phosphatase activity for FMN. Our results suggest that WxxW may function as a substrate binding motif in HAD proteins, and expand the diversity of their substrate recognition mode. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification, expression and characterization of an R-ω-transaminase from Capronia semiimmersa.

PubMed

Iglesias, César; Panizza, Paola; Rodriguez Giordano, Sonia

2017-07-01

Chiral amines are essential precursors in the production of biologically active compounds, including several important drugs. Among the biocatalytic strategies that have been developed for their synthesis, the use of ω-transaminases (ω-TA) appears as an attractive alternative allowing the stereoselective amination of prochiral ketones. However, the problems associated with narrow substrate specificity, unfavourable reaction equilibrium and expensive amine donors still hamper its industrial application. The search for novel enzymes from nature can contribute to expand the catalytic repertoire of ω-TA and help to circumvent some of these problems. A genome mining approach, based on the work described by Höhne et al., was applied for selection of potential R-ω-TA. Additional criteria were used to select an enzyme that differs from previously described ones. A candidate R-ω-TA from Capronia semiimmersa was selected, cloned and expressed in Escherichia coli. Interestingly, alignment of this enzyme with previously reported TA sequences revealed the presence of two additional amino acid residues in a loop close to the active site. The impact of this change was analysed with a structural model based on crystallized R-ω-TAs. Analysis of the substrate specificity of R-ω-TA from C. semiimmersa indicates that it accepts a diversity of ketones as substrates yielding the corresponding amine with good yields and excellent enantioselectivity. The expressed enzyme accepts isopropylamine as amine donor what makes it suitable for industrial processes.

A simple, multidimensional approach to high-throughput discovery of catalytic reactions.

PubMed

Robbins, Daniel W; Hartwig, John F

2011-09-09

Transition metal complexes catalyze many important reactions that are employed in medicine, materials science, and energy production. Although high-throughput methods for the discovery of catalysts that would mirror related approaches for the discovery of medicinally active compounds have been the focus of much attention, these methods have not been sufficiently general or accessible to typical synthetic laboratories to be adopted widely. We report a method to evaluate a broad range of catalysts for potential coupling reactions with the use of simple laboratory equipment. Specifically, we screen an array of catalysts and ligands with a diverse mixture of substrates and then use mass spectrometry to identify reaction products that, by design, exceed the mass of any single substrate. With this method, we discovered a copper-catalyzed alkyne hydroamination and two nickel-catalyzed hydroarylation reactions, each of which displays excellent functional-group tolerance.

Identification and characterization of multiple rubisco activases in chemoautotrophic bacteria

PubMed Central

Tsai, Yi-Chin Candace; Lapina, Maria Claribel; Bhushan, Shashi; Mueller-Cajar, Oliver

2015-01-01

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) is responsible for almost all biological CO2 assimilation, but forms inhibited complexes with its substrate ribulose-1,5-bisphosphate (RuBP) and other sugar phosphates. The distantly related AAA+ proteins rubisco activase and CbbX remodel inhibited rubisco complexes to effect inhibitor release in plants and α-proteobacteria, respectively. Here we characterize a third class of rubisco activase in the chemolithoautotroph Acidithiobacillus ferrooxidans. Two sets of isoforms of CbbQ and CbbO form hetero-oligomers that function as specific activases for two structurally diverse rubisco forms. Mutational analysis supports a model wherein the AAA+ protein CbbQ functions as motor and CbbO is a substrate adaptor that binds rubisco via a von Willebrand factor A domain. Understanding the mechanisms employed by nature to overcome rubisco's shortcomings will increase our toolbox for engineering photosynthetic carbon dioxide fixation. PMID:26567524

Crystal Structure of a Plant Multidrug and Toxic Compound Extrusion Family Protein.

PubMed

Tanaka, Yoshiki; Iwaki, Shigehiro; Tsukazaki, Tomoya

2017-09-05

The multidrug and toxic compound extrusion (MATE) family of proteins consists of transporters responsible for multidrug resistance in prokaryotes. In plants, a number of MATE proteins were identified by recent genomic and functional studies, which imply that the proteins have substrate-specific transport functions instead of multidrug extrusion. The three-dimensional structure of eukaryotic MATE proteins, including those of plants, has not been reported, preventing a better understanding of the molecular mechanism of these proteins. Here, we describe the crystal structure of a MATE protein from the plant Camelina sativa at 2.9 Å resolution. Two sets of six transmembrane α helices, assembled pseudo-symmetrically, possess a negatively charged internal pocket with an outward-facing shape. The crystal structure provides insight into the diversity of plant MATE proteins and their substrate recognition and transport through the membrane. Copyright © 2017 Elsevier Ltd. All rights reserved.

Organic ferroelectric evaporator with substrate cooling and in situ transport capabilities.

PubMed

Foreman, K; Labedz, C; Shearer, M; Adenwalla, S

2014-04-01

We report on the design, operation, and performance of a thermal evaporation chamber capable of evaporating organic thin films. Organic thin films are employed in a diverse range of devices and can provide insight into fundamental physical phenomena. However, growing organic thin films is often challenging and requires very specific deposition parameters. The chamber presented here is capable of cooling sample substrates to temperatures below 130 K and allows for the detachment of the sample from the cooling stage and in situ transport. This permits the use of multiple deposition techniques in separate, but connected, deposition chambers without breaking vacuum and therefore provides clean, well characterized interfaces between the organic thin film and any adjoining layers. We also demonstrate a successful thin film deposition of an organic material with a demanding set of deposition parameters, showcasing the success of this design.

A microRNA-initiated DNAzyme motor operating in living cells

NASA Astrophysics Data System (ADS)

Peng, Hanyong; Li, Xing-Fang; Zhang, Hongquan; Le, X. Chris

2017-03-01

Synthetic DNA motors have great potential to mimic natural protein motors in cells but the operation of synthetic DNA motors in living cells remains challenging and has not been demonstrated. Here we report a DNAzyme motor that operates in living cells in response to a specific intracellular target. The whole motor system is constructed on a 20 nm gold nanoparticle (AuNP) decorated with hundreds of substrate strands serving as DNA tracks and dozens of DNAzyme molecules each silenced by a locking strand. Intracellular interaction of a target molecule with the motor system initiates the autonomous walking of the motor on the AuNP. An example DNAzyme motor responsive to a specific microRNA enables amplified detection of the specific microRNA in individual cancer cells. Activated by specific intracellular targets, these self-powered DNAzyme motors will have diverse applications in the control and modulation of biological functions.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus.

PubMed

Wefers, Daniel; Dong, Jia; Abdel-Hamid, Ahmed M; Paul, Hans Müller; Pereira, Gabriel V; Han, Yejun; Dodd, Dylan; Baskaran, Ramiya; Mayer, Beth; Mackie, Roderick I; Cann, Isaac

2017-09-15

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para -nitrophenyl ( p NP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries. Copyright © 2017 American Society for Microbiology.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus

PubMed Central

Dong, Jia; Abdel-Hamid, Ahmed M.; Paul, Hans Müller; Pereira, Gabriel V.; Han, Yejun; Dodd, Dylan; Baskaran, Ramiya; Mayer, Beth; Mackie, Roderick I.

2017-01-01

ABSTRACT The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticus. IMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus. The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries. PMID:28710263

Affinity purification of the Arabidopsis 26 S proteasome reveals a diverse array of plant proteolytic complexes.

PubMed

Book, Adam J; Gladman, Nicholas P; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M; Vierstra, Richard D

2010-08-13

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes*

PubMed Central

Book, Adam J.; Gladman, Nicholas P.; Lee, Sang-Sook; Scalf, Mark; Smith, Lloyd M.; Vierstra, Richard D.

2010-01-01

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis. PMID:20516081

A UDP-Glucose:Monoterpenol Glucosyltransferase Adds to the Chemical Diversity of the Grapevine Metabolome1[W

PubMed Central

Bönisch, Friedericke; Frotscher, Johanna; Stanitzek, Sarah; Rühl, Ernst; Wüst, Matthias; Bitz, Oliver; Schwab, Wilfried

2014-01-01

Terpenoids represent one of the major classes of natural products and serve different biological functions. In grape (Vitis vinifera), a large fraction of these compounds is present as nonvolatile terpene glycosides. We have extracted putative glycosyltransferase (GT) sequences from the grape genome database that show similarity to Arabidopsis (Arabidopsis thaliana) GTs whose encoded proteins glucosylate a diversity of terpenes. Spatial and temporal expression levels of the potential VvGT genes were determined in five different grapevine varieties. Heterologous expression and biochemical assays of candidate genes led to the identification of a UDP-glucose:monoterpenol β-d-glucosyltransferase (VvGT7). The VvGT7 gene was expressed in various tissues in accordance with monoterpenyl glucoside accumulation in grape cultivars. Twelve allelic VvGT7 genes were isolated from five cultivars, and their encoded proteins were biochemically analyzed. They varied in substrate preference and catalytic activity. Three amino acids, which corresponded to none of the determinants previously identified for other plant GTs, were found to be important for enzymatic catalysis. Site-specific mutagenesis along with the analysis of allelic proteins also revealed amino acids that impact catalytic activity and substrate tolerance. These results demonstrate that VvGT7 may contribute to the production of geranyl and neryl glucoside during grape ripening. PMID:24784757

Structure-based Insights into the Catalytic Power and Conformational Dexterity of Peroxiredoxins

PubMed Central

Hall, Andrea; Nelson, Kimberly; Poole, Leslie B.

2011-01-01

Abstract Peroxiredoxins (Prxs), some of nature's dominant peroxidases, use a conserved Cys residue to reduce peroxides. They are highly expressed in organisms from all kingdoms, and in eukaryotes they participate in hydrogen peroxide signaling. Seventy-two Prx structures have been determined that cover much of the diversity of the family. We review here the current knowledge and show that Prxs can be effectively classified by a structural/evolutionary organization into six subfamilies followed by specification of a 1-Cys or 2-Cys mechanism, and for 2-Cys Prxs, the structural location of the resolving Cys. We visualize the varied catalytic structural transitions and highlight how they differ depending on the location of the resolving Cys. We also review new insights into the question of how Prxs are such effective catalysts: the enzyme activates not only the conserved Cys thiolate but also the peroxide substrate. Moreover, the hydrogen-bonding network created by the four residues conserved in all Prx active sites stabilizes the transition state of the peroxidatic SN2 displacement reaction. Strict conservation of the peroxidatic active site along with the variation in structural transitions provides a fascinating picture of how the diverse Prxs function to break down peroxide substrates rapidly. Antioxid. Redox Signal. 15, 795–815. PMID:20969484

A non-modular type B feruloyl esterase from Neurospora crassa exhibits concentration-dependent substrate inhibition.

PubMed Central

Crepin, Valerie F; Faulds, Craig B; Connerton, Ian F

2003-01-01

Feruloyl esterases, a subclass of the carboxylic acid esterases (EC 3.1.1.1), are able to hydrolyse the ester bond between the hydroxycinnamic acids and sugars present in the plant cell wall. The enzymes have been classified as type A or type B, based on their substrate specificity for aromatic moieties. We show that Neurospora crassa has the ability to produce multiple ferulic acid esterase activities depending upon the length of fermentation with either sugar beet pulp or wheat bran substrates. A gene identified on the basis of its expression on sugar beet pulp has been cloned and overexpressed in Pichia pastoris. The gene encodes a single-domain ferulic acid esterase, which represents the first report of a non-modular type B enzyme (fae-1 gene; GenBank accession no. AJ293029). The purified recombinant protein has been shown to exhibit concentration-dependent substrate inhibition (K(m) 0.048 mM, K (i) 2.5 mM and V(max) 8.2 units/mg against methyl 3,4-dihydroxycinnamate). The kinetic behaviour of the non-modular enzyme is discussed in terms of the diversity in the roles of the feruloyl esterases in the mobilization of plant cell wall materials and their respective modes of action. PMID:12435269

Targeting Cullin–RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation

PubMed Central

Bulatov, Emil; Ciulli, Alessio

2015-01-01

In the last decade, the ubiquitin–proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin–RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs. PMID:25886174

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S -Transferases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stoddard, Ethan G.; Killinger, Bryan J.; Nair, Reji N.

Glutathione S-transferases (GSTs) comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity-based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “Hmore » site”. Therefore, we developed (1) a glutathione-based photoaffinity probe (GSH-ABP) to target the “G site”, and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. The GST-ABP is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and “G” and “H” site specificity was carried out using a series of competitors in liver homogenates. Herein, we present robust tools for the novel characterization of enzyme- and active site-specific GST activity in mammalian model systems.« less

Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

PubMed

Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

2014-09-01

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Invasive exotic plants in the tropical Pacific Islands: Patterns of Diversity

Treesearch

J.S. Denslow; J.C. Space; P.A. Thomas

2009-01-01

Oceanic islands are good model systems with which to explore factors affecting exotic species diversity. Islands vary in size, topography, substrate type, degree of isolation, native species diversity, history, human population characteristics, and economic development. Moreover, islands are highly vulnerable to exotic species establishment. We used AICc analyses of...

Diversity and natural functions of antibiotics produced by beneficial and plant pathogenic bacteria.

PubMed

Raaijmakers, Jos M; Mazzola, Mark

2012-01-01

Soil- and plant-associated environments harbor numerous bacteria that produce antibiotic metabolites with specific or broad-spectrum activities against coexisting microorganisms. The function and ecological importance of antibiotics have long been assumed to yield a survival advantage to the producing bacteria in the highly competitive but resource-limited soil environments through direct suppression. Although specific antibiotics may enhance producer persistence when challenged by competitors or predators in soil habitats, at subinhibitory concentrations antibiotics exhibit a diversity of other roles in the life history of the producing bacteria. Many processes modulated by antibiotics may be inherently critical to the producing bacterium, such as the acquisition of substrates or initiation of developmental changes that will ensure survival under stressful conditions. Antibiotics may also have roles in more complex interactions, including in virulence on host plants or in shaping the outcomes of multitrophic interactions. The innate functions of antibiotics to producing bacteria in their native ecosystem are just beginning to emerge, but current knowledge already reveals a breadth of activities well beyond the historical perspective of antibiotics as weaponry in microbial conflicts.

Circuit Architecture of VTA Dopamine Neurons Revealed by Systematic Input-Output Mapping.

PubMed

Beier, Kevin T; Steinberg, Elizabeth E; DeLoach, Katherine E; Xie, Stanley; Miyamichi, Kazunari; Schwarz, Lindsay; Gao, Xiaojing J; Kremer, Eric J; Malenka, Robert C; Luo, Liqun

2015-07-30

Dopamine (DA) neurons in the midbrain ventral tegmental area (VTA) integrate complex inputs to encode multiple signals that influence motivated behaviors via diverse projections. Here, we combine axon-initiated viral transduction with rabies-mediated trans-synaptic tracing and Cre-based cell-type-specific targeting to systematically map input-output relationships of VTA-DA neurons. We found that VTA-DA (and VTA-GABA) neurons receive excitatory, inhibitory, and modulatory input from diverse sources. VTA-DA neurons projecting to different forebrain regions exhibit specific biases in their input selection. VTA-DA neurons projecting to lateral and medial nucleus accumbens innervate largely non-overlapping striatal targets, with the latter also sending extensive extra-striatal axon collaterals. Using electrophysiology and behavior, we validated new circuits identified in our tracing studies, including a previously unappreciated top-down reinforcing circuit from anterior cortex to lateral nucleus accumbens via VTA-DA neurons. This study highlights the utility of our viral-genetic tracing strategies to elucidate the complex neural substrates that underlie motivated behaviors. Copyright © 2015 Elsevier Inc. All rights reserved.

Structures of Human DPP7 Reveal the Molecular Basis of Specific Inhibition and the Architectural Diversity of Proline-Specific Peptidases

PubMed Central

Dong, Aiping; Seitova, Almagul; Crombett, Lissete; Shewchuk, Lisa M.; Hassell, Annie M.; Sweitzer, Sharon M.; Sweitzer, Thomas D.; McDevitt, Patrick J.; Johanson, Kyung O.; Kennedy-Wilson, Karen M.; Cossar, Doug; Bochkarev, Alexey; Gruber, Karl; Dhe-Paganon, Sirano

2012-01-01

Proline-specific dipeptidyl peptidases (DPPs) are emerging targets for drug development. DPP4 inhibitors are approved in many countries, and other dipeptidyl peptidases are often referred to as DPP4 activity- and/or structure-homologues (DASH). Members of the DASH family have overlapping substrate specificities, and, even though they share low sequence identity, therapeutic or clinical cross-reactivity is a concern. Here, we report the structure of human DPP7 and its complex with a selective inhibitor Dab-Pip (L-2,4-diaminobutyryl-piperidinamide) and compare it with that of DPP4. Both enzymes share a common catalytic domain (α/β-hydrolase). The catalytic pocket is located in the interior of DPP7, deep inside the cleft between the two domains. Substrates might access the active site via a narrow tunnel. The DPP7 catalytic triad is completely conserved and comprises Ser162, Asp418 and His443 (corresponding to Ser630, Asp708 and His740 in DPP4), while other residues lining the catalytic pockets differ considerably. The “specificity domains” are structurally also completely different exhibiting a β-propeller fold in DPP4 compared to a rare, completely helical fold in DPP7. Comparing the structures of DPP7 and DPP4 allows the design of specific inhibitors and thus the development of less cross-reactive drugs. Furthermore, the reported DPP7 structures shed some light onto the evolutionary relationship of prolyl-specific peptidases through the analysis of the architectural organization of their domains. PMID:22952628

CVD growth of large-area and high-quality HfS2 nanoforest on diverse substrates

NASA Astrophysics Data System (ADS)

Zheng, Binjie; Wang, Zegao; Qi, Fei; Wang, Xinqiang; Yu, Bo; Zhang, Wanli; Chen, Yuanfu

2018-03-01

Two-dimensional layered transition metal dichalcogenides (TMDs) have attracted burgeoning attention due to their various properties and wide potential applications. As a new TMD, hafnium disulfide (HfS2) is theoretically predicted to have better electrical performance than widely studied MoS2. The experimental researches also confirmed the extraordinary feature in electronics and optoelectronics. However, the maximal device performance may not be achieved due to its own limitation of planar structure and challenge of transfer without contamination. Here, through the chemical vapor deposition (CVD) technique, inch-size HfS2 nanoforest has been directly grown on diverse objective substrates covering insulating, semiconducting and conducting substrates. This direct CVD growth without conventional transfer process avoids contamination and degradation in quality, suggesting its promising and wide applications in high-quality and multifarious devices. It is noted that all the HfS2 nanoforests grown on diverse substrates are constructed with vertically aligned few-layered HfS2 nanosheets with high crystalline quality and edge orientation. Moreover, due to its unique structure, the HfS2 nanoforest owns abundant exposed edge sites and large active surface area, which is essential to apply in high-performance catalyst, sensor, and energy storage or field emitter.

Recent advances in the in silico modelling of UDP glucuronosyltransferase substrates.

PubMed

Sorich, Michael J; Smith, Paul A; Miners, John O; Mackenzie, Peter I; McKinnon, Ross A

2008-01-01

UDP glucurononosyltransferases (UGT) are a superfamily of enzymes that catalyse the conjugation of a range of structurally diverse drugs, environmental and endogenous chemicals with glucuronic acid. This process plays a significant role in the clearance and detoxification of many chemicals. Over the last decade the regulation and substrate profiles of UGT isoforms have been increasingly characterised. The resulting data has facilitated the prototyping of ligand based in silico models capable of predicting, and gaining insights into, binding affinity and the substrate- and regio- selectivity of glucuronidation by UGT isoforms. Pharmacophore modelling has produced particularly insightful models and quantitative structure-activity relationships based on machine learning algorithms result in accurate predictions. Simple structural chemical descriptors were found to capture much of the chemical information relevant to UGT metabolism. However, quantum chemical properties of molecules and the nucleophilic atoms in the molecule can enhance both the predictivity and chemical intuitiveness of structure-activity models. Chemical diversity analysis of known substrates has shown some bias towards chemicals with aromatic and aliphatic hydroxyl groups. Future progress in in silico development will depend on larger and more diverse high quality metabolic datasets. Furthermore, improved protein structure data on UGTs will enable the application of structural modelling techniques likely leading to greater insight into the binding and reactive processes of UGT catalysed glucuronidation.

Does Nature and Persistence of Substrate at a Mesohabitat Scale Matter for Chironomidae Assemblages? a Study of Two Perennial Mountain Streams in Patagonia, Argentina

PubMed Central

Epele, Luis Beltrán; Miserendino, María Laura; Brand, Cecilia

2012-01-01

Chironomid substrate—specific associations regarding the nature (organic—inorganic) and stability (stable—unstable) of different habitats were investigated at two low order Patagonian streams, during high and low water periods. Nant y Fall and Glyn rivers were visited twice (October 2007 and March 2008) and seven different habitat types were identified. A total of 60 samples were collected using a Surber sampler (0.09 m -2 and 250 µm) and a set of 23 environmental descriptors including physicochemical parameters and different fractions of particulate organic matter were assessed. 35 Chironomidae taxa were recorded with Orthocladiinae (20), Chironominae (7), and Podonominae (4) being the most well—represented subfamilies. Paratrichocladius sp. 1, Parapsectrocladius sp. 2, Parametriocnemus sp. 1, Pseudochironomus sp., and Rheotanytarsus sp. were the most abundant taxa. According to the relative preference index, at least 14 taxa showed strong affinity for a particular substrate. The structurally complex macrophyte Myriophyllum quitense supported 11 taxa compared with only five taxa found on the less complex Isoetes savatieri. Generally, stable substrates (boulders, cobbles, and rooted plants) supported significantly higher chironomids richness, abundance, and diversity than unstable ones (gravel—sand). Canonical correspondence analysis revealed that detritus (leaves, seeds, and biomass), macrophyte biomass, and secondarily hydraulic variables had high explanatory power on chironomids species composition and structure. This work suggests that more complex substrates showing persistence in the temporal dimension supported a diverse array of chironomids, meaning that the maintenance of natural habitat heterogeneity is essential for the community. Land—use practices having significant effects on ecological stream attributes such as increased turbidity, sediment deposition, and runoff patterns will alter assemblages. Understanding environmental associations of the Chironomidae assemblage at the habitat scale is significant for conservation purposes and for the management of low order streams in Patagonia. PMID:22947060

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG).

PubMed

Ghose, Kaushik; Selvaraj, Kumarakurubaran; McCallum, Jason; Kirby, Chris W; Sweeney-Nixon, Marva; Cloutier, Sylvie J; Deyholos, Michael; Datla, Raju; Fofana, Bourlaye

2014-03-28

Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Five UGT genes belonging to the glycosyltransferases' family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis.

Identification and functional characterization of a flax UDP-glycosyltransferase glucosylating secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG)

PubMed Central

2014-01-01

Background Lignans are a class of diphenolic nonsteroidal phytoestrogens often found glycosylated in planta. Flax seeds are a rich source of secoisolariciresinol diglucoside (SDG) lignans. Glycosylation is a process by which a glycosyl group is covalently attached to an aglycone substrate and is catalyzed by uridine diphosphate glycosyltransferases (UGTs). Until now, very little information was available on UGT genes that may play a role in flax SDG biosynthesis. Here we report on the identification, structural and functional characterization of 5 putative UGTs potentially involved in secoisolariciresinol (SECO) glucosylation in flax. Results Five UGT genes belonging to the glycosyltransferases’ family 1 (EC 2.4.x.y) were cloned and characterized. They fall under four UGT families corresponding to five sub-families referred to as UGT74S1, UGT74T1, UGT89B3, UGT94H1, UGT712B1 that all display the characteristic plant secondary product glycosyltransferase (PSPG) conserved motif. However, diversity was observed within this 44 amino acid sequence, especially in the two peptide sequences WAPQV and HCGWNS known to play a key role in the recognition and binding of diverse aglycone substrates and in the sugar donor specificity. In developing flax seeds, UGT74S1 and UGT94H1 showed a coordinated gene expression with that of pinoresinol-lariciresinol reductase (PLR) and their gene expression patterns correlated with SDG biosynthesis. Enzyme assays of the five heterologously expressed UGTs identified UGT74S1 as the only one using SECO as substrate, forming SECO monoglucoside (SMG) and then SDG in a sequential manner. Conclusion We have cloned and characterized five flax UGTs and provided evidence that UGT74S1 uses SECO as substrate to form SDG in vitro. This study allowed us to propose a model for the missing step in SDG lignan biosynthesis. PMID:24678929

C.R.O.P. - Combined Regenerative Organic food Production: Employing the benefits of natural communities

NASA Astrophysics Data System (ADS)

Bornemann, Gerhild; Hauslage, Jens; Hemmersbach, Ruth; Hendrik Anken, Ralf; Moeller, Ralf; Wasser, Kai; Tonat, Tim

The reutilization of nutrients bound in organic wastes for food crop cultivation is a central topic of BLSS (Bioregenerative Life Support System) research. The conversion of organic wastes into inorganic compounds utilizable by plants proceeds stepwise and each step is carried out by specific microorganisms with varying environmental demands. In BLSS design different steps are often allocated to different treatment units. Each unit is inoculated with selected microbial cultures and provides optimal growth conditions for these. The compartmented set-up is also often used in public wastewater treatment. But as wastewaters usually carry their decomposers with them, specified inoculates are only applied in special cases. Due to the highly variable composition of wastewater, diverse communities of microorganisms are found in treatment plants enabling these to cope with the unpredictable substrate. Although in isolated space habitats, microorganisms necessary for degradation will also be present on wastes and in wastewaters, their diversity will be limited to those species introduced into the system until launch. Therefore, it is crucial to establish a stable microbial community in the waste processing system that is capable to degrade all kinds of wastes, including micropollutants like pharmaceutical residues, before launch. The C.R.O.P. project aims at combining the utilization of liquid and solid organic wastes with soilless plant cultivation. The envisioned waste processing system is a trickling filter designed to join all required functions in one compartment thus reducing size and weight. To achieve this, the filter medium provides habitats with differing conditions so that a diverse microbial community grows as biofilm on its surface. We assume that, once established, such a quasi-natural community makes the system multifunctional with regard to the substrates that can be degraded, and stable with regard to invasion of undesirable microorganisms. Our current research focuses on the characterisation of the system’s capability to degrade different substrates like urine and plant wastes and the usability of the filtrate as fertilizer. Molecular analyses revealed a variety of different microorganisms in the filters. Future research will address the question how to introduce specialised microorganisms into the system to enable it to degrade micropollutants, and the question how to maintain these during periods of absence of their substrate.

Identification of microbial species present in a pesticide dissipation process in biobed systems using typical substrates from southeastern Mexico as a biomixture at a laboratory scale.

PubMed

Góngora-Echeverría, Virgilio R; Quintal-Franco, Carlos; Arena-Ortiz, María Leticia; Giácoman-Vallejos, Germán; Ponce-Caballero, Carmen

2018-07-01

Biobed systems are an important option to control point pollution in agricultural areas. Substrates used and microbial diversity present in a biomixture perform an essential function in pesticide dissipation. In this study, the effects of soil (50% of volume/volume [V/V] proportion for all biomixtures) and four soil-based biomixtures (miniaturized biobeds; addition of novel substrates from southeastern Mexico) on dissipation of high concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), atrazine, carbofuran, diazinon, and glyphosate and on microbial diversity in biomixtures were evaluated. Small residual amounts of all pesticides at 20 (<2%) and 41 (<1%) days were observed; however, the lowest efficiency rates were observed in soil. Glyphosate was the only pesticide that completely dissipated in soil and biomixtures. Archaea, bacteria, and fungi were identified in biobeds, with bacteria being the most diverse microorganisms according to the identified species. The presence of white-rot fungi (normally related to pesticide degradation in biomixtures) was observed. Effects of the pesticide type and of biomixtures on pesticide dissipation were significant (P<0.05); however, only the effect of biomixtures on microbial diversity was significant (P<0.05); microbial diversity and richness had a significant effect on the residual amount of pesticides (P<0.05). Microbial diversity in terms of phyla was directly related to physicochemical parameters such as organic matter, lignin, water-holding capacity, and pH of soil and biomixtures. Copyright © 2018 Elsevier B.V. All rights reserved.

A-Kinase Anchoring Proteins: From protein complexes to physiology and disease

PubMed Central

Carnegie, Graeme K.; Means, Christopher K.; Scott, John D.

2009-01-01

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review we focus on recent advances in the elucidation of AKAP function. PMID:19319965

A-kinase anchoring proteins: from protein complexes to physiology and disease.

PubMed

Carnegie, Graeme K; Means, Christopher K; Scott, John D

2009-04-01

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review, we focus on recent advances in the elucidation of AKAP function.

Biosynthetic machinery of ionophore polyether lasalocid: enzymatic construction of polyether skeleton.

PubMed

Minami, Atsushi; Oguri, Hiroki; Watanabe, Kenji; Oikawa, Hideaki

2013-08-01

Diversity of natural polycyclic polyethers originated from very simple yet versatile strategy consisting of epoxidation of linear polyene followed by epoxide opening cascade. To understand two-step enzymatic transformations at molecular basis, a flavin containing monooxygenase (EPX) Lsd18 and an epoxide hydrolase (EH) Lsd19 were selected as model enzymes for extensive investigation on substrate specificity, catalytic mechanism, cofactor requirement and crystal structure. This pioneering study on prototypical lasalocid EPX and EH provides insight into detailed mechanism of ionophore polyether assembly machinery and clarified remaining issues for polyether biosynthesis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Interspecific metabolic diversity of root-colonizing endophytic fungi revealed by enzyme activity tests.

PubMed

Knapp, Dániel G; Kovács, Gábor M

2016-12-01

Although dark septate endophytes (DSE) represent a worldwide dispersed form group of root-colonizing endophytic fungi, our knowledge on their role in ecosystem functioning is far limited. In this study, we aimed to test if functional diversity exists among DSE fungi representing different lineages of root endophytic fungal community of semiarid sandy grasslands. To address this question and to gain general information on function of DSE fungi, we adopted api-ZYM and BioLog FF assays to study those non-sporulating filamentous fungi and characterized the metabolic activity of 15 different DSE species. Although there were striking differences among the species, all of the substrates tested were utilized by the DSE fungi. When endophytes characteristic to grasses and non-grass host plants were separately considered, we found that the whole substrate repertoire was used by both groups. This might illustrate the complementary functional diversity of the communities root endophytic plant-associated fungi. The broad spectra of substrates utilized by these root endophytes illustrate the functional importance of their diversity, which can play role not only in nutrient mobilization and uptake of plants from with nutrient poor soils, but also in general plant performance and ecosystem functioning. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A chemical proteomics approach for global analysis of lysine monomethylome profiling.

PubMed

Wu, Zhixiang; Cheng, Zhongyi; Sun, Mingwei; Wan, Xuelian; Liu, Ping; He, Tieming; Tan, Minjia; Zhao, Yingming

2015-02-01

Methylation of lysine residues on histone proteins is known to play an important role in chromatin structure and function. However, non-histone protein substrates of this modification remain largely unknown. An effective approach for system-wide analysis of protein lysine methylation, particularly lysine monomethylation, is lacking. Here we describe a chemical proteomics approach for global screening for monomethyllysine substrates, involving chemical propionylation of monomethylated lysine, affinity enrichment of the modified monomethylated peptides, and HPLC/MS/MS analysis. Using this approach, we identified with high confidence 446 lysine monomethylation sites in 398 proteins, including three previously unknown histone monomethylation marks, representing the largest data set of protein lysine monomethylation described to date. Our data not only confirms previously discovered lysine methylation substrates in the nucleus and spliceosome, but also reveals new substrates associated with diverse biological processes. This method hence offers a powerful approach for dynamic study of protein lysine monomethylation under diverse cellular conditions and in human diseases. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Genotyping of Sulfurospirillum spp. in Mixed Cultures Allowed the Identification of a New Tetrachloroethene Reductive Dehalogenase

PubMed Central

Buttet, Géraldine F.; Holliger, Christof

2013-01-01

Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed. PMID:23995945

Large-Scale, Lineage-Specific Expansion of a Bric-a-Brac/Tramtrack/Broad Complex Ubiquitin-Ligase Gene Family in Rice[W

PubMed Central

Gingerich, Derek J.; Hanada, Kousuke; Shiu, Shin-Han; Vierstra, Richard D.

2007-01-01

Selective ubiquitination of proteins is directed by diverse families of ubiquitin-protein ligases (or E3s) in plants. One important type uses Cullin-3 as a scaffold to assemble multisubunit E3 complexes containing one of a multitude of bric-a-brac/tramtrack/broad complex (BTB) proteins that function as substrate recognition factors. We previously described the 80-member BTB gene superfamily in Arabidopsis thaliana. Here, we describe the complete BTB superfamily in rice (Oryza sativa spp japonica cv Nipponbare) that contains 149 BTB domain–encoding genes and 43 putative pseudogenes. Amino acid sequence comparisons of the rice and Arabidopsis superfamilies revealed a near equal repertoire of putative substrate recognition module types. However, phylogenetic comparisons detected numerous gene duplication and/or loss events since the rice and Arabidopsis BTB lineages split, suggesting possible functional specialization within individual BTB families. In particular, a major expansion and diversification of a subset of BTB proteins containing Meprin and TRAF homology (MATH) substrate recognition sites was evident in rice and other monocots that likely occurred following the monocot/dicot split. The MATH domain of a subset appears to have evolved significantly faster than those in a smaller core subset that predates flowering plants, suggesting that the substrate recognition module in many monocot MATH-BTB E3s are diversifying to ubiquitinate a set of substrates that are themselves rapidly changing. Intriguing possibilities include pathogen proteins attempting to avoid inactivation by the monocot host. PMID:17720868

Substrate Interactions and Promiscuity in a Viral DNA Packaging Motor

PubMed Central

Aathavan, K.; Politzer, Adam T.; Kaplan, Ariel; Moffitt, Jeffrey R.; Chemla, Yann R.; Grimes, Shelley; Jardine, Paul J.; Anderson, Dwight L.; Bustamante, Carlos

2009-01-01

The ASCE superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life1. A subset of these enzymes are multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses, and tailed bacteriophages2. While their mechanism of mechanochemical conversion is beginning to be understood3, little is known about how these motors engage their nucleic acid substrates. Do motors contact a single DNA element, such as a phosphate or a base, or are contacts distributed over multiple parts of the DNA? In addition, what role do these contacts play in the mechanochemical cycle? Here we use the genome packaging motor of the Bacillus subtilis bacteriophage φ294 to address these questions. The full mechanochemical cycle of the motor, whose ATPase is a pentameric-ring5 of gene product 16, involves two phases-- an ATP loading dwell followed by a translocation burst of four 2.5-bp steps6 triggered by hydrolysis product release7. By challenging the motor with a variety of modified DNA substrates, we find that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5’-3’ strand in the direction of packaging. In addition to providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, non-specific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily1. PMID:19794496

Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sakai, A.; Fedorov, A; Fedorov, E

2009-01-01

The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the a-proton of a carboxylate substrate that is coordinated to an essential Mg2+. The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the e-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified.more » Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221; anti, Mycobacterium smegmatis, gi 118470554) document that the conserved Lys at the end of the second e-strand in the (e/a)7e-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of 'pseudoconvergent' evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.« less

Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae

PubMed Central

Deng, Xu; Petitjean, Marjorie; Teste, Marie-Ange; Kooli, Wafa; Tranier, Samuel; François, Jean Marie; Parrou, Jean-Luc

2014-01-01

The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis–Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties. PMID:24649402

Similarities and differences in the biochemical and enzymological properties of the four isomaltases from Saccharomyces cerevisiae.

PubMed

Deng, Xu; Petitjean, Marjorie; Teste, Marie-Ange; Kooli, Wafa; Tranier, Samuel; François, Jean Marie; Parrou, Jean-Luc

2014-01-01

The yeast Saccharomyces cerevisiae IMA multigene family encodes four isomaltases sharing high sequence identity from 65% to 99%. Here, we explore their functional diversity, with exhaustive in-vitro characterization of their enzymological and biochemical properties. The four isoenzymes exhibited a preference for the α-(1,6) disaccharides isomaltose and palatinose, with Michaëlis-Menten kinetics and inhibition at high substrates concentration. They were also able to hydrolyze trisaccharides bearing an α-(1,6) linkage, but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrate ambiguity. While Ima1p and Ima2p presented almost identical characteristics, our results nevertheless showed many singularities within this protein family. In particular, Ima3p presented lower activities and thermostability than Ima2p despite only three different amino acids between the sequences of these two isoforms. The Ima3p_R279Q variant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240 significantly increased the stability of Ima3p and supported the role of prolines in thermostability. The most distant protein, Ima5p, presented the lowest optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specific amino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibition by maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitive at very low isomaltose concentrations and uncompetitive as the substrate concentration increased. Altogether, this work illustrates that a gene family encoding proteins with strong sequence similarities can lead to enzyme with notable differences in biochemical and enzymological properties.

Specificity profiling of protein-binding domains using one-bead-one-compound Peptide libraries.

PubMed

Kunys, Andrew R; Lian, Wenlong; Pei, Dehua

2012-12-01

One-bead-one-compound (OBOC) libraries consist of structurally related compounds (e.g., peptides) covalently attached to a solid support, with each resin bead carrying a unique compound. OBOC libraries of high structural diversity can be rapidly synthesized and screened without the need for any special equipment, and therefore can be employed in any chemical or biochemical laboratory. OBOC peptide libraries have been widely used to map the ligand specificity of proteins, to determine the substrate specificity of enzymes, and to develop inhibitors against macromolecular targets. They have proven particularly useful in profiling the binding specificity of protein modular domains (e.g., SH2 domains, BIR domains, and PDZ domains); subsequently, the specificity information can be used to predict the protein targets of these domains. The protocols outlined in this article describe the methodologies for synthesizing and screening OBOC peptide libraries against SH2 and PDZ domains, and the related data analysis. Curr. Protoc. Chem. Biol. 4:331-355 © 2012 by John Wiley & Sons, Inc.

Functional diversity of microbial communities in pristine aquifers inferred by PLFA- and sequencing-based approaches

NASA Astrophysics Data System (ADS)

Schwab, Valérie F.; Herrmann, Martina; Roth, Vanessa-Nina; Gleixner, Gerd; Lehmann, Robert; Pohnert, Georg; Trumbore, Susan; Küsel, Kirsten; Totsche, Kai U.

2017-05-01

Microorganisms in groundwater play an important role in aquifer biogeochemical cycles and water quality. However, the mechanisms linking the functional diversity of microbial populations and the groundwater physico-chemistry are still not well understood due to the complexity of interactions between surface and subsurface. Within the framework of Hainich (north-western Thuringia, central Germany) Critical Zone Exploratory of the Collaborative Research Centre AquaDiva, we used the relative abundances of phospholipid-derived fatty acids (PLFAs) to link specific biochemical markers within the microbial communities to the spatio-temporal changes of the groundwater physico-chemistry. The functional diversities of the microbial communities were mainly correlated with groundwater chemistry, including dissolved O2, Fet and NH4+ concentrations. Abundances of PLFAs derived from eukaryotes and potential nitrite-oxidizing bacteria (11Me16:0 as biomarker for Nitrospira moscoviensis) were high at sites with elevated O2 concentration where groundwater recharge supplies bioavailable substrates. In anoxic groundwaters more rich in Fet, PLFAs abundant in sulfate-reducing bacteria (SRB), iron-reducing bacteria and fungi increased with Fet and HCO3- concentrations, suggesting the occurrence of active iron reduction and the possible role of fungi in meditating iron solubilization and transport in those aquifer domains. In more NH4+-rich anoxic groundwaters, anammox bacteria and SRB-derived PLFAs increased with NH4+ concentration, further evidencing the dependence of the anammox process on ammonium concentration and potential links between SRB and anammox bacteria. Additional support of the PLFA-based bacterial communities was found in DNA- and RNA-based Illumina MiSeq amplicon sequencing of bacterial 16S rRNA genes, which showed high predominance of nitrite-oxidizing bacteria Nitrospira, e.g. Nitrospira moscoviensis, in oxic aquifer zones and of anammox bacteria in more NH4+-rich anoxic groundwater. Higher relative abundances of sequence reads in the RNA-based datasets affiliated with iron-reducing bacteria in more Fet-rich groundwater supported the occurrence of active dissimilatory iron reduction. The functional diversity of the microbial communities in the biogeochemically distinct groundwater assemblages can be largely attributed to the redox conditions linked to changes in bioavailable substrates and input of substrates with the seepage. Our results demonstrate the power of complementary information derived from PLFA-based and sequencing-based approaches.

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

Big River Benthos: Linking Year Round Biological Response to Altered Hydrological Regimes

DTIC Science & Technology

2017-04-02

is also home to a diversity of organisms adapted to large river habitats. Macroinvertebrates have long been used as habitat/water quality indicators...substrates, but lower abundances (Figure 5). Sand was the most frequently encountered substrate (n=52, Figure 6), and comprises approximately 80% of the...June sampling represented as percentages. 8 Because sand is predominant in the LMR, habitats containing a variety of substrates, including silt

Ada response – a strategy for repair of alkylated DNA in bacteria

PubMed Central

Mielecki, Damian; Grzesiuk, Elżbieta

2014-01-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP).

PubMed

Liu, Jun; Zou, Yang; Guan, Wanyi; Zhai, Yafei; Xue, Mengyang; Jin, Lan; Zhao, Xueer; Dong, Junkai; Wang, Wenjun; Shen, Jie; Wang, Peng George; Chen, Min

2013-07-01

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP. Copyright © 2013 Elsevier Ltd. All rights reserved.

Diversity and distribution of actinobacterial aromatic ring oxygenase genes across contrasting soil properties.

PubMed

Weidow, Christopher A; Bae, Hee-Sung; Chauhan, Ashvini; Ogram, Andrew

2015-04-01

The diversity of a gene family encoding Actinobacterial aromatic ring oxygenases (AAROs) was detected by the PCR-cloning approach using a newly designed PCR primer set. The distribution of AAROs was investigated in 11 soils representing different land management and vegetation zones and was correlated with several geochemical parameters including pH, organic matter (OM), total Kjeldahl nitrogen (TKN), and nitrogen oxides (NO(x)-N: mostly NO3(-)-N). The distribution of individual clades encoding enzymes with potentially different substrates were correlated with different environmental factors, suggesting differential environmental controls on the distribution of specific enzymes as well as sequence diversity. For example, individual clades associated with phthalate dioxygenases were either strongly negatively correlated with pH, or not correlated with pH but showed strong positive correlation with organic carbon content. A large number of clones clustering in a clade related to PAH oxygenases were positively correlated with pH and nitrogen, but not with organic matter. This analysis may yield insight into the ecological forces driving the distribution of these catabolic genes.

Re-meandering of lowland streams: will disobeying the laws of geomorphology have ecological consequences?

PubMed

Pedersen, Morten Lauge; Kristensen, Klaus Kevin; Friberg, Nikolai

2014-01-01

We evaluated the restoration of physical habitats and its influence on macroinvertebrate community structure in 18 Danish lowland streams comprising six restored streams, six streams with little physical alteration and six channelized streams. We hypothesized that physical habitats and macroinvertebrate communities of restored streams would resemble those of natural streams, while those of the channelized streams would differ from both restored and near-natural streams. Physical habitats were surveyed for substrate composition, depth, width and current velocity. Macroinvertebrates were sampled along 100 m reaches in each stream, in edge habitats and in riffle/run habitats located in the center of the stream. Restoration significantly altered the physical conditions and affected the interactions between stream habitat heterogeneity and macroinvertebrate diversity. The substrate in the restored streams was dominated by pebble, whereas the substrate in the channelized and natural streams was dominated by sand. In the natural streams a relationship was identified between slope and pebble/gravel coverage, indicating a coupling of energy and substrate characteristics. Such a relationship did not occur in the channelized or in the restored streams where placement of large amounts of pebble/gravel distorted the natural relationship. The analyses revealed, a direct link between substrate heterogeneity and macroinvertebrate diversity in the natural streams. A similar relationship was not found in either the channelized or the restored streams, which we attribute to a de-coupling of the natural relationship between benthic community diversity and physical habitat diversity. Our study results suggest that restoration schemes should aim at restoring the natural physical structural complexity in the streams and at the same time enhance the possibility of re-generating the natural geomorphological processes sustaining the habitats in streams and rivers. Documentation of restoration efforts should be intensified with continuous monitoring of geomorphological and ecological changes including surveys of reference river systems.

Re-Meandering of Lowland Streams: Will Disobeying the Laws of Geomorphology Have Ecological Consequences?

PubMed Central

Pedersen, Morten Lauge; Kristensen, Klaus Kevin; Friberg, Nikolai

2014-01-01

We evaluated the restoration of physical habitats and its influence on macroinvertebrate community structure in 18 Danish lowland streams comprising six restored streams, six streams with little physical alteration and six channelized streams. We hypothesized that physical habitats and macroinvertebrate communities of restored streams would resemble those of natural streams, while those of the channelized streams would differ from both restored and near-natural streams. Physical habitats were surveyed for substrate composition, depth, width and current velocity. Macroinvertebrates were sampled along 100 m reaches in each stream, in edge habitats and in riffle/run habitats located in the center of the stream. Restoration significantly altered the physical conditions and affected the interactions between stream habitat heterogeneity and macroinvertebrate diversity. The substrate in the restored streams was dominated by pebble, whereas the substrate in the channelized and natural streams was dominated by sand. In the natural streams a relationship was identified between slope and pebble/gravel coverage, indicating a coupling of energy and substrate characteristics. Such a relationship did not occur in the channelized or in the restored streams where placement of large amounts of pebble/gravel distorted the natural relationship. The analyses revealed, a direct link between substrate heterogeneity and macroinvertebrate diversity in the natural streams. A similar relationship was not found in either the channelized or the restored streams, which we attribute to a de-coupling of the natural relationship between benthic community diversity and physical habitat diversity. Our study results suggest that restoration schemes should aim at restoring the natural physical structural complexity in the streams and at the same time enhance the possibility of re-generating the natural geomorphological processes sustaining the habitats in streams and rivers. Documentation of restoration efforts should be intensified with continuous monitoring of geomorphological and ecological changes including surveys of reference river systems. PMID:25264627

Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution.

PubMed

Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C B; Lakshmipriya, Thangavel; Tang, Thean-Hock

2015-01-01

Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues.

Identification of Arabidopsis MYB56 as a novel substrate for CRL3BPM E3 ligases.

PubMed

Chen, Liyuan; Bernhardt, Anne; Lee, JooHyun; Hellmann, Hanjo

2014-10-24

Controlled stability of proteins is a highly efficient mechanism to direct diverse processes in living cells. A key regulatory system for protein stability is given by the ubiquitin proteasome pathway, which uses E3 ligases to mark specific proteins for degradation. In this work MYB56 is identified as a novel target of a CULLIN3 (CUL3)-based E3 ligase. Its stability depends on the presence of MATH-BTB/POZ (BPM) proteins, which function as substrate adaptors to the E3 ligase. Genetic studies pointed out that MYB56 is a negative regulator of flowering, while BPMs positively affect this developmental program. The interaction between BPMs and MYB56 occurs at the promoter of FLOWERING LOCUS T (FT), a key regulator in initiating flowering in Arabidopsis, and results in instability of MYB56. Overall the work establishes MYB transcription factors as substrates of BPM proteins, and provides novel information on components that participate in controlling the flowering time point in plants. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

Systematic review of the effects of the intestinal microbiota on selected nutrients and non-nutrients.

PubMed

Shortt, Colette; Hasselwander, Oliver; Meynier, Alexandra; Nauta, Arjen; Fernández, Estefanía Noriega; Putz, Peter; Rowland, Ian; Swann, Jonathan; Türk, Jessica; Vermeiren, Joan; Antoine, Jean-Michel

2018-02-01

There is considerable interest in the effects of the intestinal microbiota (IM) composition, its activities in relation with the metabolism of dietary substrates and the impact these effects may have in the development and prevention of certain non-communicable diseases. It is acknowledged that a complex interdependence exists between the IM and the mammalian host and that the IM possesses a far greater diversity of genes and repertoire of metabolic and enzymatic capabilities than their hosts. However, full knowledge of the metabolic activities and interactions of the IM and the functional redundancy that may exist are lacking. Thus, the current review aims to assess recent literature relating to the role played by the IM in the absorption and metabolism of key nutrients and non-nutrients. A systematic review (PROSPERO registration: CRD42015019087) was carried out focussing on energy and the following candidate dietary substrates: protein, carbohydrate, fat, fibre, resistant starch (RS), and polyphenols to further understand the effect of the IM on the dietary substrates and the resulting by-products and host impacts. Particular attention was paid to the characterisation of the IM which are predominantly implicated in each case, changes in metabolites, and indirect markers and any potential impacts on the host. Studies show that the IM plays a key role in the metabolism of the substrates studied. However, with the exception of studies focusing on fibre and polyphenols, there have been relatively few recent human studies specifically evaluating microbial metabolism. In addition, comparison of the effects of the IM across studies was difficult due to lack of specific analysis/description of the bacteria involved. Considerable animal-derived data exist, but experience suggests that care must be taken when extrapolating these results to humans. Nevertheless, it appears that the IM plays a role in energy homeostasis and that protein microbial breakdown and fermentation produced ammonia, amines, phenols and branch chain fatty acids, and a greater diversity in the microbes present. Few recent studies appear to have evaluated the effect of the IM composition and metabolism per se in relation with digestible dietary carbohydrate or fat in humans. Intakes of RS and prebiotics altered levels of specific taxa that selectively metabolised specific prebiotic/carbohydrate-type substances and levels of bifidobacteria and lactobacilli were observed to increase. In controlled human studies, consistent data exist that show a correlation between the intake of fibre and an increase in bifidobacteria and short-chain fatty acids, in particular butyrate, which leads to lower intestinal pH. Dietary polyphenols rely on modification either by host digestive enzymes or those derived from the IM for absorption to occur. In the polyphenol-related studies, a large amount of inter-individual variation was observed in the microbial metabolism and absorption of certain polyphenols. The systematic review demonstrates that the IM plays a major role in the breakdown and transformation of the dietary substrates examined. However, recent human data are limited with the exception of data from studies examining fibres and polyphenols. Results observed in relation with dietary substrates were not always consistent or coherent across studies and methodological limitations and differences in IM analyses made comparisons difficult. Moreover, non-digestible components likely to reach the colon are often not well defined or characterised in studies making comparisons between studies difficult if not impossible. Going forward, further rigorously controlled randomised human trials with well-defined dietary substrates and utilizing omic-based technologies to characterise and measure the IM and their functional activities will advance the field. Current evidence suggests that more detailed knowledge of the metabolic activities and interactions of the IM hold considerable promise in relation with host health.

Assemblage composition of fungal wood-decay species has a major influence on how climate and wood quality modify decomposition.

PubMed

Venugopal, Parvathy; Junninen, Kaisa; Edman, Mattias; Kouki, Jari

2017-03-01

The interactions among saprotrophic fungal species, as well as their interactions with environmental factors, may have a major influence on wood decay and carbon release in ecosystems. We studied the effect that decomposer diversity (species richness and assemblage composition) has on wood decomposition when the climatic variables and substrate quality vary simultaneously. We used two temperatures (16 and 21°C) and two humidity levels (70% and 90%) with two wood qualities (wood from managed and old-growth forests) of Pinus sylvestris. In a 9-month experiment, the effects of fungal diversity were tested using four wood-decaying fungi (Antrodia xantha, Dichomitus squalens, Fomitopsis pinicola and Gloeophyllum protractum) at assemblage levels of one, two and four species. Wood quality and assemblage composition affected the influence of climatic factors on decomposition rates. Fungal assemblage composition was found to be more important than fungal species richness, indicating that species-specific fungal traits are of paramount importance in driving decomposition. We conclude that models containing fungal wood-decay species (and wood-based carbon) need to take into account species-specific and assemblage composition-specific properties to improve predictive capacity in regard to decomposition-related carbon dynamics. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hard substrate in the deep ocean: How sediment features influence epibenthic megafauna on the eastern Canadian margin

NASA Astrophysics Data System (ADS)

Lacharité, Myriam; Metaxas, Anna

2017-08-01

Benthic habitats on deep continental margins (> 1000 m) are now considered heterogeneous - in particular because of the occasional presence of hard substrate in a matrix of sand and mud - influencing the distribution of megafauna which can thrive on both sedimented and rocky substrates. At these depths, optical imagery captured with high-definition cameras to describe megafauna can also describe effectively the fine-scale sediment properties in the immediate vicinity of the fauna. In this study, we determined the relationship between local heterogeneity (10-100 sm) in fine-scale sediment properties and the abundance, composition, and diversity of megafauna along a large depth gradient (1000-3000 m) in a previously-unexplored habitat: the Northeast Fan, which lies downslope of submarine canyons off the Gulf of Maine (northwest Atlantic). Substrate heterogeneity was quantified using a novel approach based on principles of computer vision. This approach proved powerful in detecting gradients in sediment, and sporadic complex features (i.e. large boulders) in an otherwise homogeneous environment because it characterizes sediment properties on a continuous scale. Sediment heterogeneity influenced megafaunal diversity (morphospecies richness and Shannon-Wiener Index) and community composition, with areas of higher substrate complexity generally supported higher diversity. However, patterns in abundance were not influenced by sediment properties, and may be best explained by gradients in food supply. Our study provides a new approach to quantify fine-scale sediment properties and assess their role in shaping megafaunal communities in the deep sea, which should be included into habitat studies given their potential ecological importance.

Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agarwal R.; Swaminathan S.; Burley, S. K.

2012-01-01

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k{sub cat} values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptidemore » chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified {sup 447}{und GG}LPQ{und K}{sup 452} as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.« less

Biomaterials Made from Coiled-Coil Peptides.

PubMed

Conticello, Vincent; Hughes, Spencer; Modlin, Charles

The development of biomaterials designed for specific applications is an important objective in personalized medicine. While the breadth and prominence of biomaterials have increased exponentially over the past decades, critical challenges remain to be addressed, particularly in the development of biomaterials that exhibit highly specific functions. These functional properties are often encoded within the molecular structure of the component molecules. Proteins, as a consequence of their structural specificity, represent useful substrates for the construction of functional biomaterials through rational design. This chapter provides an in-depth survey of biomaterials constructed from coiled-coils, one of the best-understood protein structural motifs. We discuss the utility of this structurally diverse and functionally tunable class of proteins for the creation of novel biomaterials. This discussion illustrates the progress that has been made in the development of coiled-coil biomaterials by showcasing studies that bridge the gap between the academic science and potential technological impact.

Diurnal roosts of male evening bats (Nycticeius humeralis) in diversely managed pine-hardwood forests

Treesearch

Roger W. Perry; Ronald E. Thill

2008-01-01

We examined attributes of 45 roost sites used by 17 adult male evening bats (Nycticeius humeralis) in a diverse forested landscape within the Ouachita Mountains, Arkansas. Bats roosted in a diverse array of substrates, including live or dead Pinus echinata $15 cm diam at breast height (29% of roosts) and small (,10 cm) understory or midstory...

Diversity, abundance, and size structure of bivalve assemblages in the Sipsey River, Alabama

Treesearch

Wendell R. Haag; Melvin L. Jr. Warren

2010-01-01

1. Patterns of mussel diversity and assemblage structure in the Sipsey River, Alabama, are described. Qualitative data were used to describe river-wide patterns of diversity. Quantitative data were used to describe the structure of mussel assemblages at several sites based on whole-substrate sampling that ensured all size classes were detected. 2. Major human impacts...

Synthesis of Sequence-Specific DNA-Protein Conjugates via a Reductive Amination Strategy

PubMed Central

Wickramaratne, Susith; Mukherjee, Shivam; Villalta, Peter W.; Schärer, Orlando D.; Tretyakova, Natalia

2013-01-01

DNA-protein cross-links (DPCs) are ubiquitous, structurally diverse DNA lesions formed upon exposure to bis-electrophiles, transition metals, UV light, and reactive oxygen species. Because of their super-bulky, helix distorting nature, DPCs interfere with DNA replication, transcription, and repair, potentially contributing to mutagenesis and carcinogenesis. However, the biological implications of DPC lesions have not been fully elucidated due to the difficulty of generating site-specific DNA substrates representative of DPC lesions formed in vivo. In the present study, a novel approach involving post-synthetic reductive amination has been developed to prepare a range of hydrolytically stable lesions structurally mimicking the DPCs produced between the N7 position of guanine in DNA and basic lysine or arginine side chains of proteins and peptides. PMID:23885807

Different strokes for different bugs-Examples of stridulatory diversity in the Hemiptera

USDA-ARS?s Scientific Manuscript database

Many Hemipteran insects communicate through substrate-borne vibrations produced by a diversity of stridulatory structures. Examples are presented of stridulations produced by different structures of two little-studied Hemipterans, a Rhopalid, Jadera haematoloma, and a pentatomid, Euschistus servus. ...

Biosynthesis of nanoparticles using microbes- a review.

PubMed

Hulkoti, Nasreen I; Taranath, T C

2014-09-01

The biosynthesis of nanoparticles by microorganism is a green and eco-friendly technology. This review focuses on the use of consortium of diverse microorganisms belonging to both prokaryotes and eukaryotes for the synthesis of metallic nanoparticles viz. silver, gold, platinum, zirconium, palladium, iron, cadmium and metal oxides such as titanium oxide, zinc oxide, etc. These microorganisms include bacteria, actinomycetes, fungi and algae. The synthesis of nanoparticles may be intracellular or extracellular. The several workers have reported that NADH dependent nitrate reductase enzyme plays a vital role in the conversion of metallic ions to nanoparticles. The FTIR study reveals that diverse biomolecules viz. carboxyl group, primary and secondary amines, amide I, II, and III bands etc serve as a tool for bioreduction and capping agents there by offering stability to particles by preventing agglomeration and growth. The size and shape of the nanoparticles vary with the organism employed and conditions employed during the synthesis which included pH, temperature and substrate concentration. The microorganisms provide diverse environment for biosynthesis of nanoparticles. These particles are safe and eco-friendly with a lot of applications in medicine, agriculture, cosmetic industry, drug delivery and biochemical sensors. The challenges for redressal include optimal production and minimal time to obtain desired size and shape, to enhance the stability of nanoparticles and optimization of specific microorganisms for specific application. Copyright © 2014 Elsevier B.V. All rights reserved.

Plant Biodiversity Drivers in Brazilian Campos Rupestres: Insights from Phylogenetic Structure

PubMed Central

Zappi, Daniela C.; Moro, Marcelo F.; Meagher, Thomas R.; Nic Lughadha, Eimear

2017-01-01

Old, climate-buffered infertile landscapes (Ocbils) have attracted increasing levels of interest in recent years because of their exceptionally diverse plant communities. Brazil’s campos rupestres (rupestrian grasslands) are home to almost 15% of Brazil’s native flora in less than 0.8% of Brazil’s territory: an ideal study system for exploring variation in floristic diversity and phylogenetic structure in sites differing in geology and phytophysiognomy. We found significant differences in floristic diversity and phylogenetic structure across a range of study sites encompassing open vegetation and forest on quartzite (FQ) and on ironstone substrates, commonly termed canga. Substrate and physiognomy were key in structuring floristic diversity in the Espinhaço and physiognomy was more important than substrate in structuring phylogenetic diversity, with neither substrate nor its interaction with physiognomy accounting for significant variation in phylogenetic structure. Phylogenetic clustering was significant in open vegetation on both canga and quartzite, reflecting the potential role of environmental filtering in these exposed montane communities adapted to multiple environmental stressors. In forest communities, phylogenetic clustering was significant only at relatively deep nodes of the phylogeny in FQ while no significant phylogenetic clustering was detected across forest on canga (FC), which may be attributable to proximity to the megadiverse Atlantic forest biome and/or comparatively benign environmental conditions in FC with relatively deep, nutrient-rich soils and access to edaphic water reliable in comparison to those for open vegetation on canga and open or forest communities on quartzite. Clades representing relatively old lineages are significantly over-represented in campos rupestres on quartzite, consistent with the Gondwanan Heritage Hypothesis of Ocbil theory. In contrast, forested sites on canga are recognized as Yodfels. To be effective, conservation measures must take account of the distinct communities which are encompassed within the broad term campos rupestres, and the differing vulnerabilities of Ocbils and Yodfels. PMID:29312396

Plant Biodiversity Drivers in Brazilian Campos Rupestres: Insights from Phylogenetic Structure.

PubMed

Zappi, Daniela C; Moro, Marcelo F; Meagher, Thomas R; Nic Lughadha, Eimear

2017-01-01

Old, climate-buffered infertile landscapes (Ocbils) have attracted increasing levels of interest in recent years because of their exceptionally diverse plant communities. Brazil's campos rupestres (rupestrian grasslands) are home to almost 15% of Brazil's native flora in less than 0.8% of Brazil's territory: an ideal study system for exploring variation in floristic diversity and phylogenetic structure in sites differing in geology and phytophysiognomy. We found significant differences in floristic diversity and phylogenetic structure across a range of study sites encompassing open vegetation and forest on quartzite (FQ) and on ironstone substrates, commonly termed canga . Substrate and physiognomy were key in structuring floristic diversity in the Espinhaço and physiognomy was more important than substrate in structuring phylogenetic diversity, with neither substrate nor its interaction with physiognomy accounting for significant variation in phylogenetic structure. Phylogenetic clustering was significant in open vegetation on both canga and quartzite, reflecting the potential role of environmental filtering in these exposed montane communities adapted to multiple environmental stressors. In forest communities, phylogenetic clustering was significant only at relatively deep nodes of the phylogeny in FQ while no significant phylogenetic clustering was detected across forest on canga (FC), which may be attributable to proximity to the megadiverse Atlantic forest biome and/or comparatively benign environmental conditions in FC with relatively deep, nutrient-rich soils and access to edaphic water reliable in comparison to those for open vegetation on canga and open or forest communities on quartzite. Clades representing relatively old lineages are significantly over-represented in campos rupestres on quartzite, consistent with the Gondwanan Heritage Hypothesis of Ocbil theory. In contrast, forested sites on canga are recognized as Yodfels. To be effective, conservation measures must take account of the distinct communities which are encompassed within the broad term campos rupestres , and the differing vulnerabilities of Ocbils and Yodfels.

Bacterial Community Profiling of H2/CO2 or Formate-Utilizing Acetogens Enriched from Diverse Ecosystems

NASA Astrophysics Data System (ADS)

Han, R.; Zhang, L.; Fu, B.; Liu, H.

2014-12-01

Synthetic gases are usually generated from either cellulosic agricultural waste combustion or industrial release and could be subsequently transformed into acetate, ethanol, and/or butyrate by homoacetogenic bacteria, which commonly possess reductive acetyl-CoA synthesis pathway. Homoacetogen-based syngas fermentation technology provides an alternative solution to link greenhouse gas emission control and cellulosic solid waste treatment with biofuels production. The objective of our current project is to hunt for homoacetogens with capabilities of highly efficiently converting syngases to chemical solvents. In this study, we evaluated homoacetogens population dynamics during enrichments and pinpointed dominant homoacetogens representing diverse ecosystems enriched by different substrates. We enriched homoacetogens from four different samples including waste activate sludge, freshwater sediment, anaerobic methanogenic sludge, and cow manure using H2/CO2 (4:1) or formate as substrate for homoacetogen enrichment. Along with the formyltetrahydrofolate synthetase (FTHFS) gene (fhs gene)-specific real time qPCR assay and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, 16S rRNA based 454 high-throughput pyrosequencing was applied to reveal the population dynamic and community structure during enrichment from different origins. Enrichment of homoacetogenic populations coincided with accumulations of short chain fatty acids such as acetate and butyrate. 454 high-throughput pyrosequencing revealed Firmicutes and Spirochaetes populations became dominant while the overall microbial diversity decreased after enrichment. The most abundant sequences among the four origins belonged to the following phyla: Firmicutes, Spirochaetes, Proteobacteria, and Bacteroidetes, accounting for 62.1%-99.1% of the total reads. The major putative homoacetogenic species enriched on H2/CO2 or formate belonged to Clostridium spp., Acetobacterium spp., Acetoanaerobium spp., Eubacterium spp., Sporomusa spp. This comprehensive molecular ecology study on homoacetogen enrichments provides molecular evidences for shaping homoacetogenic populations and targeting novel homoacetogenic species enriched from diverse ecosystems.

An Exploration into the Bacterial Community under Different Pasteurization Conditions during Substrate Preparation (Composting-Phase II) for Agaricus bisporus Cultivation.

PubMed

Vieira, Fabricio Rocha; Pecchia, John Andrew

2018-02-01

Substrate preparation (i.e., composting) for Agaricus bisporus cultivation is the most critical point of mushroom production. Among many factors involved in the composting process, the microbial ecology of the system is the underlying drive of composting and can be influenced by composting management techniques. Pasteurization temperature at the beginning of phase II, in theory, may influence the bacterial community and subsequently the "selectivity" and nutrition of the final substrate. Therefore, this hypothesis was tested by simulation in bioreactors under different pasteurization conditions (57 °C/6 h, 60 °C/2 h, and 68 °C/2 h), simulating conditions adopted by many producers. Bacterial diversity, based on 16S ribosomal RNA obtained by high-throughput sequencing and classified in operational taxonomic units (OTUs), was greater than previously reported using culture-dependent methods. Alpha diversity estimators show a lower diversity of OTUs under a high-temperature pasteurization condition. Bacillales order shows a relatively higher OTU abundance under a high-pasteurization temperature, which also was related to high ammonia emission measurements. On the other hand, beta diversity analysis showed no significantly changes in the bacterial community structure under different conditions. Agaricus bisporus mycelium growth during a standard spawn run period was significantly slower in the compost pasteurized at high temperature. Since the bacterial community structure was not greatly affected by different pasteurization conditions but by-products left (e.g., ammonia) at the end of compost conditioning varied, further studies need to be conducted to determine the functional role of the microbial communities found during substrate preparation for Agaricus bisporus cultivation.

Changes in methanogenic substrate utilization and communities with depth in a salt-marsh, creek sediment in southern England

NASA Astrophysics Data System (ADS)

John Parkes, R.; Brock, Fiona; Banning, Natasha; Hornibrook, Edward R. C.; Roussel, Erwan G.; Weightman, Andrew J.; Fry, John C.

2012-01-01

A combined biogeochemical and molecular genetic study of creek sediments (down to 65 cm depth) from Arne Peninsula salt-marsh (Dorset, UK) determined the substrates used for methanogenesis and the distribution of the common methanogens, Methanosarcinales and Methanomicrobiales capable of metabolising these substrates. Methane concentrations increased by 11 cm, despite pore water sulphate not being removed until 45 cm. Neither upward methane diffusion or anaerobic oxidation of methane seemed to be important in this zone. In the near-surface sulphate-reduction zone (5-25 cm) turnover time to methane for the non-competitive methanogenic substrate trimethylamine was most rapid (80 days), and were much longer for acetate (7900 days), methanol (40,500 days) and bicarbonate (361,600 days). Methylamine-utilizing Methanosarcinales were the dominant (60-95%) methanogens in this zone. In deeper sediments rates of methanogenesis from competitive substrates increased substantially, with acetate methanogenic rates becoming ˜100 times greater than H 2/CO 2 methanogenesis below 50 cm. In addition, there was a dramatic change in methanogen diversity with obligate acetate-utilizing, Methanosaeta related sequences being dominant. At a similar depth methanol turnover to methane increased to its most rapid (1700 days). This activity pattern is consistent with deeper methanogen populations (55 cm) being dominated by acetate-utilizing Methanosaeta with H 2/CO 2 and alcohol-utilizing Methanomicrobiales also present. Hence, there is close relationship between the depth distribution of methanogenic substrate utilization and specific methanogens that can utilize these compounds. It is unusual for acetate to be the dominant methanogenic substrate in coastal sediments and δ13C-CH 4 values (-74 to -71‰) were atypical for acetate methanogenesis, suggesting that common stable isotope proxy models may not apply well in this type of dynamic anoxic sediment, with multiple methanogenic substrates.

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases*

PubMed Central

Tinti, Michele; Paoluzi, Serena; Santonico, Elena; Masch, Antonia; Schutkowski, Mike

2017-01-01

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level. PMID:28159843

Broad Range Amino Acid Specificity of RNA-dependent Lipid Remodeling by Multiple Peptide Resistance Factors*

PubMed Central

Roy, Hervé; Ibba, Michael

2009-01-01

Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic deletion of the predicted membrane-inserted domains of several aaPGSs revealed that the carboxyl-terminal hydrophilic domain alone is sufficient for aminoacylphosphatidylglycerol transferase catalytic activity. In contrast to previously characterized aaPGSs, the Enterococcus faecium enzyme used an expanded repertoire of amino acids to modify PG with Ala, Arg, or Lys. Reexamination of previously characterized aaPGSs also revealed broader than anticipated substrate specificity, for example Bacillus subtilis Lys-PGS was shown to also catalyze Ala-PG synthesis. The relaxed substrate specificities of these aaPGSs allows for more elaborate remodeling of membrane lipids than previously thought, potentially providing bacteria that harbor these enzymes resistance to a broad spectrum of antibiotics and environmental stresses. PMID:19734140

Predicting the impact of mutations on the specific activity of Bacillus thermocatenulatus lipase using a combined approach of docking and molecular dynamics.

PubMed

Yukselen, Onur; Timucin, Emel; Sezerman, Ugur

2016-10-01

Lipases are important biocatalysts owing to their ability to catalyze diverse reactions with exceptional substrate specificities. A combined docking and molecular dynamics (MD) approach was applied to study the chain-length selectivity of Bacillus thermocatenulatus lipase (BTL2) towards its natural substrates (triacylglycerols). A scoring function including electrostatic, van der Waals (vdW) and desolvation energies along with conformational entropy was developed to predict the impact of mutation. The native BTL2 and its 6 mutants (F17A, V175A, V175F, D176F, T178V and I320F) were experimentally analyzed to determine their specific activities towards tributyrin (C4) or tricaprylin (C8), which were used to test our approach. Our scoring methodology predicted the chain-length selectivity of BTL2 with 85.7% (6/7) accuracy with a positive correlation between the calculated scores and the experimental activity values (r = 0.82, p = 0.0004). Additionally, the impact of mutation on activity was predicted with 75% (9/12) accuracy. The described study represents a fast and reliable approach to accurately predict the effect of mutations on the activity and selectivity of lipases and also of other enzymes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Exploring Nitrilase Sequence Space for Enantioselective Catalysis†

PubMed Central

Robertson, Dan E.; Chaplin, Jennifer A.; DeSantis, Grace; Podar, Mircea; Madden, Mark; Chi, Ellen; Richardson, Toby; Milan, Aileen; Miller, Mark; Weiner, David P.; Wong, Kelvin; McQuaid, Jeff; Farwell, Bob; Preston, Lori A.; Tan, Xuqiu; Snead, Marjory A.; Keller, Martin; Mathur, Eric; Kretz, Patricia L.; Burk, Mark J.; Short, Jay M.

2004-01-01

Nitrilases are important in the biosphere as participants in synthesis and degradation pathways for naturally occurring, as well as xenobiotically derived, nitriles. Because of their inherent enantioselectivity, nitrilases are also attractive as mild, selective catalysts for setting chiral centers in fine chemical synthesis. Unfortunately, <20 nitrilases have been reported in the scientific and patent literature, and because of stability or specificity shortcomings, their utility has been largely unrealized. In this study, 137 unique nitrilases, discovered from screening of >600 biotope-specific environmental DNA (eDNA) libraries, were characterized. Using culture-independent means, phylogenetically diverse genomes were captured from entire biotopes, and their genes were expressed heterologously in a common cloning host. Nitrilase genes were targeted in a selection-based expression assay of clonal populations numbering 106 to 1010 members per eDNA library. A phylogenetic analysis of the novel sequences discovered revealed the presence of at least five major sequence clades within the nitrilase subfamily. Using three nitrile substrates targeted for their potential in chiral pharmaceutical synthesis, the enzymes were characterized for substrate specificity and stereospecificity. A number of important correlations were found between sequence clades and the selective properties of these nitrilases. These enzymes, discovered using a high-throughput, culture-independent method, provide a catalytic toolbox for enantiospecific synthesis of a variety of carboxylic acid derivatives, as well as an intriguing library for evolutionary and structural analyses. PMID:15066841

Matrix metalloproteinase proteomics: substrates, targets, and therapy.

PubMed

Morrison, Charlotte J; Butler, Georgina S; Rodríguez, David; Overall, Christopher M

2009-10-01

Proteomics encompasses powerful techniques termed 'degradomics' for unbiased high-throughput protease substrate discovery screens that have been applied to an important family of extracellular proteases, the matrix metalloproteinases (MMPs). Together with the data generated from genetic deletion and transgenic mouse models and genomic profiling, these screens can uncover the diverse range of MMP functions, reveal which MMPs and MMP-mediated pathways exacerbate pathology, and which are involved in protection and the resolution of disease. This information can be used to identify and validate candidate drug targets and antitargets, and is critical for the development of new inhibitors of MMP function. Such inhibitors may target either the MMP directly in a specific manner or pathways upstream and downstream of MMP activity that are mediating deleterious effects in disease. Since MMPs do not operate alone but are part of the 'protease web', it is necessary to use system-wide approaches to understand MMP proteolysis in vivo, to discover new biological roles and their potential for therapeutic modification.

Biosynthesis and genetic encoding of phosphothreonine through parallel selection and deep sequencing

PubMed Central

Huguenin-Dezot, Nicolas; Liang, Alexandria D.; Schmied, Wolfgang H.; Rogerson, Daniel T.; Chin, Jason W.

2017-01-01

The phosphorylation of threonine residues in proteins regulates diverse processes in eukaryotic cells, and thousands of threonine phosphorylations have been identified. An understanding of how threonine phosphorylation regulates biological function will be accelerated by general methods to bio-synthesize defined phospho-proteins. Here we address limitations in current methods for discovering aminoacyl-tRNA synthetase/tRNA pairs for incorporating non-natural amino acids into proteins, by combining parallel positive selections with deep sequencing and statistical analysis, to create a rapid approach for directly discovering aminoacyl-tRNA synthetase/tRNA pairs that selectively incorporate non-natural substrates. Our approach is scalable and enables the direct discovery of aminoacyl-tRNA synthetase/tRNA pairs with mutually orthogonal substrate specificity. We biosynthesize phosphothreonine in cells, and use our new selection approach to discover a phosphothreonyl-tRNA synthetase/tRNACUA pair. By combining these advances we create an entirely biosynthetic route to incorporating phosphothreonine in proteins and biosynthesize several phosphoproteins; enabling phosphoprotein structure determination and synthetic protein kinase activation. PMID:28553966

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

PubMed

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Evolution of an intricate J-protein network driving protein disaggregation in eukaryotes.

PubMed

Nillegoda, Nadinath B; Stank, Antonia; Malinverni, Duccio; Alberts, Niels; Szlachcic, Anna; Barducci, Alessandro; De Los Rios, Paolo; Wade, Rebecca C; Bukau, Bernd

2017-05-15

Hsp70 participates in a broad spectrum of protein folding processes extending from nascent chain folding to protein disaggregation. This versatility in function is achieved through a diverse family of J-protein cochaperones that select substrates for Hsp70. Substrate selection is further tuned by transient complexation between different classes of J-proteins, which expands the range of protein aggregates targeted by metazoan Hsp70 for disaggregation. We assessed the prevalence and evolutionary conservation of J-protein complexation and cooperation in disaggregation. We find the emergence of a eukaryote-specific signature for interclass complexation of canonical J-proteins. Consistently, complexes exist in yeast and human cells, but not in bacteria, and correlate with cooperative action in disaggregation in vitro. Signature alterations exclude some J-proteins from networking, which ensures correct J-protein pairing, functional network integrity and J-protein specialization. This fundamental change in J-protein biology during the prokaryote-to-eukaryote transition allows for increased fine-tuning and broadening of Hsp70 function in eukaryotes.

Detection of Methanotroph Diversity on Roots of Submerged Rice Plants by Molecular Retrieval of pmoA, mmoX, mxaF, and 16S rRNA and Ribosomal DNA, Including pmoA-Based Terminal Restriction Fragment Length Polymorphism Profiling

PubMed Central

Horz, Hans-Peter; Yimga, Merlin Tchawa; Liesack, Werner

2001-01-01

The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA. PMID:11526021

Levels of analysis in neuroscientific studies of emotion: Comment on "The quartet theory of human emotions: an integrative and neurofunctional model" by S. Koelsch et al.

NASA Astrophysics Data System (ADS)

Kuiken, Don; Douglas, Shawn

2015-06-01

In the conduct of neuroscience research, methodological choices and theoretical claims often reveal underlying metamethodological and ontological commitments. Koelsch et al. [1] accentuate such commitments in their description of four "neuroanatomically distinct systems," each the substrate of "a specific class of affects" (p. 1). Explication of those classes of affect require theoretical integration across methodologically diverse disciplines, including "psychology, neurobiology, sociology, anthropology, and psycholinguistics" (p. 3). (Philosophy is noticeably missing from this list, but several aspects of the authors' stance indicate that it is not ignored.)

Strategies in the design of small-molecule fluorescent probes for peptidases.

PubMed

Chen, Laizhong; Li, Jing; Du, Lupei; Li, Minyong

2014-11-01

Peptidases, which can cleave specific peptide bonds in innumerable categories of substrates, usually present pivotal positions in protein activation, cell signaling and regulation as well as in the origination of amino acids for protein generation or application in other metabolic pathways. They are also involved in many pathological conditions, such as cancer, atherosclerosis, arthritis, and neurodegenerative disorders. This review article aims to conduct a wide-ranging survey on the development of small-molecule fluorescent probes for peptidases, as well as to realize the state of the art in the tailor-made probes for diverse types of peptidases. © 2014 Wiley Periodicals, Inc.

Targeting of insect epicuticular lipids by the entomopathogenic fungus Beauveria bassiana: hydrocarbon oxidation within the context of a host-pathogen interaction

PubMed Central

Pedrini, Nicolás; Ortiz-Urquiza, Almudena; Huarte-Bonnet, Carla; Zhang, Shizhu; Keyhani, Nemat O.

2013-01-01

Broad host range entomopathogenic fungi such as Beauveria bassiana attack insect hosts via attachment to cuticular substrata and the production of enzymes for the degradation and penetration of insect cuticle. The outermost epicuticular layer consists of a complex mixture of non-polar lipids including hydrocarbons, fatty acids, and wax esters. Long chain hydrocarbons are major components of the outer waxy layer of diverse insect species, where they serve to protect against desiccation and microbial parasites, and as recognition molecules or as a platform for semiochemicals. Insect pathogenic fungi have evolved mechanisms for overcoming this barrier, likely with sets of lipid degrading enzymes with overlapping substrate specificities. Alkanes and fatty acids are substrates for a specific subset of fungal cytochrome P450 monooxygenases involved in insect hydrocarbon degradation. These enzymes activate alkanes by terminal oxidation to alcohols, which are further oxidized by alcohol and aldehyde dehydrogenases, whose products can enter β-oxidation pathways. B. bassiana contains at least 83 genes coding for cytochrome P450s (CYP), a subset of which are involved in hydrocarbon oxidation, and several of which represent new CYP subfamilies/families. Expression data indicated differential induction by alkanes and insect lipids and four CYP proteins have been partially characterized after heterologous expression in yeast. Gene knockouts revealed a phenotype for only one (cyp52X1) out of six genes examined to date. CYP52X1 oxidizes long chain fatty acids and participates in the degradation of specific epicuticular lipid components needed for breaching the insect waxy layer. Examining the hydrocarbon oxidizing CYP repertoire of pathogens involved in insect epicuticle degradation can lead to the characterization of enzymes with novel substrate specificities. Pathogen targeting may also represent an important co-evolutionary process regarding insect cuticular hydrocarbon synthesis. PMID:23422735

Fungal chitinases: diversity, mechanistic properties and biotechnological potential.

PubMed

Hartl, Lukas; Zach, Simone; Seidl-Seiboth, Verena

2012-01-01

Chitin derivatives, chitosan and substituted chito-oligosaccharides have a wide spectrum of applications ranging from medicine to cosmetics and dietary supplements. With advancing knowledge about the substrate-binding properties of chitinases, enzyme-based production of these biotechnologically relevant sugars from biological resources is becoming increasingly interesting. Fungi have high numbers of glycoside hydrolase family 18 chitinases with different substrate-binding site architectures. As presented in this review, the large diversity of fungal chitinases is an interesting starting point for protein engineering. In this review, recent data about the architecture of the substrate-binding clefts of fungal chitinases, in connection with their hydrolytic and transglycolytic abilities, and the development of chitinase inhibitors are summarized. Furthermore, the biological functions of chitinases, chitin and chitosan utilization by fungi, and the effects of these aspects on biotechnological applications, including protein overexpression and autolysis during industrial processes, are discussed in this review.

Genetic and Metabolic Intraspecific Biodiversity of Ganoderma lucidum

PubMed Central

Pawlik, Anna; Janusz, Grzegorz; Dębska, Iwona; Siwulski, Marek; Frąc, Magdalena; Rogalski, Jerzy

2015-01-01

Fourteen Ganoderma lucidum strains from different geographic regions were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship between the analyzed strains was determined. All G. lucidum strains were also genetically characterized using the AFLP technique. G. lucidum strains included in the analysis displayed an AFLP profile similarity level in the range from 9.6 to 33.9%. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mitochondrial activity. The analysis allowed comparison of functional diversity of the fungal strains. The substrate utilization profiles for the isolates tested revealed a broad variability within the analyzed G. lucidum species and proved to be a good profiling technology for studying the diversity in fungi. Significant differences have been demonstrated in substrate richness values. Interestingly, the analysis of growth and biomass production also differentiated the strains based on the growth rate on the agar and sawdust substrate. In general, the mycelial growth on the sawdust substrate was more balanced and the fastest fungal growth was observed for GRE3 and FCL192. PMID:25815332

Systems and methods for the combinatorial synthesis of novel materials

DOEpatents

Wu, Xin Di; Wang, Youqi; Goldwasser, Isy

2000-01-01

Methods and apparatus for the preparation of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by depositing components of target materials to predefined regions on the substrate, and, in some embodiments, simultaneously reacting the components to form at least two resulting materials. In particular, the present invention provides novel masking systems and methods for applying components of target materials onto a substrate in a combinatorial fashion, thus creating arrays of resulting materials that differ slightly in composition, stoichiometry, and/or thickness. Using the novel masking systems of the present invention, components can be delivered to each site in a uniform distribution, or in a gradient of stoichiometries, thicknesses, compositions, etc. Resulting materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. Once prepared, these resulting materials can be screened sequentially, or in parallel, for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical and other properties.

Community analysis of hydrogen-producing extreme thermophilic anaerobic microflora enriched from cow manure with five substrates.

PubMed

Yokoyama, Hiroshi; Moriya, Naoko; Ohmori, Hideyuki; Waki, Miyoko; Ogino, Akifumi; Tanaka, Yasuo

2007-11-01

The present study analyzed the community structures of anaerobic microflora producing hydrogen under extreme thermophilic conditions by two culture-independent methods: denaturing gradient gel electrophoresis (DGGE) and clone library analyses. Extreme thermophilic microflora (ETM) was enriched from cow manure by repeated batch cultures at 75 degrees C, using a substrate of xylose, glucose, lactose, cellobiose, or soluble starch, and produced hydrogen at yields of 0.56, 2.65, 2.17, 2.68, and 1.73 mol/mol-monosaccharide degraded, respectively. The results from the DGGE and clone library analyses were consistent and demonstrated that the community structures of ETM enriched with the four hexose-based substrates (glucose, lactose, cellobiose, and soluble starch) consisted of a single species, closely related to a hydrogen-producing extreme thermophile, Caldoanaerobacter subterraneus, with diversity at subspecies levels. The ETM enriched with xylose was more diverse than those enriched with the other substrates, and contained the bacterium related to C. subterraneus and an unclassified bacterium, distantly related to a xylan-degrading and hydrogen-producing extreme thermophile, Caloramator fervidus.

Characterization of the macrocyclase involved in the biosynthesis of RiPP cyclic peptides in plants.

PubMed

Chekan, Jonathan R; Estrada, Paola; Covello, Patrick S; Nair, Satish K

2017-06-20

Enzymes that can catalyze the macrocyclization of linear peptide substrates have long been sought for the production of libraries of structurally diverse scaffolds via combinatorial gene assembly as well as to afford rapid in vivo screening methods. Orbitides are plant ribosomally synthesized and posttranslationally modified peptides (RiPPs) of various sizes and topologies, several of which are shown to be biologically active. The diversity in size and sequence of orbitides suggests that the corresponding macrocyclases may be ideal catalysts for production of cyclic peptides. Here we present the biochemical characterization and crystal structures of the plant enzyme PCY1 involved in orbitide macrocyclization. These studies demonstrate how the PCY1 S9A protease fold has been adapted for transamidation, rather than hydrolysis, of acyl-enzyme intermediates to yield cyclic products. Notably, PCY1 uses an unusual strategy in which the cleaved C-terminal follower peptide from the substrate stabilizes the enzyme in a productive conformation to facilitate macrocyclization of the N-terminal fragment. The broad substrate tolerance of PCY1 can be exploited as a biotechnological tool to generate structurally diverse arrays of macrocycles, including those with nonproteinogenic elements.

Investigating functional redundancy versus complementarity in Hawaiian herbivorous coral reef fishes.

PubMed

Kelly, Emily L A; Eynaud, Yoan; Clements, Samantha M; Gleason, Molly; Sparks, Russell T; Williams, Ivor D; Smith, Jennifer E

2016-12-01

Patterns of species resource use provide insight into the functional roles of species and thus their ecological significance within a community. The functional role of herbivorous fishes on coral reefs has been defined through a variety of methods, but from a grazing perspective, less is known about the species-specific preferences of herbivores on different groups of reef algae and the extent of dietary overlap across an herbivore community. Here, we quantified patterns of redundancy and complementarity in a highly diverse community of herbivores at a reef on Maui, Hawaii, USA. First, we tracked fish foraging behavior in situ to record bite rate and type of substrate bitten. Second, we examined gut contents of select herbivorous fishes to determine consumption at a finer scale. Finally, we placed foraging behavior in the context of resource availability to determine how fish selected substrate type. All species predominantly (73-100 %) foraged on turf algae, though there were differences among the types of macroalgae and other substrates bitten. Increased resolution via gut content analysis showed the composition of turf algae consumed by fishes differed across herbivore species. Consideration of foraging behavior by substrate availability revealed 50 % of herbivores selected for turf as opposed to other substrate types, but overall, there were variable foraging portfolios across all species. Through these three methods of investigation, we found higher complementarity among herbivorous fishes than would be revealed using a single metric. These results suggest differences across species in the herbivore "rain of bites" that graze and shape benthic community composition.

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Diversity and bioprospection of fungal community present in oligotrophic soil of continental Antarctica

USDA-ARS?s Scientific Manuscript database

The diversity of fungal communities from different substrates in Antarctica were studied and their capability to produce bioactive compounds. A one hundred and one fungal isolates were identified by molecular analysis in 35 different fungal taxa from 20 genera. Pseudogymnoascus sp. 3, Pseudogymnoasc...

Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate

PubMed Central

Gaudy, Anthony F.; Obayashi, Alan; Gaudy, Elizabeth T.

1971-01-01

The hyperbolic relationship between specific growth rate, μ, and substrate concentration, proposed by Monod and used since as the basis for the theory of steady-state growth in continuous-flow systems, was tested experimentally in batch cultures. Use of a Flavobacterium sp. exhibiting a high saturation constant for growth in glucose minimal medium allowed direct measurement of growth rate and substrate concentration throughout the growth cycle in medium containing a rate-limiting initial concentration of glucose. Specific growth rates were also measured for a wide range of initial glucose concentrations. A plot of specific growth rate versus initial substrate concentration was found to fit the hyperbolic equation. However, the instantaneous relationship between specific growth rate and substrate concentration during growth, which is stated by the equation, was not observed. Well defined exponential growth phases were developed at initial substrate concentrations below that required for support of the maximum exponential growth rate and a constant doubling time was maintained until 50% of the substrate had been used. It is suggested that the external substrate concentration initially present “sets” the specific growth rate by establishing a steady-state internal concentration of substrate, possibly through control of the number of permeation sites. PMID:5137579

Colonization of plant substrates at hydrothermal vents and cold seeps in the northeast Atlantic and Mediterranean and occurrence of symbiont-related bacteria

PubMed Central

Szafranski, Kamil M.; Deschamps, Philippe; Cunha, Marina R.; Gaudron, Sylvie M.; Duperron, Sébastien

2015-01-01

Reducing conditions with elevated sulfide and methane concentrations in ecosystems such as hydrothermal vents, cold seeps or organic falls, are suitable for chemosynthetic primary production. Understanding processes driving bacterial diversity, colonization and dispersal is of prime importance for deep-sea microbial ecology. This study provides a detailed characterization of bacterial assemblages colonizing plant-derived substrates using a standardized approach over a geographic area spanning the North-East Atlantic and Mediterranean. Wood and alfalfa substrates in colonization devices were deployed for different periods at 8 deep-sea chemosynthesis-based sites in four distinct geographic areas. Pyrosequencing of a fragment of the 16S rRNA-encoding gene was used to describe bacterial communities. Colonization occurred within the first 14 days. The diversity was higher in samples deployed for more than 289 days. After 289 days, no relation was observed between community richness and deployment duration, suggesting that diversity may have reached saturation sometime in between. Communities in long-term deployments were different, and their composition was mainly influenced by the geographical location where devices were deployed. Numerous sequences related to horizontally-transmitted chemosynthetic symbionts of metazoans were identified. Their potential status as free-living forms of these symbionts was evaluated based on sequence similarity with demonstrated symbionts. Results suggest that some free-living forms of metazoan symbionts or their close relatives, such as Epsilonproteobacteria associated with the shrimp Rimicaris exoculata, are efficient colonizers of plant substrates at vents and seeps. PMID:25774156

Effects of garlic oil, nitrate, saponin and their combinations supplemented to different substrates on in vitro fermentation, ruminal methanogenesis, and abundance and diversity of microbial populations.

PubMed

Patra, A K; Yu, Z

2015-07-01

To investigate the effect of garlic oil (G), nitrate (N), saponin (S) and their combinations supplemented to different forage to concentrate substrates on methanogenesis, fermentation, diversity and abundances of bacteria and Archaea in vitro. The study was conducted in an 8 × 2 factorial design with eight treatments and two substrates using mixed ruminal batch cultures obtained. Quillaja S (0·6 g l(-1) ), N (5 mmol l(-1) ) and G (0·27 g l(-1) ) were used separately or in binary and tertiary combinations. The two substrates contained grass hay and a dairy concentrate mixture at a 70 : 30 (high-forage substrate) ratio or a 30 : 70 (high-concentrate substrate) ratio. Ruminal fermentation and cellulolytic bacterial populations were affected by interaction between substrate and anti-methanogenic compounds. The inhibitor combinations decreased the methane production additively regardless of substrate. For the high-concentrate substrate, S decreased methane production to a greater extent, so did G and N individually for the high-forage substrate. Feed degradability and total volatile fatty acid (VFA) concentrations were not decreased by any of the treatments. Fibre degradability was actually improved by N+S for the high-forage substrate. VFA concentrations and profiles were affected differently by different anti-methanogenic inhibitors and their combinations. All treatments inhibited the growth of Archaea, but the effect on Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens varied. The results suggest that substrate influences the efficacy of these inhibitors when they are used separately, but in combinations, they can lower methanogenesis additively without much influence from the substrate. The presented research provided evidence that binary and tertiary combination of garlic oil, nitrate and saponin can lower the methane production additively without adversely impacting rumen fermentation and degradability, and forage to concentrate ratio does not change the above effects. These anti-methanogenic inhibitors in combination may have practical application to mitigate methane emission from ruminants. © 2015 The Society for Applied Microbiology.

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires' disease.

PubMed

Gomez-Valero, Laura; Rusniok, Christophe; Rolando, Monica; Neou, Mario; Dervins-Ravault, Delphine; Demirtas, Jasmin; Rouy, Zoe; Moore, Robert J; Chen, Honglei; Petty, Nicola K; Jarraud, Sophie; Etienne, Jerome; Steinert, Michael; Heuner, Klaus; Gribaldo, Simonetta; Médigue, Claudine; Glöckner, Gernot; Hartland, Elizabeth L; Buchrieser, Carmen

2014-01-01

The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans. We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains. Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Quantum mechanical approaches to in silico enzyme characterization and drug design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nilmeier, J P; Fattebert, J L; Jacobson, M P

2012-01-17

The astonishing, exponentially increasing rates of genome sequencing has led to one of the most significant challenges for the biological and computational sciences in the 21st century: assigning the likely functions of the encoded proteins. Enzymes represent a particular challenge, and a critical one, because the universe of enzymes is likely to contain many novel functions that may be useful for synthetic biology, or as drug targets. Current approaches to protein annotation are largely based on bioinformatics. At the simplest level, this annotation involves transferring the annotations of characterized enzymes to related sequences. In practice, however, there is no simple,more » sequence based criterion for transferring annotations, and bioinformatics alone cannot propose new enzymatic functions. Structure-based computational methods have the potential to address these limitations, by identifying potential substrates of enzymes, as we and others have shown. One successful approach has used in silico 'docking' methods, more commonly applied in structure-based drug design, to identify possible metabolite substrates. A major limitation of this approach is that it only considers substrate binding, and does not directly assess the potential of the enzyme to catalyze a particular reaction using a particular substrate. That is, substrate binding affinity is necessary but not sufficient to assign function. A reaction profile is ultimately what is needed for a more complete quantitative description of function. To address this rather fundamental limitation, they propose to use quantum mechanical methods to explicitly compute transition state barriers that govern the rates of catalysis. Although quantum mechanical, and mixed quantum/classical (QM/MM), methods have been used extensively to investigate enzymatic reactions, the focus has been primarily on elucidating complex reaction mechanisms. Here, the key catalytic steps are known, and they use these methods quantify substrate specificity. That is, we bring the power of quantum mechanics to bear on the problem of annotating enzyme function, which is a novel approach. Although it has been clear to us at the Jacobson group for some time that enzyme specificity may be encoded in transition states, rather than simply substrate recognition, the main limitation has always been computational expense. Using a hierarchy of different methods, they can reduce the list of plausible substrates of an enzyme to a small number in most cases, but even identifying the transition states for a dozen plausible substrates requires significant computational effort, beyond what is practical using standard QM/MM methods. For this project, they have chosen two enzyme superfamilies which they have used as 'model systems' for functional assignment. The enolase superfamily is a large group of {alpha}-{beta} barrel enzymes with highly diverse substrates and chemical transformations. Despite decades of work, over a third of the superfamily remains unassigned, which means that the remaining cases are by definition difficult to assign. They have focused on acid sugar dehydratases, and have considerable expertise on the matter. They are also interested in the isoprenoid synthase superfamily, which is of central interest to the synthetic biology community, because these enzymes are used by nature to create complex rare natural products of medicinal value. the most notable example of this is the artemisinin, an antimalarial compound that is found in trace amounts in the wormwod root. From the standpoint of enzyme function assignment, these enzymes are intriguing because they use a small number of chemically simple substrates to generate, potentially, tens of thousands of different products. Hence, substrate binding specificity is only a small part of the challenge; the key is determining how the enzyme directs the carbocation chemistry to specific products. These more complex modeling approaches clearly require quantum mechanical methods.« less

Quantifying fish habitat associated with stream simulation design culverts in northern Wisconsin

Treesearch

A. Timm; D. Higgins; J. Stanovick; R. Kolka; S. Eggert

2017-01-01

This study investigated the effects of culvert replacement design on fish habitat and fish weight by comparing substrate diversity and weight at three stream simulation (SS)-design and three bankfull and backwater (BB)-design sites on the Chequamegon-Nicolet National Forest, Wisconsin. Stream channel cross-sections, Wolman substrate particle counts, and single-pass...

Composite polymer systems with control of local substrate elasticity and their effect on cytoskeletal and morphological characteristics of adherent cells.

PubMed

Chou, Szu-Yuan; Cheng, Chao-Min; LeDuc, Philip R

2009-06-01

At the interface between extracellular substrates and biological materials, substrate elasticity strongly influences cell morphology and function. The associated biological ramifications comprise a diversity of critical responses including apoptosis, differentiation, and motility, which can affect medical devices such as stents. The interactions of the extracellular environment with the substrate are also affected by local properties wherein cells sense and respond to different physical inputs. To investigate the effects of having localized elasticity control of substrate microenvironments on cell response, we have developed a method to control material interface interactions with cells by dictating local substrate elasticity. This system is created by generating a composite material system with alternating, linear regions of polymers that have distinct stiffness characteristics. This approach was used to examine cytoskeletal and morphological changes in NIH 3T3 fibroblasts with emphasis on both local and global properties, noting that cells sense and respond to distinct material elasticities. Isolated cells sense and respond to these local differences in substrate elasticity by extending processes along the interface. Also, cells grown on softer elastic regions at higher densities (in contact with each other) have a higher projected area than isolated cells. Furthermore, when using chemical agents such as cytochalasin-D to disrupt the actin cytoskeleton, there is a significant increase in projected area for cells cultured on softer elastic regions This method has the potential to promote understanding of biomaterial-affected responses in a diversity of areas including morphogenesis, mechanotransduction, stents, and stem cell differentiation.

Dynamic succession of substrate-associated bacterial composition and function during Ganoderma lucidum growth

PubMed Central

Li, Qiang; Zou, Jie; Tan, Hao; Tan, Wei; Peng, Weihong

2018-01-01

Background Ganoderma lucidum, a valuable medicinal fungus, is widely distributed in China. It grows alongside with a complex microbial ecosystem in the substrate. As sequencing technology advances, it is possible to reveal the composition and functions of substrate-associated bacterial communities. Methods We analyzed the bacterial community dynamics in the substrate during the four typical growth stages of G. lucidum using next-generation sequencing. Results The physicochemical properties of the substrate (e.g. acidity, moisture, total nitrogen, total phosphorus and total potassium) changed between different growth stages. A total of 598,771 sequences from 12 samples were obtained and assigned to 22 bacterial phyla. Proteobacteria and Firmicutes were the dominant phyla. Bacterial community composition and diversity significantly differed between the elongation stage and the other three growth stages. LEfSe analysis revealed a large number of bacterial taxa (e.g. Bacteroidetes, Acidobacteria and Nitrospirae) with significantly higher abundance at the elongation stage. Functional pathway prediction uncovered significant abundance changes of a number of bacterial functional pathways between the elongation stage and other growth stages. At the elongation stage, the abundance of the environmental information processing pathway (mainly membrane transport) decreased, whereas that of the metabolism-related pathways increased. Discussion The changes in bacterial community composition, diversity and predicted functions were most likely related to the changes in the moisture and nutrient conditions in the substrate with the growth of G. lucidum, particularly at the elongation stage. Our findings shed light on the G. lucidum-bacteria-substrate relationships, which should facilitate the industrial cultivation of G. lucidum. PMID:29915697

Lack of Influence of Substrate on Ligand Interaction with the Human Multidrug and Toxin Extruder, MATE1

PubMed Central

Martínez-Guerrero, Lucy J.; Morales, Mark; Ekins, Sean

2016-01-01

Multidrug and toxin extruder (MATE) 1 plays a central role in mediating renal secretion of organic cations, a structurally diverse collection of compounds that includes ∼40% of prescribed drugs. Because inhibition of transport activity of other multidrug transporters, including the organic cation transporter (OCT) 2, is influenced by the structure of the transported substrate, the present study screened over 400 drugs as inhibitors of the MATE1-mediated transport of four structurally distinct organic cation substrates: the commonly used drugs: 1) metformin and 2) cimetidine; and two prototypic cationic substrates, 3) 1-methyl-4-phenylpyridinium (MPP), and 4) the novel fluorescent probe, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium iodide. Transport was measured in Chinese hamster ovary cells that stably expressed the human ortholog of MATE1. Comparison of the resulting inhibition profiles revealed no systematic influence of substrate structure on inhibitory efficacy. Similarly, IC50 values for 26 structurally diverse compounds revealed no significant influence of substrate structure on the kinetic interaction of inhibitor with MATE1. The IC50 data were used to generate three-dimensional quantitative pharmacophores that identified hydrophobic regions, H-bond acceptor sites, and an ionizable (cationic) feature as key determinants for ligand binding to MATE1. In summary, in contrast to the behavior observed with some other multidrug transporters, including OCT2, the results suggest that substrate identity exerts comparatively little influence on ligand interaction with MATE1. PMID:27418674

Engineering of a conditional allele reveals multiple roles of XRN2 in Caenorhabditis elegans development and substrate specificity in microRNA turnover.

PubMed

Miki, Takashi S; Rüegger, Stefan; Gaidatzis, Dimos; Stadler, Michael B; Großhans, Helge

2014-04-01

Although XRN2 proteins are highly conserved eukaryotic 5'→3' exonucleases, little is known about their function in animals. Here, we characterize Caenorhabditis elegans XRN2, which we find to be a broadly and constitutively expressed nuclear protein. An xrn-2 null mutation or loss of XRN2 catalytic activity causes a molting defect and early larval arrest. However, by generating a conditionally mutant xrn-2ts strain de novo through an approach that may be also applicable to other genes of interest, we reveal further functions in fertility, during embryogenesis and during additional larval stages. Consistent with the known role of XRN2 in controlling microRNA (miRNA) levels, we can demonstrate that loss of XRN2 activity stabilizes some rapidly decaying miRNAs. Surprisingly, however, other miRNAs continue to decay rapidly in xrn-2ts animals. Thus, XRN2 has unanticipated miRNA specificity in vivo, and its diverse developmental functions may relate to distinct substrates. Finally, our global analysis of miRNA stability during larval stage 1 reveals that miRNA passenger strands (miR*s) are substantially less stable than guide strands (miRs), supporting the notion that the former are mostly byproducts of biogenesis rather than a less abundant functional species.

Kinetic characterization of Vibrio cholerae ApbE: Substrate specificity and regulatory mechanisms

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fang, Xuan; Liang, Pingdong; Raba, Daniel Alexander

ApbE is a member of a novel family of flavin transferases that incorporates flavin mononucleotide (FMN) to subunits of diverse respiratory complexes, which fulfill important homeostatic functions. In this work a detailed characterization of Vibrio cholerae ApbE physiologic activity, substrate specificity and pH dependency was carried out. The data obtained show novel characteristics of the regulation and function of this family. For instance, our experiments indicate that divalent cations are essential for ApbE function, and that the selectivity depends largely on size and the coordination sphere of the cation. Our data also show that ApbE regulation by pH, ADP andmore » potassium is an important mechanism that enhances the adaptation, survival and colonization of V. cholerae in the small intestine. Moreover, studies of the pH-dependency of the activity show that the reaction is favored under alkaline conditions, with a pKa of 8.4. These studies, together with sequence and structure analysis allowed us to identify His257, which is absolutely conserved in the family, as a candidate for the residue whose deprotonation controls the activity. Remarkably, the mutant H257G abolished the flavin transfer activity, strongly indicating that this residue plays an important role in the catalytic mechanism of ApbE.« less

Directed evolution of new and improved enzyme functions using an evolutionary intermediate and multidirectional search.

PubMed

Porter, Joanne L; Boon, Priscilla L S; Murray, Tracy P; Huber, Thomas; Collyer, Charles A; Ollis, David L

2015-02-20

The ease with which enzymes can be adapted from their native roles and engineered to function specifically for industrial or commercial applications is crucial to enabling enzyme technology to advance beyond its current state. Directed evolution is a powerful tool for engineering enzymes with improved physical and catalytic properties and can be used to evolve enzymes where lack of structural information may thwart the use of rational design. In this study, we take the versatile and diverse α/β hydrolase fold framework, in the form of dienelactone hydrolase, and evolve it over three unique sequential evolutions with a total of 14 rounds of screening to generate a series of enzyme variants. The native enzyme has a low level of promiscuous activity toward p-nitrophenyl acetate but almost undetectable activity toward larger p-nitrophenyl esters. Using p-nitrophenyl acetate as an evolutionary intermediate, we have generated variants with altered specificity and catalytic activity up to 3 orders of magnitude higher than the native enzyme toward the larger nonphysiological p-nitrophenyl ester substrates. Several variants also possess increased stability resulting from the multidimensional approach to screening. Crystal structure analysis and substrate docking show how the enzyme active site changes over the course of the evolutions as either a direct or an indirect result of mutations.

Characterization of transgenic tobacco plants containing bacterial bphC gene and study of their phytoremediation ability.

PubMed

Viktorovtá, Jitka; Novakova, Martina; Trbolova, Ladislava; Vrchotova, Blanka; Lovecka, Petra; Mackova, Martina; Macek, Tomas

2014-01-01

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility*

PubMed Central

Buechner, Claudia N.; Heil, Korbinian; Michels, Gudrun; Carell, Thomas; Kisker, Caroline; Tessmer, Ingrid

2014-01-01

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5′-3′ helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a “final” verification state, which may then trigger the recruitment of further NER proteins. PMID:24338567

Ada response - a strategy for repair of alkylated DNA in bacteria.

PubMed

Mielecki, Damian; Grzesiuk, Elżbieta

2014-06-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N(3)-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N(1)-methyladenine (1meA) and N(3)-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O(6)-methylguanine (O(6) meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. © 2014 The Authors. FEMS Microbiology Letters published by John Wiley & Sons Ltd on behalf of Federation of European Microbiological Societies.

AAV Vectorization of DSB-mediated Gene Editing Technologies.

PubMed

Moser, Rachel J; Hirsch, Matthew L

2016-01-01

Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

Thioredoxin 1-Mediated Post-Translational Modifications: Reduction, Transnitrosylation, Denitrosylation, and Related Proteomics Methodologies

PubMed Central

Wu, Changgong; Parrott, Andrew M.; Fu, Cexiong; Liu, Tong; Marino, Stefano M.; Gladyshev, Vadim N.; Jain, Mohit R.; Baykal, Ahmet T.; Li, Qing; Oka, Shinichi; Sadoshima, Junichi; Beuve, Annie; Simmons, William J.

2011-01-01

Abstract Despite the significance of redox post-translational modifications (PTMs) in regulating diverse signal transduction pathways, the enzymatic systems that catalyze reversible and specific oxidative or reductive modifications have yet to be firmly established. Thioredoxin 1 (Trx1) is a conserved antioxidant protein that is well known for its disulfide reductase activity. Interestingly, Trx1 is also able to transnitrosylate or denitrosylate (defined as processes to transfer or remove a nitric oxide entity to/from substrates) specific proteins. An intricate redox regulatory mechanism has recently been uncovered that accounts for the ability of Trx1 to catalyze these different redox PTMs. In this review, we will summarize the available evidence in support of Trx1 as a specific disulfide reductase, and denitrosylation and transnitrosylation agent, as well as the biological significance of the diverse array of Trx1-regulated pathways and processes under different physiological contexts. The dramatic progress in redox proteomics techniques has enabled the identification of an increasing number of proteins, including peroxiredoxin 1, whose disulfide bond formation and nitrosylation status are regulated by Trx1. This review will also summarize the advancements of redox proteomics techniques for the identification of the protein targets of Trx1-mediated PTMs. Collectively, these studies have shed light on the mechanisms that regulate Trx1-mediated reduction, transnitrosylation, and denitrosylation of specific target proteins, solidifying the role of Trx1 as a master regulator of redox signal transduction. Antioxid. Redox Signal. 15, 2565–2604. PMID:21453190

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE PAGES

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.; ...

2017-02-01

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

ERIC Educational Resources Information Center

Kin, Ng Hong; Ling, Tan Aik

2016-01-01

The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology.

PubMed

Gupta, Rani; Kumari, Arti; Syal, Poonam; Singh, Yogesh

2015-01-01

Lipase catalyzes hydrolysis of fats in lipid water interphase and perform variety of biotransformation reactions under micro aqueous conditions. The major sources include microbial lipases; among these yeast and fungal lipases are of special interest because they can carry out various stereoselective reactions. These lipases are highly diverse and are categorized into three classes on the basis of oxyanion hole: GX, GGGX and Y. The detailed phylogenetic analysis showed that GX family is more diverse than GGGX and Y family. Sequence and structural comparisons revealed that lipases are conserved only in the signature sequence region. Their characteristic structural determinants viz. lid, binding pocket and oxyanion hole are hotspots for mutagenesis. Few examples are cited in this review to highlight the multidisciplinary approaches for designing novel enzyme variants with improved thermo stability and substrate specificity. In addition, we present a brief account on biotechnological applications of lipases. Lipases have also gained attention as virulence factors, therefore, we surveyed the role of lipases in yeast physiology related to colonization, adhesion, biofilm formation and pathogenesis. The new genomic era has opened numerous possibilities to genetically manipulate lipases for food, fuel and pharmaceuticals. Copyright © 2014 Elsevier Ltd. All rights reserved.

A Modern Understanding of the Traditional and Nontraditional Biological Functions of Angiotensin-Converting Enzyme

PubMed Central

Ong, Frank S.; Blackwell, Wendell-Lamar B.; Shah, Kandarp H.; Giani, Jorge F.; Gonzalez-Villalobos, Romer A.; Shen, Xiao Z.; Fuchs, Sebastien

2013-01-01

Angiotensin-converting enzyme (ACE) is a zinc-dependent peptidase responsible for converting angiotensin I into the vasoconstrictor angiotensin II. However, ACE is a relatively nonspecific peptidase that is capable of cleaving a wide range of substrates. Because of this, ACE and its peptide substrates and products affect many physiologic processes, including blood pressure control, hematopoiesis, reproduction, renal development, renal function, and the immune response. The defining feature of ACE is that it is composed of two homologous and independently catalytic domains, the result of an ancient gene duplication, and ACE-like genes are widely distributed in nature. The two ACE catalytic domains contribute to the wide substrate diversity of ACE and, by extension, the physiologic impact of the enzyme. Several studies suggest that the two catalytic domains have different biologic functions. Recently, the X-ray crystal structure of ACE has elucidated some of the structural differences between the two ACE domains. This is important now that ACE domain-specific inhibitors have been synthesized and characterized. Once widely available, these reagents will undoubtedly be powerful tools for probing the physiologic actions of each ACE domain. In turn, this knowledge should allow clinicians to envision new therapies for diseases not currently treated with ACE inhibitors. PMID:23257181

One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates.

PubMed

Yu, Hai; Chen, Xi

2016-03-14

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with glycosyltransferases in one pot for efficient production of the target glycans from simple monosaccharides and acceptors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitated the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modifications (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequence for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of the glycosyltransferases define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. This Perspective summarizes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed.

One-pot multienzyme (OPME) systems for chemoenzymatic synthesis of carbohydrates

PubMed Central

Yu, Hai; Chen, Xi

2016-01-01

Glycosyltransferase-catalyzed enzymatic and chemoenzymatic syntheses are powerful approaches for the production of oligosaccharides, polysaccharides, glycoconjugates, and their derivatives. Enzymes involved in the biosynthesis of sugar nucleotide donors can be combined with the glycosyltransferases in one pot for efficient production of target glycans from simple monosaccharides and accpetors. The identification of enzymes involved in the salvage pathway of sugar nucleotide generation has greatly facilitate the development of simplified and efficient one-pot multienzyme (OPME) systems for synthesizing major glycan epitopes in mammalian glycomes. The applications of OPME methods are steadily gaining popularity mainly due to the increasing availability of wild-type and engineered enzymes. Substrate promiscuity of these enzymes and their mutants allows OPME synthesis of carbohydrates with naturally occurring post-glycosylational modificiation (PGMs) and their non-natural derivatives using modified monosaccharides as precursors. The OPME systems can be applied in sequential for synthesizing complex carbohydrates. The sequence of the sequential OPME processes, the glycosyltransferase used, and the substrate specificities of glycosyltransferasese define the structures of the products. The OPME and sequential OPME strategies can be extended to diverse glycans in other glycomes when suitable enzymes with substrate promiscuity become available. The Perspective summariezes the work of the authors and collaborators on the development of glycosyltransferase-based OPME systems for carbohydrate synthesis. Future directions are also discussed. PMID:26881499

Lipase-Secreting Bacillus Species in an Oil-Contaminated Habitat: Promising Strains to Alleviate Oil Pollution

PubMed Central

Lee, Li Pin; Karbul, Hudzaifah Mohamed; Citartan, Marimuthu; Gopinath, Subash C. B.; Lakshmipriya, Thangavel; Tang, Thean-Hock

2015-01-01

Lipases are of great interest for different industrial applications due to their diversity and versatility. Among different lipases, microbial lipases are preferable due to their broad substrate specificity, and higher stability with lower production costs compared to the lipases from plants and animals. In the past, a vast number of bacterial species have been reported as potential lipases producers. In this study, the lipases-producing bacterial species were isolated from an oil spillage area in the conventional night market. Isolated species were identified as Bacillus species by biochemical tests which indicate their predominant establishment, and further screened on the agar solid surfaces using lipid and gelatin as the substrates. Out of the ten strains tested, four potential strains were subjected to comparison analysis of the lipolytic versus proteolytic activities. Strain 10 exhibited the highest lipolytic and proteolytic activity. In all the strains, the proteolytic activity is higher than the lipolytic activity except for strain 8, suggesting the possibility for substrate-based extracellular gene induction. The simultaneous secretion of both the lipase and protease is a mean of survival. The isolated bacterial species which harbour both lipase and protease enzymes could render potential industrial-based applications and solve environmental issues. PMID:26180812

Insights into Penicillium roqueforti Morphological and Genetic Diversity

PubMed Central

Gillot, Guillaume; Jany, Jean-Luc; Coton, Monika; Le Floch, Gaétan; Debaets, Stella; Ropars, Jeanne; López-Villavicencio, Manuela; Dupont, Joëlle; Branca, Antoine; Giraud, Tatiana; Coton, Emmanuel

2015-01-01

Fungi exhibit substantial morphological and genetic diversity, often associated with cryptic species differing in ecological niches. Penicillium roqueforti is used as a starter culture for blue-veined cheeses, being responsible for their flavor and color, but is also a common spoilage organism in various foods. Different types of blue-veined cheeses are manufactured and consumed worldwide, displaying specific organoleptic properties. These features may be due to the different manufacturing methods and/or to the specific P. roqueforti strains used. Substantial morphological diversity exists within P. roqueforti and, although not taxonomically valid, several technological names have been used for strains on different cheeses (e.g., P. gorgonzolae, P. stilton). A worldwide P. roqueforti collection from 120 individual blue-veined cheeses and 21 other substrates was analyzed here to determine (i) whether P. roqueforti is a complex of cryptic species, by applying the Genealogical Concordance Phylogenetic Species Recognition criterion (GC-PSR), (ii) whether the population structure assessed using microsatellite markers correspond to blue cheese types, and (iii) whether the genetic clusters display different morphologies. GC-PSR multi-locus sequence analyses showed no evidence of cryptic species. The population structure analysis using microsatellites revealed the existence of highly differentiated populations, corresponding to blue cheese types and with contrasted morphologies. This suggests that the population structure has been shaped by different cheese-making processes or that different populations were recruited for different cheese types. Cheese-making fungi thus constitute good models for studying fungal diversification under recent selection. PMID:26091176

The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis

PubMed Central

Rautengarten, Carsten; Ebert, Berit; Moreno, Ignacio; Temple, Henry; Herter, Thomas; Link, Bruce; Doñas-Cofré, Daniela; Moreno, Adrián; Saéz-Aguayo, Susana; Blanco, Francisca; Mortimer, Jennifer C.; Schultink, Alex; Reiter, Wolf-Dieter; Dupree, Paul; Pauly, Markus; Heazlewood, Joshua L.; Scheller, Henrik V.; Orellana, Ariel

2014-01-01

Plant cells are surrounded by a cell wall that plays a key role in plant growth, structural integrity, and defense. The cell wall is a complex and diverse structure that is mainly composed of polysaccharides. The majority of noncellulosic cell wall polysaccharides are produced in the Golgi apparatus from nucleotide sugars that are predominantly synthesized in the cytosol. The transport of these nucleotide sugars from the cytosol into the Golgi lumen is a critical process for cell wall biosynthesis and is mediated by a family of nucleotide sugar transporters (NSTs). Numerous studies have sought to characterize substrate-specific transport by NSTs; however, the availability of certain substrates and a lack of robust methods have proven problematic. Consequently, we have developed a novel approach that combines reconstitution of NSTs into liposomes and the subsequent assessment of nucleotide sugar uptake by mass spectrometry. To address the limitation of substrate availability, we also developed a two-step reaction for the enzymatic synthesis of UDP–l-rhamnose (Rha) by expressing the two active domains of the Arabidopsis UDP–l-Rha synthase. The liposome approach and the newly synthesized substrates were used to analyze a clade of Arabidopsis NSTs, resulting in the identification and characterization of six bifunctional UDP–l-Rha/UDP–d-galactose (Gal) transporters (URGTs). Further analysis of loss-of-function and overexpression plants for two of these URGTs supported their roles in the transport of UDP–l-Rha and UDP–d-Gal for matrix polysaccharide biosynthesis. PMID:25053812

Diversity in ABC transporters: Type I, II and III importers

PubMed Central

Rice, Austin J.; Park, Aekyung

2014-01-01

ATP-binding cassette transporters are multi-subunit membrane pumps that transport substrates across membranes. While significant in the transport process, transporter architecture exhibits a range of diversity that we are only beginning to recognize. This divergence may provide insight into the mechanisms of substrate transport and homeostasis. Until recently, ABC importers have been classified into two types, but with the emergence of energy-coupling factor (ECF) transporters there are potentially three types of ABC importers. In this review, we summarize an expansive body of research on the three types of importers with an emphasis on the basics that underlie ABC importers, such as structure, subunit composition and mechanism. PMID:25155087

Comparative Chloroplast Genomes of Pinaceae: Insights into the Mechanism of Diversified Genomic Organizations

PubMed Central

Wu, Chung-Shien; Lin, Ching-Ping; Hsu, Chi-Yao; Wang, Rui-Jiang; Chaw, Shu-Miaw

2011-01-01

Abstract Pinaceae, the largest family of conifers, has diversified organizations of chloroplast genomes (cpDNAs) with the two typical inverted repeats (IRs) highly reduced. To unravel the mechanism of this genomic diversification, we examined the cpDNA organizations from 53 species of the ten Pinaceous genera, including those of Larix decidua (122,474 bp), Picea morrisonicola (124,168 bp), and Pseudotsuga wilsoniana (122,513 bp), which were firstly elucidated. The results uncovered four distinct cpDNA forms (A−C and P) that are due to rearrangements of two ∼20 and ∼21 kb specific fragments. The C form was documented for the first time and the A form might be the most ancestral one. In addition, only the individuals of Ps. macrocarpa and Ps. wilsoniana were detected to have isomeric cpDNA forms. Three types (types 1−3) of Pinaceae-specific repeats situated nearby the rearranged fragments were found to be syntenic. We hypothesize that type 1 (949 ± 343 bp) and type 3 (608 ± 73 bp) repeats are substrates for homologous recombination (HR), whereas type 2 repeats are likely inactive for HR because of their relatively short sizes (151 ± 30 bp). Conversions among the four distinct forms may be achieved by HR and mediated by type 1 or 3 repeats, thus resulting in increased diversity of cpDNA organizations. We propose that in the Pinaceae cpDNAs, the reduced IRs have lost HR activity, then decreasing the diversity of cpDNA organizations, but the specific repeats that the evolution endowed Pinaceae complement the reduced IRs and increase the diversity of cpDNA organizations. PMID:21402866

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Fungicides degradation in an organic biomixture: impact on microbial diversity.

PubMed

Coppola, Laura; Comitini, Francesca; Casucci, Cristiano; Milanovic, Vesna; Monaci, Elga; Marinozzi, Maria; Taccari, Manuela; Ciani, Maurizio; Vischetti, Costantino

2011-12-15

Biological systems are being developed all over EU countries to protect water-bodies from pesticide contamination at farm level. A laboratory experiment was carried out to test the efficiency of a mixture of compost and straw in bio-degrading different mixtures of fungicides usually applied in vineyards. At the same time the effects of fungicide applications on microbial community of biomixture were also evaluated. Results showed that the biomixture had a good capability of degrading pesticides. Indeed, at the end of the experiment (112 days), the concentration of most of the pesticides was close to complete degradation. Denaturing gradient gel electrophoresis (DGGE) analysis showed an evident modification of microbial diversity after the addition of fungicides. However, at the end of degradation process, no significant changes in the composition of microbial community were seen. In this specific substrate used in the biomixture, yeast flora and ascomycete filamentous fungi seemed to be involved in the degradation activity. Copyright © 2011 Elsevier B.V. All rights reserved.

Glyoxalase diversity in parasitic protists.

PubMed

Deponte, Marcel

2014-04-01

Our current knowledge of the isomerase glyoxalase I and the thioesterase glyoxalase II is based on a variety of prokaryotic and eukaryotic (model) systems with an emphasis on human glyoxalases. During the last decade, important insights on glyoxalase catalysis and structure-function relationships have also been obtained from parasitic protists. These organisms, including kinetoplastid and apicomplexan parasites, are particularly interesting, both because of their relevance as pathogens and because of their phylogenetic diversity and host-parasite co-evolution which has led to specialized organellar and metabolic adaptations. Accordingly, the glyoxalase repertoire and properties vary significantly among parasitic protists of different major eukaryotic lineages (and even between closely related organisms). For example, several protists have an insular or non-canonical glyoxalase. Furthermore, the structures and the substrate specificities of glyoxalases display drastic variations. The aim of the present review is to highlight such differences as well as similarities between the glyoxalases of parasitic protists and to emphasize the power of comparative studies for gaining insights into fundamental principles and alternative glyoxalase functions.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis.

PubMed

Ang, Ee L; Obbard, Jeffrey P; Zhao, Huimin

2007-02-01

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Degeneracy allows for both apparent homogeneity and diversification in populations

PubMed Central

Whitacre, James M.; Atamas, Sergei P.

2013-01-01

Trait diversity – the substrate for natural selection – is necessary for adaptation through selection, particularly in populations faced with environmental changes that diminish population fitness. In habitats that remain unchanged for many generations, stabilizing selection maximizes exploitation of resources by reducing trait diversity to a narrow optimal range. One might expect that such ostensibly homogeneous populations would have a reduced potential for heritable adaptive responses when faced with fitness-reducing environmental changes. However, field studies have documented populations that, even after long periods of evolutionary stasis, can still rapidly evolve in response to changed environmental conditions. We argue that degeneracy, the ability of diverse population elements to function similarly, can satisfy both the current need to maximize fitness and the future need for diversity. Degenerate ensembles appear functionally redundant in certain environmental contexts and functionally diverse in others. We propose that genetic variation not contributing to the observed range of phenotypes in a current population, also known as cryptic genetic variation (CGV), is a specific case of degeneracy. We argue that CGV, which gradually accumulates in static populations in stable environments, reveals hidden trait differences when environments change. By allowing CGV accumulation, static populations prepare themselves for future rapid adaptations to environmental novelty. A greater appreciation of degeneracy’s role in resolving the inherent tension between current stabilizing selection and future directional selection has implications in conservation biology and may be applied in social and technological systems to maximize current performance while strengthening the potential for future changes. PMID:22910487

A Compact Viral Processing Proteinase/Ubiquitin Hydrolase from the OTU Family

PubMed Central

Chenon, Mélanie; Andreani, Jessica; Guerois, Raphaël; Jupin, Isabelle; Bressanelli, Stéphane

2013-01-01

Turnip yellow mosaic virus (TYMV) - a member of the alphavirus-like supergroup of viruses - serves as a model system for positive-stranded RNA virus membrane-bound replication. TYMV encodes a precursor replication polyprotein that is processed by the endoproteolytic activity of its internal cysteine proteinase domain (PRO). We recently reported that PRO is actually a multifunctional enzyme with a specific ubiquitin hydrolase (DUB) activity that contributes to viral infectivity. Here, we report the crystal structure of the 150-residue PRO. Strikingly, PRO displays no homology to other processing proteinases from positive-stranded RNA viruses, including that of alphaviruses. Instead, the closest structural homologs of PRO are DUBs from the Ovarian tumor (OTU) family. In the crystal, one molecule's C-terminus inserts into the catalytic cleft of the next, providing a view of the N-terminal product complex in replication polyprotein processing. This allows us to locate the specificity determinants of PRO for its proteinase substrates. In addition to the catalytic cleft, at the exit of which the active site is unusually pared down and solvent-exposed, a key element in molecular recognition by PRO is a lobe N-terminal to the catalytic domain. Docking models and the activities of PRO and PRO mutants in a deubiquitylating assay suggest that this N-terminal lobe is also likely involved in PRO's DUB function. Our data thus establish that DUBs can evolve to specifically hydrolyze both iso- and endopeptide bonds with different sequences. This is achieved by the use of multiple specificity determinants, as recognition of substrate patches distant from the cleavage sites allows a relaxed specificity of PRO at the sites themselves. Our results thus shed light on how such a compact protein achieves a diversity of key functions in viral genome replication and host-pathogen interaction. PMID:23966860

Holocene forest development and maintenance on different substrates in the Klamath mountains, northern California, USA

Treesearch

Christy E. Briles; Cathy Whitlock; Carl N. Skinner; Jerry Mohr

2011-01-01

The influence of substrate on long-term vegetation dynamics has received little attention, and yet nutrient-limited ecosystems have some of the highest levels of endemism in the world. The diverse geology of the Klamath Mountains of northern California (USA) allows examination of the long-term influence of edaphic constraints in subalpine forests through a comparison...

The Microbial Community of Tardigrades: Environmental Influence and Species Specificity of Microbiome Structure and Composition.

PubMed

Vecchi, Matteo; Newton, Irene L G; Cesari, Michele; Rebecchi, Lorena; Guidetti, Roberto

2018-01-15

Symbiotic associations of metazoans with bacteria strongly influence animal biology since bacteria are ubiquitous and virtually no animal is completely free from them. Tardigrades are micrometazoans famous for their ability to undergo ametabolic states (cryptobiosis) but very little information is available on potential microbial associations. We characterized the microbiomes of six limnoterrestrial tardigrade species belonging to several phylogenetic lines in tandem with the microbiomes of their respective substrates. The experimental design enabled us to determine the effects of both the environment and the host genetic background on the tardigrade microbiome; we were able to define the microbial community of the same species sampled from different environments, and the communities of different species from the same environment. Our 16S rRNA gene amplicon approach indicated that the tardigrade microbiome is species-specific and well differentiated from the environment. Tardigrade species showed a much lower microbial diversity compared to their substrates, with only one significant exception. Forty-nine common OTUs (operational taxonomic units) were classified into six bacterial phyla, while four common OTUs were unclassified and probably represent novel bacterial taxa. Specifically, the tardigrade microbiome appears dominated by Proteobacteria and Bacteroidetes. Some OTUs were shared between different species from geographically distant samples, suggesting the associated bacteria may be widespread. Putative endosymbionts of tardigrades from the order Rickettsiales were identified. Our results indicated that like all other animals, tardigrades have their own microbiota that is different among species, and its assembly is determined by host genotype and environmental influences.

Advances in photonic MOEMS-MEMS device thinning and polishing

NASA Astrophysics Data System (ADS)

McAneny, James J.; Kennedy, Mark; McGroggan, Tom

2010-02-01

As devices continue to increase in density and complexity, ever more stringent specifications are placed on the wafer scale equipment manufacturers to produce higher quality and higher output. This results in greater investment and more resource being diverted into producing tools and processes which can meet the latest demanding criteria. Substrate materials employed in the fabrication process range from Silicon through InP and include GaAs, InSb and other optical networking or waveguide materials. With this diversity of substrate materials presented, controlling the geometries and surfaces grows progressively more challenging. This article highlights the key parameters which require close monitoring and control in order to produce highly precise wafers as part of the fabrication process. Several as cut and commercially available standard polished wafer materials were used in empirical trials to test tooling options in generating high levels of geometric control over the dimensions while producing high quality surface finishes. Specific attention was given to the measurement and control of: flatness; parallelism/TTV; surface roughness and final target thickness as common specifications required by the industry. By combining the process variables of: plate speed, download pressure, slurry flow rate and concentration, pad type and wafer travel path across the polish pad, the effect of altering these variables was recorded and analysed to realize the optimum process conditions for the materials under test. The results being then used to design improved methods and tooling for the thinning and polishing of photonic materials applied to MOEMS-MEMS device fabrication.

Flavonoid transport mechanisms: how to go, and with whom.

PubMed

Zhao, Jian

2015-09-01

Subcellular flavonoid transport and its underlying regulatory mechanisms are still poorly understood, but are fascinating research frontiers in plant science. Recent studies support and further extend previous hypotheses indicating that vacuolar sequestration of flavonoids involves vesicle trafficking, membrane transporters, and glutathione S-transferase (GST). However, the question remains to be addressed of how three distinct but nonexclusive mechanisms are functionally integrated into diverse but redundant transport routes for vacuolar sequestration or extracellular secretion of flavonoids. In this review, I highlight recent progress in understanding flavonoid-transporting vesicle behavior and properties, GST and membrane transporter functions and mechanisms, and flavonoid transport substrate specificity and preference. Copyright © 2015 Elsevier Ltd. All rights reserved.

From hatching to dispatching: the multiple cellular roles of the Hsp70 molecular chaperone machinery.

PubMed

Meimaridou, Eirini; Gooljar, Sakina B; Chapple, J Paul

2009-01-01

Molecular chaperones are best recognized for their roles in de novo protein folding and the cellular response to stress. However, many molecular chaperones, and in particular the Hsp70 chaperone machinery, have multiple diverse cellular functions. At the molecular level, chaperones are mediators of protein conformational change. To facilitate conformational change of client/substrate proteins, in manifold contexts, chaperone power must be closely regulated and harnessed to specific cellular locales--this is controlled by cochaperones. This review considers specialized functions of the Hsp70 chaperone machinery mediated by its cochaperones. We focus on vesicular trafficking, protein degradation and a potential role in G protein-coupled receptor processing.

A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

PubMed Central

Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

2014-01-01

Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

Functional diversity for biomass deconstruction in family 5 subfamily 5 (GH5_5) of fungal endo-β1,4-glucanases.

PubMed

Li, Bingyao; Walton, Jonathan D

2017-05-01

Endo-β1,4-glucanases in glycosyl hydrolase family 5 (GH5) are ubiquitous enzymes in the multicellular fungi and are common components of enzyme cocktails for biomass conversion. We recently showed that an endo-glucanase of subfamily 5 of GH5 (GH5_5) from Sporotrichum thermophile (StCel5A) was more effective at releasing glucose from pretreated corn stover, when part of an eight-component synthetic enzyme mixture, compared to its closely related counterpart from Trichoderma reesei, TrCel5A. StCel5A and TrCel5A belong to different clades of GH5_5 (GH5_5_1 and GH5_5_2, respectively). To test whether the superior activity of StCel5A was a general property of all enzymes in the GH5_5_2 clade, StCel5A, TrCel5A, and two additional members of each subfamily were expressed in a common host that had been engineered to suppress its native cellulases (T. reesei Δxyr1) and compared against each other alone on pure substrates, in synthetic mixtures on pure substrates, and against each other in synthetic mixtures on real biomass. The results indicated that superiority is a unique property of StCel5A and not of GH5_5_2 generally. The six Cel5A enzymes had significant differences in relative activities on different substrates, in specific activities, and in sensitivities to mannan inhibition. Importantly, the behavior of the six endo-glucanases on pure cellulose substrates did not predict their behavior in combination with other cellulolytic enzymes on a real lignocellulosic biomass substrate.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

Grazing exclusion, substrate type, and drought frequency affect plant community structure in rangelands of the arid unpredictable Arabian Deserts

NASA Astrophysics Data System (ADS)

El-Keblawy, Ali; El-Sheikh, Mohamed

2017-04-01

Grazing and drought can adversely affect the ecology and management of rangeland ecosystems. Several management actions have been applied to restore species diversity and community structure in degraded rangelands of the unpredictable arid environment. Protection from grazing is considered as a proper approach for restoration of degraded rangelands, but this depends on substrate type and sometime is hindered with water deficiency (drought). In this study, the effect of protection from grazing animals on species diversity and plant community structure was assessed after a dry and wet periods in both sandy and gravelly substrates in the Dubai Desert Conservation reserve (DDCR), United Arab Emirates. Two sites were selected during November 2012 on the two substrate types (fixed sandy flat and gravel plain) in the arid DDCR. An enclosure was established in each site. Plant community attributes (plant cover, density, frequency, species composition, and diversity indices) were assessed in a number of permanent plots laid inside and outside each enclosure during November 2012, April 2014 and April 2016. The results showed that protection improved clay content, but decreased the organic matters. Interestingly, the protection reduced the concentrations of most estimated nutrients, which could be attributed to the high turnover rate of nutrients associated grazing and low decomposition of accumulated dry plants of non-protected sites. Protection significantly increased all plant community attributes, but the only significant effect was for plant density. Plant density was almost twice greater inside than outside the enclosures. During the dry period, protection resulted in significantly greater deterioration in cover, density and all diversity indices in gravel, compared to sandy sites. Most of the grasses and shrubby plants had died in the gravel plains. However, plant community of the gravel plains was significantly restored after receiving considerable rainfalls. The overall results indicate that rangelands of the gravel plains are more fragile and should receive proper management through conservation and restoration.

Effects of grade control structures on the macroinvertebrate assemblage of an agriculturally impacted stream

USGS Publications Warehouse

Litvan, M.E.; Stewart, T.W.; Pierce, C.L.; Larson, C.J.

2008-01-01

Nearly 400 rock rip-rap grade control structures (hereafter GCS) were recently placed in streams of western Iowa, USA to reduce streambank erosion and protect bridge infrastructure and farmland. In this region, streams are characterized by channelized reaches, highly incised banks and silt and sand substrates that normally support low macroinvertebrate abundance and diversity. Therefore, GCS composed of rip-rap provide the majority of coarse substrate habitat for benthic macroinvertebrates in these streams. We sampled 20 sites on Walnut Creek, Montgomery County, Iowa to quantify macroinvertebrate assemblage characteristics (1) on GCS rip-rap and at sites located (2) 5-50 m upstream of GCS, (3) 5-50 m downstream of GCS and (4) at least 1 km from any GCS (five sites each). Macroinvertebrate biomass, numerical densities and diversity were greatest at sites with coarse substrates, including GCS sites and one natural riffle site and relatively low at remaining sites with soft substrates. Densities of macroinvertebrates in the orders Ephemeroptera, Trichoptera, Diptera, Coleoptera and Acariformes were abundant on GCS rip-rap. Increases in macroinvertebrate biomass, density and diversity at GCS may improve local efficiency of breakdown of organic matter and nutrient and energy flow, and provide enhanced food resources for aquatic vertebrates. However, lack of positive macroinvertebrate responses immediately upstream and downstream of GCS suggest that positive effects might be restricted to the small areas of streambed covered by GCS. Improved understanding of GCS effects at both local and ecosystem scales is essential for stream management when these structures are present. Copyright ?? 2007 John Wiley & Sons, Ltd.

Screening combinatorial arrays of inorganic materials with spectroscopy or microscopy

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2004-02-03

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial synthesis and screening of non-biological polymers

DOEpatents

Schultz, Peter G.; Xiang, Xiao-Dong; Goldwasser, Isy; Briceno, Gabriel; Sun, Xiao-Dong; Wang, Kai-An

2006-04-25

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial synthesis of novel materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

1999-01-01

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial sythesis of organometallic materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2002-07-16

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Polymer arrays from the combinatorial synthesis of novel materials

DOEpatents

Schultz, Peter G.; Xiang, Xiao-Dong; Goldwasser, Isy; Briceno, Gabriel; Sun, Xiao-Dong

2004-09-21

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Giant magnetoresistive cobalt oxide compounds

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

1998-01-01

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial synthesis of novel materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2002-02-12

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Giant magnetoresistive cobalt oxide compounds

DOEpatents

Schultz, P.G.; Xiang, X.; Goldwasser, I.

1998-07-07

Methods and apparatus are disclosed for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties. 58 figs.

Preparation and screening of crystalline inorganic materials

DOEpatents

Schultz, Peter G [La Jolla, CA; Xiang, Xiaodong [Danville, CA; Goldwasser, Isy [Palo Alto, CA; Brice{hacek over }o, Gabriel; Sun, Xiao-Dong [Fremont, CA; Wang, Kai-An [Cupertino, CA

2008-10-28

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Synthesis and screening combinatorial arrays of zeolites

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2003-11-18

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial synthesis of novel materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

1999-12-21

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial synthesis of novel materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2001-01-01

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Combinatorial screening of inorganic and organometallic materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy

2002-01-01

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Preparation and screening of crystalline zeolite and hydrothermally-synthesized materials

DOEpatents

Schultz, Peter G.; Xiang, Xiaodong; Goldwasser, Isy; Briceno, Gabriel; Sun, Xiao-Dong; Wang, Kai-An

2005-03-08

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, non-biological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Multifunctional Mitochondrial AAA Proteases

PubMed Central

Glynn, Steven E.

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

Multifunctional Mitochondrial AAA Proteases.

PubMed

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Effects of Gelling Agent and Extracellular Signaling Molecules on the Culturability of Marine Bacteria

PubMed Central

Rygaard, Anita Mac; Thøgersen, Mariane Schmidt; Nielsen, Kristian Fog; Gram, Lone

2017-01-01

ABSTRACT Only 1% of marine bacteria are currently culturable using standard laboratory procedures, and this is a major obstacle for our understanding of the biology of marine microorganisms and for the discovery of novel microbial natural products. Therefore, the purpose of this study was to investigate if improved cultivation conditions, including the use of an alternative gelling agent and supplementation with signaling molecules, improve the culturability of bacteria from seawater. Replacing agar with gellan gum improved viable counts 3- to 40-fold, depending on medium composition and incubation conditions, with a maximum of 6.6% culturability relative to direct cell counts. Through V4 amplicon sequencing we found that culturable diversity was also affected by a change in gelling agent, facilitating the growth of orders not culturable on agar-based substrates. Community analyses showed that communities grown on gellan gum substrates were significantly different from communities grown on agar and that they covered a larger fraction of the seawater community. Other factors, such as incubation temperature and time, had less obvious effects on viable counts and culturable diversity. Supplementation with acylated homoserine lactones (AHLs) did not have a positive effect on total viable counts or a strong effect on culturable diversity. However, low concentrations of AHLs increased the relative abundance of sphingobacteria. Hence, with alternative growth substrates, it is possible to significantly increase the number and diversity of cultured marine bacteria. IMPORTANCE Serious challenges to human health, such as the occurrence and spread of antibiotic resistance and an aging human population in need of bioactive pharmaceuticals, have revitalized the search for natural microbial products. The marine environment, representing the largest ecosystem in the biosphere, harbors an immense and virtually untapped microbial diversity producing unique bioactive compounds. However, we are currently able to cultivate only a minute fraction of this diversity. The lack of cultivated microbes is hampering not only bioprospecting efforts but also our general understanding of marine microbes. In this study, we present a means to increase the number and diversity of cultured bacteria from seawater, showing that relatively simple changes to medium components may facilitate the isolation and growth of hitherto unknown bacterial orders. PMID:28213548

Rice to vegetables: short- versus long-term impact of land-use change on the indigenous soil microbial community.

PubMed

Sun, Bo; Dong, Zhi-Xing; Zhang, Xue-Xian; Li, Yun; Cao, Hui; Cui, Zong-Li

2011-08-01

Land-use change is known to have a significant effect on the indigenous soil microbial community, but it is unknown if there are any general trends regarding how this effect varies over time. Here, we describe a comparative analysis of microbial communities from three adjacent agricultural fields: one-century-old paddy field (OP) and two vegetable fields (new vegetable field (NV) and old vegetable field (OV)) that were established on traditional paddy fields 10 and 100 years ago, respectively. Soil chemical and physical analysis showed that both vegetable fields were more nutrient rich than the paddy field in terms of organic C, total N, total P, and available K. The vegetable fields possessed relatively higher abundance of culturable bacteria, fungi, and specific groups of bacteria (Actinomyces, nitrifying bacteria, and cellulose-decomposing bacteria) but lower levels of microbial biomass C and N. Notably, the decrease of biomass was further confirmed by analysis of seven additional soils in chronosequence sampled from the same area. Next we examined the metabolic diversity of the microbial community using the EcoPlate(TM) system from Biolog Inc. (Hayward, CA, USA). The utilization patterns of 31 unique C substrates (i.e., community-level physiological profile) showed that microorganisms in vegetable soil and paddy soil prefer to use different C substrates (polymeric compounds for NV and OV soils, phenolic acids for OP soil). Principal component analysis and the average well color development data showed that the NV is metabolically more distinct from the OV and OP. The effect was likely attributable to the elevated soil pH in NV soil. Furthermore, we assessed the diversity of soil bacterial populations using the cultivation-independent technology of amplified ribosomal DNA restriction analysis (ARDRA). Results showed that levels of bacterial diversity in OP and NV soils were similar (Shannon's diversity index H = 4.83 and 4.79, respectively), whereas bacteria in OV soil have the lowest score of diversity (H = 3.48). The low level of bacterial diversity in OV soil was supported by sequencing of ten randomly selected 16S rDNA clones from each of the three rDNA libraries. Phylogenetic analysis showed that all the ten OV clones belonged to Proteobacteria with eight in the gamma-subdivision and two in the alpha-subdivision. In contrast, the ten clones from NV and OP soils were classified into four and eight bacterial classes or unclassified groups, respectively. Taken together, our data suggest that land-use change from rice to vegetables resulted in a decrease of bacterial diversity and soil biomass despite an increase in the abundance of culturable microorganisms and, moreover, the decrease of bacterial diversity occurred during long-term rather than short-term vegetable cultivation.

Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Westfall, Corey S.; Zubieta, Chloe; Herrmann, Jonathan

Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid-specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how amore » highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.« less

A neural circuit encoding sexual preference in humans

PubMed Central

Poeppl, Timm B.; Langguth, Berthold; Rupprecht, Rainer; Laird, Angela R; Eickhoff, Simon B.

2016-01-01

Sexual preference determines mate choice for reproduction and hence guarantees conservation of species in mammals. Despite this fundamental role in human behavior, current knowledge on its target-specific neurofunctional substrate is based on lesion studies and therefore limited. We used meta-analytic remodeling of neuroimaging data from 364 human subjects with diverse sexual interests during sexual stimulation to quantify neural regions associated with sexual preference manipulations. We found that sexual preference is encoded by four phylogenetically old, subcortical brain structures. More specifically, sexual preference is controlled by the anterior and preoptic area of the hypothalamus, the anterior and mediodorsal thalamus, the septal area, and the perirhinal parahippocampus including the dentate gyrus. In contrast, sexual non-preference is regulated by the substantia innominata. We anticipate the identification of a core neural circuit for sexual preferences to be a starting point for further sophisticated investigations into the neural principles of sexual behavior and particularly of its aberrations. PMID:27339689

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Phylogeny and Expression Analyses Reveal Important Roles for Plant PKS III Family during the Conquest of Land by Plants and Angiosperm Diversification

PubMed Central

Xie, Lulu; Liu, Pingli; Zhu, Zhixin; Zhang, Shifan; Zhang, Shujiang; Li, Fei; Zhang, Hui; Li, Guoliang; Wei, Yunxiao; Sun, Rifei

2016-01-01

Polyketide synthases (PKSs) utilize the products of primary metabolism to synthesize a wide array of secondary metabolites in both prokaryotic and eukaryotic organisms. PKSs can be grouped into three distinct classes, types I, II, and III, based on enzyme structure, substrate specificity, and catalytic mechanisms. The type III PKS enzymes function as homodimers, and are the only class of PKS that do not require acyl carrier protein. Plant type III PKS enzymes, also known as chalcone synthase (CHS)-like enzymes, are of particular interest due to their functional diversity. In this study, we mined type III PKS gene sequences from the genomes of six aquatic algae and 25 land plants (1 bryophyte, 1 lycophyte, 2 basal angiosperms, 16 core eudicots, and 5 monocots). PKS III sequences were found relatively conserved in all embryophytes, but not exist in algae. We also examined gene expression patterns by analyzing available transcriptome data, and identified potential cis-regulatory elements in upstream sequences. Phylogenetic trees of dicots angiosperms showed that plant type III PKS proteins fall into three clades. Clade A contains CHS/STS-type enzymes coding genes with diverse transcriptional expression patterns and enzymatic functions, while clade B is further divided into subclades b1 and b2, which consist of anther-specific CHS-like enzymes. Differentiation regions, such as amino acids 196-207 between clades A and B, and predicted positive selected sites within α-helixes in late appeared branches of clade A, account for the major diversification in substrate choice and catalytic reaction. The integrity and location of conserved cis-elements containing MYB and bHLH binding sites can affect transcription levels. Potential binding sites for transcription factors such as WRKY, SPL, or AP2/EREBP may contribute to tissue- or taxon-specific differences in gene expression. Our data shows that gene duplications and functional diversification of plant type III PKS enzymes played a critical role in the ancient conquest of the land by early plants and angiosperm diversification. PMID:27625671

Comparison of three tannases cloned from closely related lactobacillus species: L. Plantarum, L. Paraplantarum, and L. Pentosus

PubMed Central

2014-01-01

Background Tannase (tannin acyl hydrolase, EC 3.1.1.20) specifically catalyzes the hydrolysis of the galloyl ester bonds in hydrolyzable tannins to release gallic acid. The enzyme was found not only in fungal species but also many bacterial species including Lactobacillus plantarum, L. paraplantarum, and L. pentosus. Recently, we identified and expressed a tannase gene of L. plantarum, tanLpl, to show remarkable differences to characterized fungal tannases. However, little is known about genes responsible for tannase activities of L. paraplantarum and L. pentosus. We here identify the tannase genes (i.e. tanLpa and tanLpe) of the above lactobacilli species, and describe their molecular diversity among the strains as well as enzymological difference between species inclusive of L. plantarum. Results The genes encoding tannase, designated tanLpa and tanLpe, were cloned from Lactobacillus paraplantarum NSO120 and Lactobacillus pentosus 21A-3, which shared 88% and 72% amino acid identity with TanLpl, cloned from Lactobacillus plantarum ATCC 14917T, respectively. These three enzymes could comprise a novel tannase subfamily of independent lineage, because no other tannases in the databases share significant sequence similarity with them. Each of tanLpl, tanLpa, and tanLpe was expressed in Bacillus subtilis RIK 1285 and recombinant enzymes were secreted and purified. The Km values of the enzymes on each galloyl ester were comparable; however, the kcat/Km values of TanLpa for EGCg, ECg, Cg, and GCg were markedly higher than those for TanLpl and TanLpe. Their enzymological properties were compared to reveal differences at least in substrate specificity. Conclusion Two tannase genes responsible for tannase activities of L. paraplantarum and L. pentosus were identified and characterized. TanLpl, TanLpa and TanLpe forming a phylogenetic cluster in the known bacterial tannase genes and had a limited diversity in each other. Their enzymological properties were compared to reveal differences at least in substrate specificity. This is the first comparative study of closely related bacterial tannases. PMID:24708557

A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway1[OPEN

PubMed Central

2016-01-01

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. PMID:27356974

Mutant Allele-Specific Uncoupling of PENETRATION3 Functions Reveals Engagement of the ATP-Binding Cassette Transporter in Distinct Tryptophan Metabolic Pathways1[OPEN

PubMed Central

Lu, Xunli; Dittgen, Jan; Piślewska-Bednarek, Mariola; Molina, Antonio; Schneider, Bernd; Doubský, Jan; Schneeberger, Korbinian; Schulze-Lefert, Paul

2015-01-01

Arabidopsis (Arabidopsis thaliana) PENETRATION (PEN) genes quantitatively contribute to the execution of different forms of plant immunity upon challenge with diverse leaf pathogens. PEN3 encodes a plasma membrane-resident pleiotropic drug resistance-type ATP-binding cassette transporter and is thought to act in a pathogen-inducible and PEN2 myrosinase-dependent metabolic pathway in extracellular defense. This metabolic pathway directs the intracellular biosynthesis and activation of tryptophan-derived indole glucosinolates for subsequent PEN3-mediated efflux across the plasma membrane at pathogen contact sites. However, PEN3 also functions in abiotic stress responses to cadmium and indole-3-butyric acid (IBA)-mediated auxin homeostasis in roots, raising the possibility that PEN3 exports multiple functionally unrelated substrates. Here, we describe the isolation of a pen3 allele, designated pen3-5, that encodes a dysfunctional protein that accumulates in planta like wild-type PEN3. The specific mutation in pen3-5 uncouples PEN3 functions in IBA-stimulated root growth modulation, callose deposition induced with a conserved peptide epitope of bacterial flagellin (flg22), and pathogen-inducible salicylic acid accumulation from PEN3 activity in extracellular defense, indicating the engagement of multiple PEN3 substrates in different PEN3-dependent biological processes. We identified 4-O-β-d-glucosyl-indol-3-yl formamide (4OGlcI3F) as a pathogen-inducible, tryptophan-derived compound that overaccumulates in pen3 leaf tissue and has biosynthesis that is dependent on an intact PEN2 metabolic pathway. We propose that a precursor of 4OGlcI3F is the PEN3 substrate in extracellular pathogen defense. These precursors, the shared indole core present in IBA and 4OGlcI3F, and allele-specific uncoupling of a subset of PEN3 functions suggest that PEN3 transports distinct indole-type metabolites in distinct biological processes. PMID:26023163

Geckoprinting: assembly of microelectronic devices on unconventional surfaces by transfer printing with isolated gecko setal arrays

PubMed Central

Jeong, Jaeyoung; Kim, Juho; Song, Kwangsun; Autumn, Kellar; Lee, Jongho

2014-01-01

Developing electronics in unconventional forms provides opportunities to expand the use of electronics in diverse applications including bio-integrated or implanted electronics. One of the key challenges lies in integrating semiconductor microdevices onto unconventional substrates without glue, high pressure or temperature that may cause damage to microdevices, substrates or interfaces. This paper describes a solution based on natural gecko setal arrays that switch adhesion mechanically on and off, enabling pick and place manipulation of thin microscale semiconductor materials onto diverse surfaces including plants and insects whose surfaces are usually rough and irregular. A demonstration of functional ‘geckoprinted’ microelectronic devices provides a proof of concept of our results in practical applications. PMID:25056216

Control of seizures by ketogenic diet-induced modulation of metabolic pathways.

PubMed

Clanton, Ryan M; Wu, Guoyao; Akabani, Gamal; Aramayo, Rodolfo

2017-01-01

Epilepsy is too complex to be considered as a disease; it is more of a syndrome, characterized by seizures, which can be caused by a diverse array of afflictions. As such, drug interventions that target a single biological pathway will only help the specific individuals where that drug's mechanism of action is relevant to their disorder. Most likely, this will not alleviate all forms of epilepsy nor the potential biological pathways causing the seizures, such as glucose/amino acid transport, mitochondrial dysfunction, or neuronal myelination. Considering our current inability to test every individual effectively for the true causes of their epilepsy and the alarming number of misdiagnoses observed, we propose the use of the ketogenic diet (KD) as an effective and efficient preliminary/long-term treatment. The KD mimics fasting by altering substrate metabolism from carbohydrates to fatty acids and ketone bodies (KBs). Here, we underscore the need to understand the underlying cellular mechanisms governing the KD's modulation of various forms of epilepsy and how a diverse array of metabolites including soluble fibers, specific fatty acids, and functional amino acids (e.g., leucine, D-serine, glycine, arginine metabolites, and N-acetyl-cysteine) may potentially enhance the KD's ability to treat and reverse, not mask, these neurological disorders that lead to epilepsy.

Non-Cell-Adhesive Substrates for Printing of Arrayed Biomaterials

PubMed Central

Appel, Eric A.; Larson, Benjamin L.; Luly, Kathryn M.; Kim, Jinseong D.

2015-01-01

Cellular microarrays have become extremely useful in expediting the investigation of large libraries of (bio)materials for both in vitro and in vivo biomedical applications. We have developed an exceedingly simple strategy for the fabrication of non-cell-adhesive substrates supporting the immobilization of diverse (bio)material features, including both monomeric and polymeric adhesion molecules (e.g. RGD and polylysine), hydrogels, and polymers. PMID:25430948

Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase.

PubMed

Weber, Annika; Cohen, Itamar; Popp, Oliver; Dittmar, Gunnar; Reiss, Yuval; Sommer, Thomas; Ravid, Tommer; Jarosch, Ernst

2016-09-01

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system. Copyright © 2016 Elsevier Inc. All rights reserved.

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis*

PubMed Central

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M.

2010-01-01

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed. PMID:20382731

Single-molecule force spectroscopy approach to enzyme catalysis.

PubMed

Alegre-Cebollada, Jorge; Perez-Jimenez, Raul; Kosuri, Pallav; Fernandez, Julio M

2010-06-18

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

PubMed Central

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

Introduction to the IWA task group on biofilm modeling.

PubMed

Noguera, D R; Morgenroth, E

2004-01-01

An International Water Association (IWA) Task Group on Biofilm Modeling was created with the purpose of comparatively evaluating different biofilm modeling approaches. The task group developed three benchmark problems for this comparison, and used a diversity of modeling techniques that included analytical, pseudo-analytical, and numerical solutions to the biofilm problems. Models in one, two, and three dimensional domains were also compared. The first benchmark problem (BM1) described a monospecies biofilm growing in a completely mixed reactor environment and had the purpose of comparing the ability of the models to predict substrate fluxes and concentrations for a biofilm system of fixed total biomass and fixed biomass density. The second problem (BM2) represented a situation in which substrate mass transport by convection was influenced by the hydrodynamic conditions of the liquid in contact with the biofilm. The third problem (BM3) was designed to compare the ability of the models to simulate multispecies and multisubstrate biofilms. These three benchmark problems allowed identification of the specific advantages and disadvantages of each modeling approach. A detailed presentation of the comparative analyses for each problem is provided elsewhere in these proceedings.

Structural Elements Regulating AAA+ Protein Quality Control Machines.

PubMed

Chang, Chiung-Wen; Lee, Sukyeong; Tsai, Francis T F

2017-01-01

Members of the ATPases Associated with various cellular Activities (AAA+) superfamily participate in essential and diverse cellular pathways in all kingdoms of life by harnessing the energy of ATP binding and hydrolysis to drive their biological functions. Although most AAA+ proteins share a ring-shaped architecture, AAA+ proteins have evolved distinct structural elements that are fine-tuned to their specific functions. A central question in the field is how ATP binding and hydrolysis are coupled to substrate translocation through the central channel of ring-forming AAA+ proteins. In this mini-review, we will discuss structural elements present in AAA+ proteins involved in protein quality control, drawing similarities to their known role in substrate interaction by AAA+ proteins involved in DNA translocation. Elements to be discussed include the pore loop-1, the Inter-Subunit Signaling (ISS) motif, and the Pre-Sensor I insert (PS-I) motif. Lastly, we will summarize our current understanding on the inter-relationship of those structural elements and propose a model how ATP binding and hydrolysis might be coupled to polypeptide translocation in protein quality control machines.

Impact of oxygen on the coexistence of nitrification, denitrification, and sulfate reduction in oxygen-based membrane aerated biofilm.

PubMed

Liu, Hong; Tan, Shuying; Sheng, Zhiya; Yu, Tong; Liu, Yang

2015-03-01

Membrane aerated biofilms (MABs) are subject to "counter diffusion" of oxygen and substrates. In a membrane aerated biofilm reactor, gases (e.g., oxygen) diffuse through the membrane into the MAB, and liquid substrates pass from the bulk liquid into the MAB. This behavior can result in a unique biofilm structure in terms of microbial composition, distribution, and community activity in the MAB. Previous studies have shown simultaneous aerobic oxidation, nitrification, and denitrification within a single MAB. Using molecular techniques, we investigated the growth of sulfate-reducing bacteria (SRB) in the oxygen-based MAB attached to a flat sheet membrane. Denaturing gradient gel electrophoresis of the amplified 16S rRNA gene fragments and functional gene fragments specific for ammonia-oxidizing bacteria (amoA), denitrifying bacteria (nirK), and SRB (dsrB) demonstrated the coexistence of nitrifiers, denitrifiers, and SRB communities within a single MAB. The functional diversities of SRB and denitrifiers decreased with an increase in the oxygen concentration in the bulk water of the reactor.

Phylogenetic and Metagenomic Analyses of Substrate-Dependent Bacterial Temporal Dynamics in Microbial Fuel Cells

PubMed Central

Zhang, Husen; Chen, Xi; Braithwaite, Daniel; He, Zhen

2014-01-01

Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate. PMID:25202990

Phylogenetic and metagenomic analyses of substrate-dependent bacterial temporal dynamics in microbial fuel cells.

PubMed

Zhang, Husen; Chen, Xi; Braithwaite, Daniel; He, Zhen

2014-01-01

Understanding the microbial community structure and genetic potential of anode biofilms is key to improve extracellular electron transfers in microbial fuel cells. We investigated effect of substrate and temporal dynamics of anodic biofilm communities using phylogenetic and metagenomic approaches in parallel with electrochemical characterizations. The startup non-steady state anodic bacterial structures were compared for a simple substrate, acetate, and for a complex substrate, landfill leachate, using a single-chamber air-cathode microbial fuel cell. Principal coordinate analysis showed that distinct community structures were formed with each substrate type. The bacterial diversity measured as Shannon index decreased with time in acetate cycles, and was restored with the introduction of leachate. The change of diversity was accompanied by an opposite trend in the relative abundance of Geobacter-affiliated phylotypes, which were acclimated to over 40% of total Bacteria at the end of acetate-fed conditions then declined in the leachate cycles. The transition from acetate to leachate caused a decrease in output power density from 243±13 mW/m2 to 140±11 mW/m2, accompanied by a decrease in Coulombic electron recovery from 18±3% to 9±3%. The leachate cycles selected protein-degrading phylotypes within phylum Synergistetes. Metagenomic shotgun sequencing showed that leachate-fed communities had higher cell motility genes including bacterial chemotaxis and flagellar assembly, and increased gene abundance related to metal resistance, antibiotic resistance, and quorum sensing. These differentially represented genes suggested an altered anodic biofilm community in response to additional substrates and stress from the complex landfill leachate.

Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

PubMed Central

2015-01-01

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

A Small Number of Low-abundance Bacteria Dominate Plant Species-specific Responses during Rhizosphere Colonization

PubMed Central

Dawson, Wayne; Hör, Jens; Egert, Markus; van Kleunen, Mark; Pester, Michael

2017-01-01

Plant growth can be affected by soil bacteria. In turn, plants are known to influence soil bacteria through rhizodeposits and changes in abiotic conditions. We aimed to quantify the phylotype richness and relative abundance of rhizosphere bacteria that are actually influenced in a plant species-specific manner and to determine the role of the disproportionately large diversity of low-abundance bacteria belonging to the rare biosphere (<0.1 relative abundance) in this process. In addition, we aimed to determine whether plant phylogeny has an influence on the plant species-specific rhizosphere bacterial community. For this purpose, 19 herbaceous plant species from five different plant orders were grown in a common soil substrate. Bacterial communities in the initial soil substrate and the established rhizosphere soils were compared by 16S rRNA gene amplicon sequencing. Only a small number of bacterial operational taxonomic units (OTUs, 97% sequence identity) responded either positively (ca. 1%) or negatively (ca. 1%) to a specific plant species. On average, 91% of plant-specific positive response OTUs comprised bacteria belonging to the rare biosphere, highlighting that low-abundance populations are metabolically active in the rhizosphere. In addition, low-abundance OTUs were in terms of their summed relative abundance major drivers of the bacterial phyla composition across the rhizosphere of all tested plant species. However, no effect of plant phylogeny could be observed on the established rhizosphere bacterial communities, neither when considering differences in the overall established rhizosphere communities nor when considering plant species-specific responders only. Our study provides a quantitative assessment of the effect of plants on their rhizosphere bacteria across multiple plant orders. Plant species-specific effects on soil bacterial communities involved only 18–111 bacterial OTUs out of several 1000s; this minority may potentially impact plant growth in plant–bacteria interactions. PMID:28611765

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Metagenomic Virome Analysis of Culex Mosquitoes from Kenya and China

PubMed Central

Atoni, Evans; Wang, Yujuan; Karungu, Samuel; Waruhiu, Cecilia; Zohaib, Ali; Obanda, Vincent; Agwanda, Bernard; Mutua, Morris; Xia, Han; Yuan, Zhiming

2018-01-01

Many blood-feeding arthropods are known vectors of viruses that are a source of unprecedented global health concern. Mosquitoes are an integral part of these arthropod vectors. Advancements in next-generation sequencing and bioinformatics has expanded our knowledge on the richness of viruses harbored by arthropods. In the present study, we applied a metagenomic approach to determine the intercontinental virome diversity of Culex quinquefasciatus and Culex tritaeniorhynchus in Kwale, Kenya and provinces of Hubei and Yunnan in China. Our results showed that viromes from the three locations were strikingly diverse and comprised 30 virus families specific to vertebrates, invertebrates, plants, and protozoa as well as unclassified group of viruses. Though sampled at different times, both Kwale and Hubei mosquito viromes were dominated by vertebrate viruses, in contrast to the Yunnan mosquito virome, which was dominated by insect-specific viruses. However, each virome was unique in terms of virus proportions partly influenced by type of ingested meals (blood, nectar, plant sap, environment substrates). The dominant vertebrate virus family in the Kwale virome was Papillomaviridae (57%) while in Hubei it was Herpesviridae (30%) and the Yunnan virome was dominated by an unclassified viruses group (27%). Given that insect-specific viruses occur naturally in their hosts, they should be the basis for defining the viromes. Hence, the dominant insect-specific viruses in Kwale, Hubei, and Yunnan were Baculoviridae, Nimaviridae and Iflaviridae, respectively. Our study is preliminary but contributes to growing and much needed knowledge, as mosquito viromes could be manipulated to prevent and control pathogenic arboviruses. PMID:29329230

Construction and in vivo assembly of a catalytically proficient and hyperthermostable de novo enzyme.

PubMed

Watkins, Daniel W; Jenkins, Jonathan M X; Grayson, Katie J; Wood, Nicola; Steventon, Jack W; Le Vay, Kristian K; Goodwin, Matthew I; Mullen, Anna S; Bailey, Henry J; Crump, Matthew P; MacMillan, Fraser; Mulholland, Adrian J; Cameron, Gus; Sessions, Richard B; Mann, Stephen; Anderson, J L Ross

2017-08-25

Although catalytic mechanisms in natural enzymes are well understood, achieving the diverse palette of reaction chemistries in re-engineered native proteins has proved challenging. Wholesale modification of natural enzymes is potentially compromised by their intrinsic complexity, which often obscures the underlying principles governing biocatalytic efficiency. The maquette approach can circumvent this complexity by combining a robust de novo designed chassis with a design process that avoids atomistic mimicry of natural proteins. Here, we apply this method to the construction of a highly efficient, promiscuous, and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 . The maquette exhibits kinetics that match and even surpass those of certain natural peroxidases, retains its activity at elevated temperature and in the presence of organic solvents, and provides a simple platform for interrogating catalytic intermediates common to natural heme-containing enzymes.Catalytic mechanisms of enzymes are well understood, but achieving diverse reaction chemistries in re-engineered proteins can be difficult. Here the authors show a highly efficient and thermostable artificial enzyme that catalyzes a diverse array of substrate oxidations coupled to the reduction of H 2 O 2 .

Fungal Taxa Target Different Carbon Substrates in Harvard Forest Soils

NASA Astrophysics Data System (ADS)

Hanson, C. A.; Allison, S. D.; Wallenstein, M. D.; Mellilo, J. M.; Treseder, K. K.

2006-12-01

The mineralization of soil organic carbon is a major component of the global carbon cycle and is largely controlled by soil microbial communities. However, little is known about the functional roles of soil microbes or whether different microbial taxa target different carbon substrates under natural conditions. To examine this possibility, we assessed the community composition of active fungi by using a novel nucleotide analog technique in soils from the Harvard Forest. We hypothesized that fungal community composition would shift in response to the addition of different substrates and that specific fungal taxa would respond differentially to particular carbon sources. To test this hypothesis, we added a nucleotide analog probe directly to soils in conjunction with one of five carbon compounds of increasing recalcitrance: glycine, sucrose, cellulose, tannin-protein complex, and lignin. During 48 hour incubations, the nucleotide analog was incorporated into newly replicated DNA of soil organisms that proliferated following the addition of the substrates. In this way, we labeled the DNA of microbes that respond to a particular carbon source. Labeled DNA was isolated and fungal Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) were sequenced and analyzed to identify active fungi to near-species resolution. Diversity analyses at the ≥97% sequence similarity level indicated that taxonomic richness was greater under cellulose (Shannon Index: 3.23 ± 0.11 with ± 95% CI) and lignin (2.87 ± 0.15) additions than the other treatments (2.34 ± 0.16 to 2.64 ± 0.13). In addition, community composition of active fungi shifted under glycine, sucrose, and cellulose additions. Specifically, the community under glycine was significantly different from communities under control, cellulose, and tannin-protein (P<0.05). Additionally, the sucrose and cellulose communities were marginally different from the control community (P = 0.059 and 0.054, respectively) and each other (P = 0.058). Together these results support our hypothesis that fungal communities change in response to different carbon sources. We found 11 fungal operational taxonomic units (OTUs) whose relative abundances differed at least marginally significantly among substrates. One OTU related to Mortierella increased in abundance under cellulose, but was absent or rare under the other substrates. Another OTU related to an unidentified Basidiomycete was only present under lignin addition, while yet another OTU closely related to Mortierella macrocystis greatly increased in abundance under tannin-protein and slightly increased in response to lignin and sucrose. This confirms our hypothesis that particular taxa respond differently to specific carbon substrates and suggests that some fungal taxa may specialize in the break-down of particular carbon sources in soils. Overall, our results imply that microbes have varying roles in the mineralization of soil carbon, and thus microbial community composition may be an important control over ecosystem carbon dynamics and storage, especially in relation to global change.

Diversity of function in the isocitrate lyase enzyme superfamily: the Dianthus caryophyllus petal death protein cleaves alpha-keto and alpha-hydroxycarboxylic acids.

PubMed

Lu, Zhibing; Feng, Xiaohua; Song, Ling; Han, Ying; Kim, Alexander; Herzberg, Osnat; Woodson, William R; Martin, Brian M; Mariano, Patrick S; Dunaway-Mariano, Debra

2005-12-20

The work described in this paper was carried out to define the chemical function a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. This protein (Swiss-Prot entry Q05957) is synthesized in the senescent flower petals and is named the "petal death protein" or "PDP". On the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily, a substrate screen that employed a (2R)-malate core structure was designed. Accordingly, stereochemically defined C(2)- and C(3)-substituted malates were synthesized and tested as substrates for PDP-catalyzed cleavage of the C(2)-C(3) bond. The screen identified (2R)-ethyl, (3S)-methylmalate, and oxaloacetate [likely to bind as the hydrate, C(2)(OH)(2) gem-diol] as the most active substrates (for each, k(cat)/K(m) = 2 x 10(4) M(-)(1) s(-)(1)). In contrast to the stringent substrate specificities previously observed for the Escherichia coli isocitrate and 2-methylisocitrate lyases, the PDP tolerated hydrogen, methyl, and to a much lesser extent acetate substituents at the C(3) position (S configuration only) and hydoxyl, methyl, ethyl, propyl, and to a much lesser extent isobutyl substituents at C(2) (R configuration only). It is hypothesized that PDP functions in oxalate production in Ca(2+) sequestering and/or in carbon scavenging from alpha-hydroxycarboxylate catabolites during the biochemical transition accompanying petal senescence.

Functionally relevant diversity of closely related Nitrospira in activated sludge.

PubMed

Gruber-Dorninger, Christiane; Pester, Michael; Kitzinger, Katharina; Savio, Domenico F; Loy, Alexander; Rattei, Thomas; Wagner, Michael; Daims, Holger

2015-03-01

Nitrospira are chemolithoautotrophic nitrite-oxidizing bacteria that catalyze the second step of nitrification in most oxic habitats and are important for excess nitrogen removal from sewage in wastewater treatment plants (WWTPs). To date, little is known about their diversity and ecological niche partitioning within complex communities. In this study, the fine-scale community structure and function of Nitrospira was analyzed in two full-scale WWTPs as model ecosystems. In Nitrospira-specific 16S rRNA clone libraries retrieved from each plant, closely related phylogenetic clusters (16S rRNA identities between clusters ranged from 95.8% to 99.6%) within Nitrospira lineages I and II were found. Newly designed probes for fluorescence in situ hybridization (FISH) allowed the specific detection of several of these clusters, whose coexistence in the WWTPs was shown for prolonged periods of several years. In situ ecophysiological analyses based on FISH, relative abundance and spatial arrangement quantification, as well as microautoradiography revealed functional differences of these Nitrospira clusters regarding the preferred nitrite concentration, the utilization of formate as substrate and the spatial coaggregation with ammonia-oxidizing bacteria as symbiotic partners. Amplicon pyrosequencing of the nxrB gene, which encodes subunit beta of nitrite oxidoreductase of Nitrospira, revealed in one of the WWTPs as many as 121 species-level nxrB operational taxonomic units with highly uneven relative abundances in the amplicon library. These results show a previously unrecognized high diversity of Nitrospira in engineered systems, which is at least partially linked to niche differentiation and may have important implications for process stability.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

The abundance and diversity of heterotrophic bacteria as a function of harvesting frequency of duckweed (Lemna minor L.) in recirculating aquaculture systems.

PubMed

Ardiansyah, A; Fotedar, R

2016-07-01

Duckweed (Lemna minor L.) is a potential biofilter for nutrient removal and acts as a substrate for heterotrophic bacteria in recirculating aquaculture systems (RAS). Here, we determined the effects of harvesting frequency of duckweed on heterotrophic bacteria in RAS. Twelve independent RAS consisting of fish-rearing tank, biofilter tank and waste-collection tank were used to study the interactions between duckweed harvest frequencies up to 6 days and the composition, abundance and diversity of heterotrophic bacteria. After 36 days, heterotrophic bacteria in the biofilter tank were primarily Gram-negative cocci or ovoid, coccobacilli, Gram-negative bacilli and Gram-positive bacilli. Most bacterial genera were Bacillus and Pseudomonas while the least common was Acinetobacter. Duckweed harvested after every 2 days produced the highest specific growth rates (SGR) and total harvested biomass of duckweed, but harboured less abundant bacteria, whereas 6-day harvests had a higher growth index (GI) of duckweed than 2-day harvests, but caused a poor relationship between SGR and biomass harvest with the abundance and diversity of heterotrophic bacteria. Stronger correlations (R(2)  > 0·65) between duckweed SGR and biomass harvest with the heterotrophic bacteria diversity were observed at 4-day harvest frequency and the control. This study provides significant information on the interaction between the harvest frequency of duckweed and the composition, abundance and diversity of heterotrophic bacteria in recirculating aquaculture systems (RAS). Different harvest frequencies significantly influence the abundance and diversity of heterotrophic bacteria, which in turn may influence the nitrogen uptake efficiency of the system. The research is useful in improving the efficiency of removing nitrogenous metabolites in RAS directly by the duckweed and associated heterotrophic bacteria. © 2016 The Society for Applied Microbiology.

Degeneracy-Driven Self-Structuring Dynamics in Selective Repertoires

PubMed Central

Atamas, Sergei P.; Bell, Jonathan

2013-01-01

Numerous biological interactions, such as interactions between T cell receptors or antibodies with antigens, interactions between enzymes and substrates, or interactions between predators and prey are often not strictly specific. In such less specific, or “sloppy,” systems, referred to here as degenerate systems, a given unit of a diverse resource (antigens, enzymatic substrates, prey) is at risk of being recognized and consumed by multiple consumers (lymphocytes, enzymes, predators). In this study, we model generalized degenerate consumer-resource systems of Lotka–Volterra and Verhulst types. In the degenerate systems of Lotka–Volterra, there is a continuum of types of consumer and resource based on variation of a single trait (characteristic, or preference). The consumers experience competition for a continuum of resource types. This non-local interaction system is modeled with partial differential-integral equations and shows spontaneous self-structuring of the consumer population that depends on the degree of interaction degeneracy between resource and consumer, but does not mirror the distribution of resource. We also show that the classical Verhulst (i.e. logistic) single population model can be generalized to a degenerate model, which shows qualitative behavior similar to that in the degenerate Lotka–Volterra model. These results provide better insight into the dynamics of selective systems in biology, suggesting that adaptation of degenerate repertoires is not a simple “mirroring” of the environment by the “fittest” elements of population. PMID:19337776

Degeneracy-driven self-structuring dynamics in selective repertoires.

PubMed

Atamas, Sergei P; Bell, Jonathan

2009-08-01

Numerous biological interactions, such as interactions between T cell receptors or antibodies with antigens, interactions between enzymes and substrates, or interactions between predators and prey are often not strictly specific. In such less specific, or "sloppy," systems, referred to here as degenerate systems, a given unit of a diverse resource (antigens, enzymatic substrates, prey) is at risk of being recognized and consumed by multiple consumers (lymphocytes, enzymes, predators). In this study, we model generalized degenerate consumer-resource systems of Lotka-Volterra and Verhulst types. In the degenerate systems of Lotka-Volterra, there is a continuum of types of consumer and resource based on variation of a single trait (characteristic, or preference). The consumers experience competition for a continuum of resource types. This non-local interaction system is modeled with partial differential-integral equations and shows spontaneous self-structuring of the consumer population that depends on the degree of interaction degeneracy between resource and consumer, but does not mirror the distribution of resource. We also show that the classical Verhulst (i.e. logistic) single population model can be generalized to a degenerate model, which shows qualitative behavior similar to that in the degenerate Lotka-Volterra model. These results provide better insight into the dynamics of selective systems in biology, suggesting that adaptation of degenerate repertoires is not a simple "mirroring" of the environment by the "fittest" elements of population.

Fabrication of nanowire electronics on nonconventional substrates by water-assisted transfer printing method

NASA Astrophysics Data System (ADS)

Lee, Chi Hwan; Kim, Dong Rip; Zheng, Xiaolin

2015-06-01

We report a simple, versatile, and wafer-scale water-assisted transfer printing method (WTP) that enables the transfer of nanowire devices onto diverse nonconventional substrates that were not easily accessible before, such as paper, plastics, tapes, glass, polydimethylsiloxane (PDMS), aluminum foil, and ultrathin polymer substrates. The WTP method relies on the phenomenon of water penetrating into the interface between Ni and SiO2. The transfer yield is nearly 100%, and the transferred devices, including NW resistors, diodes, and field effect transistors, maintain their original geometries and electronic properties with high fidelity.

Matrix and Backstage: Cellular Substrates for Viral Vaccines

PubMed Central

Jordan, Ingo; Sandig, Volker

2014-01-01

Vaccines are complex products that are manufactured in highly dynamic processes. Cellular substrates are one critical component that can have an enormous impact on reactogenicity of the final preparation, level of attenuation of a live virus, yield of infectious units or antigens, and cost per vaccine dose. Such parameters contribute to feasibility and affordability of vaccine programs both in industrialized countries and developing regions. This review summarizes the diversity of cellular substrates for propagation of viral vaccines from primary tissue explants and embryonated chicken eggs to designed continuous cell lines of human and avian origin. PMID:24732259

Burial and exhumation of temperate bedrock reefs as elucidated by repetitive high-resolution sea floor sonar surveys: Spatial patterns and impacts to species' richness and diversity

USGS Publications Warehouse

Storlazzi, Curt D.; Fregoso, Theresa A.; Figurski, Jared D.; Freiwald, Jan; Lonhart, Steve I.; Finlayson, David P.

2013-01-01

To understand how chronic sediment burial and scour contribute to variation in the structure of algal and invertebrate communities on temperate bedrock reefs, the dynamics of the substrate and communities were monitored at locations that experience sand inundation and adjacent areas that do not. Co-located benthic scuba-transect surveys and high-resolution swath-sonar surveys were completed on bedrock reefs on the inner shelf of northern Monterey Bay, CA, in early winter 2009, spring 2010, and summer 2010. Analysis of the sonar surveys demonstrates that during the 8 months over which the surveys were conducted, 19.6% of the study area was buried by sand while erosion resulted in the exposure of bedrock over 13.8% of the study area; the remainder underwent no change between the surveys. Substrate classifications from the benthic transect surveys correlated with classifications generated from the sonar surveys, demonstrating the capacity of high-resolution sonar surveys to detect burial of bedrock reefs by sediment. On bedrock habitat that underwent burial and exhumation, species' diversity and richness of rock-associated sessile and mobile organisms were 50–66% lower as compared to adjacent stable bedrock habitat. While intermediate levels of disturbance can increase the diversity and richness of communities, these findings demonstrate that burial and exhumation of bedrock habitat are sources of severe disturbance. We suggest that substrate dynamics must be considered when developing predictions of benthic community distributions based on sea floor imagery. These results highlight the need for predictive models of substrate dynamics and for a better understanding of how burial and exhumation shape benthic communities.

Microbial Community Responses to Glycine Addition in Kansas Prairie Soils

NASA Astrophysics Data System (ADS)

Bottos, E.; Roy Chowdhury, T.; White, R. A., III; Brislawn, C.; Fansler, S.; Kim, Y. M.; Metz, T. O.; McCue, L. A.; Jansson, J.

2015-12-01

Advances in sequencing technologies are rapidly expanding our abilities to unravel aspects of microbial community structure and function in complex systems like soil; however, characterizing the highly diverse communities is problematic, due primarily to challenges in data analysis. To tackle this problem, we aimed to constrain the microbial diversity in a soil by enriching for particular functional groups within a community through addition of "trigger substrates". Such trigger substrates, characterized by low molecular weight, readily soluble and diffusible in soil solution, representative of soil organic matter derivatives, would also be rapidly degradable. A relatively small energy investment to maintain the cell in a state of metabolic alertness for such substrates would be a better evolutionary strategy and presumably select for a cohort of microorganisms with the energetics and cellular machinery for utilization and growth. We chose glycine, a free amino acid (AA) known to have short turnover times (in the range of hours) in soil. As such, AAs are a good source of nitrogen and easily degradable, and can serve as building blocks for microbial proteins and other biomass components. We hypothesized that the addition of glycine as a trigger substrate will decrease microbial diversity and evenness, as taxa capable of metabolizing it are enriched in relation to those that are not. We tested this hypothesis by incubating three Kansas native prairie soils with glycine for 24 hours at 21 degree Celsius, and measured community level responses by 16S rRNA gene sequencing, metagenomics, and metatranscriptomics. Preliminary evaluation of 16S rRNA gene sequences revealed minor changes in bacterial community composition in response to glycine addition. We will also present data on functional gene abundance and expression. The results of these analyses will be useful in designing sequencing strategies aimed at dissecting and deciphering complex microbial communities.

Effects of Management on Lichen Species Richness, Ecological Traits and Community Structure in the Rodnei Mountains National Park (Romania).

PubMed

Ardelean, Ioana Violeta; Keller, Christine; Scheidegger, Christoph

2015-01-01

Lichens are valuable bio-indicators for evaluating the consequences of human activities that are increasingly changing the earth's ecosystems. Since a major objective of national parks is the preservation of biodiversity, our aim is to analyse how natural resource management, the availability of lichen substrates and environmental parameters influence lichen diversity in Rodnei Mountains National Park situated in the Eastern Carpathians. Three main types of managed vegetation were investigated: the transhumance systems in alpine meadows, timber exploitation in mixed and pure spruce forests, and the corresponding conserved sites. The data were sampled following a replicated design. For the analysis, we considered not only all lichen species, but also species groups from different substrates such as soil, trees and deadwood. The lichen diversity was described according to species richness, red-list status and substrate-specialist species richness. The variation in species composition was related to the environmental variables. Habitat management was found to negatively influence species richness and alter the lichen community composition, particularly for threatened and substrate-specialist species. It reduced the mean level of threatened species richness by 59%, when all lichen species were considered, and by 81%, when only epiphytic lichens were considered. Management-induced disturbance significantly decreased lichen species richness in forest landscapes with long stand continuity. The diversity patterns of the lichens indicate a loss of species richness and change in species composition in areas where natural resources are still exploited inside the borders of the national park. It is thus imperative for protected areas, in particular old-growth forests and alpine meadows, to receive more protection than they have received in the past to ensure populations of the characteristic species remain viable in the future.

Secretomic survey of Trichoderma harzianum grown on plant biomass substrates.

PubMed

Gómez-Mendoza, Diana Paola; Junqueira, Magno; do Vale, Luis Henrique Ferreira; Domont, Gilberto Barbosa; Ferreira Filho, Edivaldo Ximenes; Sousa, Marcelo Valle de; Ricart, Carlos André Ornelas

2014-04-04

The present work aims at characterizing T. harzianum secretome when the fungus is grown in synthetic medium supplemented with one of the four substrates: glucose, cellulose, xylan, and sugarcane bagasse (SB). The characterization was done by enzymatic assays and proteomic analysis using 2-DE/MALDI-TOF and gel-free shotgun LC-MS/MS. The results showed that SB induced the highest cellulolytic and xylanolytic activities when compared with the other substrates, while remarkable differences in terms of number and distribution of protein spots in 2-DE gels were also observed among the samples. Additionally, treatment of the secretomes with PNGase F revealed that most spot trails in 2-DE gels corresponded to N-glycosylated proteoforms. The LC-MS/MS analysis of the samples identified 626 different protein groups, including carbohydrate-active enzymes and accessory, noncatalytic, and cell-wall-associated proteins. Although the SB-induced secretome displayed the highest cellulolytic and xylanolytic activities, it did not correspond to a higher proteome complexity because CM-cellulose-induced secretome was significantly more diverse. Among the identified proteins, 73% were exclusive to one condition, while only 5% were present in all samples. Therefore, this study disclosed the variation of T. harzianum secretome in response to different substrates and revealed the diversity of the fungus enzymatic toolbox.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Principles of motivation revealed by the diverse functions of neuropharmacological and neuroanatomical substrates underlying feeding behavior

PubMed Central

Baldo, Brian A.; Pratt, Wayne E.; Will, Matthew J.; Hanlon, Erin C.; Bakshi, Vaishali P.; Cador, Martine

2013-01-01

Circuits that participate in specific subcomponents of feeding (e.g., gustatory perception, peripheral feedback relevant to satiety and energy balance, reward coding, etc.) are found at all levels of the neural axis. Further complexity is conferred by the wide variety of feeding-modulatory neurotransmitters and neuropeptides that act within these circuits. An ongoing challenge has been to refine the understanding of the functional specificity of these neurotransmitters and circuits, and there have been exciting advances in recent years. We focus here on foundational work of Dr. Ann Kelley that identified distinguishable actions of striatal opioid peptide modulation and dopamine transmission in subcomponents of reward processing. We also discuss her work in overlaying these neuropharmacological effects upon anatomical pathways that link the telencephalon (cortex and basal ganglia) with feeding-control circuits in the hypothalamus. Using these seminal contributions as a starting point, we will discuss new findings that expand our understanding of (1) the specific, differentiable motivational processes that are governed by central dopamine and opioid transmission, (2) the manner in which other striatal neuromodulators, specifically acetylcholine, endocannabinoids and adenosine, modulate these motivational processes (including via interactions with opioid systems), and (3) the organization of the cortical-subcortical network that subserves opioid-driven feeding. The findings discussed here strengthen the view that incentive-motivational properties of food are coded by substrates and neural circuits that are distinguishable from those that mediate the acute hedonic experience of food reward. Striatal opioid transmission modulates reward processing by engaging frontotemporal circuits, possibly via a hypothalamic-thalamic axis, that ultimately impinges upon hypothalamic modules dedicated to autonomic function and motor pattern control. We will conclude by discussing implications for understanding disorders of “non-homeostatic” feeding. PMID:23466532

Principles of motivation revealed by the diverse functions of neuropharmacological and neuroanatomical substrates underlying feeding behavior.

PubMed

Baldo, Brian A; Pratt, Wayne E; Will, Matthew J; Hanlon, Erin C; Bakshi, Vaishali P; Cador, Martine

2013-11-01

Circuits that participate in specific subcomponents of feeding (e.g., gustatory perception, peripheral feedback relevant to satiety and energy balance, reward coding, etc.) are found at all levels of the neural axis. Further complexity is conferred by the wide variety of feeding-modulatory neurotransmitters and neuropeptides that act within these circuits. An ongoing challenge has been to refine the understanding of the functional specificity of these neurotransmitters and circuits, and there have been exciting advances in recent years. We focus here on foundational work of Dr. Ann Kelley that identified distinguishable actions of striatal opioid peptide modulation and dopamine transmission in subcomponents of reward processing. We also discuss her work in overlaying these neuropharmacological effects upon anatomical pathways that link the telencephalon (cortex and basal ganglia) with feeding-control circuits in the hypothalamus. Using these seminal contributions as a starting point, we will discuss new findings that expand our understanding of (1) the specific, differentiable motivational processes that are governed by central dopamine and opioid transmission, (2) the manner in which other striatal neuromodulators, specifically acetylcholine, endocannabinoids and adenosine, modulate these motivational processes (including via interactions with opioid systems), and (3) the organization of the cortical-subcortical network that subserves opioid-driven feeding. The findings discussed here strengthen the view that incentive-motivational properties of food are coded by substrates and neural circuits that are distinguishable from those that mediate the acute hedonic experience of food reward. Striatal opioid transmission modulates reward processing by engaging frontotemporal circuits, possibly via a hypothalamic-thalamic axis, that ultimately impinges upon hypothalamic modules dedicated to autonomic function and motor pattern control. We will conclude by discussing implications for understanding disorders of "non-homeostatic" feeding. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure of a bacterial toxin-activating acyltransferase.

PubMed

Greene, Nicholas P; Crow, Allister; Hughes, Colin; Koronakis, Vassilis

2015-06-09

Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host-cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove.

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE PAGES

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

2016-03-31

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

Degeneracy allows for both apparent homogeneity and diversification in populations.

PubMed

Whitacre, James M; Atamas, Sergei P

2012-10-01

Trait diversity - the substrate for natural selection - is necessary for adaptation through selection, particularly in populations faced with environmental changes that diminish population fitness. In habitats that remain unchanged for many generations, stabilizing selection maximizes exploitation of resources by reducing trait diversity to a narrow optimal range. One might expect that such ostensibly homogeneous populations would have a reduced potential for heritable adaptive responses when faced with fitness-reducing environmental changes. However, field studies have documented populations that, even after long periods of evolutionary stasis, can still rapidly evolve in response to changed environmental conditions. We argue that degeneracy, the ability of diverse population elements to function similarly, can satisfy both the current need to maximize fitness and the future need for diversity. Degenerate ensembles appear functionally redundant in certain environmental contexts and functionally diverse in others. We propose that genetic variation not contributing to the observed range of phenotypes in a current population, also known as cryptic genetic variation (CGV), is a specific case of degeneracy. We argue that CGV, which gradually accumulates in static populations in stable environments, reveals hidden trait differences when environments change. By allowing CGV accumulation, static populations prepare themselves for future rapid adaptations to environmental novelty. A greater appreciation of degeneracy's role in resolving the inherent tension between current stabilizing selection and future directional selection has implications in conservation biology and may be applied in social and technological systems to maximize current performance while strengthening the potential for future changes. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Nitric oxide-based protein modification: formation and site-specificity of protein S-nitrosylation

PubMed Central

Kovacs, Izabella; Lindermayr, Christian

2013-01-01

Nitric oxide (NO) is a reactive free radical with pleiotropic functions that participates in diverse biological processes in plants, such as germination, root development, stomatal closing, abiotic stress, and defense responses. It acts mainly through redox-based modification of cysteine residue(s) of target proteins, called protein S-nitrosylation.In this way NO regulates numerous cellular functions and signaling events in plants. Identification of S-nitrosylated substrates and their exact target cysteine residue(s) is very important to reveal the molecular mechanisms and regulatory roles of S-nitrosylation. In addition to the necessity of protein–protein interaction for trans-nitrosylation and denitrosylation reactions, the cellular redox environment and cysteine thiol micro-environment have been proposed important factors for the specificity of protein S-nitrosylation. Several methods have recently been developed for the proteomic identification of target proteins. However, the specificity of NO-based cysteine modification is still less defined. In this review, we discuss formation and specificity of S-nitrosylation. Special focus will be on potential S-nitrosylation motifs, site-specific proteomic analyses, computational predictions using different algorithms, and on structural analysis of cysteine S-nitrosylation. PMID:23717319

Substrate Diffusion Heterogeneity Controls Bacterial Competition and Coexistence

NASA Astrophysics Data System (ADS)

Dechesne, A.; Or, D.; Smets, B. F.

2005-12-01

Diffusion has long been recognized as a key process affecting bacterial physiological functions ranging from nutrient uptake to removal of metabolic waste products. In the vadose zone, significant convective flows are limited and bacteria rely primarily on diffusion for nutrient supply. Even under relatively "wet" conditions (e.g. matric potentials -20 J/kg), soil water is fragmented and exists as thin liquid films or held in crevices imposing constraints on substrate diffusion. Our objective was to investigate the role of diffusion on soil microbial diversity, by focusing on one of the processes that shapes the structure of bacterial communities: competitive interactions. We used a simplified setup, in which the substrate (citrate) fluxes were controlled by different agar gels thicknesses and spatially heterogeneous diffusive pathways were created by an impermeable film with prescribed hole sizes and patterns. Our competition experiments involved two soil bacteria: Burkholderia xenovorans LB400 and Pseudomonas putida KT2440, which were tagged with different constitutive fluorescent markers, allowing for their on line microscopic detection. The growth parameters on citrate of these strains were thoroughly assessed. B. xenovorans LB400 is the weaker competitor. As a result, this strain was outcompeted by KT2440 under high substrate diffusivity and homogeneous conditions. Conversely, the disadvantage of the weakest competitor was not so marked under low substrate diffusivity condition. These results suggest that dry conditions in soil would provide conditions allowing the sustaining of weak bacterial competitors, resulting in the maintenance of high bacterial diversity.

Diversity and wiring variability of visual local neurons in the Drosophila medulla M6 stratum.

PubMed

Chin, An-Lun; Lin, Chih-Yung; Fu, Tsai-Feng; Dickson, Barry J; Chiang, Ann-Shyn

2014-12-01

Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information and in shaping bipolar-to-ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons that project to the medulla M6 stratum, with relatively little known of this downstream substrate. Here, using R7 terminals as references, we generated a 3D volume model of the M6 stratum, which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, we compiled and analyzed the spatial distributions of more than 200 single M6-specific local neurons (M6-LNs). Based on the segregation of putative dendrites and axons, these local neurons were classified into two families, directional and nondirectional. Neurotransmitter immunostaining suggested a signal routing model in which some visual information is relayed by directional M6-LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of nondirectional M6-LNs covering the entire M6 stratum. Our findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer. © 2014 Wiley Periodicals, Inc.

Plant P450s as versatile drivers for evolution of species-specific chemical diversity

PubMed Central

Hamberger, Björn; Bak, Søren

2013-01-01

The irreversible nature of reactions catalysed by P450s makes these enzymes landmarks in the evolution of plant metabolic pathways. Founding members of P450 families are often associated with general (i.e. primary) metabolic pathways, restricted to single copy or very few representatives, indicative of purifying selection. Recruitment of those and subsequent blooms into multi-member gene families generates genetic raw material for functional diversification, which is an inherent characteristic of specialized (i.e. secondary) metabolism. However, a growing number of highly specialized P450s from not only the CYP71 clan indicate substantial contribution of convergent and divergent evolution to the observed general and specialized metabolite diversity. We will discuss examples of how the genetic and functional diversification of plant P450s drives chemical diversity in light of plant evolution. Even though it is difficult to predict the function or substrate of a P450 based on sequence similarity, grouping with a family or subfamily in phylogenetic trees can indicate association with metabolism of particular classes of compounds. Examples will be given that focus on multi-member gene families of P450s involved in the metabolic routes of four classes of specialized metabolites: cyanogenic glucosides, glucosinolates, mono- to triterpenoids and phenylpropanoids. PMID:23297350

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

PubMed Central

Lee, Rachel S.; House, Colin M.; Cristiano, Briony E.; Hannan, Ross D.; Pearson, Richard B.; Hannan, Katherine M.

2011-01-01

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ. PMID:21869924

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

PubMed Central

Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

2017-01-01

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

[Effect of mechanical grinding of Sphagnum on the structure and physiological state of bacterial communities].

PubMed

Dobrovol'skaya, T G; Golovchenko, A V; Yakushev, A V; Manucharova, N A; Yurchenko, E N

2014-01-01

The microcosm method was used to demonstrate an increase in bacterial numbers and drastic changes in the taxonomic structure of saprotrophic bacteria as a result of mechanical grinding of Sphagnum moss. Ekkrisotrophic agrobacteria predominant in untreated moss were replaced by hydrolytic bacteria. Molecular biological approaches revealed such specific hydrolytic bacteria as Janthinobacterium agaricum and Streptomyces purpurascens among the dominant taxa. The application of kinetic technique for determination of the physiological state of bacteria in situ revealed higher functional diversity of hydrolytic bacteria in ground moss than in untreated samples. A considerable decrease of the C/N ratio in ground samples of living Sphagnum incubated using the microcosm technique indicated decomposition of this substrate.

Insight into the stereospecificity of short-chain thermus thermophilus alcohol dehydrogenase showing pro-S hydride transfer and prelog enantioselectivity.

PubMed

Pennacchio, Angela; Giordano, Assunta; Esposito, Luciana; Langella, Emma; Rossi, Mosè; Raia, Carlo A

2010-04-01

The stereochemistry of the hydride transfer in reactions catalyzed by NAD(H)-dependent alcohol dehydrogenase from Thermus thermophilus HB27 was determined by means of (1)H-NMR spectroscopy. The enzyme transfers the pro-S hydrogen of [4R-(2)H]NADH and exhibits Prelog specificity. Enzyme-substrate docking calculations provided structural details about the enantioselectivity of this thermophilic enzyme. These results give additional insights into the diverse active site architectures of the largely versatile short-chain dehydrogenase superfamily enzymes. A feasible protocol for the synthesis of [4R-(2)H]NADH with high yield was also set up by enzymatic oxidation of 2-propanol-d(8) catalyzed by Bacillus stearothermophilus alcohol dehydrogenase.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Activity and stability of a complex bacterial soil community under simulated Martian conditions

NASA Astrophysics Data System (ADS)

Hansen, Aviaja Anna; Merrison, Jonathan; Nørnberg, Per; Aagaard Lomstein, Bente; Finster, Kai

2005-04-01

A simulation experiment with a complex bacterial soil community in a Mars simulation chamber was performed to determine the effect of Martian conditions on community activity, stability and survival. At three different depths in the soil core short-term effects of Martian conditions with and without ultraviolet (UV) exposure corresponding to 8 Martian Sol were compared. Community metabolic activities and functional diversity, measured as glucose respiration and versatility in substrate utilization, respectively, decreased after UV exposure, whereas they remained unaffected by Martian conditions without UV exposure. In contrast, the numbers of culturable bacteria and the genetic diversity were unaffected by the simulated Martian conditions both with and without UV exposure. The genetic diversity of the soil community and of the colonies grown on agar plates were evaluated by denaturant gradient gel electrophoresis (DGGE) on DNA extracts. Desiccation of the soil prior to experimentation affected the functional diversity by decreasing the versatility in substrate utilization. The natural dominance of endospores and Gram-positive bacteria in the investigated Mars-analogue soil may explain the limited effect of the Mars incubations on the survival and community structure. Our results suggest that UV radiation and desiccation are major selecting factors on bacterial functional diversity in terrestrial bacterial communities incubated under simulated Martian conditions. Furthermore, these results suggest that forward contamination of Mars is a matter of great concern in future space missions.

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants

PubMed Central

Balachandar, D.; Raja, P.; Sundaram, SP.

2008-01-01

Diversity of Pink-Pigmented Facultative Methylotrophs (PPFMs) in phyllosphere of cotton, maize and sunflower was determined based on differential carbon-substrate utilization profile and Random Amplified Polymorphic DNA data. Results indicate that six diversified groups of PPFMs are found in these crops. Sunflower and maize phyllosphere harbor four different groups of methylobacteria while cotton has only two groups. PMID:24031182

Genetic and metabolic diversity of pink-pigmented facultative methylotrophs in phyllosphere of tropical plants.

PubMed

Balachandar, D; Raja, P; Sundaram, Sp

2008-01-01

Diversity of Pink-Pigmented Facultative Methylotrophs (PPFMs) in phyllosphere of cotton, maize and sunflower was determined based on differential carbon-substrate utilization profile and Random Amplified Polymorphic DNA data. Results indicate that six diversified groups of PPFMs are found in these crops. Sunflower and maize phyllosphere harbor four different groups of methylobacteria while cotton has only two groups.

Effect of pig manure on the chemical composition and microbial diversity during co-composting with spent mushroom substrate and rice husks.

PubMed

Meng, Xingyao; Liu, Bin; Xi, Chen; Luo, Xiaosha; Yuan, Xufeng; Wang, Xiaofen; Zhu, Wanbin; Wang, Hongliang; Cui, Zongjun

2018-03-01

In this study, the impact of pig manure on the maturity of compost consisting of spent mushroom substrate and rice husks was accessed. The results showed that the addition of pig manure (SMS-PM) reached 50°C 5days earlier and lasted 15days longer than without pig manure (SMS). Furthermore, the addition of pig manure improved nutrition and germination index. High-throughput 16S rRNA pyrosequencing was used to evaluate the bacterial and fungal composition during the composting process of SMS-PM compared to SMS alone. The SMS treatment showed a relatively higher abundance of carbon-degrading microbes (Bacillaceae and Thermomyces) and plant pathogenic fungi (Sordariomycetes_unclassified) at the end of the compost. In contrast, the SMS-PM showed an increased bacterial diversity with anti-pathogen (Pseudomonas). The results indicated that the addition of pig manure improved the decomposition of refractory carbon from the spent mushroom substrate and promoted the maturity and nutritional content of the compost product. Copyright © 2017. Published by Elsevier Ltd.

Operational Plasticity Enables Hsp104 to Disaggregate Diverse Amyloid and Non-Amyloid Clients

PubMed Central

DeSantis, Morgan E.; Leung, Eunice H.; Sweeny, Elizabeth A.; Jackrel, Meredith E.; Cushman-Nick, Mimi; Neuhaus-Follini, Alexandra; Vashist, Shilpa; Sochor, Matthew A.; Knight, M. Noelle; Shorter, James

2012-01-01

Summary It is not understood how Hsp104, a hexameric AAA+ ATPase from yeast, disaggregates diverse structures including stress-induced aggregates, prions, and α-synuclein conformers connected to Parkinson disease. Here, we establish that Hsp104 hexamers adapt different mechanisms of intersubunit collaboration to disaggregate stress-induced aggregates versus amyloid. To resolve disordered aggregates, Hsp104 subunits collaborate non-co-operatively via probabilistic substrate binding and ATP hydrolysis. To disaggregate amyloid, several subunits co-operatively engage substrate and hydrolyze ATP. Importantly, Hsp104 variants with impaired intersubunit communication dissolve disordered aggregates but not amyloid. Unexpectedly, prokaryotic ClpB subunits collaborate differently than Hsp104 and couple probabilistic substrate binding to cooperative ATP hydrolysis, which enhances disordered aggregate dissolution but sensitizes ClpB to inhibition and diminishes amyloid disaggregation. Finally, we establish that Hsp104 hexamers deploy more subunits to disaggregate Sup35 prion strains with more stable ‘cross-β’ cores. Thus, operational plasticity enables Hsp104 to robustly dissolve amyloid and non-amyloid clients, which impose distinct mechanical demands. PMID:23141537

Combinatorial synthesis of inorganic or composite materials

DOEpatents

Goldwasser, Isy; Ross, Debra A.; Schultz, Peter G.; Xiang, Xiao-Dong; Briceno, Gabriel; Sun, Xian-Dong; Wang, Kai-An

2010-08-03

Methods and apparatus for the preparation and use of a substrate having an array of diverse materials in predefined regions thereon. A substrate having an array of diverse materials thereon is generally prepared by delivering components of materials to predefined regions on a substrate, and simultaneously reacting the components to form at least two materials or, alternatively, allowing the components to interact to form at least two different materials. Materials which can be prepared using the methods and apparatus of the present invention include, for example, covalent network solids, ionic solids and molecular solids. More particularly, materials which can be prepared using the methods and apparatus of the present invention include, for example, inorganic materials, intermetallic materials, metal alloys, ceramic materials, organic materials, organometallic materials, nonbiological organic polymers, composite materials (e.g., inorganic composites, organic composites, or combinations thereof), etc. Once prepared, these materials can be screened for useful properties including, for example, electrical, thermal, mechanical, morphological, optical, magnetic, chemical, or other properties. Thus, the present invention provides methods for the parallel synthesis and analysis of novel materials having useful properties.

Influence of red mud on soil microbial communities: Application and comprehensive evaluation of the Biolog EcoPlate approach as a tool in soil microbiological studies.

PubMed

Feigl, Viktória; Ujaczki, Éva; Vaszita, Emese; Molnár, Mónika

2017-10-01

Red mud can be applied as soil ameliorant to acidic, sandy and micronutrient deficient soils. There are still knowledge gaps regarding the effects of red mud on the soil microbial community. The Biolog EcoPlate technique is a promising tool for community level physiological profiling. This study presents a detailed evaluation of Biolog EcoPlate data from two case studies. In experiment "A" red mud from Ajka (Hungary) was mixed into acidic sandy soil in soil microcosms at 5-50 w/w%. In experiement "B" red mud soil mixture was mixed into low quality subsoil in a field experiment at 5-50 w/w%. According to average well color development, substrate average well color development and substrate richness 5-20% red mud increased the microbial activity of the acidic sandy soil over the short term, but the effect did not last for 10months. Shannon diversity index showed that red mud at up to 20% did not change microbial diversity over the short term, but the diversity decreased by the 10th month. 30-50% red mud had deteriorating effect on the soil microflora. 5-20% red mud soil mixture in the low quality subsoil had a long lasting enhancing effect on the microbial community based on all Biolog EcoPlate parameters. However, 50% red mud soil mixture caused a decrease in diversity and substrate richness. With the Biolog EcoPlate we were able to monitor the changes of the microbial community in red mud affected soils and to assess the amount of red mud and red mud soil mixture applicable for soil treatment in these cases. Copyright © 2017 Elsevier B.V. All rights reserved.

One plant, two plants, three plants, four: does soil carbon respond to diversifying by one plant more? (Invited)

NASA Astrophysics Data System (ADS)

Grandy, S.

2013-12-01

Plant diversity is known to strongly influence aboveground ecosystem functions, but our understanding of its effects on belowground carbon (C) cycling has not kept pace. We know in broad terms that the belowground implications of reducing plant diversity include changes in soil nutrient cycling and biological communities, but remain uncertain about the specific links between plant diversity, soil microbial communities, and soil C cycling. Our knowledge gap is especially wide in agricultural systems, which comprise ~50% of the contiguous U.S. and differ from non-managed systems because diversity: (1) occurs primarily over time (i.e. crop rotations) rather than in space (i.e. inter-cropping); (2) exists as one of multiple management factors that potentially regulates soil C dynamics; and (3) is almost always low, with the addition or subtraction of a single plant species often representing a substantial change in diversity. I have been addressing the uncertain relationships between agricultural plant diversity and soil C cycling with a multi-tiered approach that includes a global meta-analysis, site-specific field manipulations, and intensive laboratory analyses. The meta-analysis using 122 studies shows that compared to single-crop monocultures, rotations increased soil microbial biomass C by 20.7% and microbial biomass N by 26.1% as well as total soil C and N. In a complimentary field study at the W.K. Kellogg Biological Station LTER Cropping Biodiversity Gradient Experiment we examined microbial communities, C cycling processes, and trace gas emissions in five rotation sequences varying in complexity from continuous corn monoculture to a five crop three-year rotation. Finding striking differences between monocultures and systems with more complex plant communities, these results confirm our meta-analysis, and highlight the strong effects of diversifying plant communities in agricultural systems. A complimentary lab study examining decomposition processes in monocultures and more diverse rotations shows that rotation soils process chemically complex C more rapidly. My studies point to complex relationships between the chemistry of substrate inputs and their fate in soils, while also emphasizing an important management consideration: maintaining soil biological functions and ecosystem services in managed agricultural systems requires the rotation of different crops, rather than the production of single crop monocultures.

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Chromatin-Bound Cullin-Ring Ligases: Regulatory Roles in DNA Replication and Potential Targeting for Cancer Therapy

PubMed Central

Jang, Sang-Min; Redon, Christophe E.; Aladjem, Mirit I.

2018-01-01

Cullin-RING (Really Interesting New Gene) E3 ubiquitin ligases (CRLs), the largest family of E3 ubiquitin ligases, are functional multi-subunit complexes including substrate receptors, adaptors, cullin scaffolds, and RING-box proteins. CRLs are responsible for ubiquitination of ~20% of cellular proteins and are involved in diverse biological processes including cell cycle progression, genome stability, and oncogenesis. Not surprisingly, cullins are deregulated in many diseases and instances of cancer. Recent studies have highlighted the importance of CRL-mediated ubiquitination in the regulation of DNA replication/repair, including specific roles in chromatin assembly and disassembly of the replication machinery. The development of novel therapeutics targeting the CRLs that regulate the replication machinery and chromatin in cancer is now an attractive therapeutic strategy. In this review, we summarize the structure and assembly of CRLs and outline their cellular functions and their diverse roles in cancer, emphasizing the regulatory functions of nuclear CRLs in modulating the DNA replication machinery. Finally, we discuss the current strategies for targeting CRLs against cancer in the clinic. PMID:29594129

Why geodiversity matters in valuing nature's stage.

PubMed

Hjort, Jan; Gordon, John E; Gray, Murray; Hunter, Malcolm L

2015-06-01

Geodiversity--the variability of Earth's surface materials, forms, and physical processes-is an integral part of nature and crucial for sustaining ecosystems and their services. It provides the substrates, landform mosaics, and dynamic physical processes for habitat development and maintenance. By determining the heterogeneity of the physical environment in conjunction with climate interactions, geodiversity has a crucial influence on biodiversity across a wide range of scales. From a literature review, we identified the diverse values of geodiversity; examined examples of the dependencies of biodiversity on geodiversity at a site-specific scale (for geosites <1 km(2) in area); and evaluated various human-induced threats to geosites and geodiversity. We found that geosites are important to biodiversity because they often support rare or unique biota adapted to distinctive environmental conditions or create a diversity of microenvironments that enhance species richness. Conservation of geodiversity in the face of a range of threats is critical both for effective management of nature's stage and for its own particular values. This requires approaches to nature conservation that integrate climate, biodiversity, and geodiversity at all spatial scales. © 2015 Society for Conservation Biology.

Effects of inorganic carbon on the nitrous oxide emissions and microbial diversity of an anaerobic ammonia oxidation reactor.

PubMed

Zhang, Wenjie; Wang, Dunqiu; Jin, Yue

2018-02-01

Inorganic carbon (IC) is important for anaerobic ammonium oxidation (anammox). In this study, the effects of the IC concentration on N 2 O emissions and microbial diversity in an anammox reactor were investigated. N 2 O emissions were positively correlated with IC concentrations, and IC concentrations in the range of 55-130 mg/L were optimal, considering the nitrogen removal rate and N 2 O emissions. High IC concentrations resulted in the formation of CaCO 3 on the surface of anammox granules, which impacted the diffusion conditions of the substrate. Microbial community analysis indicated that high IC concentrations decreased the populations of specific bacteria, such as Achromobacter spanius strain YJART-7, Achromobacter xylosoxidans strain IHB B 6801, and Denitratisoma oestradiolicum clone 20b_15. D. oestradiolicum clone 20b_15 appeared to be the key contributor to N 2 O emissions. High N 2 O emissions may result from changes in organic carbon sources, which lead to denitrification by D. oestradiolicum clone 20b_15. Copyright © 2017 Elsevier Ltd. All rights reserved.

Establishing a Role for Bacterial Cellulose in Environmental Interactions: Lessons Learned from Diverse Biofilm-Producing Proteobacteria

PubMed Central

Augimeri, Richard V.; Varley, Andrew J.; Strap, Janice L.

2015-01-01

Bacterial cellulose (BC) serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation, and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC-containing biofilms is to establish close contact with a preferred host to facilitate efficient host–bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology, and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3′→5′)-cyclic diguanylate (c-di-GMP) levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal, or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host–bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, its regulation and its ecophysiological roles. PMID:26635751

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

LacSubPred: predicting subtypes of Laccases, an important lignin metabolism-related enzyme class, using in silico approaches

PubMed Central

2014-01-01

Background Laccases (E.C. 1.10.3.2) are multi-copper oxidases that have gained importance in many industries such as biofuels, pulp production, textile dye bleaching, bioremediation, and food production. Their usefulness stems from the ability to act on a diverse range of phenolic compounds such as o-/p-quinols, aminophenols, polyphenols, polyamines, aryl diamines, and aromatic thiols. Despite acting on a wide range of compounds as a family, individual Laccases often exhibit distinctive and varied substrate ranges. This is likely due to Laccases involvement in many metabolic roles across diverse taxa. Classification systems for multi-copper oxidases have been developed using multiple sequence alignments, however, these systems seem to largely follow species taxonomy rather than substrate ranges, enzyme properties, or specific function. It has been suggested that the roles and substrates of various Laccases are related to their optimal pH. This is consistent with the observation that fungal Laccases usually prefer acidic conditions, whereas plant and bacterial Laccases prefer basic conditions. Based on these observations, we hypothesize that a descriptor-based unsupervised learning system could generate homology independent classification system for better describing the functional properties of Laccases. Results In this study, we first utilized unsupervised learning approach to develop a novel homology independent Laccase classification system. From the descriptors considered, physicochemical properties showed the best performance. Physicochemical properties divided the Laccases into twelve subtypes. Analysis of the clusters using a t-test revealed that the majority of the physicochemical descriptors had statistically significant differences between the classes. Feature selection identified the most important features as negatively charges residues, the peptide isoelectric point, and acidic or amidic residues. Secondly, to allow for classification of new Laccases, a supervised learning system was developed from the clusters. The models showed high performance with an overall accuracy of 99.03%, error of 0.49%, MCC of 0.9367, precision of 94.20%, sensitivity of 94.20%, and specificity of 99.47% in a 5-fold cross-validation test. In an independent test, our models still provide a high accuracy of 97.98%, error rate of 1.02%, MCC of 0.8678, precision of 87.88%, sensitivity of 87.88% and specificity of 98.90%. Conclusion This study provides a useful classification system for better understanding of Laccases from their physicochemical properties perspective. We also developed a publically available web tool for the characterization of Laccase protein sequences (http://lacsubpred.bioinfo.ucr.edu/). Finally, the programs used in the study are made available for researchers interested in applying the system to other enzyme classes (https://github.com/tweirick/SubClPred). PMID:25350584

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

PubMed Central

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

PubMed

Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

2015-04-15

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Transposon-driven transcription is a conserved feature of vertebrate spermatogenesis and transcript evolution.

PubMed

Davis, Matthew P; Carrieri, Claudia; Saini, Harpreet K; van Dongen, Stijn; Leonardi, Tommaso; Bussotti, Giovanni; Monahan, Jack M; Auchynnikava, Tania; Bitetti, Angelo; Rappsilber, Juri; Allshire, Robin C; Shkumatava, Alena; O'Carroll, Dónal; Enright, Anton J

2017-07-01

Spermatogenesis is associated with major and unique changes to chromosomes and chromatin. Here, we sought to understand the impact of these changes on spermatogenic transcriptomes. We show that long terminal repeats (LTRs) of specific mouse endogenous retroviruses (ERVs) drive the expression of many long non-coding transcripts (lncRNA). This process occurs post-mitotically predominantly in spermatocytes and round spermatids. We demonstrate that this transposon-driven lncRNA expression is a conserved feature of vertebrate spermatogenesis. We propose that transposon promoters are a mechanism by which the genome can explore novel transcriptional substrates, increasing evolutionary plasticity and allowing for the genesis of novel coding and non-coding genes. Accordingly, we show that a small fraction of these novel ERV-driven transcripts encode short open reading frames that produce detectable peptides. Finally, we find that distinct ERV elements from the same subfamilies act as differentially activated promoters in a tissue-specific context. In summary, we demonstrate that LTRs can act as tissue-specific promoters and contribute to post-mitotic spermatogenic transcriptome diversity. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adhikary, Suraj; Eichman, Brandt F.

DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity,more » although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.« less

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

Two fatty acyl reductases involved in moth pheromone biosynthesis

PubMed Central

Antony, Binu; Ding, Bao-Jian; Moto, Ken’Ichi; Aldosari, Saleh A.; Aldawood, Abdulrahman S.

2016-01-01

Fatty acyl reductases (FARs) constitute an evolutionarily conserved gene family found in all kingdoms of life. Members of the FAR gene family play diverse roles, including seed oil synthesis, insect pheromone biosynthesis, and mammalian wax biosynthesis. In insects, FAR genes dedicated to sex pheromone biosynthesis (pheromone-gland-specific fatty acyl reductase, pgFAR) form a unique clade that exhibits substantial modifications in gene structure and possesses unique specificity and selectivity for fatty acyl substrates. Highly selective and semi-selective ‘single pgFARs’ produce single and multicomponent pheromone signals in bombycid, pyralid, yponomeutid and noctuid moths. An intriguing question is how a ‘single reductase’ can direct the synthesis of several fatty alcohols of various chain lengths and isomeric forms. Here, we report two active pgFARs in the pheromone gland of Spodoptera, namely a semi-selective, C14:acyl-specific pgFAR and a highly selective, C16:acyl-specific pgFAR, and demonstrate that these pgFARs play a pivotal role in the formation of species-specific signals, a finding that is strongly supported by functional gene expression data. The study envisages a new area of research for disclosing evolutionary changes associated with C14- and C16-specific FARs in moth pheromone biosynthesis. PMID:27427355

Spatial and seasonal variation in diversity and structure of microbial biofilms on marine plastics in Northern European waters.

PubMed

Oberbeckmann, Sonja; Loeder, Martin G J; Gerdts, Gunnar; Osborn, A Mark

2014-11-01

Plastic pollution is now recognised as a major threat to marine environments and marine biota. Recent research highlights that diverse microbial species are found to colonise plastic surfaces (the plastisphere) within marine waters. Here, we investigate how the structure and diversity of marine plastisphere microbial community vary with respect to season, location and plastic substrate type. We performed a 6-week exposure experiment with polyethylene terephthalate (PET) bottles in the North Sea (UK) as well as sea surface sampling of plastic polymers in Northern European waters. Scanning electron microscopy revealed diverse plastisphere communities comprising prokaryotic and eukaryotic microorganisms. Denaturing gradient gel electrophoresis (DGGE) and sequencing analysis revealed that plastisphere microbial communities on PET fragments varied both with season and location and comprised of bacteria belonging to Bacteroidetes, Proteobacteria, Cyanobacteria and members of the eukaryotes Bacillariophyceae and Phaeophyceae. Polymers sampled from the sea surface mainly comprised polyethylene, polystyrene and polypropylene particles. Variation within plastisphere communities on different polymer types was observed, but communities were primarily dominated by Cyanobacteria. This research reveals that the composition of plastisphere microbial communities in marine waters varies with season, geographical location and plastic substrate type. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Nucleophile Promiscuity of Natural and Engineered Aldolases.

PubMed

Clapes, Pere; Hernández, Karel; Szekrenyi, Anna

2018-04-12

Asymmetric aldol addition reaction mediated by aldolases is recognized as a green and sustainable way for carbon-carbon bond formation. Research in this line has unveiled their unprecedented synthetic potentiality toward diverse new chemical structures, novel product families and even as a technology for industrial manufacturing processes. Despite that, aldolases have long been regarded as strictly selective catalysts, particularly for the nucleophilic substrate, limiting their broad applicability. In recent years, the advances in screening technologies and metagenomics uncovered novel C-C biocatalysts from superfamilies of widely known lyases. Moreover, protein engineering revealed the extraordinary malleability of different carboligases, offering a toolbox of biocatalysts active towards a large structural diversity of nucleophile substrates. In this paper, the nucleophile ambiguity of native and engineered aldolases is discussed with recent examples proving this novel concept. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of Benzannulated Medium-ring Lactams via a Tandem Oxidative Dearomatization-Ring Expansion Reaction.

PubMed

Guney, Tezcan; Wenderski, Todd A; Boudreau, Matthew W; Tan, Derek S

2018-06-23

Medium-ring natural products exhibit diverse biological activities but such scaffolds are underrepresented in probe and drug discovery efforts due to the limitations of classical macrocyclization reactions. We report herein a tandem oxidative dearomatization-ring-expanding rearoma¬tization (ODRE) reaction that generates benzannulated medium-ring lactams directly from simple bicyclic substrates. The reaction accommodates diverse aryl substrates (haloarenes, aryl ethers, aryl amides, heterocycles) and strategic incorporation of a bridgehead alcohol generates a versatile ketone moiety in the products amenable to downstream modifications. Cheminformatic analysis indicates that these medium rings access regions of chemical space that overlap with related natural products and are distinct from synthetic drugs, setting the stage for their use in discovery screening against novel biological targets. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The organization and contribution of helicases to RNA splicing.

PubMed

De, Inessa; Schmitzová, Jana; Pena, Vladimir

2016-01-01

Splicing is an essential step of gene expression. It occurs in two consecutive chemical reactions catalyzed by a large protein-RNA complex named the spliceosome. Assembled on the pre-mRNA substrate from five small nuclear proteins, the spliceosome acts as a protein-controlled ribozyme to catalyze the two reactions and finally dissociates into its components, which are re-used for a new round of splicing. Upon following this cyclic pathway, the spliceosome undergoes numerous intermediate stages that differ in composition as well as in their internal RNA-RNA and RNA-protein contacts. The driving forces and control mechanisms of these remodeling processes are provided by specific molecular motors called RNA helicases. While eight spliceosomal helicases are present in all organisms, higher eukaryotes contain five additional ones potentially required to drive a more intricate splicing pathway and link it to an RNA metabolism of increasing complexity. Spliceosomal helicases exhibit a notable structural diversity in their accessory domains and overall architecture, in accordance with the diversity of their task-specific functions. This review summarizes structure-function knowledge about all spliceosomal helicases, including the latter five, which traditionally are treated separately from the conserved ones. The implications of the structural characteristics of helicases for their functions, as well as for their structural communication within the multi-subunits environment of the spliceosome, are pointed out. © 2016 Wiley Periodicals, Inc.

Structure and function of nucleotide sugar transporters: Current progress.

PubMed

Hadley, Barbara; Maggioni, Andrea; Ashikov, Angel; Day, Christopher J; Haselhorst, Thomas; Tiralongo, Joe

2014-06-01

The proteomes of eukaryotes, bacteria and archaea are highly diverse due, in part, to the complex post-translational modification of protein glycosylation. The diversity of glycosylation in eukaryotes is reliant on nucleotide sugar transporters to translocate specific nucleotide sugars that are synthesised in the cytosol and nucleus, into the endoplasmic reticulum and Golgi apparatus where glycosylation reactions occur. Thirty years of research utilising multidisciplinary approaches has contributed to our current understanding of NST function and structure. In this review, the structure and function, with reference to various disease states, of several NSTs including the UDP-galactose, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine, GDP-fucose, UDP-N-acetylglucosamine/UDP-glucose/GDP-mannose and CMP-sialic acid transporters will be described. Little is known regarding the exact structure of NSTs due to difficulties associated with crystallising membrane proteins. To date, no three-dimensional structure of any NST has been elucidated. What is known is based on computer predictions, mutagenesis experiments, epitope-tagging studies, in-vitro assays and phylogenetic analysis. In this regard the best-characterised NST to date is the CMP-sialic acid transporter (CST). Therefore in this review we will provide the current state-of-play with respect to the structure-function relationship of the (CST). In particular we have summarised work performed by a number groups detailing the affect of various mutations on CST transport activity, efficiency, and substrate specificity.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Spatial Segregation of γ-Secretase and Substrates in Distinct Membrane Domains*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Kim, Seong-Hun; Chen, Ying; Barnes, Natalie Y.; Parent, Ange‘le T.; Sisodia, Sangram S.; Thinakaran, Gopal

2005-01-01

γ-Secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional γ-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and γ-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking γ-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic im-munoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active γ-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that γ-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of γ-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates. PMID:15886206

Spatial segregation of gamma-secretase and substrates in distinct membrane domains.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Kim, Seong-Hun; Chen, Ying; Barnes, Natalie Y; Parent, Angèle T; Sisodia, Sangram S; Thinakaran, Gopal

2005-07-08

Gamma-secretase facilitates the regulated intramembrane proteolysis of select type I membrane proteins that play diverse physiological roles in multiple cell types and tissue. In this study, we used biochemical approaches to examine the distribution of amyloid precursor protein (APP) and several additional gamma-secretase substrates in membrane microdomains. We report that APP C-terminal fragments (CTFs) and gamma-secretase reside in Lubrol WX detergent-insoluble membranes (DIM) of cultured cells and adult mouse brain. APP CTFs that accumulate in cells lacking gamma-secretase activity preferentially associate with DIM. Cholesterol depletion and magnetic immunoisolation studies indicate recruitment of APP CTFs into cholesterol- and sphingolipid-rich lipid rafts, and co-residence of APP CTFs, PS1, and syntaxin 6 in DIM patches derived from the trans-Golgi network. Photoaffinity cross-linking studies provided evidence for the preponderance of active gamma-secretase in lipid rafts of cultured cells and adult brain. Remarkably, unlike the case of APP, CTFs derived from Notch1, Jagged2, deleted in colorectal cancer (DCC), and N-cadherin remain largely detergent-soluble, indicative of their spatial segregation in non-raft domains. In embryonic brain, the majority of PS1 and nicastrin is present in Lubrol WX-soluble membranes, wherein the CTFs derived from APP, Notch1, DCC, and N-cadherin also reside. We suggest that gamma-secretase residence in non-raft membranes facilitates proteolysis of diverse substrates during embryonic development but that the translocation of gamma-secretase to lipid rafts in adults ensures processing of certain substrates, including APP CTFs, while limiting processing of other potential substrates.

The Use of Bryophytes as Indicators of Hydric Soils and Wetland Hydrology during Wetland Delineations in the United States

DTIC Science & Technology

2010-09-01

wetland delineations generally involves separating plants into layers or strata based on life form: tree, vine , shrub , and herb (USACE 1987). ERDC/CRREL...trunks, roots, and coarse woody debris in many wetland types, including floodplain forests (Muhle and LeBlanc 1975), cypress domes (Carr et al. 2006...depositing woody debris, rock, and sediments. Because many bryophytes exhibit strong preferences for a particular substrate, substrate diversity

Short-term microbial effects of a large-scale mine-tailing storage facility collapse on the local natural environment

PubMed Central

Baldwin, Susan A.; Taylor, Jon; Gurr, David B.; Denesiuk, Daniel R.; Van Hamme, Jonathan D.; Fraser, Lauchlan H.

2018-01-01

We investigated the impacts of the Mount Polley tailings impoundment failure on chemical, physical, and microbial properties of substrates within the affected watershed, comprised of 70 hectares of riparian wetlands and 40 km of stream and lake shore. We established a biomonitoring network in October of 2014, two months following the disturbance, and evaluated riparian and wetland substrates for microbial community composition and function via 16S and full metagenome sequencing. A total of 234 samples were collected from substrates at 3 depths and 1,650,752 sequences were recorded in a geodatabase framework. These data revealed a wealth of information regarding watershed-scale distribution of microbial community members, as well as community composition, structure, and response to disturbance. Substrates associated with the impact zone were distinct chemically as indicated by elevated pH, nitrate, and sulphate. The microbial community exhibited elevated metabolic capacity for selenate and sulfate reduction and an abundance of chemolithoautotrophs in the Thiobacillus thiophilus/T. denitrificans/T. thioparus clade that may contribute to nitrate attenuation within the affected watershed. The most impacted area (a 6 km stream connecting two lakes) exhibited 30% lower microbial diversity relative to the remaining sites. The tailings impoundment failure at Mount Polley Mine has provided a unique opportunity to evaluate functional and compositional diversity soon after a major catastrophic disturbance to assess metabolic potential for ecosystem recovery. PMID:29694379

Genesis Contingency Planning and Mishap Recovery: The Sample Curation View

NASA Technical Reports Server (NTRS)

Stansbery, E. K.; Allton, J. H.; Allen, C. C.; McNamara, K. M.; Calaway, M.; Rodriques, M. C.

2007-01-01

Planning for sample preservation and curation was part of mission design from the beginning. One of the scientific objectives for Genesis included collecting samples of three regimes of the solar wind in addition to collecting bulk solar wind during the mission. Collectors were fabricated in different thicknesses for each regime of the solar wind and attached to separate frames exposed to the solar wind during specific periods of solar activity associated with each regime. The original plan to determine the solar regime sampled for specific collectors was to identify to which frame the collector was attached. However, the collectors were dislodged during the hard landing making identification by frame attachment impossible. Because regimes were also identified by thickness of the collector, the regime sampled is identified by measuring fragment thickness. A variety of collector materials and thin films applied to substrates were selected and qualified for flight. This diversity provided elemental measurement in more than one material, mitigating effects of diffusion rates and/or radiation damage. It also mitigated against different material and substrate strengths resulting in differing effects of the hard landing. For example, silicon crystal substrates broke into smaller fragments than sapphire-based substrates and diamond surfaces were more resilient to flying debris damage than gold. The primary responsibility of the curation team for recovery was process documentation. Contingency planning for the recovery phase expanded this responsibility to include not only equipment to document, but also gather, contain and identify samples from the landing area and the recovered spacecraft. The team developed contingency plans for various scenarios as part of mission planning that included topographic maps to aid in site recovery and identification of different modes of transport and purge capability depending on damage. A clean tent, set-up at Utah Test & Training Range to control the environment for processing the sample return capsule and cleanly installing a nitrogen purge to the canister, was used to control the environment for extracting collector fragments from the damaged canister and to document and package over 10,000 collector fragments.

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate

PubMed Central

2012-01-01

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ∼2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ∼150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes. PMID:22894855

Substrate interactions and promiscuity in a viral DNA packaging motor.

PubMed

Aathavan, K; Politzer, Adam T; Kaplan, Ariel; Moffitt, Jeffrey R; Chemla, Yann R; Grimes, Shelley; Jardine, Paul J; Anderson, Dwight L; Bustamante, Carlos

2009-10-01

The ASCE (additional strand, conserved E) superfamily of proteins consists of structurally similar ATPases associated with diverse cellular activities involving metabolism and transport of proteins and nucleic acids in all forms of life. A subset of these enzymes consists of multimeric ringed pumps responsible for DNA transport in processes including genome packaging in adenoviruses, herpesviruses, poxviruses and tailed bacteriophages. Although their mechanism of mechanochemical conversion is beginning to be understood, little is known about how these motors engage their nucleic acid substrates. Questions remain as to whether the motors contact a single DNA element, such as a phosphate or a base, or whether contacts are distributed over several parts of the DNA. Furthermore, the role of these contacts in the mechanochemical cycle is unknown. Here we use the genome packaging motor of the Bacillus subtilis bacteriophage varphi29 (ref. 4) to address these questions. The full mechanochemical cycle of the motor, in which the ATPase is a pentameric-ring of gene product 16 (gp16), involves two phases-an ATP-loading dwell followed by a translocation burst of four 2.5-base-pair (bp) steps triggered by hydrolysis product release. By challenging the motor with a variety of modified DNA substrates, we show that during the dwell phase important contacts are made with adjacent phosphates every 10-bp on the 5'-3' strand in the direction of packaging. As well as providing stable, long-lived contacts, these phosphate interactions also regulate the chemical cycle. In contrast, during the burst phase, we find that DNA translocation is driven against large forces by extensive contacts, some of which are not specific to the chemical moieties of DNA. Such promiscuous, nonspecific contacts may reflect common translocase-substrate interactions for both the nucleic acid and protein translocases of the ASCE superfamily.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew

Here, many species of Gram-positive bacteria use sortase transpeptidases to covalently affix proteins to their cell wall or to assemble pili. Sortase-displayed proteins perform critical and diverse functions for cell survival, including cell adhesion, nutrient acquisition, and morphological development, among others. Based on their amino acid sequences, there are at least six types of sortases (class A to F enzymes); however, class E enzymes have not been extensively studied. Class E sortases are used by soil and freshwater-dwelling Actinobacteria to display proteins that contain a non-canonical LAXTG sorting signal, which differs from 90% of known sorting signals by substitution ofmore » alanine for proline. Here we report the first crystal structure of a class E sortase, the 1.93 Å resolution structure of the SrtE1 enzyme from Streptomyces coelicolor. The active site is bound to a tripeptide, providing insight into the mechanism of substrate binding. SrtE1 possesses β3/β4 and β6/β7 active site loops that contact the LAXTG substrate and are structurally distinct from other classes. We propose that SrtE1 and other class E sortases employ a conserved tyrosine residue within their β3/β4 loop to recognize the amide nitrogen of alanine at position P3 of the sorting signal through a hydrogen bond, as seen here. Incapability of hydrogen-bonding with canonical proline-containing sorting signals likely contributes to class E substrate specificity. Furthermore, we demonstrate that surface anchoring of proteins involved in aerial hyphae formation requires an N-terminal segment in SrtE1 that is presumably positioned within the cytoplasm. Combined, our results reveal unique features within class E enzymes that enable them to recognize distinct sorting signals, and could facilitate the development of substrate-based inhibitors of this important enzyme family.« less

Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

PubMed

Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

2015-11-01

Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

PubMed

Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak

2018-01-01

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Microplastics alter composition of fungal communities in aquatic ecosystems.

PubMed

Kettner, Marie Therese; Rojas-Jimenez, Keilor; Oberbeckmann, Sonja; Labrenz, Matthias; Grossart, Hans-Peter

2017-11-01

Despite increasing concerns about microplastic (MP) pollution in aquatic ecosystems, there is insufficient knowledge on how MP affect fungal communities. In this study, we explored the diversity and community composition of fungi attached to polyethylene (PE) and polystyrene (PS) particles incubated in different aquatic systems in north-east Germany: the Baltic Sea, the River Warnow and a wastewater treatment plant. Based on next generation 18S rRNA gene sequencing, 347 different operational taxonomic units assigned to 81 fungal taxa were identified on PE and PS. The MP-associated communities were distinct from fungal communities in the surrounding water and on the natural substrate wood. They also differed significantly among sampling locations, pointing towards a substrate and location specific fungal colonization. Members of Chytridiomycota, Cryptomycota and Ascomycota dominated the fungal assemblages, suggesting that both parasitic and saprophytic fungi thrive in MP biofilms. Thus, considering the worldwide increasing accumulation of plastic particles as well as the substantial vector potential of MP, especially these fungal taxa might benefit from MP pollution in the aquatic environment with yet unknown impacts on their worldwide distribution, as well as biodiversity and food web dynamics at large. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

A high throughput mutagenic analysis of yeast sumo structure and function

PubMed Central

Newman, Heather A.; Lu, Jian; Carson, Caryn; Boeke, Jef D.

2017-01-01

Sumoylation regulates a wide range of essential cellular functions through diverse mechanisms that remain to be fully understood. Using S. cerevisiae, a model organism with a single essential SUMO gene (SMT3), we developed a library of >250 mutant strains with single or multiple amino acid substitutions of surface or core residues in the Smt3 protein. By screening this library using plate-based assays, we have generated a comprehensive structure-function based map of Smt3, revealing essential amino acid residues and residues critical for function under a variety of genotoxic and proteotoxic stress conditions. Functionally important residues mapped to surfaces affecting Smt3 precursor processing and deconjugation from protein substrates, covalent conjugation to protein substrates, and non-covalent interactions with E3 ligases and downstream effector proteins containing SUMO-interacting motifs. Lysine residues potentially involved in formation of polymeric chains were also investigated, revealing critical roles for polymeric chains, but redundancy in specific chain linkages. Collectively, our findings provide important insights into the molecular basis of signaling through sumoylation. Moreover, the library of Smt3 mutants represents a valuable resource for further exploring the functions of sumoylation in cellular stress response and other SUMO-dependent pathways. PMID:28166236

Selective Inactivation of Functional RNAs by Ribozyme-Catalyzed Covalent Modification.

PubMed

Poudyal, Raghav R; Benslimane, Malak; Lokugamage, Melissa P; Callaway, Mackenzie K; Staller, Seth; Burke, Donald H

2017-03-17

The diverse functions of RNA provide numerous opportunities for programming biological circuits. We describe a new strategy that uses ribozyme K28min to covalently tag a specific nucleobase within an RNA or DNA target strand to regulate and selectively inactivate those nucleic acids. K28min variants with appropriately reprogrammed internal guide sequences efficiently tagged multiple sites from an mRNA and from aptamer and ribozyme targets. Upon covalent modification by the corresponding K28min variant, an ATP-binding aptamer lost all affinity for ATP, and the fluorogenic Mango aptamer lost its ability to activate fluorescence of its dye ligand. Modifying a hammerhead ribozyme near the catalytic core led to loss of almost all of its substrate-cleaving activity, but modifying the same hammerhead ribozyme within a tertiary stabilizing element that reduces magnesium dependence only impaired substrate cleavage at low magnesium concentration. Thus, ribozyme-mediated covalent modification can be used both to selectively inactivate and to fine-tune the activities of targeted functional RNAs, analogous to the effects of post-translational modifications of proteins. Ribozyme-catalyzed covalent modification could therefore be developed to regulate nucleic acids components of synthetic and natural circuits.

Analysis of Protein Phosphorylation and Its Functional Impact on Protein-Protein Interactions via Text Mining of the Scientific Literature.

PubMed

Wang, Qinghua; Ross, Karen E; Huang, Hongzhan; Ren, Jia; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

2017-01-01

Post-translational modifications (PTMs) are one of the main contributors to the diversity of proteoforms in the proteomic landscape. In particular, protein phosphorylation represents an essential regulatory mechanism that plays a role in many biological processes. Protein kinases, the enzymes catalyzing this reaction, are key participants in metabolic and signaling pathways. Their activation or inactivation dictate downstream events: what substrates are modified and their subsequent impact (e.g., activation state, localization, protein-protein interactions (PPIs)). The biomedical literature continues to be the main source of evidence for experimental information about protein phosphorylation. Automatic methods to bring together phosphorylation events and phosphorylation-dependent PPIs can help to summarize the current knowledge and to expose hidden connections. In this chapter, we demonstrate two text mining tools, RLIMS-P and eFIP, for the retrieval and extraction of kinase-substrate-site data and phosphorylation-dependent PPIs from the literature. These tools offer several advantages over a literature search in PubMed as their results are specific for phosphorylation. RLIMS-P and eFIP results can be sorted, organized, and viewed in multiple ways to answer relevant biological questions, and the protein mentions are linked to UniProt identifiers.

Life in the "plastisphere": microbial communities on plastic marine debris.

PubMed

Zettler, Erik R; Mincer, Tracy J; Amaral-Zettler, Linda A

2013-07-02

Plastics are the most abundant form of marine debris, with global production rising and documented impacts in some marine environments, but the influence of plastic on open ocean ecosystems is poorly understood, particularly for microbial communities. Plastic marine debris (PMD) collected at multiple locations in the North Atlantic was analyzed with scanning electron microscopy (SEM) and next-generation sequencing to characterize the attached microbial communities. We unveiled a diverse microbial community of heterotrophs, autotrophs, predators, and symbionts, a community we refer to as the "Plastisphere". Pits visualized in the PMD surface conformed to bacterial shapes suggesting active hydrolysis of the hydrocarbon polymer. Small-subunit rRNA gene surveys identified several hydrocarbon-degrading bacteria, supporting the possibility that microbes play a role in degrading PMD. Some Plastisphere members may be opportunistic pathogens (the authors, unpublished data) such as specific members of the genus Vibrio that dominated one of our plastic samples. Plastisphere communities are distinct from surrounding surface water, implying that plastic serves as a novel ecological habitat in the open ocean. Plastic has a longer half-life than most natural floating marine substrates, and a hydrophobic surface that promotes microbial colonization and biofilm formation, differing from autochthonous substrates in the upper layers of the ocean.

A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil1[C][W][OA

PubMed Central

Berim, Anna; Hyatt, David C.; Gang, David R.

2012-01-01

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 4′- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4′-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation. PMID:22923679

Beyond Vmax and Km: How details of enzyme function influence geochemical cycles

NASA Astrophysics Data System (ADS)

Steen, A. D.

2015-12-01

Enzymes catalyze the vast majority of chemical reactions relevant to geomicrobiology. Studies of the activities of enzymes in environmental systems often report Vmax (the maximum possible rate of reaction; often proportional to the concentration of enzymes in the system) and sometimes Km (a measure of the affinity between enzymes and their substrates). However, enzyme studies - particularly those related to enzymes involved in organic carbon oxidation - are often limited to only those parameters, and a relatively limited and mixed set of enzymes. Here I will discuss some novel methods to assay and characterize the specific sets of enzymes that may be important to the carbon cycle in aquatic environments. First, kinetic experiments revealed the collective properties of the complex mixtures of extracellular peptidases that occur where microbial communities are diverse. Crystal structures combined with biochemical characterization of specific enzymes can yield more detailed information about key steps in organic carbon transformations. These new techniques have the potential to provide mechanistic grounding to geomicrobiological models.

The multiple functions of plant serine protease inhibitors

PubMed Central

Giri, Ashok P; Kaur, Harleen; Baldwin, Ian T

2011-01-01

Plant protease inhibitors (PIs) are a diverse group of proteins which have been intensely investigated due to their potential function in protecting plants against herbivorous insects by inhibiting digestive proteases. Although this mechanism has been well documented for a number of single PIs and their target enzymes, whether this mechanism protects plants in nature remains unclear. Moreover, many plants express a number of different PIs and it was unknown if these proteins work synergistically as defenses or if they also have other functions. We recently identified four serine PIs (SPI) of Solanum nigrum and demonstrated that they differ substantially in substrate specificity, accumulation patterns, and their effect against different natural herbivorous insects in field- and glasshouse experiments. These differences suggest that SPIs have at least partially diversified to provide protection against different attackers. Although we could not detect effects on plant development or growth when silencing SPIs, gene- and tissue-specific expression patterns suggest multiple functions in generative tissues, including a possible involvement in development. PMID:22004998

Expanding the Bioactive Chemical Space of Anthrabenzoxocinones through Engineering the Highly Promiscuous Biosynthetic Modification Steps.

PubMed

Mei, Xianyi; Yan, Xiaoli; Zhang, Hui; Yu, Mingjia; Shen, Guangqing; Zhou, Linjun; Deng, Zixin; Lei, Chun; Qu, Xudong

2018-01-19

Anthrabenzoxocinones (ABXs) including (-)-ABXs and (+)-ABXs are a group of bacterial FabF-specific inhibitors with potent antimicrobial activity of resistant strains. Optimization of their chemical structures is a promising method to develop potent antibiotics. Through biosynthetic investigation, we herein identified and characterized two highly promiscuous enzymes involved in the (-)-ABX structural modification. The promiscuous halogenase and methyltransferase can respectively introduce halogen-modifications into various positions of the ABX scaffolds and methylation to highly diverse substrates. Manipulation of their activity in both of the (-)-ABXs and (+)-ABXs biosyntheses led to the generation of 14 novel ABX analogues of both enantiomers. Bioactivity assessment revealed that a few of the analogues showed significantly improved antimicrobial activity, with the C3-hydroxyl and chlorine substitutions critical for their activity. This study enormously expands the bioactive chemical space of the ABX family and FabF-specific inhibitors. The disclosed broad-selective biosynthetic machineries and structure-activity relationship provide a solid basis for further generation of potent antimicrobial agents.

ADAM-17: The Enzyme That Does It All

PubMed Central

Gooz, Monika

2010-01-01

This review focuses on the role of ADAM-17 in disease. Since its debut as the tumor necrosis factor converting enzyme or TACE, ADAM-17 has been reported to be an indispensible regulator of almost every cellular event from proliferation to migration. The central role of ADAM-17 in cell regulation is rooted in its diverse array of substrates: cytokines, growth factors, and their receptors as well as adhesion molecules are activated or inactivated by their cleavage with ADAM-17. It is therefore not surprising that ADAM-17 is implicated in numerous human diseases including cancer, heart disease, diabetes, rheumatoid arthritis, kidney fibrosis, Alzheimer’s disease, and is a promising target for future treatments. The specific role of ADAM-17 in the pathophysiology of these diseases is very complex and depends on the cellular context. To exploit the therapeutic potential of ADAM-17, it is important to understand how its activity is regulated and how specific organs and cells can be targeted to inactivate or activate the enzyme. PMID:20184396

Ecologic and Morphologic Analysis of a Proposed Network of Sediment Diversions

NASA Astrophysics Data System (ADS)

Meselhe, E. A.; Sadid, K. M.; Jung, H.; Messina, F.; Esposito, C.; Liang, M.

2017-12-01

Deltaic processes are governed by factors including the characteristics of inflowing sediment (e.g., temporal variability of the load and size class distribution), receiving basins (e.g., water depth, tidal range, circulation pattern, and wind field), and substrate (e.g., sediment type and soil strength). These factors influence the deltaic growth as well as the size and pattern of channel bifurcations. This topic is of importance to deltas experiencing land loss due to subsidence and sea level rise. The Mississippi River Delta is an example where a number of sediment diversions are being considered in conjunction with other restoration actions to minimize loss of wetlands. Historically, the Mississippi River played a significant role in providing sediment, nutrients, and fresh water to support Louisiana's coastal wetland system. As such, a systems perspective for regional-scale implementation of diversions is important. Field observations coupled with numerical modeling at various temporal and spatial scales, has provided insights toward a system-scale approach to design, evaluate and operate sediment diversions. These research activities investigate the uncertainties associated with morphodynamic processes both on the river and receiving basin sides and identify parameters influencing the magnitude and rate of building new land and sustaining existing wetland areas. Specifically, this presentation discusses the impact of extracting sediment and water from fluvial rivers, the ability to convey (and retain) sediment to the receiving basins. In addition to delivering sediment to receiving basins, some proposed sediment diversions could discharge high volumes of nutrient-rich fresh water into existing wetlands and bays. A goal of the analysis presented here is to improve our understanding of morphodynamic responses of the receiving basins and the ecosystem effects of discharges of freshwater and nutrients at this scale.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Molecular Library Synthesis Using Complex Substrates: Expanding the Framework of Triterpenoids

PubMed Central

Ignatenko, Vasily A.; Han, Yong; Tochtrop, Gregory P.

2013-01-01

The remodelling of a natural product core framework by means of diversity-oriented synthesis (DOS) is a valuable approach to access diverse/biologically relevant chemical space and to overcome the limitations of combinatorial-type compounds. Here we provide proof of principle and a thorough conformational analysis for a general strategy whereby the inherent complexity of a starting material is used to define the regio- and stereochemical outcomes of reactions in chemical library construction. This is in contrast to the traditional DOS logic employing reaction development and catalysis to drive library diversity PMID:23245400

Identification of a common neurobiological substrate for mental illness.

PubMed

Goodkind, Madeleine; Eickhoff, Simon B; Oathes, Desmond J; Jiang, Ying; Chang, Andrew; Jones-Hagata, Laura B; Ortega, Brissa N; Zaiko, Yevgeniya V; Roach, Erika L; Korgaonkar, Mayuresh S; Grieve, Stuart M; Galatzer-Levy, Isaac; Fox, Peter T; Etkin, Amit

2015-04-01

Psychiatric diagnoses are currently distinguished based on sets of specific symptoms. However, genetic and clinical analyses find similarities across a wide variety of diagnoses, suggesting that a common neurobiological substrate may exist across mental illness. To conduct a meta-analysis of structural neuroimaging studies across multiple psychiatric diagnoses, followed by parallel analyses of 3 large-scale healthy participant data sets to help interpret structural findings in the meta-analysis. PubMed was searched to identify voxel-based morphometry studies through July 2012 comparing psychiatric patients to healthy control individuals for the meta-analysis. The 3 parallel healthy participant data sets included resting-state functional magnetic resonance imaging, a database of activation foci across thousands of neuroimaging experiments, and a data set with structural imaging and cognitive task performance data. Studies were included in the meta-analysis if they reported voxel-based morphometry differences between patients with an Axis I diagnosis and control individuals in stereotactic coordinates across the whole brain, did not present predominantly in childhood, and had at least 10 studies contributing to that diagnosis (or across closely related diagnoses). The meta-analysis was conducted on peak voxel coordinates using an activation likelihood estimation approach. We tested for areas of common gray matter volume increase or decrease across Axis I diagnoses, as well as areas differing between diagnoses. Follow-up analyses on other healthy participant data sets tested connectivity related to regions arising from the meta-analysis and the relationship of gray matter volume to cognition. Based on the voxel-based morphometry meta-analysis of 193 studies comprising 15 892 individuals across 6 diverse diagnostic groups (schizophrenia, bipolar disorder, depression, addiction, obsessive-compulsive disorder, and anxiety), we found that gray matter loss converged across diagnoses in 3 regions: the dorsal anterior cingulate, right insula, and left insula. By contrast, there were few diagnosis-specific effects, distinguishing only schizophrenia and depression from other diagnoses. In the parallel follow-up analyses of the 3 independent healthy participant data sets, we found that the common gray matter loss regions formed a tightly interconnected network during tasks and at resting and that lower gray matter in this network was associated with poor executive functioning. We identified a concordance across psychiatric diagnoses in terms of integrity of an anterior insula/dorsal anterior cingulate-based network, which may relate to executive function deficits observed across diagnoses. This concordance provides an organizing model that emphasizes the importance of shared neural substrates across psychopathology, despite likely diverse etiologies, which is currently not an explicit component of psychiatric nosology.

Microbial ecology of the skin in the era of metagenomics and molecular microbiology.

PubMed

Hannigan, Geoffrey D; Grice, Elizabeth A

2013-12-01

The skin is the primary physical barrier between the body and the external environment and is also a substrate for the colonization of numerous microbes. Previously, dermatological microbiology research was dominated by culture-based techniques, but significant advances in genomic technologies have enabled the development of less-biased, culture-independent approaches to characterize skin microbial communities. These molecular microbiology approaches illustrate the great diversity of microbiota colonizing the skin and highlight unique features such as site specificity, temporal dynamics, and interpersonal variation. Disruptions in skin commensal microbiota are associated with the progression of many dermatological diseases. A greater understanding of how skin microbes interact with each other and with their host, and how we can therapeutically manipulate those interactions, will provide powerful tools for treating and preventing dermatological disease.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

PubMed

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

Marine Microcosm Experiments on Effects of Copper and Tributylin-Based Antifouling Paint Leachates

DTIC Science & Technology

1988-06-01

consumers in HliOrg microcosms were probably the thick carpets of green algae ( Cladophora socialis) that developed in those tanks during leachate and...and bottom substrate in the SPG-4 tanks were dominated by a low-diversity community composed primarily of Cladophora socialis, a filamentous green...coverage and diversity of species after 4 weeks of’ exposure. Moderate to thick growths of filamentous green algae ( Cladophora socialis) covered with

Dissecting the function of Cullin-RING ubiquitin ligase complex genes in planarian regeneration.

PubMed

Strand, Nicholas S; Allen, John M; Ghulam, Mahjoobah; Taylor, Matthew R; Munday, Roma K; Carrillo, Melissa; Movsesyan, Artem; Zayas, Ricardo M

2018-01-15

The ubiquitin system plays a role in nearly every aspect of eukaryotic cell biology. The enzymes responsible for transferring ubiquitin onto specific substrates are the E3 ubiquitin ligases, a large and diverse family of proteins, for which biological roles and target substrates remain largely undefined. Studies using model organisms indicate that ubiquitin signaling mediates key steps in developmental processes and tissue regeneration. Here, we used the freshwater planarian, Schmidtea mediterranea, to investigate the role of Cullin-RING ubiquitin ligase (CRL) complexes in stem cell regulation during regeneration. We identified six S. mediterranea cullin genes, and used RNAi to uncover roles for homologs of Cullin-1, -3 and -4 in planarian regeneration. The cullin-1 RNAi phenotype included defects in blastema formation, organ regeneration, lesions, and lysis. To further investigate the function of cullin-1-mediated cellular processes in planarians, we examined genes encoding the adaptor protein Skp1 and F-box substrate-recognition proteins that are predicted to partner with Cullin-1. RNAi against skp1 resulted in phenotypes similar to cullin-1 RNAi, and an RNAi screen of the F-box genes identified 19 genes that recapitulated aspects of cullin-1 RNAi, including ones that in mammals are involved in stem cell regulation and cancer biology. Our data provides evidence that CRLs play discrete roles in regenerative processes and provide a platform to investigate how CRLs regulate stem cells in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Contrasting trends in distribution of four major planktonic betaproteobacterial groups along a pH gradient of epilimnia of 72 freshwater habitats.

PubMed

Jezbera, Jan; Jezberová, Jitka; Koll, Ulrike; Horňák, Karel; Šimek, Karel; Hahn, Martin W

2012-08-01

The distribution and abundance of Betaproteobacteria and three of its genera - Limnohabitans (R-BT065 lineage), Polynucleobacter (including subclusters Polynucleobacter necessarius and Polynucleobacter acidiphobus/Polynucleobacter difficilis), and Methylophilus - across the epilimnia of 72 limnologically diverse freshwater habitats were investigated using fluorescence in situ hybridization. Moreover, seasonal development of Betaproteobacteria subgroups along the longitudinal axis of a reservoir was followed. Betaproteobacteria comprised on average 29.1%, Polynucleobacter 11.6%, P. necessarius 10.1%, P. acidiphobus/difficilis 0.5%, Limnohabitans 8.9%, and Methylophilus 0.9% of total bacterioplankton cells in the investigated habitats. Polynucleobacter necessarius and Limnohabitans coexisted in the majority of habitats but showed contrasting abundance patterns along the pH gradient of habitats (pH, 3.8-8.5). The observed distribution patterns could theoretically be explained by different preferences for substrate sources, that is, substances of humic origin in acidic waters and algal-derived substances in alkaline waters. However, substrate utilization patterns observed in laboratory experiments indicate no coherent group-specific differences in substrate preferences. Interestingly, similar distribution patterns were revealed for Limnohabitans and P. acidiphobus/difficilis, suggesting similar ecological adaptations of these distantly related taxa. Our findings further emphasize that at least two taxa of freshwater Betaproteobacteria represent ecologically diversified groups. Investigations at higher phylogenetic resolution are required for obtaining further insights into their ecology. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Positive selection sites in tertiary structure of Leguminosae chalcone isomerase 1.

PubMed

Wang, R K; Zhan, S F; Zhao, T J; Zhou, X L; Wang, C E

2015-03-20

Isoflavonoids and the related synthesis enzyme, chalcone isomerase 1 (CHI1), are unique in the Leguminosae, with diverse biological functions. Among the Leguminosae, the soybean is an important oil, protein crop, and model plant. In this study, we aimed to detect the generation pattern of Leguminosae CHI1. Genome-wide sequence analysis of CHI in 3 Leguminosae and 3 other closely related model plants was performed; the expression levels of soybean chalcone isomerases were also analyzed. By comparing positively selected sites and their protein structures, we retrieved the evolution patterns for Leguminosae CHI1. A total of 28 CHI and 7 FAP3 (CHI4) genes were identified and separated into 4 clades: CHI1, CHI2, CHI3, and FAP3. Soybean genes belonging to the same chalcone isomerase subfamily had similar expression patterns. CHI1, the unique chalcone isomerase subfamily in Leguminosae, showed signs of significant positive selection as well as special expression characteristics, indicating an accelerated evolution throughout its divergence. Eight sites were identified as undergoing positive selection with high confidence. When mapped onto the tertiary structure of CHI1, these 8 sites were observed surrounding the enzyme substrate only; some of them connected to the catalytic core of CHI. Thus, we inferred that the generation of Leguminosae CHI1 is dependent on the positively selected amino acids surrounding its catalytic substrate. In other words, the evolution of CHI1 was driven by specific selection or processing conditions within the substrate.

The integrase of the long terminal repeat-retrotransposon tf1 has a chromodomain that modulates integrase activities.

PubMed

Hizi, Amnon; Levin, Henry L

2005-11-25

Chromodomains in a variety of proteins mediate the formation of heterochromatin by interacting directly with histone H3, DNA, or RNA. A diverse family of long terminal repeat (LTR)-retrotransposons possesses chromodomains in their integrases (IN), suggesting that the chromodomains may control integration. The LTR-retrotransposon Tf1 of Schizosaccharomyces pombe is highly active and possesses a chromodomain in the COOH terminus of its IN. To test this chromodomain for a role in integration, recombinant INs with and without the chromodomain were assayed for activity in in vitro reactions. The full-length IN had integration activity with oligonucleotide substrates that modeled both the insertion reaction and a reverse reaction known as disintegration. The INs of retroviruses possess an additional activity termed 3' processing that must remove 2-3 nucleotides from the 3' ends of the viral cDNA before insertion can occur. These additional nucleotides are added during reverse transcription because of the position of the minus strand primer downstream of the LTR. The position of the primer for Tf1 suggests no nucleotides are added 3' of the LTR. It was therefore surprising that Tf1 IN was capable of 3' cleavage. The most unexpected result reported here was that the IN lacking the chromodomain had significantly higher activity and substantially reduced substrate specificity. These results reveal that both the activity and specificity of enzymes can be modulated by their chromodomains.

Lightweight Electrode For Nickel/Hydrogen Cell

NASA Technical Reports Server (NTRS)

Britton, Doris L.

1994-01-01

Improved substrate for nickel electrode increases specific energy of nickel/hydrogen cell. Consists of 50 percent by weight nickel fiber, 35 percent nickel powder, and 15 percent cobalt powder. Porosity and thickness of nickel electrodes affect specific energy, initial performance, and cycle life of cell. Substrate easily manufactured with much larger porosities than those of heavy-sintered state-of-art nickel substrate.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

Enrichment and cultivation of prokaryotes associated with the sulphate-methane transition zone of diffusion-controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions.

PubMed

Webster, Gordon; Sass, Henrik; Cragg, Barry A; Gorra, Roberta; Knab, Nina J; Green, Christopher J; Mathes, Falko; Fry, John C; Weightman, Andrew J; Parkes, R John

2011-08-01

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10 mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (~2 mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Impacts of rapid urbanization on the water quality and macroinvertebrate communities of streams: A case study in Liangjiang New Area, China.

PubMed

Luo, Kun; Hu, Xuebin; He, Qiang; Wu, Zhengsong; Cheng, Hao; Hu, Zhenlong; Mazumder, Asit

2018-04-15

Rapid urbanization in China has dramatically deteriorated the water quality of streams and threatening aquatic ecosystem health. This study aims to 1) assess the impacts of urbanization on water quality and macroinvertebrate composition and 2) address the question of how urbanization affects macroinvertebrate distribution patterns. Environmental variables over multispatial scales and macroinvertebrate community data were collected on April (dry season) and September (wet season) of 2014 and 2015 at 19 sampling sites, of which nine had a high urbanization level (HUL), six had moderate urbanization level (MUL) and four had low urbanization level (LUL), in the Liangjiang New Area. The results of this study showed that macroinvertebrate assemblages significantly varied across the three urbanization levels. The sensitive species (e.g., EPT taxa) were mainly centralized at LUL sites, whereas tolerant species, such as Tubificidae (17.3%), Chironomidae (12.1%), and Physidae (4.61%), reached highest relative abundance at LUL sites. The values of family biotic index (FBI) and biological monitoring working party (BMWP) indicated the deterioration of water quality along urbanization gradient. Seasonal and inter - annual changes in macroinvertebrate communities were not observed. The results of variation partitioning analyses (CCAs) showed that habitat scale variables explained the major variation in macroinvertebrate community composition. Specifically, the increased nutrient concentrations favored tolerant species, whereas high water flow and substrate coarseness benefitted community taxa richness, diversity and EPT richness. Considering the interactions between scale-related processes, the results of this study suggested that urbanization resulted in less diverse and more tolerant stream macroinvertebrate assemblages mainly via increased nutrient concentrations and reduced substrate coarseness. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural and Functional Characterization of a Ruminal β-Glycosidase Defines a Novel Subfamily of Glycoside Hydrolase Family 3 with Permuted Domain Topology*

PubMed Central

Ramírez-Escudero, Mercedes; del Pozo, Mercedes V.; Marín-Navarro, Julia; González, Beatriz; Golyshin, Peter N.; Polaina, Julio; Ferrer, Manuel; Sanz-Aparicio, Julia

2016-01-01

Metagenomics has opened up a vast pool of genes for putative, yet uncharacterized, enzymes. It widens our knowledge on the enzyme diversity world and discloses new families for which a clear classification is still needed, as is exemplified by glycoside hydrolase family-3 (GH3) proteins. Herein, we describe a GH3 enzyme (GlyA1) from resident microbial communities in strained ruminal fluid. The enzyme is a β-glucosidase/β-xylosidase that also shows β-galactosidase, β-fucosidase, α-arabinofuranosidase, and α-arabinopyranosidase activities. Short cello- and xylo-oligosaccharides, sophorose and gentibiose, are among the preferred substrates, with the large polysaccharide lichenan also being hydrolyzed by GlyA1. The determination of the crystal structure of the enzyme in combination with deletion and site-directed mutagenesis allowed identification of its unusual domain composition and the active site architecture. Complexes of GlyA1 with glucose, galactose, and xylose allowed picturing the catalytic pocket and illustrated the molecular basis of the substrate specificity. A hydrophobic platform defined by residues Trp-711 and Trp-106, located in a highly mobile loop, appears able to allocate differently β-linked bioses. GlyA1 includes an additional C-terminal domain previously unobserved in GH3 members, but crystallization of the full-length enzyme was unsuccessful. Therefore, small angle x-ray experiments have been performed to investigate the molecular flexibility and overall putative shape. This study provided evidence that GlyA1 defines a new subfamily of GH3 proteins with a novel permuted domain topology. Phylogenetic analysis indicates that this topology is associated with microbes inhabiting the digestive tracts of ruminants and other animals, feeding on chemically diverse plant polymeric materials. PMID:27679487

Isolating causal pathways between flow and fish in the regulated river hierarchy

DOE Office of Scientific and Technical Information (OSTI.GOV)

McManamay, Ryan A.; Peoples, Brandon K.; Orth, Donald J.

Unregulated river systems are organized in a hierarchy in which large-scale factors (i.e., landscape and segment scales) influence local habitats (i.e., reach, meso-, and microhabitat scales), and both differentially exert selective pressures on biota. Dams, however, create discontinua in these processes and change the hierarchical structure. We examined the relative roles of hydrology and other instream factors, within a hierarchical landscape context, in organizing fish communities in regulated and unregulated tributaries to the Upper Tennessee River, USA. We also used multivariate regression trees to identify factors that partition fish assemblages based on trait similarities, irrespective of spatial scale. Then, wemore » used classical path analysis and structural equation modeling to evaluate the most plausible hierarchical causal structure of specific trait-based community components, given the data. Both statistical approaches suggested that river regulation affects stream fishes through a variety of reach-scale variables, not always through hydrology itself. Though we observed different changes in flow, temperature, and biotic responses according to regulation types, the most predominant path in which dam regulation affected biota was via temperature alterations. Diversion dams had the strongest effects on fish assemblages. Diversion dams reduced flow magnitudes, leading to declines in fish richness but increased temperatures, leading to lower abundances in equilibrium species and nest guarders. Peaking and run-of-river dams increased flow variability, leading to lower abundances in nest-guarding fishes. Flow displayed direct relationships with biotic responses; however, results indicated that changes in temperature and substrate had equal, if not stronger, effects on fish assemblage composition. The strength and nature of relationships depended on whether flow metrics were standardized for river size. Here, we suggest that restoration efforts in regulated rivers focus on improving flow conditions in conjunction with temperature and substrate restoration.« less

Isolating causal pathways between flow and fish in the regulated river hierarchy

DOE PAGES

McManamay, Ryan A.; Peoples, Brandon K.; Orth, Donald J.; ...

2015-07-07

Unregulated river systems are organized in a hierarchy in which large-scale factors (i.e., landscape and segment scales) influence local habitats (i.e., reach, meso-, and microhabitat scales), and both differentially exert selective pressures on biota. Dams, however, create discontinua in these processes and change the hierarchical structure. We examined the relative roles of hydrology and other instream factors, within a hierarchical landscape context, in organizing fish communities in regulated and unregulated tributaries to the Upper Tennessee River, USA. We also used multivariate regression trees to identify factors that partition fish assemblages based on trait similarities, irrespective of spatial scale. Then, wemore » used classical path analysis and structural equation modeling to evaluate the most plausible hierarchical causal structure of specific trait-based community components, given the data. Both statistical approaches suggested that river regulation affects stream fishes through a variety of reach-scale variables, not always through hydrology itself. Though we observed different changes in flow, temperature, and biotic responses according to regulation types, the most predominant path in which dam regulation affected biota was via temperature alterations. Diversion dams had the strongest effects on fish assemblages. Diversion dams reduced flow magnitudes, leading to declines in fish richness but increased temperatures, leading to lower abundances in equilibrium species and nest guarders. Peaking and run-of-river dams increased flow variability, leading to lower abundances in nest-guarding fishes. Flow displayed direct relationships with biotic responses; however, results indicated that changes in temperature and substrate had equal, if not stronger, effects on fish assemblage composition. The strength and nature of relationships depended on whether flow metrics were standardized for river size. Here, we suggest that restoration efforts in regulated rivers focus on improving flow conditions in conjunction with temperature and substrate restoration.« less

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Endophytic fungus Phomopsis liquidambari and different doses of N-fertilizer alter microbial community structure and function in rhizosphere of rice

PubMed Central

Siddikee, Md. Ashaduzzaman; Zereen, Mst Israt; Li, Cai-Feng; Dai, Chuan-Chao

2016-01-01

Microbial community structure and functions of rhizosphere soil of rice were investigated after applying low and high doses of nitrogenous fertilizer and Phomopsis liquidambari. Average well color development, substrate richness, catabolic diversity and soil enzymes activities varied after applying N-fertilizer and P. liquidambari and were greater in P. liquidambari treated soil than only N-fertilization. Multivariate analysis distinctly separated the catabolic and enzymes activity profile which statistically proved alteration of microbial functional diversity. Nitrogen fertilizer altered microbial community structure revealed by the increased content of total PLFAs, specific subgroup marker PLFAs except fungal PLFAs and by the decreased ratio of G+/G−, sat/monunsat, iso/anteiso, F/B except trans/cis while P. liquidambari inoculation enhanced N-fertilization effect except increased fungal PLFA and decreased trans/cis. PCA using identified marker PLFAs revealed definite discrimination among the treatments which further statistically confirmed structural changed of microbial community. Nitrogenase activity representative of N-fixing community decreased in N-fertilizer treatment while P. liquidambari inoculation increased. In short, application of P. liquidambari with low doses of N-fertilizer improved rice growth and reduced N-fertilizer requirement by increasing enzymes activities involved in C, N and P cycling, structural and functional diversity of microbes, nitrogenase activity involved in N2 fixation and accumulation of total-N. PMID:27596935

Diversity and wiring variability of visual local neurons in the Drosophila medulla M6 stratum

PubMed Central

Chin, An-Lun; Lin, Chih-Yung; Fu, Tsai-Feng; Dickson, Barry J; Chiang, Ann-Shyn

2014-01-01

Local neurons in the vertebrate retina are instrumental in transforming visual inputs to extract contrast, motion, and color information and in shaping bipolar-to-ganglion cell transmission to the brain. In Drosophila, UV vision is represented by R7 inner photoreceptor neurons that project to the medulla M6 stratum, with relatively little known of this downstream substrate. Here, using R7 terminals as references, we generated a 3D volume model of the M6 stratum, which revealed a retinotopic map for UV representations. Using this volume model as a common 3D framework, we compiled and analyzed the spatial distributions of more than 200 single M6-specific local neurons (M6-LNs). Based on the segregation of putative dendrites and axons, these local neurons were classified into two families, directional and nondirectional. Neurotransmitter immunostaining suggested a signal routing model in which some visual information is relayed by directional M6-LNs from the anterior to the posterior M6 and all visual information is inhibited by a diverse population of nondirectional M6-LNs covering the entire M6 stratum. Our findings suggest that the Drosophila medulla M6 stratum contains diverse LNs that form repeating functional modules similar to those found in the vertebrate inner plexiform layer. J. Comp. Neurol. 522:3795–3816, 2014. © 2014 Wiley Periodicals, Inc. PMID:24782245

Glycyl radical activating enzymes: Structure, mechanism, and substrate interactions☆

PubMed Central

Shisler, Krista A.; Broderick, Joan B.

2014-01-01

The glycyl radical enzyme activating enzymes (GRE–AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe–4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE–AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE–AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE–AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes. PMID:24486374

Binding of Substrate Locks the Electrochemistry of CRY-DASH into DNA Repair.

PubMed

Gindt, Yvonne M; Messyasz, Adriana; Jumbo, Pamela I

2015-05-12

VcCry1, a member of the CRY-DASH family, may serve two diverse roles in vivo, including blue-light signaling and repair of UV-damaged DNA. We have discovered that the electrochemistry of the flavin adenine dinucleotide cofactor of VcCry1 is locked to cycle only between the hydroquinone and neutral semiquinone states when UV-damaged DNA is present. Other potential substrates, including undamaged DNA and ATP, have no discernible effect on the electrochemistry, and the kinetics of the reduction is unaffected by damaged DNA. Binding of the damaged DNA substrate determines the role of the protein and prevents the presumed photochemistry required for blue-light signaling.

Plastic Responses of a Sessile Prey to Multiple Predators: A Field and Experimental Study

PubMed Central

Hirsch, Philipp Emanuel; Cayon, David; Svanbäck, Richard

2014-01-01

Background Theory predicts that prey facing a combination of predators with different feeding modes have two options: to express a response against the feeding mode of the most dangerous predator, or to express an intermediate response. Intermediate phenotypes protect equally well against several feeding modes, rather than providing specific protection against a single predator. Anti-predator traits that protect against a common feeding mode displayed by all predators should be expressed regardless of predator combination, as there is no need for trade-offs. Principal Findings We studied phenotypic anti-predator responses of zebra mussels to predation threat from a handling-time-limited (crayfish) and a gape-size-limited (roach) predator. Both predators dislodge mussels from the substrate but diverge in their further feeding modes. Mussels increased expression of a non-specific defense trait (attachment strength) against all combinations of predators relative to a control. In response to roach alone, mussels showed a tendency to develop a weaker and more elongated shell. In response to crayfish, mussels developed a harder and rounder shell. When exposed to either a combination of predators or no predator, mussels developed an intermediate phenotype. Mussel growth rate was positively correlated with an elongated weaker shell and negatively correlated with a round strong shell, indicating a trade-off between anti-predator responses. Field observations of prey phenotypes revealed the presence of both anti-predator phenotypes and the trade-off with growth, but intra-specific population density and bottom substrate had a greater influence than predator density. Conclusions Our results show that two different predators can exert both functionally equivalent and inverse selection pressures on a single prey. Our field study suggests that abiotic factors and prey population density should be considered when attempting to explain phenotypic diversity in the wild. PMID:25517986

A new class of HIV-1 protease inhibitor: the crystallographic structure, inhibition and chemical synthesis of an aminimide peptide isostere.

PubMed

Rutenber, E E; McPhee, F; Kaplan, A P; Gallion, S L; Hogan, J C; Craik, C S; Stroud, R M

1996-09-01

The essential role of HIV-1 protease (HIV-1 PR) in the viral life cycle makes it an attractive target for the development of substrate-based inhibitors that may find efficacy as anti-AIDS drugs. However, resistance has arisen to potent peptidomimetic drugs necessitating the further development of novel chemical backbones for diversity based chemistry focused on probing the active site for inhibitor interactions and binding modes that evade protease resistance. AQ148 is a potent inhibitor of HIV-1 PR and represents a new class of transition state analogues incorporating an aminimide peptide isostere. A 3-D crystallographic structure of AQ148, a tetrapeptide isostere, has been determined in complex with its target HIV-1 PR to a resolution of 2.5 A and used to evaluate the specific structural determinants of AQ148 potency and to correlate structure-activity relationships within the class of related compounds. AQ148 is a competitive inhibitor of HIV-1 PR with a Ki value of 137 nM. Twenty-nine derivatives have been synthesized and chemical modifications have been made at the P1, P2, P1', and P2' sites. The atomic resolution structure of AQ148 bound to HIV-1 PR reveals both an inhibitor binding mode that closely resembles that of other peptidomimetic inhibitors and specific protein/inhibitor interactions that correlate with structure-activity relationships. The structure provides the basis for the design, synthesis and evaluation of the next generation of hydroxyethyl aminimide inhibitors. The aminimide peptide isostere is a scaffold with favorable biological properties well suited to both the combinatorial methods of peptidomimesis and the rational design of potent and specific substrate-based analogues.