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.

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.

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.

LRAT-specific domain facilitates vitamin A metabolism by domain swapping in HRASLS3

DOE PAGES

Golczak, Marcin; Sears, Avery E.; Kiser, Philip D.; ...

2014-11-10

Cellular uptake of vitamin A, production of visual chromophore and triglyceride homeostasis in adipocytes depend on two representatives of the vertebrate N1pC/P60 protein family, lecithin:retinol acyltransferase (LRAT) and HRAS-like tumor suppressor 3 (HRASLS3). Both proteins function as lipid-metabolizing enzymes but differ in their substrate preferences and dominant catalytic activity. The mechanism of this catalytic diversity is not understood. In this paper, by using a gain-of-function approach, we identified a specific sequence responsible for the substrate specificity of N1pC/P60 proteins. A 2.2-Å crystal structure of the HRASLS3-LRAT chimeric enzyme in a thioester catalytic intermediate state revealed a major structural rearrangement accompaniedmore » by three-dimensional domain swapping dimerization not observed in native HRASLS proteins. Structural changes affecting the active site environment contributed to slower hydrolysis of the catalytic intermediate, supporting efficient acyl transfer. Finally, these findings reveal structural adaptation that facilitates selective catalysis and mechanism responsible for diverse substrate specificity within the LRAT-like enzyme family.« less

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.

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.

Different substrates and starter inocula govern microbial community structures in biogas reactors.

PubMed

Satpathy, Preseela; Steinigeweg, Sven; Cypionka, Heribert; Engelen, Bert

2016-01-01

The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.

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

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.

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.

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.

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

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.

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.

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.

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.

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.

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

In situ environment rather than substrate type dictates microbial community structure of biofilms in a cold seep system

PubMed Central

Lee, On On; Wang, Yong; Tian, Renmao; Zhang, Weipeng; Shek, Chun Shum; Bougouffa, Salim; Al-Suwailem, Abdulaziz; Batang, Zenon B.; Xu, Wei; Wang, Guang Chao; Zhang, Xixiang; Lafi, Feras F.; Bajic, Vladmir B.; Qian, Pei-Yuan

2014-01-01

Using microscopic and molecular techniques combined with computational analysis, this study examined the structure and composition of microbial communities in biofilms that formed on different artificial substrates in a brine pool and on a seep vent of a cold seep in the Red Sea to test our hypothesis that initiation of the biofilm formation and spreading mode of microbial structures differs between the cold seep and the other aquatic environments. Biofilms on different substrates at two deployment sites differed morphologically, with the vent biofilms having higher microbial abundance and better structural features than the pool biofilms. Microbes in the pool biofilms were more taxonomically diverse and mainly composed of various sulfate-reducing bacteria whereas the vent biofilms were exclusively dominated by sulfur-oxidizing Thiomicrospira. These results suggest that the redox environments at the deployment sites might have exerted a strong selection on microbes in the biofilms at two sites whereas the types of substrates had limited effects on the biofilm development. PMID:24399144

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.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

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.

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.

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.

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Diversity-oriented synthesis of a library of substituted tetrahydropyrones using oxidative carbon-hydrogen bond activation and click chemistry.

PubMed

Zaware, Nilesh; Laporte, Matthew G; Farid, Ramy; Liu, Lei; Wipf, Peter; Floreancig, Paul E

2011-05-02

Eighteen (2RS,6RS)-2-(4-methoxyphenyl)-6-(substituted ethyl)dihydro-2H-pyran-4(3H)ones were synthesized via a DDQ-mediated oxidative carbon-hydrogen bond activation reaction. Fourteen of these tetrahydropyrans were substituted with triazoles readily assembled via azide-alkyne click-chemistry reactions. Examples of a linked benzotriazole and pyrazole motif were also prepared. To complement the structural diversity, the alcohol substrates were obtained from stereoselective reductions of the tetrahydropyrone. This library provides rapid access to structurally diverse non-natural compounds to be screened against a variety of biological targets.

Molecular Basis of Substrate Recognition and Degradation by Human Presequence Protease

PubMed Central

King, John V.; Liang, Wenguang G.; Scherpelz, Kathryn P.; Schilling, Alexander B.; Meredith, Stephen C.; Tang, Wei-Jen

2014-01-01

Summary Human Presequence Protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing a ∼13,300Å3 catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and amyloid-β (Aβ), the latter of which contributes to Alzheimer's disease pathogenesis. Here we report crystal structures for hPreP alone and in complex with Aβ, which show that hPreP uses size-exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aβ binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP. PMID:24931469

Structural basis for dynamic mechanism of nitrate/nitrite antiport by NarK

NASA Astrophysics Data System (ADS)

Fukuda, Masahiro; Takeda, Hironori; Kato, Hideaki E.; Doki, Shintaro; Ito, Koichi; Maturana, Andrés D.; Ishitani, Ryuichiro; Nureki, Osamu

2015-05-01

NarK belongs to the nitrate/nitrite porter (NNP) family in the major facilitator superfamily (MFS) and plays a central role in nitrate uptake across the membrane in diverse organisms, including archaea, bacteria, fungi and plants. Although previous studies provided insight into the overall structure and the substrate recognition of NarK, its molecular mechanism, including the driving force for nitrate transport, remained elusive. Here we demonstrate that NarK is a nitrate/nitrite antiporter, using an in vitro reconstituted system. Furthermore, we present the high-resolution crystal structures of NarK from Escherichia coli in the nitrate-bound occluded, nitrate-bound inward-open and apo inward-open states. The integrated structural, functional and computational analyses reveal the nitrate/nitrite antiport mechanism of NarK, in which substrate recognition is coupled to the transport cycle by the concomitant movement of the transmembrane helices and the key tyrosine and arginine residues in the substrate-binding site.

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.

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

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.

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.

Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

PubMed Central

Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

2013-01-01

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

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

Brettanomyces bruxellensis population survey reveals a diploid-triploid complex structured according to substrate of isolation and geographical distribution.

PubMed

Avramova, Marta; Cibrario, Alice; Peltier, Emilien; Coton, Monika; Coton, Emmanuel; Schacherer, Joseph; Spano, Giuseppe; Capozzi, Vittorio; Blaiotta, Giuseppe; Salin, Franck; Dols-Lafargue, Marguerite; Grbin, Paul; Curtin, Chris; Albertin, Warren; Masneuf-Pomarede, Isabelle

2018-03-07

Brettanomyces bruxellensis is a unicellular fungus of increasing industrial and scientific interest over the past 15 years. Previous studies revealed high genotypic diversity amongst B. bruxellensis strains as well as strain-dependent phenotypic characteristics. Genomic assemblies revealed that some strains harbour triploid genomes and based upon prior genotyping it was inferred that a triploid population was widely dispersed across Australian wine regions. We performed an intraspecific diversity genotypic survey of 1488 B. bruxellensis isolates from 29 countries, 5 continents and 9 different fermentation niches. Using microsatellite analysis in combination with different statistical approaches, we demonstrate that the studied population is structured according to ploidy level, substrate of isolation and geographical origin of the strains, underlying the relative importance of each factor. We found that geographical origin has a different contribution to the population structure according to the substrate of origin, suggesting an anthropic influence on the spatial biodiversity of this microorganism of industrial interest. The observed clustering was correlated to variable stress response, as strains from different groups displayed variation in tolerance to the wine preservative sulfur dioxide (SO 2 ). The potential contribution of the triploid state for adaptation to industrial fermentations and dissemination of the species B. bruxellensis is discussed.

Evaluating the Substrate Selectivity of Alkyladenine DNA Glycosylase: The Synergistic Interplay of Active Site Flexibility and Water Reorganization.

PubMed

Lenz, Stefan A P; Wetmore, Stacey D

2016-02-09

Human alkyladenine DNA glycosylase (AAG) functions as part of the base excision repair (BER) pathway by cleaving the N-glycosidic bond that connects nucleobases to the sugar-phosphate backbone in DNA. AAG targets a range of structurally diverse purine lesions using nonspecific DNA-protein π-π interactions. Nevertheless, the enzyme discriminates against the natural purines and is inhibited by pyrimidine lesions. This study uses molecular dynamics simulations and seven different neutral or charged substrates, inhibitors, or canonical purines to probe how the bound nucleotide affects the conformation of the AAG active site, and the role of active site residues in dictating substrate selectivity. The neutral substrates form a common DNA-protein hydrogen bond, which results in a consistent active site conformation that maximizes π-π interactions between the aromatic residues and the nucleobase required for catalysis. Nevertheless, subtle differences in DNA-enzyme contacts for different neutral substrates explain observed differential catalytic efficiencies. In contrast, the exocyclic amino groups of the natural purines clash with active site residues, which leads to catalytically incompetent DNA-enzyme complexes due to significant reorganization of active site water. Specifically, water resides between the A nucleobase and the active site aromatic amino acids required for catalysis, while a shift in the position of the general base (E125) repositions (potentially nucleophilic) water away from G. Despite sharing common amino groups, the methyl substituents in cationic purine lesions (3MeA and 7MeG) exhibit repulsion with active site residues, which repositions the damaged bases in the active site in a manner that promotes their excision. Overall, we provide a structural explanation for the diverse yet discriminatory substrate selectivity of AAG and rationalize key kinetic data available for the enzyme. Specifically, our results highlight the complex interplay of many different DNA-protein interactions used by AAG to facilitate BER, as well as the crucial role of the general base and water (nucleophile) positioning. The insights gained from our work will aid the understanding of the function of other enzymes that use flexible active sites to exhibit diverse substrate specificity.

Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

PubMed Central

Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

2015-01-01

Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403

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.

Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

PubMed

Shisler, Krista A; Broderick, Joan B

2014-03-15

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. Copyright © 2014. Published by Elsevier Inc.

Illumina MiSeq sequencing reveals microbial community in HA process for dyeing wastewater treatment fed with different co-substrates.

PubMed

Xie, Xuehui; Liu, Na; Ping, Jing; Zhang, Qingyun; Zheng, Xiulin; Liu, Jianshe

2018-06-01

In present study, a hydrolysis acidification (HA) reactor was used for simulated dyeing wastewater treatment. Co-substrates included starch, glucose, sucrose, yeast extract (YE) and peptone were fed sequentially into the HA reactor to enhance the HA process effects. The performance of the HA reactor and the microbial community structure in HA process were investigated under different co-substrates conditions. Results showed that different co-substrates had different influences on the performance of HA reactor. The highest decolorization (50.64%) and COD removal rate (60.73%) of the HA reactor were obtained when sucrose was as the co-substrate. And it found that carbon co-substrates starch, glucose and sucrose exhibited better decolorization and higher COD removal efficiency of the HA reactor than the nitrogen co-substrates YE and peptone. Microbial community structure in the HA process was analyzed by Illumina MiSeq sequencing. Results revealed different co-substrates had different influences on the community structure and microbial diversity in HA process. It was considered that sucrose could enrich the species such as Raoultella, Desulfovibrio, Tolumonas, Clostridium, which might be capable of degrading the dyes. Sucrose was considered to be the best co-substrate of enhancing the HA reactor's performance in this study. This work would provide deep insight into the influence of many different co-substrates on HA reactor performance and microbial communities in HA process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Microbial-Catalyzed Biotransformation of Multifunctional Triterpenoids Derived from Phytonutrients

PubMed Central

Shah, Syed Adnan Ali; Tan, Huey Ling; Sultan, Sadia; Mohd Faridz, Muhammad Afifi Bin; Mohd Shah, Mohamad Azlan Bin; Nurfazilah, Sharifah; Hussain, Munawar

2014-01-01

Microbial-catalyzed biotransformations have considerable potential for the generation of an enormous variety of structurally diversified organic compounds, especially natural products with complex structures like triterpenoids. They offer efficient and economical ways to produce semi-synthetic analogues and novel lead molecules. Microorganisms such as bacteria and fungi could catalyze chemo-, regio- and stereospecific hydroxylations of diverse triterpenoid substrates that are extremely difficult to produce by chemical routes. During recent years, considerable research has been performed on the microbial transformation of bioactive triterpenoids, in order to obtain biologically active molecules with diverse structures features. This article reviews the microbial modifications of tetranortriterpenoids, tetracyclic triterpenoids and pentacyclic triterpenoids. PMID:25003642

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

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.

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

Detecting Below-Ground Processes, Diversity, and Ecosystem Function in a Savanna Ecosystem Using Spectroscopy Across Different Vegetation Layers

NASA Astrophysics Data System (ADS)

Cavender-Bares, J.; Schweiger, A. K.; Madritch, M. D.; Gamon, J. A.; Hobbie, S. E.; Montgomery, R.; Townsend, P. A.

2017-12-01

Above-and below-ground plant traits are important for substrate input to the rhizosphere. The substrate composition of the rhizosphere, in turn, affects the diversity of soil organisms, influences soil biochemistry, and water content, and resource availability for plant growth. This has substantial consequences for ecosystem functions, such as above-ground productivity and stability. Above-ground plant chemical and structural traits can be linked to the characteristics of other plant organs, including roots. Airborne imaging spectroscopy has been successfully used to model and predict chemical and structural traits of the above-ground vegetation. However, remotely sensed images capture, almost exclusively, signals from the top of the canopy, providing limited direct information about understory vegetation. Here, we use a data set collected in a savanna ecosystem consisting of spectral measurements gathered at the leaf, the whole plant, and vegetation canopy level to test for hypothesized linkages between above- and below-ground processes that influence root biomass, soil biochemistry, and the diversity of the soil community. In this environment, consisting of herbaceous vegetation intermixed with shrubs and trees growing at variable densities, we investigate the contribution of different vegetation strata to soil characteristics and test the ability of imaging spectroscopy to detect these in plant communities with contrasting vertical structure.

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

Greatest soil microbial diversity found in micro-habitats

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

Bach, Elizabeth M.; Williams, Ryan J.; Hargreaves, Sarah K.

Microbial interactions occur in habitats much smaller than typically considered in classic ecological studies. This study uses soil aggregates to examine soil microbial community composition and structure of both bacteria and fungi at a microbially relevant scale. Aggregates were isolated from three land management systems in central Iowa, USA to test if aggregate-level microbial responses were sensitive to large-scale shifts in plant community and management practices. Bacteria and fungi exhibited similar patterns of community structure and diversity among soil aggregates, regardless of land management. Microaggregates supported more diverse microbial communities, both taxonomically and functionally. Calculation of a weighted proportional wholemore » soil diversity, which accounted for microbes found in aggregate fractions, resulted in 65% greater bacterial richness and 100% greater fungal richness over independently sampled whole soil. Our results show microaggregates support a previously unrecognized diverse microbial community that likely effects microbial access and metabolism of soil substrates.« less

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

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.

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

Influence of substrate type on microbial community structure in vertical-flow constructed wetland treating polluted river water.

PubMed

Guan, Wei; Yin, Min; He, Tao; Xie, Shuguang

2015-10-01

Microorganisms attached on the surfaces of substrate materials in constructed wetland play crucial roles in the removal of organic and inorganic pollutants. However, the impact of substrate material on wetland microbial community structure remains unclear. Moreover, little is known about microbial community in constructed wetland purifying polluted surface water. In this study, Illumina high-throughput sequencing was applied to profile the spatial variation of microbial communities in three pilot-scale surface water constructed wetlands with different substrate materials (sand, zeolite, and gravel). Bacterial community diversity and structure showed remarkable spatial variation in both sand and zeolite wetland systems, but changed slightly in gravel wetland system. Bacterial community was found to be significantly influenced by wetland substrate type. A number of bacterial groups were detected in wetland systems, including Proteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria, Cyanobacteria, Nitrospirae, Planctomycetes, Actinobacteria, Firmicutes, Chlorobi, Spirochaetae, Gemmatimonadetes, Deferribacteres, OP8, WS3, TA06, and OP3, while Proteobacteria (accounting for 29.1-62.3 %), mainly composed of Alpha-, Beta-, Gamma-, and Deltaproteobacteria, showed the dominance and might contribute to the effective reduction of organic pollutants. In addition, Nitrospira-like microorganisms were abundant in surface water constructed wetlands.

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

Impacts of supplementing chemical fertilizers with organic fertilizers manufactured using pig manure as a substrate on the spread of tetracycline resistance genes in soil.

PubMed

Kang, Yijun; Hao, Yangyang; Shen, Min; Zhao, Qingxin; Li, Qing; Hu, Jian

2016-08-01

Using pig manure (PM) compost as a partial substitute for the conventional chemical fertilizers (CFs) is considered an effective approach in sustainable agricultural systems. This study aimed to analyze the impacts of supplementing CF with organic fertilizers (OFs) manufactured using pig manure as a substrate on the spread of tetracycline resistance genes (TRGs) as well as the community structures and diversities of tetracycline-resistant bacteria (TRB) in bulk and cucumber rhizosphere soils. In this study, three organic fertilizers manufactured using the PM as a substrate, namely fresh PM, common OF, and bio-organic fertilizer (BF), were supplemented with a CF. Composted manures combined with a CF did not significantly increase TRB compared with the CF alone, but PM treatment resulted in the long-term survival of TRB in soil. The use of CF+PM also increased the risk of spreading TRGs in soil. As beneficial microorganisms in BF may function as reservoirs for the spread of antibiotic resistance genes, care should be taken when adding them to the OF matrix. The PM treatment significantly altered the community structures and increased the species diversity of TRB, especially in the rhizosphere soil. BF treatment caused insignificant changes in the community structure of TRB compared with CF treatment, yet it reduced the species diversities of TRB in soil. Thus, the partial use of fresh PM as a substitute for CF could increase the risk of spread of TRGs. Apart from plant growth promotion, BF was a promising fertilizer owing to its potential ability to control TRGs. Copyright © 2016 Elsevier Inc. All rights reserved.

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

Structural basis of reverse nucleotide polymerization

PubMed Central

Nakamura, Akiyoshi; Nemoto, Taiki; Heinemann, Ilka U.; Yamashita, Keitaro; Sonoda, Tomoyo; Komoda, Keisuke; Tanaka, Isao; Söll, Dieter; Yao, Min

2013-01-01

Nucleotide polymerization proceeds in the forward (5′-3′) direction. This tenet of the central dogma of molecular biology is found in diverse processes including transcription, reverse transcription, DNA replication, and even in lagging strand synthesis where reverse polymerization (3′-5′) would present a “simpler” solution. Interestingly, reverse (3′-5′) nucleotide addition is catalyzed by the tRNA maturation enzyme tRNAHis guanylyltransferase, a structural homolog of canonical forward polymerases. We present a Candida albicans tRNAHis guanylyltransferase-tRNAHis complex structure that reveals the structural basis of reverse polymerization. The directionality of nucleotide polymerization is determined by the orientation of approach of the nucleotide substrate. The tRNA substrate enters the enzyme’s active site from the opposite direction (180° flip) compared with similar nucleotide substrates of canonical 5′-3′ polymerases, and the finger domains are on opposing sides of the core palm domain. Structural, biochemical, and phylogenetic data indicate that reverse polymerization appeared early in evolution and resembles a mirror image of the forward process. PMID:24324136

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.

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

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

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

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.

Animal social networks as substrate for cultural behavioural diversity.

PubMed

Whitehead, Hal; Lusseau, David

2012-02-07

We used individual-based stochastic models to examine how social structure influences the diversity of socially learned behaviour within a non-human population. For continuous behavioural variables we modelled three forms of dyadic social learning, averaging the behavioural value of the two individuals, random transfer of information from one individual to the other, and directional transfer from the individual with highest behavioural value to the other. Learning had potential error. We also examined the transfer of categorical behaviour between individuals with random directionality and two forms of error, the adoption of a randomly chosen existing behavioural category or the innovation of a new type of behaviour. In populations without social structuring the diversity of culturally transmitted behaviour increased with learning error and population size. When the populations were structured socially either by making individuals members of permanent social units or by giving them overlapping ranges, behavioural diversity increased with network modularity under all scenarios, although the proportional increase varied considerably between continuous and categorical behaviour, with transmission mechanism, and population size. Although functions of the form e(c)¹(m)⁻(c)² + (c)³(Log(N)) predicted the mean increase in diversity with modularity (m) and population size (N), behavioural diversity could be highly unpredictable both between simulations with the same set of parameters, and within runs. Errors in social learning and social structuring generally promote behavioural diversity. Consequently, social learning may be considered to produce culture in populations whose social structure is sufficiently modular. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

NASA Astrophysics Data System (ADS)

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C. K.; Wu, Qiaqing; Zhu, Dunming

2016-05-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines.

New recombinant cyclohexylamine oxidase variants for deracemization of secondary amines by orthogonally assaying designed mutants with structurally diverse substrates

PubMed Central

Li, Guangyue; Yao, Peiyuan; Cong, Peiqian; Ren, Jie; Wang, Lei; Feng, Jinhui; Lau, Peter C.K.; Wu, Qiaqing; Zhu, Dunming

2016-01-01

To further expand the substrate range of the cyclohexylamine oxidase (CHAO) from Brevibacterium oxydans, a library of diverse mutants was created and assayed toward a group of structurally diverse substrates. Among them, mutants T198A and M226A exhibited enhanced activity relative to wt CHAO for most (S)-enantiomers of primary amines and some secondary amines. While mutants T198I, L199I, L199F, M226I and M226T were more active than wt CHAO toward the primary amines, mutants T198F, L199T, Y321A, Y321T, Y321I and Y321F enhanced the enzyme activity toward the secondary amines. In particular, mutant Y321I displayed an enhanced catalytic efficiency toward 1-(4-methoxybenzyl)-1, 2, 3, 4, 5, 6, 7, 8-octahydroisoquinoline (13). Whereas a double mutant, Y321I/M226T, acted on (S)-N-(prop-2-yn-1-yl)-2, 3-dihydro-1H-inden-1-amine [(S)-8]. Since (R)-8 is an irreversible inhibitor of monoamine oxidase and (S)-13 is an intermediate of dextromethorphan, a cough suppressant drug, deracemizations of 8 and 13 were carried out with crude enzyme extracts of the respective mutants. This resulted in 51% and 78% isolated yields of (R)-8 and (S)-13, respectively, each with high enantiomeric excess (93% and 99% ee). The results demonstrated the application potential of the evolved CHAO mutants in drug synthesis requiring chiral secondary amines. PMID:27138090

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.

Structure of Full-length Drosophila Cryptochrome

PubMed Central

Zoltowski, Brian D.; Vaidya, Anand T.; Top, Deniz; Widom, Joanne; Young, Michael W.; Crane, Brian R.

2011-01-01

The Cryptochrome/Photolyase (CRY/PL) family of photoreceptors mediates adaptive responses to UV and blue light exposure in all kingdoms of life 1; 2; 3; 4; 5. Whereas PLs function predominantly in DNA repair of cyclobutane pyrimidine dimers (CPDs)and 6-4 photolesions caused by UV radiation, CRYs transduce signals important for growth, development, magnetosensitivity and circadian clocks1; 2; 3; 4; 5. Despite these diverse functions, PLs/CRYs preserve a common structural fold, a dependence on flavin adenine dinucleotide (FAD) and an internal photoactivation mechanism3; 6. However, members of the CRY/PL family differ in the substrates recognized (protein or DNA), photochemical reactions catalyzed and involvement of an antenna cofactor. It is largely unknown how the animal CRYs that regulate circadian rhythms act on their substrates. CRYs contain a variable C-terminal tail that appends the conserved PL homology domain (PHD) and is important for function 7; 8; 9; 10; 11; 12. Herein, we report a 2.3 Å resolution crystal structure of Drosophila CRY with an intact C-terminus. The C-terminal helix docks in the analogous groove that binds DNA substrates in PLs. Conserved Trp536 juts into the CRY catalytic center to mimic PL recognition of DNA photolesions. The FAD anionic semiquinone found in the crystals assumes a conformation to facilitate restructuring of the tail helix. These results help reconcile the diverse functions of the CRY/PL family by demonstrating how conserved protein architecture, and photochemistry can be elaborated into a range of light-driven functions. PMID:22080955

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

PubMed Central

Cuthbertson, Leslie; Kos, Veronica; Whitfield, Chris

2010-01-01

Summary: Complex glycoconjugates play critical roles in the biology of microorganisms. Despite the remarkable diversity in glycan structures and the bacteria that produce them, conserved themes are evident in the biosynthesis-export pathways. One of the primary pathways involves representatives of the ATP-binding cassette (ABC) transporter superfamily. These proteins are responsible for the export of a wide variety of cell surface oligo- and polysaccharides in both Gram-positive and Gram-negative bacteria. Recent investigations of the structure and function of ABC transporters involved in the export of lipopolysaccharide O antigens have revealed two fundamentally different strategies for coupling glycan polymerization to export. These mechanisms are distinguished by the presence (or absence) of characteristic nonreducing terminal modifications on the export substrates, which serve as chain termination and/or export signals, and by the presence (or absence) of a discrete substrate-binding domain in the nucleotide-binding domain polypeptide of the ABC transporter. A bioinformatic survey examining ABC exporters from known oligo- and polysaccharide biosynthesis loci identifies conserved nucleotide-binding domain protein families that correlate well with themes in the structures and assembly of glycans. The familial relationships among the ABC exporters generate hypotheses concerning the biosynthesis of structurally diverse oligo- and polysaccharides, which play important roles in the biology of bacteria with different lifestyles. PMID:20805402

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

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis*

PubMed Central

Goblirsch, Brandon R.; Jensen, Matthew R.; Mohamed, Fatuma A.; Wackett, Lawrence P.; Wilmot, Carrie M.

2016-01-01

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety—unusual for a thiolase—are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation. PMID:27815501

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

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

Goblirsch, Brandon R.; Jensen, Matthew R.; Mohamed, Fatuma A.

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry aremore » precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety—unusual for a thiolase—are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys143) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C12 and C14) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ117) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.« less

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

Evolving marine biomimetics for regenerative dentistry.

PubMed

Green, David W; Lai, Wing-Fu; Jung, Han-Sung

2014-05-13

New products that help make human tissue and organ regeneration more effective are in high demand and include materials, structures and substrates that drive cell-to-tissue transformations, orchestrate anatomical assembly and tissue integration with biology. Marine organisms are exemplary bioresources that have extensive possibilities in supporting and facilitating development of human tissue substitutes. Such organisms represent a deep and diverse reserve of materials, substrates and structures that can facilitate tissue reconstruction within lab-based cultures. The reason is that they possess sophisticated structures, architectures and biomaterial designs that are still difficult to replicate using synthetic processes, so far. These products offer tantalizing pre-made options that are versatile, adaptable and have many functions for current tissue engineers seeking fresh solutions to the deficiencies in existing dental biomaterials, which lack the intrinsic elements of biofunctioning, structural and mechanical design to regenerate anatomically correct dental tissues both in the culture dish and in vivo.

Evolving Marine Biomimetics for Regenerative Dentistry

PubMed Central

Green, David W.; Lai, Wing-Fu; Jung, Han-Sung

2014-01-01

New products that help make human tissue and organ regeneration more effective are in high demand and include materials, structures and substrates that drive cell-to-tissue transformations, orchestrate anatomical assembly and tissue integration with biology. Marine organisms are exemplary bioresources that have extensive possibilities in supporting and facilitating development of human tissue substitutes. Such organisms represent a deep and diverse reserve of materials, substrates and structures that can facilitate tissue reconstruction within lab-based cultures. The reason is that they possess sophisticated structures, architectures and biomaterial designs that are still difficult to replicate using synthetic processes, so far. These products offer tantalizing pre-made options that are versatile, adaptable and have many functions for current tissue engineers seeking fresh solutions to the deficiencies in existing dental biomaterials, which lack the intrinsic elements of biofunctioning, structural and mechanical design to regenerate anatomically correct dental tissues both in the culture dish and in vivo. PMID:24828293

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.

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

Structure of the Epiphyte Community in a Tropical Montane Forest in SW China

PubMed Central

Zhao, Mingxu; Geekiyanage, Nalaka; Xu, Jianchu; Khin, Myo Myo; Nurdiana, Dian Ridwan; Paudel, Ekananda; Harrison, Rhett Daniel

2015-01-01

Vascular epiphytes are an understudied and particularly important component of tropical forest ecosystems. However, owing to the difficulties of access, little is known about the properties of epiphyte-host tree communities and the factors structuring them, especially in Asia. We investigated factors structuring the vascular epiphyte-host community and its network properties in a tropical montane forest in Xishuangbanna, SW China. Vascular epiphytes were surveyed in six plots located in mature forests. Six host and four micro-site environmental factors were investigated. Epiphyte diversity was strongly correlated with host size (DBH, diameter at breast height), while within hosts the highest epiphyte diversity was in the middle canopy and epiphyte diversity was significantly higher in sites with canopy soil or a moss mat than on bare bark. DBH, elevation and stem height explained 22% of the total variation in the epiphyte species assemblage among hosts, and DBH was the most important factor which alone explained 6% of the variation. Within hosts, 51% of the variation in epiphyte assemblage composition was explained by canopy position and substrate, and the most important single factor was substrate which accounted for 16% of the variation. Analysis of network properties indicated that the epiphyte host community was highly nested, with a low level of epiphyte specialization, and an almost even interaction strength between epiphytes and host trees. Together, these results indicate that large trees harbor a substantial proportion of the epiphyte community in this forest. PMID:25856457

Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members.

PubMed

Dohrmann, Anja B; Baumert, Susann; Klingebiel, Lars; Weiland, Peter; Tebbe, Christoph C

2011-03-01

Microbial conversion of organic waste or harvested plant material into biogas has become an attractive technology for energy production. Biogas is produced in reactors under anaerobic conditions by a consortium of microorganisms which commonly include bacteria of the genus Clostridium. Since the genus Clostridium also harbors some highly pathogenic members in its phylogenetic cluster I, there has been some concern that an unintended growth of such pathogens might occur during the fermentation process. Therefore this study aimed to follow how process parameters affect the diversity of Bacteria in general, and the diversity of Clostridium cluster I members in particular. The development of both communities was followed in model biogas reactors from start-up during stable methanogenic conditions. The biogas reactors were run with either cattle or pig manures as substrates, and both were operated at mesophilic and thermophilic conditions. The structural diversity was analyzed independent of cultivation using a PCR-based detection of 16S rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP). Genetic profiles indicated that both bacterial and clostridial communities evolved in parallel, and the community structures were highly influenced by both substrate and temperature. Sequence analysis of 16S rRNA genes recovered from prominent bands from SSCP profiles representing Clostridia detected no pathogenic species. Thus, this study gave no indication that pathogenic clostridia would be enriched as dominant community members in biogas reactors fed with manure.

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.

Switchable regioselectivity in amine-catalysed asymmetric cycloadditions

NASA Astrophysics Data System (ADS)

Zhou, Zhi; Wang, Zhou-Xiang; Zhou, Yuan-Chun; Xiao, Wei; Ouyang, Qin; Du, Wei; Chen, Ying-Chun

2017-06-01

Building small-molecule libraries with structural and stereogenic diversity plays an important role in drug discovery. The development of switchable intermolecular cycloaddition reactions from identical substrates in different regioselective fashions would provide an attractive protocol. However, this also represents a challenge in organic chemistry, because it is difficult to control regioselectivity to afford the products exclusively and at the same time achieve high levels of stereoselectivity. Here, we report the diversified cycloadditions of α‧-alkylidene-2-cyclopentenones catalysed by cinchona-derived primary amines. An asymmetric γ,β‧-regioselective intermolecular [6+2] cycloaddition reaction with 3-olefinic (7-aza)oxindoles is realized through the in situ generation of formal 4-aminofulvenes, while a different β,γ-regioselective [2+2] cycloaddition reaction with maleimides to access fused cyclobutanes is disclosed. In contrast, an intriguing α,γ-regioselective [4+2] cycloaddition reaction is uncovered with the same set of substrates, by employing an unprecedented dual small-molecule catalysis of amines and thiols. All of the cycloaddition reactions exhibit excellent regio- and stereoselectivity, producing a broad spectrum of chiral architectures with high structural diversity and molecular complexity.

Photoelectron spectroscopy study of the electronic structures at CoPc/Bi(111) interface

NASA Astrophysics Data System (ADS)

Sun, Haoliang; Liang, Zhaofeng; Shen, Kongchao; Hu, Jinbang; Ji, Gengwu; Li, Zheshen; Li, Haiyang; Zhu, Zhiyuan; Li, Jiong; Gao, Xingyu; Han, Huang; Jiang, Zheng; Song, Fei

2017-07-01

Self-assembly of functional molecules on solid substrate has been recognized as an appealing approach for the fabrication of diverse nanostructures for nanoelectronics. Herein, we investigate the growth of cobalt phthalocyanine (CoPc) on a Bi(111) surface with focus on the interface electronic structures utilizing photoelectron spectroscopy. While charge transfer from bismuth substrate to the molecule results in the emergence of an interface component in the Co 3p core level at lower binding energy, core-levels associated to the molecular ligand (C 1s and N 1s) are less influenced by the adsorption. In addition, density functional theory (DFT) calculations also support the empirical inference that the molecule-substrate interaction mainly involves the out-of-plane empty Co 3d orbital and bismuth states. Finally, valence band spectra demonstrate the molecule-substrate interaction is induced by interface charge transfer, agreeing well with core level measurements. Charge transfer is shown to be mainly from the underlying bismuth substrate to the empty states located at the central Co atom in the CoPc molecules. This report may provide a fundamental basis to the on-surface engineering of interfaces for molecular devices and spintronics.

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

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

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

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

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.

The compositional and evolutionary logic of metabolism

NASA Astrophysics Data System (ADS)

Braakman, Rogier; Smith, Eric

2013-02-01

Metabolism is built on a foundation of organic chemistry, and employs structures and interactions at many scales. Despite these sources of complexity, metabolism also displays striking and robust regularities in the forms of modularity and hierarchy, which may be described compactly in terms of relatively few principles of composition. These regularities render metabolic architecture comprehensible as a system, and also suggests the order in which layers of that system came into existence. In addition metabolism also serves as a foundational layer in other hierarchies, up to at least the levels of cellular integration including bioenergetics and molecular replication, and trophic ecology. The recapitulation of patterns first seen in metabolism, in these higher levels, motivates us to interpret metabolism as a source of causation or constraint on many forms of organization in the biosphere. Many of the forms of modularity and hierarchy exhibited by metabolism are readily interpreted as stages in the emergence of catalytic control by living systems over organic chemistry, sometimes recapitulating or incorporating geochemical mechanisms. We identify as modules, either subsets of chemicals and reactions, or subsets of functions, that are re-used in many contexts with a conserved internal structure. At the small molecule substrate level, module boundaries are often associated with the most complex reaction mechanisms, catalyzed by highly conserved enzymes. Cofactors form a biosynthetically and functionally distinctive control layer over the small-molecule substrate. The most complex members among the cofactors are often associated with the reactions at module boundaries in the substrate networks, while simpler cofactors participate in widely generalized reactions. The highly tuned chemical structures of cofactors (sometimes exploiting distinctive properties of the elements of the periodic table) thereby act as ‘keys’ that incorporate classes of organic reactions within biochemistry. Module boundaries provide the interfaces where change is concentrated, when we catalogue extant diversity of metabolic phenotypes. The same modules that organize the compositional diversity of metabolism are argued, with many explicit examples, to have governed long-term evolution. Early evolution of core metabolism, and especially of carbon-fixation, appears to have required very few innovations, and to have used few rules of composition of conserved modules, to produce adaptations to simple chemical or energetic differences of environment without diverse solutions and without historical contingency. We demonstrate these features of metabolism at each of several levels of hierarchy, beginning with the small-molecule metabolic substrate and network architecture, continuing with cofactors and key conserved reactions, and culminating in the aggregation of multiple diverse physical and biochemical processes in cells.

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse.

PubMed

Agarwal, Vinayak; Miles, Zachary D; Winter, Jaclyn M; Eustáquio, Alessandra S; El Gamal, Abrahim A; Moore, Bradley S

2017-04-26

Naturally produced halogenated compounds are ubiquitous across all domains of life where they perform a multitude of biological functions and adopt a diversity of chemical structures. Accordingly, a diverse collection of enzyme catalysts to install and remove halogens from organic scaffolds has evolved in nature. Accounting for the different chemical properties of the four halogen atoms (fluorine, chlorine, bromine, and iodine) and the diversity and chemical reactivity of their organic substrates, enzymes performing biosynthetic and degradative halogenation chemistry utilize numerous mechanistic strategies involving oxidation, reduction, and substitution. Biosynthetic halogenation reactions range from simple aromatic substitutions to stereoselective C-H functionalizations on remote carbon centers and can initiate the formation of simple to complex ring structures. Dehalogenating enzymes, on the other hand, are best known for removing halogen atoms from man-made organohalogens, yet also function naturally, albeit rarely, in metabolic pathways. This review details the scope and mechanism of nature's halogenation and dehalogenation enzymatic strategies, highlights gaps in our understanding, and posits where new advances in the field might arise in the near future.

A one-pot, microwave-influenced synthesis of diverse small molecules by multicomponent reaction cascades.

PubMed

Santra, Soumava; Andreana, Peter R

2007-11-22

Small molecule diversity can be achieved in a single synthetic operation from bifunctional substrates in the absence of additives and under the influence of microwaves with complete control of pathway selectivity. The preliminary Ugi four-component coupling products give rise to three structurally distinct scaffolds that are dependent on solvent effects and sterics. 2,5-Diketopiperazines (Type A), 2-azaspiro[4.5]deca-6,9-diene-3,8-diones (Type B), and thiophene-derived Diels-Alder tricyclic lactams (Type C) predominate in this reaction cascade.

Influence of a dominant consumer species reverses at increased diversity.

PubMed

Brandt, Margarita; Witman, Jon D; Chiriboga, Angel I

2012-04-01

Theory and experiments indicate that changes in consumer diversity affect benthic community structure and ecosystem functioning. Although the effects of consumer diversity have been tested in the laboratory and the field, little is known about effects of consumer diversity in the subtidal zone, one of the largest marine habitats. We investigated the grazing effects of sea urchins on algal abundance and benthic community structure in a natural subtidal habitat of the Galápagos Islands. Three species of urchins (Eucidaris, Lytechinus, and Tripneustes) were manipulated in inclusion cages following a replacement design with three levels of species richness (one, two, and three species) with all possible two-species urchin combinations. Identity was the main factor accounting for changes in the percentage of substrate grazed and benthic community structure. Two out of the three two-species assemblages grazed more than expected, suggesting a richness effect, but analyses revealed that this increased grazing was due to a sampling effect of the largest and commercially valued urchin species, Tripneustes. Benthic community structure in treatments with Eucidaris, Lytechinus, and Tripneustes alone was significantly different at the end of the experiment, suggesting that resource use differentiation occurred. Communities in Tripneustes enclosures were characterized by abundant crustose coralline algae and grazed substrate, while those without it contained abundant green foliose algae (Ulva sp.). An unexpected emergent property of the system was that the most species-rich urchin assemblage underyielded, grazing less than any other assemblage with Tripneustes, effectively reversing its dominant influence observed in the two-species treatments. While further experiments are needed to discern the mechanisms of underyielding, it may be related to changing interspecific interactions as richness increases from two to three species or to density-dependent Tripneustes grazing. This study highlights the general importance of evaluating consumer richness effects across the entire range of species richness considered, as the performance of the most species-rich consumer assemblage could not be predicted by manipulations of intermediate levels of consumer species richness.

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.

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

Structure and Spatial Distribution of the Chironomidae Community in Mesohabitats in a First Order Stream at the Poço D'Anta Municipal Biological Reserve in Brazil

PubMed Central

Vescovi Rosa, Beatriz Figueiraujo Jabour; de Oliveira, Vívian Campos; Alves, Roberto da Gama

2011-01-01

The Chironomidae occupy different habitats along the lotic system with their distribution determined by different factors such as the substrate characteristics and water speed. The input of vegetable material from the riparian forest allows a higher habitat diversity and food to the benthic fauna. The main aim of this paper is to verify the structure and spatial distribution of the Chironomidae fauna in different mesohabitats in a first order stream located at a Biological Reserve in the southeast of Brazil. In the months of July, August, and September 2007, and in January, February, and March 2008, samples were collected with a hand net (250 µm) in the following mesohabitats: litter from riffles, litter from pools, and sediment from pools. The community structure of each mesohabitat was analyzed through the abundance of organisms, taxa richness, Pielou's evenness, Shannon's diversity, and taxa dominance. Similarity among the mesohabitats was obtained by Cluster analysis, and Chironomidae larvae distribution through the Correspondence analysis. Indicator species analysis was used to identify possible taxa preference for a determined mesohabitat. The analyzed mesohabitats showed high species richness and diversity favored by the large environmental heterogeneity. Some taxa were indicators of the type of mesohabitat. The substrate was the main factor that determined taxa distribution in relation to water flow differences (riffle and pool). Stream characteristics such as low water speed and the presence of natural mechanisms of retention may have provided a higher faunistic similarity between the areas with different flows. The results showed that the physical characteristics of each environment presented a close relationship with the structure and spatial distribution of the Chironomidae fauna in lotic systems. PMID:21529258

Agricultural management and labile carbon additions affect soil microbial community structure and interact with carbon and nitrogen cycling.

PubMed

Berthrong, Sean T; Buckley, Daniel H; Drinkwater, Laurie E

2013-07-01

We investigated how conversion from conventional agriculture to organic management affected the structure and biogeochemical function of soil microbial communities. We hypothesized the following. (1) Changing agricultural management practices will alter soil microbial community structure driven by increasing microbial diversity in organic management. (2) Organically managed soil microbial communities will mineralize more N and will also mineralize more N in response to substrate addition than conventionally managed soil communities. (3) Microbial communities under organic management will be more efficient and respire less added C. Soils from organically and conventionally managed agroecosystems were incubated with and without glucose ((13)C) additions at constant soil moisture. We extracted soil genomic DNA before and after incubation for TRFLP community fingerprinting of soil bacteria and fungi. We measured soil C and N pools before and after incubation, and we tracked total C respired and N mineralized at several points during the incubation. Twenty years of organic management altered soil bacterial and fungal community structure compared to continuous conventional management with the bacterial differences caused primarily by a large increase in diversity. Organically managed soils mineralized twice as much NO3 (-) as conventionally managed ones (44 vs. 23 μg N/g soil, respectively) and increased mineralization when labile C was added. There was no difference in respiration, but organically managed soils had larger pools of C suggesting greater efficiency in terms of respiration per unit soil C. These results indicate that the organic management induced a change in community composition resulting in a more diverse community with enhanced activity towards labile substrates and greater capacity to mineralize N.

High-fidelity large area nano-patterning of silicon with femtosecond light sheet

NASA Astrophysics Data System (ADS)

Sidhu, Mehra S.; Munjal, Pooja; Singh, Kamal P.

2018-01-01

We employ a femtosecond light sheet generated by a cylindrical lens to rapidly produce high-fidelity nano-structures over large area on silicon surface. The Fourier analysis of electron microscopy images of the laser-induced surface structures reveals sharp peaks indicating good homogeneity. We observed an emergence of second-order spatial periodicity on increasing the scan speed. Our reliable approach may rapidly nano-pattern curved solid surfaces and tiny objects for diverse potential applications in optical devices, structural coloring, plasmonic substrates and in high-harmonic generation.

Structural Basis of J Cochaperone Binding and Regulation of Hsp70

PubMed Central

Jiang, Jianwen; Maes, E. Guy; Taylor, Alex B; Wang, Liping; Hinck, Andrew P; Lafer, Eileen M; Sousa, Rui

2007-01-01

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) towards a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, while both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles. PMID:17996706

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.

Crystal Structures of Active Fully Assembled Substrate- and Product-Bound Complexes of UDP-N-Acetylmuramic Acid:l-Alanine Ligase (MurC) from Haemophilus influenzae

PubMed Central

Mol, Clifford D.; Brooun, Alexei; Dougan, Douglas R.; Hilgers, Mark T.; Tari, Leslie W.; Wijnands, Robert A.; Knuth, Mark W.; McRee, Duncan E.; Swanson, Ronald V.

2003-01-01

UDP-N-acetylmuramic acid:l-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg2+ and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-l-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn2+ have been determined to 1.85- and 1.7-Å resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the γ-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates. PMID:12837790

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

PubMed

Mol, Clifford D; Brooun, Alexei; Dougan, Douglas R; Hilgers, Mark T; Tari, Leslie W; Wijnands, Robert A; Knuth, Mark W; McRee, Duncan E; Swanson, Ronald V

2003-07-01

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Mesophotic bioerosion: Variability and structural impact on U.S. Virgin Island deep reefs

NASA Astrophysics Data System (ADS)

Weinstein, David K.; Smith, Tyler B.; Klaus, James S.

2014-10-01

Mesophotic reef corals, found 30-150 m below sea level, build complex structures that provide habitats for diverse ecosystems. Whereas bioerosion is known to impact the development and persistence of shallow reef structures, little is known regarding the extent of mesophotic bioerosion or how it might affect deeper reef geomorphology and carbonate accretion. Originally pristine experimental coral substrates and collected coral rubble were both used to investigate the variation and significance of mesophotic coral reef bioerosion south of St. Thomas, U.S. Virgin Islands. Bioerosion rates were calculated from experimental coral substrates exposed as framework for 1 and 2 years at four structurally distinct mesophotic coral reef habitats (between 30 and 45 m) as well as at a mid-shelf patch reef (21 m) and a shallow fringing patch reef (9 m). The long-term effects of macroboring were assessed by examining coral rubble collected at all sites. Overall, differences in bioerosional processes were found between shallow and mesophotic reefs. Increases in bioerosion on experimental substrates (amount of weight lost) were related to both decreasing seawater depth and increasing biomass of bioeroding parrotfish. Significant differences in coral skeleton bioerosion rates were also found between the transitional mesophotic reef zone (30-35 m) and the upper mesophotic reef zone (35-50 m) after 2 years of exposure, ranging from - 19.6 to 3.7 g/year. Total coral rubble macroboring was greater at most deep sites compared to shallower sites. Bioerosional grazing was found to dominate initial substrate modification in reefs 30.7 m and shallower, but sponges are believed to act as the main time-averaged long-term substrate bioeroders in reefs between 35 and 50 m. Although initial substrate bioerosion rates of a uniform substrate were relatively homogeneous in the 35-50 m depth zone, comparison of site composition suggests that mesophotic bioerosion will vary depending on the amount, location, and type of available substrate, and the duration both coral rubble and in situ coral framework are exposed on the seafloor. These variations may exaggerate pronounced structural differences in mesophotic reef habitats that experience few other methods of erosion.

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.

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

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

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis.

PubMed

Goblirsch, Brandon R; Jensen, Matthew R; Mohamed, Fatuma A; Wackett, Lawrence P; Wilmot, Carrie M

2016-12-23

Phylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a β-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolase-are unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys 143 ) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C 12 and C 14 ) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Gluβ 117 ) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural variability and species diversity of a dwarf Caribbean dry forest

Treesearch

E. Medina; E. Cuevas; S. Molina; A.E. Lugo; O. Ramos

2010-01-01

Low stature woody vegetation of the south-west coast of Puerto Rico grows on a rocky calcareous substrate where plants can only root in holes, cracks, and crevices accumulating water and sediments that allow seed germination and seedling development. Being in a coastal location these communities are influenced by steady onshore winds, high solar radiation, and salt...

Direct visualization of critical hydrogen atoms in a pyridoxal 5'-phosphate enzyme.

PubMed

Dajnowicz, Steven; Johnston, Ryne C; Parks, Jerry M; Blakeley, Matthew P; Keen, David A; Weiss, Kevin L; Gerlits, Oksana; Kovalevsky, Andrey; Mueser, Timothy C

2017-10-16

Enzymes dependent on pyridoxal 5'-phosphate (PLP, the active form of vitamin B 6 ) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. Quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.Pyridoxal 5'-phosphate (PLP) is a ubiquitous co factor for diverse enzymes, among them aspartate aminotransferase. Here the authors use neutron crystallography, which allows the visualization of the positions of hydrogen atoms, and computation to characterize the catalytic mechanism of the enzyme.

Roles of type II thioesterases and their application for secondary metabolite yield improvement.

PubMed

Kotowska, Magdalena; Pawlik, Krzysztof

2014-09-01

A large number of antibiotics and other industrially important microbial secondary metabolites are synthesized by polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs). These multienzymatic complexes provide an enormous flexibility in formation of diverse chemical structures from simple substrates, such as carboxylic acids and amino acids. Modular PKSs and NRPSs, often referred to as megasynthases, have brought about a special interest due to the colinearity between enzymatic domains in the proteins working as an "assembly line" and the chain elongation and modification steps. Extensive efforts toward modified compound biosynthesis by changing organization of PKS and NRPS domains in a combinatorial manner laid good grounds for rational design of new structures and their controllable biosynthesis as proposed by the synthetic biology approach. Despite undeniable progress made in this field, the yield of such "unnatural" natural products is often not satisfactory. Here, we focus on type II thioesterases (TEIIs)--discrete hydrolytic enzymes often encoded within PKS and NRPS gene clusters which can be used to enhance product yield. We review diverse roles of TEIIs (removal of aberrant residues blocking the megasynthase, participation in substrate selection, intermediate, and product release) and discuss their application in new biosynthetic systems utilizing PKS and NRPS parts.

Structural analysis of a putative SAM-dependent methyltransferase, YtqB, from Bacillus subtilis.

PubMed

Park, Sun Cheol; Song, Wan Seok; Yoon, Sung-il

2014-04-18

S-adenosyl-L-methionine (SAM)-dependent methyltransferases (MTases) methylate diverse biological molecules using a SAM cofactor. The ytqB gene of Bacillus subtilis encodes a putative MTase and its biological function has never been characterized. To reveal the structural features and the cofactor binding mode of YtqB, we have determined the crystal structures of YtqB alone and in complex with its cofactor, SAM, at 1.9 Å and 2.2 Å resolutions, respectively. YtqB folds into a β-sheet sandwiched by two α-helical layers, and assembles into a dimeric form. Each YtqB monomer contains one SAM binding site, which shapes SAM into a slightly curved conformation and exposes the reactive methyl group of SAM potentially to a substrate. Our comparative structural analysis of YtqB and its homologues indicates that YtqB is a SAM-dependent class I MTase, and provides insights into the substrate binding site of YtqB. Copyright © 2014 Elsevier Inc. All rights reserved.

Structural and functional diversity of microbial communities from a lake sediment contaminated with trenbolone, an endocrine-disrupting chemical.

PubMed

Radl, Viviane; Pritsch, Karin; Munch, Jean Charles; Schloter, Michael

2005-09-01

Effects of trenbolone (TBOH), a hormone used in cattle production, on the structure and function of microbial communities in a fresh water sediment from a lake in Southern Germany were studied in a microcosm experiment. The microbial community structure and the total gene pool of the sediment, assessed by 16S rRNA/rDNA and RAPD fingerprint analysis, respectively, were not significantly affected by TBOH. In contrast, the N-acetyl-glucosaminidase activity was almost 50% lower in TBOH treated samples (P<0.05). Also, the substrate utilization potential, measured using the BIOLOG system, was reduced after TBOH treatment. Interestingly, this potential did not recover at the end of the experiment, i.e. 19 days after the addition of the chemical. Repeated application of TBOH did not lead to an additional reduction in the substrate utilization potential. Overall results indicate that microbial community function was more sensitive to TBOH treatment than the community structure and the total gene pool.

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

Cityscape genetics: structural vs. functional connectivity of an urban lizard population.

PubMed

Beninde, Joscha; Feldmeier, Stephan; Werner, Maike; Peroverde, Daniel; Schulte, Ulrich; Hochkirch, Axel; Veith, Michael

2016-10-01

Functional connectivity is essential for the long-term persistence of populations. However, many studies assess connectivity with a focus on structural connectivity only. Cityscapes, namely urban landscapes, are particularly dynamic and include numerous potential anthropogenic barriers to animal movements, such as roads, traffic or buildings. To assess and compare structural connectivity of habitats and functional connectivity of gene flow of an urban lizard, we here combined species distribution models (SDMs) with an individual-based landscape genetic optimization procedure. The most important environmental factors of the SDMs are structural diversity and substrate type, with high and medium levels of structural diversity as well as open and rocky/gravel substrates contributing most to structural connectivity. By contrast, water cover was the best model of all environmental factors following landscape genetic optimization. The river is thus a major barrier to gene flow, while of the typical anthropogenic factors only buildings showed an effect. Nonetheless, using SDMs as a basis for landscape genetic optimization provided the highest ranked model for functional connectivity. Optimizing SDMs in this way can provide a sound basis for models of gene flow of the cityscape, and elsewhere, while presence-only and presence-absence modelling approaches showed differences in performance. Additionally, interpretation of results based on SDM factor importance can be misleading, dictating more thorough analyses following optimization of SDMs. Such approaches can be adopted for management strategies, for example aiming to connect native common wall lizard populations or disconnect them from non-native introduced populations, which are currently spreading in many cities in Central Europe. © 2016 John Wiley & Sons Ltd.

Large-Scale Noniridescent Structural Color Printing Enabled by Infiltration-Driven Nonequilibrium Colloidal Assembly.

PubMed

Bai, Ling; Mai, Van Cuong; Lim, Yun; Hou, Shuai; Möhwald, Helmuth; Duan, Hongwei

2018-03-01

Structural colors originating from interaction of light with intricately arranged micro-/nanostructures have stimulated considerable interest because of their inherent photostability and energy efficiency. In particular, noniridescent structural color with wide viewing angle has been receiving increasing attention recently. However, no method is yet available for rapid and large-scale fabrication of full-spectrum structural color patterns with wide viewing angles. Here, infiltration-driven nonequilibrium assembly of colloidal particles on liquid-permeable and particle-excluding substrates is demonstrated to direct the particles to form amorphous colloidal arrays (ACAs) within milliseconds. The infiltration-assisted (IFAST) colloidal assembly opens new possibilities for rapid manufacture of noniridescent structural colors of ACAs and straightforward structural color mixing. Full-spectrum noniridescent structural colors are successfully produced by mixing primary structural colors of red, blue, and yellow using a commercial office inkjet printer. Rapid fabrication of large-scale structural color patterns with sophisticated color combination/layout by IFAST printing is realized. The IFAST technology is versatile for developing structural color patterns with wide viewing angles, as colloidal particles, inks, and substrates are flexibly designable for diverse applications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Diversity of microbes and potential exoelectrogenic bacteria on anode surface in microbial fuel cells.

PubMed

Sun, Yujiao; Zuo, Jiane; Cui, Longtao; Deng, Qian; Dang, Yan

2010-02-01

Single-chamber microbial fuel cells (MFCs), inoculated with anaerobic sludge and continuously run with two kinds of organic wastewater influents, were systemically investigated. The diversity of microbes, determined by 16S rDNA analysis, was analyzed on three anodes under different conditions. One anode was in a closed circuit in synthetic wastewater containing glucose. The other two anodes, in open or closed circuits, were fed effluent from an anaerobic reactor treating starch wastewater. The chemical oxygen demand (COD) removal efficiency was about 70%, and the exported voltages were about 450 mV. The 16S rDNA molecular clones of microbes on anode surfaces showed significant changes in Eubacterial structure under different conditions. gamma-Proteobacteria and the high G+C gram-positive groups were predominant in the synthetic wastewater, while epsilon-Proteobacteria predominated in the anaerobic reactor effluent. Known exoelectrogenic bacterial species composition also changed greatly depending on substrate. On the artificial substrate, 28% of the bacterial sequences were affiliated with Aeromonas, Pseudomonas, Geobacter, and Desulfobulbus. On the anaerobic effluent, only 6% were affiliated with Geobacter or Clostridium. Because only a few exoelectrogenic bacteria from MFCs have been directly isolated and studied, we compared the community structures of two bacterial anodes, in open and closed circuits, under the same substrate of anaerobic effluent in order to identify additional exoelectrogenic bacterial strains. Alcaligenes monasteriensis, Comamonas denitrificans, and Dechloromonas sp. were found to be potential exoelectrogenic bacteria worthy of further research.

Assessing the Regioselectivity of OleD-Catalyzed Glycosylation with a Diverse Set of Acceptors

PubMed Central

Zhou, Maoquan; Hamza, Adel; Zhan, Chang-Guo; Thorson, Jon S.

2013-01-01

To explore the acceptor regioselectivity of OleD-catalyzed glucosylation, the products of OleD-catalyzed reactions with the six structurally diverse acceptors - flavones (daidzein), isoflavones (flavopiridol), stilbenes (resveratrol), indole alkaloids (10-hydroxycamptothecin), and steroids (2-methoxyestradiol) - were determined. This study highlights the first synthesis of flavopiridol and 2-methoxyestradiol glucosides and confirms the ability of OleD to glucosylate both aromatic and aliphatic nucleophiles. In all cases, molecular dynamics simulations were consistent with the determined product distribution and suggest the potential to develop a virtual screening model to identify additional OleD substrates. PMID:23360118

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.

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.

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.

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.

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

The evolution of Lachancea thermotolerans is driven by geographical determination, anthropisation and flux between different ecosystems

PubMed Central

Bely, Marina; Masneuf-Pomarede, Isabelle; Jiranek, Vladimir; Albertin, Warren

2017-01-01

The yeast Lachancea thermotolerans (formerly Kluyveromyces thermotolerans) is a species with remarkable, yet underexplored, biotechnological potential. This ubiquist occupies a range of natural and anthropic habitats covering a wide geographic span. To gain an insight into L. thermotolerans population diversity and structure, 172 isolates sourced from diverse habitats worldwide were analysed using a set of 14 microsatellite markers. The resultant clustering revealed that the evolution of L. thermotolerans has been driven by the geography and ecological niche of the isolation sources. Isolates originating from anthropic environments, in particular grapes and wine, were genetically close, thus suggesting domestication events within the species. The observed clustering was further validated by several means including, population structure analysis, F-statistics, Mantel’s test and the analysis of molecular variance (AMOVA). Phenotypic performance of isolates was tested using several growth substrates and physicochemical conditions, providing added support for the clustering. Altogether, this study sheds light on the genotypic and phenotypic diversity of L. thermotolerans, contributing to a better understanding of the population structure, ecology and evolution of this non-Saccharomyces yeast. PMID:28910346

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

Similar PAH Fate in Anaerobic Digesters Inoculated with Three Microbial Communities Accumulating Either Volatile Fatty Acids or Methane

PubMed Central

Braun, Florence; Hamelin, Jérôme; Bonnafous, Anaïs; Delgenès, Nadine; Steyer, Jean-Philippe; Patureau, Dominique

2015-01-01

Urban sludge produced on wastewater treatment plants are often contaminated by organic pollutants such as polycyclic aromatic hydrocarbons (PAH). Their removal under methanogenic conditions was already reported, but the factors influencing this removal remain unclear. Here, we determined the influence of microbial communities on PAH removal under controlled physico-chemical conditions. Twelve mesophilic anaerobic digesters were inoculated with three microbial communities extracted from ecosystems with contrasting pollution histories: a PAH contaminated soil, a PCB contaminated sediment and a low contaminated anaerobic sludge. These anaerobic digesters were operated during 100 days in continuous mode. A sterilised activated sludge, spiked with 13 PAH at concentrations usually encountered in full-scale wastewater treatment plants, was used as substrate. The dry matter and volatile solid degradation, the biogas production rate and composition, the volatile fatty acids (VFA) production and the PAH removals were monitored. Bacterial and archaeal communities were compared in abundance (qPCR), in community structure (SSCP fingerprinting) and in dominant microbial species (454-pyrosequencing). The bioreactors inoculated with the community extracted from low contaminated anaerobic sludge showed the greater methane production. The PAH removals ranged from 10 % to 30 %, respectively, for high and low molecular weight PAH, whatever the inoculums tested, and were highly correlated with the dry matter and volatile solid removals. The microbial community structure and diversity differed with the inoculum source; this difference was maintained after the 100 days of digestion. However, the PAH removal was not correlated to these diverse structures and diversities. We hence obtained three functional stable consortia with two contrasted metabolic activities, and three different pictures of microbial diversity, but similar PAH and matter removals. These results confirm that PAH removal depends on the molecule type and on the solid matter removal. But, as PAH elimination is similar whether the solid substrate is degraded into VFA or into methane, it seems that the fermentative communities are responsible for their elimination. PMID:25874750

Similar PAH fate in anaerobic digesters inoculated with three microbial communities accumulating either volatile fatty acids or methane.

PubMed

Braun, Florence; Hamelin, Jérôme; Bonnafous, Anaïs; Delgenès, Nadine; Steyer, Jean-Philippe; Patureau, Dominique

2015-01-01

Urban sludge produced on wastewater treatment plants are often contaminated by organic pollutants such as polycyclic aromatic hydrocarbons (PAH). Their removal under methanogenic conditions was already reported, but the factors influencing this removal remain unclear. Here, we determined the influence of microbial communities on PAH removal under controlled physico-chemical conditions. Twelve mesophilic anaerobic digesters were inoculated with three microbial communities extracted from ecosystems with contrasting pollution histories: a PAH contaminated soil, a PCB contaminated sediment and a low contaminated anaerobic sludge. These anaerobic digesters were operated during 100 days in continuous mode. A sterilised activated sludge, spiked with 13 PAH at concentrations usually encountered in full-scale wastewater treatment plants, was used as substrate. The dry matter and volatile solid degradation, the biogas production rate and composition, the volatile fatty acids (VFA) production and the PAH removals were monitored. Bacterial and archaeal communities were compared in abundance (qPCR), in community structure (SSCP fingerprinting) and in dominant microbial species (454-pyrosequencing). The bioreactors inoculated with the community extracted from low contaminated anaerobic sludge showed the greater methane production. The PAH removals ranged from 10% to 30%, respectively, for high and low molecular weight PAH, whatever the inoculums tested, and were highly correlated with the dry matter and volatile solid removals. The microbial community structure and diversity differed with the inoculum source; this difference was maintained after the 100 days of digestion. However, the PAH removal was not correlated to these diverse structures and diversities. We hence obtained three functional stable consortia with two contrasted metabolic activities, and three different pictures of microbial diversity, but similar PAH and matter removals. These results confirm that PAH removal depends on the molecule type and on the solid matter removal. But, as PAH elimination is similar whether the solid substrate is degraded into VFA or into methane, it seems that the fermentative communities are responsible for their elimination.

Can misfolded proteins be beneficial? The HAMLET case.

PubMed

Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina

2009-01-01

By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation.

PubMed

Ariyawutthiphan, Orapin; Ose, Toyoyuki; Minami, Atsushi; Shinde, Sandip; Sinde, Sandip; Tsuda, Muneya; Gao, Yong-Gui; Yao, Min; Oikawa, Hideaki; Tanaka, Isao

2012-11-01

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Rapid change with depth in megabenthic structure-forming communities of the Makapu'u deep-sea coral bed

NASA Astrophysics Data System (ADS)

Long, Dustin J.; Baco, Amy R.

2014-01-01

Seamounts are largely unexplored undersea mountains rising abruptly from the ocean floor, which can support an increased abundance and diversity of organisms. Deep-sea corals are important benthic structure-formers on current-swept hard substrates in these habitats. While depth is emerging as a factor structuring the fauna of seamounts on a large spatial scale, most work addressing deep-sea coral and seamount community structure has not considered the role of small-scale variation in species distributions. Video from six ROV dives over a depth range of ~320-530 m were analyzed to assess the diversity and density of benthic megafaunal invertebrates across the Makapu'u deep-sea coral bed, offshore of Oahu, Hawaii. At the same time, the physical environment along the dive track was surveyed to relate biotic patterns with abiotic variables including depth, aspect, rugosity, substrate, slope and relief to test the factors structuring community assemblages. Despite the narrow range examined, depth was found to be the strongest structuring gradient, and six unique macrobenthic communities were found, with a 93% faunal dissimilarity over the depth surveyed. Relief, rugosity and slope were also factors in the final model. Alcyonacean octocorals were the dominant macrofaunal invertebrates at all but the deepest depth zone. The commercially harvested precious coral C. secundum was the dominant species at depths 370-470 m, with a distribution that is on average deeper than similar areas. This may be artificial due to the past harvesting of this species on the shallower portion of its range. Primnoid octocorals were the most abundant octocoral family overall. This work yields new insight on the spatial ecology of seamounts, pointing out that community changes can occur over narrow depth ranges and that communities can be structured by small-scale physiography.

Functional and structural diversity in GH62 α-L-arabinofuranosidases from the thermophilic fungus Scytalidium thermophilum

DOE PAGES

Kaur, Amrit Pal; Nocek, Boguslaw P.; Xu, Xiaohui; ...

2015-05-01

The genome of the thermophilic fungus Scytalidium thermophilum (strain CBS 625.91) harbours a wide range of genes involved in carbohydrate degradation, including three genes, abf62A, abf62B and abf62C, predicted to encode glycoside hydrolase family 62 (GH62) enzymes. Transcriptome analysis showed that only abf62A and abf62C are actively expressed during growth on diverse substrates including straws from barley, alfalfa, triticale and canola. The abf62A and abf62C genes were expressed in Escherichia coli and the resulting recombinant proteins were characterized. Calcium-free crystal structures of Abf62C in apo and xylotriose bound forms were determined to 1.23 and 1.48 Å resolution respectively. Site-directed mutagenesismore » confirmed Asp55, Asp171 and Glu230 as catalytic triad residues, and revealed the critical role of non-catalytic residues Asp194, Trp229 and Tyr338 in positioning the scissile α-L-arabinofuranoside bond at the catalytic site. Further, the +2R substrate-binding site residues Tyr168 and Asn339, as well as the +2NR residue Tyr226, are involved in accommodating long-chain xylan polymers. Overall, our structural and functional analysis highlights characteristic differences between Abf62A and Abf62C, which represent divergent subgroups in the GH62 family.« less

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

Variable distributional characteristics of substrate utilization patterns in activated sludge plants in Kuwait.

PubMed

Al-Mutairi, N Z

2009-02-01

The objective of this study was to determine the magnitude of microbial functional potential and community structure between three different WWTPs using the Lorenz curve method and to find the effect of seasonal variation on patterns of substrate utilization. Lorenz curve method was sensitive enough to detect short-term changes in microbial functional diversity between Riqqa, Umm Al-Haiman and Al-Jahra activated sludge systems and showed seasonal variations of the utilized carbon sources. Gini coefficient ranged from 0.21 to 0.8. Lorenz curves seemed particularly suitable to present microbial heterogeneity in term of inequality and to highlight the relative contribution of low-and high functional diversity for the three different types of mixed liquors. Correlation analysis of the experimental data show that the complement of the Gini coefficient was strongly and positively correlated with the Shannon index (r(xy)=0.89), evenness (r(xy)=0.91), and AWCD (r(xy)=0.95) at the 95% level of significance (alpha=0.05).

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.

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

Controls on bacterial and archaeal community structure and greenhouse gas production in natural, mined, and restored Canadian peatlands

PubMed Central

Basiliko, Nathan; Henry, Kevin; Gupta, Varun; Moore, Tim R.; Driscoll, Brian T.; Dunfield, Peter F.

2013-01-01

Northern peatlands are important global C reservoirs, largely because of their slow rates of microbial C mineralization. Particularly in sites that are heavily influenced by anthropogenic disturbances, there is scant information about microbial ecology and whether or not microbial community structure influences greenhouse gas production. This work characterized communities of bacteria and archaea using terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis of 16S rRNA and functional genes across eight natural, mined, or restored peatlands in two locations in eastern Canada. Correlations were explored among chemical properties of peat, bacterial and archaeal community structure, and carbon dioxide (CO2) and methane (CH4) production rates under oxic and anoxic conditions. Bacteria and archaea similar to those found in other peat soil environments were detected. In contrast to other reports, methanogen diversity was low in our study, with only 2 groups of known or suspected methanogens. Although mining and restoration affected substrate availability and microbial activity, these land-uses did not consistently affect bacterial or archaeal community composition. In fact, larger differences were observed between the two locations and between oxic and anoxic peat samples than between natural, mined, and restored sites, with anoxic samples characterized by less detectable bacterial diversity and stronger dominance by members of the phylum Acidobacteria. There were also no apparent strong linkages between prokaryote community structure and CH4 or CO2 production, suggesting that different organisms exhibit functional redundancy and/or that the same taxa function at very different rates when exposed to different peat substrates. In contrast to other earlier work focusing on fungal communities across similar mined and restored peatlands, bacterial and archaeal communities appeared to be more resistant or resilient to peat substrate changes brought about by these land uses. PMID:23914185

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.

Microbial eukaryotic distributions and diversity patterns in a deep-sea methane seep ecosystem.

PubMed

Pasulka, Alexis L; Levin, Lisa A; Steele, Josh A; Case, David H; Landry, Michael R; Orphan, Victoria J

2016-09-01

Although chemosynthetic ecosystems are known to support diverse assemblages of microorganisms, the ecological and environmental factors that structure microbial eukaryotes (heterotrophic protists and fungi) are poorly characterized. In this study, we examined the geographic, geochemical and ecological factors that influence microbial eukaryotic composition and distribution patterns within Hydrate Ridge, a methane seep ecosystem off the coast of Oregon using a combination of high-throughput 18S rRNA tag sequencing, terminal restriction fragment length polymorphism fingerprinting, and cloning and sequencing of full-length 18S rRNA genes. Microbial eukaryotic composition and diversity varied as a function of substrate (carbonate versus sediment), activity (low activity versus active seep sites), sulfide concentration, and region (North versus South Hydrate Ridge). Sulfide concentration was correlated with changes in microbial eukaryotic composition and richness. This work also revealed the influence of oxygen content in the overlying water column and water depth on microbial eukaryotic composition and diversity, and identified distinct patterns from those previously observed for bacteria, archaea and macrofauna in methane seep ecosystems. Characterizing the structure of microbial eukaryotic communities in response to environmental variability is a key step towards understanding if and how microbial eukaryotes influence seep ecosystem structure and function. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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.

Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

PubMed Central

Louie, Gordon; Noel, Joseph P.; Baran, Phil S.; Palfey, Bruce; Moore, Bradley S.

2013-01-01

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization. PMID:24162851

Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator

NASA Astrophysics Data System (ADS)

Deng, Zengqin; Wang, Qing; Liu, Zhao; Zhang, Manfeng; Machado, Ana Carolina Dantas; Chiu, Tsu-Pei; Feng, Chong; Zhang, Qi; Yu, Lin; Qi, Lei; Zheng, Jiangge; Wang, Xu; Huo, Xinmei; Qi, Xiaoxuan; Li, Xiaorong; Wu, Wei; Rohs, Remo; Li, Ying; Chen, Zhongzhou

2015-07-01

Ferric uptake regulator (Fur) plays a key role in the iron homeostasis of prokaryotes, such as bacterial pathogens, but the molecular mechanisms and structural basis of Fur-DNA binding remain incompletely understood. Here, we report high-resolution structures of Magnetospirillum gryphiswaldense MSR-1 Fur in four different states: apo-Fur, holo-Fur, the Fur-feoAB1 operator complex and the Fur-Pseudomonas aeruginosa Fur box complex. Apo-Fur is a transition metal ion-independent dimer whose binding induces profound conformational changes and confers DNA-binding ability. Structural characterization, mutagenesis, biochemistry and in vivo data reveal that Fur recognizes DNA by using a combination of base readout through direct contacts in the major groove and shape readout through recognition of the minor-groove electrostatic potential by lysine. The resulting conformational plasticity enables Fur binding to diverse substrates. Our results provide insights into metal ion activation and substrate recognition by Fur that suggest pathways to engineer magnetotactic bacteria and antipathogenic drugs.

Multisensor Analysis of Spectral Dimensionality and Soil Diversity in the Great Central Valley of California.

PubMed

Sousa, Daniel; Small, Christopher

2018-02-14

Planned hyperspectral satellite missions and the decreased revisit time of multispectral imaging offer the potential for data fusion to leverage both the spectral resolution of hyperspectral sensors and the temporal resolution of multispectral constellations. Hyperspectral imagery can also be used to better understand fundamental properties of multispectral data. In this analysis, we use five flight lines from the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) archive with coincident Landsat 8 acquisitions over a spectrally diverse region of California to address the following questions: (1) How much of the spectral dimensionality of hyperspectral data is captured in multispectral data?; (2) Is the characteristic pyramidal structure of the multispectral feature space also present in the low order dimensions of the hyperspectral feature space at comparable spatial scales?; (3) How much variability in rock and soil substrate endmembers (EMs) present in hyperspectral data is captured by multispectral sensors? We find nearly identical partitions of variance, low-order feature space topologies, and EM spectra for hyperspectral and multispectral image composites. The resulting feature spaces and EMs are also very similar to those from previous global multispectral analyses, implying that the fundamental structure of the global feature space is present in our relatively small spatial subset of California. Finally, we find that the multispectral dataset well represents the substrate EM variability present in the study area - despite its inability to resolve narrow band absorptions. We observe a tentative but consistent physical relationship between the gradation of substrate reflectance in the feature space and the gradation of sand versus clay content in the soil classification system.

Multisensor Analysis of Spectral Dimensionality and Soil Diversity in the Great Central Valley of California

PubMed Central

Small, Christopher

2018-01-01

Planned hyperspectral satellite missions and the decreased revisit time of multispectral imaging offer the potential for data fusion to leverage both the spectral resolution of hyperspectral sensors and the temporal resolution of multispectral constellations. Hyperspectral imagery can also be used to better understand fundamental properties of multispectral data. In this analysis, we use five flight lines from the Airborne Visible/Infrared Imaging Spectrometer (AVIRIS) archive with coincident Landsat 8 acquisitions over a spectrally diverse region of California to address the following questions: (1) How much of the spectral dimensionality of hyperspectral data is captured in multispectral data?; (2) Is the characteristic pyramidal structure of the multispectral feature space also present in the low order dimensions of the hyperspectral feature space at comparable spatial scales?; (3) How much variability in rock and soil substrate endmembers (EMs) present in hyperspectral data is captured by multispectral sensors? We find nearly identical partitions of variance, low-order feature space topologies, and EM spectra for hyperspectral and multispectral image composites. The resulting feature spaces and EMs are also very similar to those from previous global multispectral analyses, implying that the fundamental structure of the global feature space is present in our relatively small spatial subset of California. Finally, we find that the multispectral dataset well represents the substrate EM variability present in the study area – despite its inability to resolve narrow band absorptions. We observe a tentative but consistent physical relationship between the gradation of substrate reflectance in the feature space and the gradation of sand versus clay content in the soil classification system. PMID:29443900

Environmental Impact Research Program. Gravel Bar Mussel Communities: A Community Model.

DTIC Science & Technology

1987-10-01

flowing waters, where all but the coarse substrate has washed away, have stone flies, mayflies, blackflies, and caddis flies adapted for attachment and...bivalve genera are very important in the faunas of estuarine and fresh waters, as are the more diverse groups of freshwater gastropods . The only...relatively permanent river systems. In - their soft parts most freshwater mussels are structural>y rather stereotyped. UnIonaceans show adaptive

Diversity and biogeochemical structuring of bacterial communities across the Porangahau ridge accretionary prism, New Zealand

USGS Publications Warehouse

Hamdan, L.J.; Gillevet, P.M.; Pohlman, J.W.; Sikaroodi, M.; Greinert, J.; Coffin, R.B.

2011-01-01

Sediments from the Porangahau ridge, located off the northeastern coast of New Zealand, were studied to describe bacterial community structure in conjunction with differing biogeochemical regimes across the ridge. Low diversity was observed in sediments from an eroded basin seaward of the ridge and the community was dominated by uncultured members of the Burkholderiales. Chloroflexi/GNS and Deltaproteobacteria were abundant in sediments from a methane seep located landward of the ridge. Gas-charged and organic-rich sediments further landward had the highest overall diversity. Surface sediments, with the exception of those from the basin, were dominated by Rhodobacterales sequences associated with organic matter deposition. Taxa related to the Desulfosarcina/Desulfococcus and the JS1 candidates were highly abundant at the sulfate-methane transition zone (SMTZ) at three sites. To determine how community structure was influenced by terrestrial, pelagic and in situ substrates, sequence data were statistically analyzed against geochemical data (e.g. sulfate, chloride, nitrogen, phosphorous, methane, bulk inorganic and organic carbon pools) using the Biota-Environmental matching procedure. Landward of the ridge, sulfate was among the most significant structuring factors. Seaward of the ridge, silica and ammonium were important structuring factors. Regardless of the transect location, methane was the principal structuring factor on SMTZ communities. FEMS Microbiology Ecology ?? 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. No claim to original US government works.

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.

Hierarchical Materials Design by Pattern Transfer Printing of Self-Assembled Binary Nanocrystal Superlattices

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

Paik, Taejong; Yun, Hongseok; Fleury, Blaise

We demonstrate the fabrication of hierarchical materials by controlling the structure of highly ordered binary nanocrystal superlattices (BNSLs) on multiple length scales. Combinations of magnetic, plasmonic, semiconducting, and insulating colloidal nanocrystal (NC) building blocks are self-assembled into BNSL membranes via the liquid–interfacial assembly technique. Free-standing BNSL membranes are transferred onto topographically structured poly(dimethylsiloxane) molds via the Langmuir–Schaefer technique and then deposited in patterns onto substrates via transfer printing. BNSLs with different structural motifs are successfully patterned into various meso- and microstructures such as lines, circles, and even three-dimensional grids across large-area substrates. A combination of electron microscopy and grazing incidencemore » small-angle X-ray scattering (GISAXS) measurements confirm the ordering of NC building blocks in meso- and micropatterned BNSLs. This technique demonstrates structural diversity in the design of hierarchical materials by assembling BNSLs from NC building blocks of different composition and size by patterning BNSLs into various size and shape superstructures of interest for a broad range of applications.« less

Volcanic ash supports a diverse bacterial community in a marine mesocosm

USGS Publications Warehouse

Verena Witt,; Paul M Ayris,; Damby, David; Corrado Cimarelli,; Ulrich Kueppers,; Donald B Dingwell,; Gert Wörheide,

2017-01-01

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.

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.

Microbial Community Profiles in Wastewaters from Onsite Wastewater Treatment Systems Technology

PubMed Central

Jałowiecki, Łukasz; Chojniak, Joanna Małgorzata; Dorgeloh, Elmar; Hegedusova, Berta; Ejhed, Helene; Magnér, Jörgen; Płaza, Grażyna Anna

2016-01-01

The aim of the study was to determine the potential of community-level physiological profiles (CLPPs) methodology as an assay for characterization of the metabolic diversity of wastewater samples and to link the metabolic diversity patterns to efficiency of select onsite biological wastewater facilities. Metabolic fingerprints obtained from the selected samples were used to understand functional diversity implied by the carbon substrate shifts. Three different biological facilities of onsite wastewater treatment were evaluated: fixed bed reactor (technology A), trickling filter/biofilter system (technology B), and aerated filter system (the fluidized bed reactor, technology C). High similarities of the microbial community functional structures were found among the samples from the three onsite wastewater treatment plants (WWTPs), as shown by the diversity indices. Principal components analysis (PCA) showed that the diversity and CLPPs of microbial communities depended on the working efficiency of the wastewater treatment technologies. This study provided an overall picture of microbial community functional structures of investigated samples in WWTPs and discerned the linkages between microbial communities and technologies of onsite WWTPs used. The results obtained confirmed that metabolic profiles could be used to monitor treatment processes as valuable biological indicators of onsite wastewater treatment technologies efficiency. This is the first step toward understanding relations of technology types with microbial community patterns in raw and treated wastewaters. PMID:26807728

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

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

PubMed Central

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

Response of estuarine biofilm microbial community development to changes in dissolved oxygen and nutrient concentrations.

PubMed

Nocker, Andreas; Lepo, Joe Eugene; Martin, Linda Lin; Snyder, Richard Allan

2007-10-01

The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.

Degradation of Potassium Rock by Earthworms and Responses of Bacterial Communities in Its Gut and Surrounding Substrates after Being Fed with Mineral

PubMed Central

Liu, Dianfeng; Lian, Bin; Wang, Bin; Jiang, Guofang

2011-01-01

Background Earthworms are an ecosystem's engineers, contributing to a wide range of nutrient cycling and geochemical processes in the ecosystem. Their activities can increase rates of silicate mineral weathering. Their intestinal microbes usually are thought to be one of the key drivers of mineral degradation mediated by earthworms,but the diversities of the intestinal microorganisms which were relevant with mineral weathering are unclear. Methodology/Principal Findings In this report, we show earthworms' effect on silicate mineral weathering and the responses of bacterial communities in their gut and surrounding substrates after being fed with potassium-bearing rock powder (PBRP). Determination of water-soluble and HNO3-extractable elements indicated some elements such as Al, Fe and Ca were significantly released from mineral upon the digestion of earthworms. The microbial communities in earthworms' gut and the surrounding substrates were investigated by amplified ribosomal DNA restriction analysis (ARDRA) and the results showed a higher bacterial diversity in the guts of the earthworms fed with PBRP and the PBRP after being fed to earthworms. UPGMA dendrogram with unweighted UniFrac analysis, considering only taxa that are present, revealed that earthworms' gut and their surrounding substrate shared similar microbiota. UPGMA dendrogram with weighted UniFrac, considering the relative abundance of microbial lineages, showed the two samples from surrounding substrate and the two samples from earthworms' gut had similarity in microbial community, respectively. Conclusions/Significance Our results indicated earthworms can accelerate degradation of silicate mineral. Earthworms play an important role in ecosystem processe since they not only have some positive effects on soil structure, but also promote nutrient cycling of ecosystem by enhancing the weathering of minerals. PMID:22174903

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

In search of actionable targets for agrigenomics and microalgal biofuel production: sequence-structural diversity studies on algal and higher plants with a focus on GPAT protein.

PubMed

Misra, Namrata; Panda, Prasanna Kumar

2013-04-01

The triacylglycerol (TAG) pathway provides several targets for genetic engineering to optimize microalgal lipid productivity. GPAT (glycerol-3-phosphate acyltransferase) is a crucial enzyme that catalyzes the initial step of TAG biosynthesis. Despite many recent biochemical studies, a comprehensive sequence-structure analysis of GPAT across diverse lipid-yielding organisms is lacking. Hence, we performed a comparative genomic analysis of plastid-located GPAT proteins from 7 microalgae and 3 higher plants species. The close evolutionary relationship observed between red algae/diatoms and green algae/plant lineages in the phylogenetic tree were further corroborated by motif and gene structure analysis. The predicted molecular weight, amino acid composition, Instability Index, and hydropathicity profile gave an overall representation of the biochemical features of GPAT protein across the species under study. Furthermore, homology models of GPAT from Chlamydomonas reinhardtii, Arabidopsis thaliana, and Glycine max provided deep insights into the protein architecture and substrate binding sites. Despite low sequence identity found between algal and plant GPATs, the developed models exhibited strikingly conserved topology consisting of 14α helices and 9β sheets arranged in two domains. However, subtle variations in amino acids of fatty acyl binding site were identified that might influence the substrate selectivity of GPAT. Together, the results will provide useful resources to understand the functional and evolutionary relationship of GPAT and potentially benefit in development of engineered enzyme for augmenting algal biofuel production.

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.

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

Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

PubMed

Sunde, Margaret; Pham, Chi L L; Kwan, Ann H

2017-06-20

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

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.

A study on arrangement characteristics of microparticles in sedimentation on flat and round substrates

NASA Astrophysics Data System (ADS)

Yeo, Eunju; Son, Minhee; Kim, Kwanoh; Kim, Jeong Hwan; Yoo, Yeong-Eun; Choi, Doo-Sun; Kim, Jungchul; Yoon, Seok Ho; Yoon, Jae Sung

2017-12-01

Recent advances of microfabrication techniques have enabled diverse structures and devices on the microscale. This fabrication method using microparticles is one of the most promising technologies because it can provide a cost effective process for large areas. So, many researchers are studying modulation and manipulation of the microparticles in solution to obtain a proper arrangement. However, the microparticles are in sedimentation status during the process in many cases, which makes it difficult to control their arrangement. In this study, droplets containing microparticles were placed on a substrate with minimal force and we investigated the arrangement of these microparticles after evaporation of the liquid. Experiments have been performed with upward and downward substrates to change the direction of gravity. The geometry of substrates was also changed, which were flat or round. The results show that the arrangement depends on the size of particles and gravity and geometry of the substrate. The arrangement also depends on the movement of the contact line of the droplets, which may recede or be pinned during evaporation. This study is expected to provide a method of the fabrication process for microparticles which may not be easily manipulated due to sedimentation.

Phylum Level Change in the Cecal and Fecal Gut Communities of Rats Fed Diets Containing Different Fermentable Substrates Supports a Role for Nitrogen as a Factor Contributing to Community Structure

PubMed Central

Kalmokoff, Martin; Franklin, Jeff; Petronella, Nicholas; Green, Judy; Brooks, Stephen P.J.

2015-01-01

Fermentation differs between the proximal and distal gut but little is known regarding how the bacterial communities differ or how they are influenced by diet. In order to investigate this, we compared community diversity in the cecum and feces of rats by 16S rRNA gene content and DNA shot gun metagenomics after feeding purified diets containing different fermentable substrates. Gut community composition was dependent on the source of fermentable substrate included in the diet. Cecal communities were dominated by Firmicutes, and contained a higher abundance of Lachnospiraceae compared to feces. In feces, community structure was shifted by varying degrees depending on diet towards the Bacteroidetes, although this change was not always evident from 16S rRNA gene data. Multi-dimensional scaling analysis (PCoA) comparing cecal and fecal metagenomes grouped by location within the gut rather than by diet, suggesting that factors in addition to substrate were important for community change in the distal gut. Differentially abundant genes in each environment supported this shift away from the Firmicutes in the cecum (e.g., motility) towards the Bacteroidetes in feces (e.g., Bacteroidales transposons). We suggest that this phylum level change reflects a shift to ammonia as the primary source of nitrogen used to support continued microbial growth in the distal gut. PMID:25954902

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.

Phylum level change in the cecal and fecal gut communities of rats fed diets containing different fermentable substrates supports a role for nitrogen as a factor contributing to community structure.

PubMed

Kalmokoff, Martin; Franklin, Jeff; Petronella, Nicholas; Green, Judy; Brooks, Stephen P J

2015-05-06

Fermentation differs between the proximal and distal gut but little is known regarding how the bacterial communities differ or how they are influenced by diet. In order to investigate this, we compared community diversity in the cecum and feces of rats by 16S rRNA gene content and DNA shot gun metagenomics after feeding purified diets containing different fermentable substrates. Gut community composition was dependent on the source of fermentable substrate included in the diet. Cecal communities were dominated by Firmicutes, and contained a higher abundance of Lachnospiraceae compared to feces. In feces, community structure was shifted by varying degrees depending on diet towards the Bacteroidetes, although this change was not always evident from 16S rRNA gene data. Multi-dimensional scaling analysis (PCoA) comparing cecal and fecal metagenomes grouped by location within the gut rather than by diet, suggesting that factors in addition to substrate were important for community change in the distal gut. Differentially abundant genes in each environment supported this shift away from the Firmicutes in the cecum (e.g., motility) towards the Bacteroidetes in feces (e.g., Bacteroidales transposons). We suggest that this phylum level change reflects a shift to ammonia as the primary source of nitrogen used to support continued microbial growth in the distal gut.

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.

Plasmonic nanostructures through DNA-assisted lithography

PubMed Central

Shen, Boxuan; Linko, Veikko; Tapio, Kosti; Pikker, Siim; Lemma, Tibebe; Gopinath, Ashwin; Gothelf, Kurt V.; Kostiainen, Mauri A.; Toppari, J. Jussi

2018-01-01

Programmable self-assembly of nucleic acids enables the fabrication of custom, precise objects with nanoscale dimensions. These structures can be further harnessed as templates to build novel materials such as metallic nanostructures, which are widely used and explored because of their unique optical properties and their potency to serve as components of novel metamaterials. However, approaches to transfer the spatial information of DNA constructions to metal nanostructures remain a challenge. We report a DNA-assisted lithography (DALI) method that combines the structural versatility of DNA origami with conventional lithography techniques to create discrete, well-defined, and entirely metallic nanostructures with designed plasmonic properties. DALI is a parallel, high-throughput fabrication method compatible with transparent substrates, thus providing an additional advantage for optical measurements, and yields structures with a feature size of ~10 nm. We demonstrate its feasibility by producing metal nanostructures with a chiral plasmonic response and bowtie-shaped nanoantennas for surface-enhanced Raman spectroscopy. We envisage that DALI can be generalized to large substrates, which would subsequently enable scale-up production of diverse metallic nanostructures with tailored plasmonic features. PMID:29423446

C-terminal domains of bacterial proteases: structure, function and the biotechnological applications.

PubMed

Huang, J; Wu, C; Liu, D; Yang, X; Wu, R; Zhang, J; Ma, C; He, H

2017-01-01

C-terminal domains widely exist in the C-terminal region of multidomain proteases. As a β-sandwich domain in multidomain protease, the C-terminal domain plays an important role in proteolysis including regulation of the secretory process, anchoring and swelling the substrate molecule, presenting as an inhibitor for the preprotease and adapting the protein structural flexibility and stability. In this review, the diversity, structural characteristics and biological function of C-terminal protease domains are described. Furthermore, the application prospects of C-terminal domains, including polycystic kidney disease, prepeptidase C-terminal and collagen-binding domain, in the area of medicine and biological artificial materials are also discussed. © 2016 The Society for Applied Microbiology.

CVD synthesis of large-area, highly crystalline MoSe2 atomic layers on diverse substrates and application to photodetectors.

PubMed

Xia, Jing; Huang, Xing; Liu, Ling-Zhi; Wang, Meng; Wang, Lei; Huang, Ben; Zhu, Dan-Dan; Li, Jun-Jie; Gu, Chang-Zhi; Meng, Xiang-Min

2014-08-07

Synthesis of large-area, atomically thin transition metal dichalcogenides (TMDs) on diverse substrates is of central importance for the large-scale fabrication of flexible devices and heterojunction-based devices. In this work, we successfully synthesized a large area of highly-crystalline MoSe2 atomic layers on SiO2/Si, mica and Si substrates using a simple chemical vapour deposition (CVD) method at atmospheric pressure. Atomic force microscopy (AFM) and Raman spectroscopy reveal that the as-grown ultrathin MoSe2 layers change from a single layer to a few layers. Photoluminescence (PL) spectroscopy demonstrates that while the multi-layer MoSe2 shows weak emission peaks, the monolayer has a much stronger emission peak at ∼ 1.56 eV, indicating the transition from an indirect to a direct bandgap. Transmission electron microscopy (TEM) analysis confirms the single-crystallinity of MoSe2 layers with a hexagonal structure. In addition, the photoresponse performance of photodetectors based on MoSe2 monolayer was studied for the first time. The devices exhibit a rapid response of ∼ 60 ms and a good photoresponsivity of ∼ 13 mA/W (using a 532 nm laser at an intensity of 1 mW mm(-2) and a bias of 10 V), suggesting that MoSe2 monolayer is a promising material for photodetection applications.

Deviation of the typical AAA substrate-threading pore prevents fatal protein degradation in yeast Cdc48.

PubMed

Esaki, Masatoshi; Islam, Md Tanvir; Tani, Naoki; Ogura, Teru

2017-07-14

Yeast Cdc48 is a well-conserved, essential chaperone of ATPases associated with diverse cellular activity (AAA) proteins, which recognizes substrate proteins and modulates their conformations to carry out many cellular processes. However, the fundamental mechanisms underlying the diverse pivotal roles of Cdc48 remain unknown. Almost all AAA proteins form a ring-shaped structure with a conserved aromatic amino acid residue that is essential for proper function. The threading mechanism hypothesis suggests that this residue guides the intrusion of substrate proteins into a narrow pore of the AAA ring, thereby becoming unfolded. By contrast, the aromatic residue in one of the two AAA rings of Cdc48 has been eliminated through evolution. Here, we show that artificial retrieval of this aromatic residue in Cdc48 is lethal, and essential features to support the threading mechanism are required to exhibit the lethal phenotype. In particular, genetic and biochemical analyses of the Cdc48 lethal mutant strongly suggested that when in complex with the 20S proteasome, essential proteins are abnormally forced to thread through the Cdc48 pore to become degraded, which was not detected in wild-type Cdc48. Thus, the widely applicable threading model is less effective for wild-type Cdc48; rather, Cdc48 might function predominantly through an as-yet-undetermined mechanism.

In situ Generated Ruthenium Catalyst Systems Bearing Diverse N-Heterocyclic Carbene Precursors for Atom-Economic Amide Synthesis from Alcohols and Amines.

PubMed

Cheng, Hua; Xiong, Mao-Qian; Cheng, Chuan-Xiang; Wang, Hua-Jing; Lu, Qiang; Liu, Hong-Fu; Yao, Fu-Bin; Chen, Cheng; Verpoort, Francis

2018-02-16

The transition-metal-catalyzed direct synthesis of amides from alcohols and amines is herein demonstrated as a highly environmentally benign and atom-economic process. Among various catalyst systems, in situ generated N-heterocyclic carbene (NHC)-based ruthenium (Ru) halide catalyst systems have been proven to be active for this transformation. However, these existing catalyst systems usually require an additional ligand to achieve satisfactory results. In this work, through extensive screening of a diverse variety of NHC precursors, we discovered an active in situ catalyst system for efficient amide synthesis without any additional ligand. Notably, this catalyst system was found to be insensitive to the electronic effects of the substrates, and various electron-deficient substrates, which were not highly reactive with our previous catalyst systems, could be employed to afford the corresponding amides efficiently. Furthermore, mechanistic investigations were performed to provide a rationale for the high activity of the optimized catalyst system. NMR-scale reactions indicated that the rapid formation of a Ru hydride intermediate (signal at δ=-7.8 ppm in the 1 H NMR spectrum) after the addition of the alcohol substrate should be pivotal in establishing the high catalyst activity. Besides, HRMS analysis provided possible structures of the in situ generated catalyst system. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The t-Butylsulfinamide Lynchpin in Transition Metal-Mediated Multiscaffold Library Synthesis

PubMed Central

Bauer, Renato A.; DiBlasi, Christine M.; Tan, Derek S.

2010-01-01

A unified synthetic approach to diverse polycyclic scaffolds has been developed using transition metal-mediated cycloaddition and cyclization reactions of enynes and diynes. The t-butylsulfinamide group has been identified as a particularly versatile lynchpin in these reactions, with a reactivity profile uniquely suited for efficient, stereoselective substrate synthesis and downstream transformations. This approach provides ten distinct, functionalized scaffold classes related to common core structures in alkaloid and terpenoid natural products. PMID:20356070

Diversity of an aerial phototrophic coating of historic buildings in the former Auschwitz II-Birkenau concentration camp.

PubMed

Nowicka-Krawczyk, Paulina; Żelazna-Wieczorek, Joanna; Otlewska, Anna; Koziróg, Anna; Rajkowska, Katarzyna; Piotrowska, Małgorzata; Gutarowska, Beata; Żydzik-Białek, Agnieszka

2014-09-15

Aerial phototrophs colonize materials of anthropogenic origin, thus contributing to their biodeterioration. Structures preserved at the former Auschwitz II-Birkenau concentration and extermination camp show signs of degradation by cyanobacteria and algae. In order to protect the Auschwitz-Birkenau Memorial Site, diversity of aerial phototrophs growing on the historic buildings has been studied. Analyses of cyanobacterial and algal biofilms growing on various construction substrates were carried out in summer and winter. Multivariate data analyses were used to: characterize the diversity of cyanobacteria and algae growing in brick and wooden camp buildings depending on the research season, indicate preferences of cyanobacteria and algae in colonizing substrates, and to predict the environmental factor that most determines the growth of phototrophs. The biofilms were formed mainly by cyanobacteria, green algae and diatoms. The amount of cyanobacteria and algae in the biofilms was varied, which resulted from changes in climatic conditions, the type of substrate and the height at which the biofilms developed. In the summer, the ratio of cyanobacteria and algae groups was balanced, while in the winter, green algae and diatoms were dominant. Green algae showed a preference for colonizing plaster, wood and concrete, of which the walls and doors of the buildings were made. Their participation was correlated with a height gradient. Cyanobacteria and diatoms grew on bricks and soil on the floor of the buildings and temperature and relative humidity were the factors that modified their amount. Green algae were more cosmopolitan-occurred in dry places, potentially inaccessible to other organisms; therefore, they have been identified as the pioneer group in the prevailing climatic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Understanding microbial ecology can help improve biogas production in AD.

PubMed

Ferguson, Robert M W; Coulon, Frédéric; Villa, Raffaella

2018-06-16

454-Pyrosequencing and lipid fingerprinting were used to link anaerobic digestion (AD) process parameters (pH, alkalinity, volatile fatty acids (VFAs), biogas production and methane content) with the reactor microbial community structure and composition. AD microbial communities underwent stress conditions after changes in organic loading rate and digestion substrates. 454-Pyrosequencing analysis showed that, irrespectively of the substrate digested, methane content and pH were always significantly, and positively, correlated with community evenness. In AD, microbial communities with more even distributions of diversity are able to use parallel metabolic pathways and have greater functional stability; hence, they are capable of adapting and responding to disturbances. In all reactors, a decrease in methane content to <30% was always correlated with a 50% increase of Firmicutes sequences (particularly in operational taxonomic units (OTUs) related to Ruminococcaceae and Veillonellaceae). Whereas digesters producing higher methane content (above 60%), contained a high number of sequences related to Synergistetes and unidentified bacterial OTUs. Finally, lipid fingerprinting demonstrated that, under stress, the decrease in archaeal biomass was higher than the bacterial one, and that archaeal Phospholipid etherlipids (PLEL) levels were correlated to reactor performances. These results demonstrate that, across a number of parameters such as lipids, alpha and beta diversity, and OTUs, knowledge of the microbial community structure can be used to predict, monitor, or optimise AD performance. Copyright © 2018 Elsevier B.V. All rights reserved.

Novel proteases from the genome of the carnivorous plant Drosera capensis: structural prediction and comparative analysis

PubMed Central

Butts, Carter T.; Bierma, Jan C.; Martin, Rachel W.

2016-01-01

In his 1875 monograph on insectivorous plants, Darwin described the feeding reactions of Drosera flypaper traps and predicted that their secretions contained a “ferment” similar to mammalian pepsin, an aspartic protease. Here we report a high-quality draft genome sequence for the cape sundew, Drosera capensis, the first genome of a carnivorous plant from order Caryophyllales, which also includes the Venus flytrap (Dionaea) and the tropical pitcher plants (Nepenthes). This species was selected in part for its hardiness and ease of cultivation, making it an excellent model organism for further investigations of plant carnivory. Analysis of predicted protein sequences yields genes encoding proteases homologous to those found in other plants, some of which display sequence and structural features that suggest novel functionalities. Because the sequence similarity to proteins of known structure is in most cases too low for traditional homology modeling, 3D structures of representative proteases are predicted using comparative modeling with all-atom refinement. Although the overall folds and active residues for these proteins are conserved, we find structural and sequence differences consistent with a diversity of substrate recognition patterns. Finally, we predict differences in substrate specificities using in silico experiments, providing targets for structure/function studies of novel enzymes with biological and technological significance. PMID:27353064

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.

Physical soil architectural traits are functionally linked to carbon decomposition and bacterial diversity.

PubMed

Rabbi, S M F; Daniel, H; Lockwood, P V; Macdonald, C; Pereg, L; Tighe, M; Wilson, B R; Young, I M

2016-09-12

Aggregates play a key role in protecting soil organic carbon (SOC) from microbial decomposition. The objectives of this study were to investigate the influence of pore geometry on the organic carbon decomposition rate and bacterial diversity in both macro- (250-2000 μm) and micro-aggregates (53-250 μm) using field samples. Four sites of contrasting land use on Alfisols (i.e. native pasture, crop/pasture rotation, woodland) were investigated. 3D Pore geometry of the micro-aggregates and macro-aggregates were examined by X-ray computed tomography (μCT). The occluded particulate organic carbon (oPOC) of aggregates was measured by size and density fractionation methods. Micro-aggregates had 54% less μCT observed porosity but 64% more oPOC compared with macro-aggregates. In addition, the pore connectivity in micro-aggregates was lower than macro-aggregates. Despite both lower μCT observed porosity and pore connectivity in micro-aggregates, the organic carbon decomposition rate constant (Ksoc) was similar in both aggregate size ranges. Structural equation modelling showed a strong positive relationship of the concentration of oPOC with bacterial diversity in aggregates. We use these findings to propose a conceptual model that illustrates the dynamic links between substrate, bacterial diversity, and pore geometry that suggests a structural explanation for differences in bacterial diversity across aggregate sizes.

Physical soil architectural traits are functionally linked to carbon decomposition and bacterial diversity

PubMed Central

Rabbi, S. M. F.; Daniel, H.; Lockwood, P. V.; Macdonald, C.; Pereg, L.; Tighe, M.; Wilson, B. R.; Young, I. M.

2016-01-01

Aggregates play a key role in protecting soil organic carbon (SOC) from microbial decomposition. The objectives of this study were to investigate the influence of pore geometry on the organic carbon decomposition rate and bacterial diversity in both macro- (250–2000 μm) and micro-aggregates (53–250 μm) using field samples. Four sites of contrasting land use on Alfisols (i.e. native pasture, crop/pasture rotation, woodland) were investigated. 3D Pore geometry of the micro-aggregates and macro-aggregates were examined by X-ray computed tomography (μCT). The occluded particulate organic carbon (oPOC) of aggregates was measured by size and density fractionation methods. Micro-aggregates had 54% less μCT observed porosity but 64% more oPOC compared with macro-aggregates. In addition, the pore connectivity in micro-aggregates was lower than macro-aggregates. Despite both lower μCT observed porosity and pore connectivity in micro-aggregates, the organic carbon decomposition rate constant (Ksoc) was similar in both aggregate size ranges. Structural equation modelling showed a strong positive relationship of the concentration of oPOC with bacterial diversity in aggregates. We use these findings to propose a conceptual model that illustrates the dynamic links between substrate, bacterial diversity, and pore geometry that suggests a structural explanation for differences in bacterial diversity across aggregate sizes. PMID:27615807

High process yield rates of thermoplastic nanofluidic devices using a hybrid thermal assembly technique.

PubMed

Uba, Franklin I; Hu, Bo; Weerakoon-Ratnayake, Kumuditha; Oliver-Calixte, Nyote; Soper, Steven A

2015-02-21

Over the past decade, thermoplastics have been used as alternative substrates to glass and Si for microfluidic devices because of the diverse and robust fabrication protocols available for thermoplastics that can generate high production rates of the desired structures at low cost and with high replication fidelity, the extensive array of physiochemical properties they possess, and the simple surface activation strategies that can be employed to tune their surface chemistry appropriate for the intended application. While the advantages of polymer microfluidics are currently being realized, the evolution of thermoplastic-based nanofluidic devices is fraught with challenges. One challenge is assembly of the device, which consists of sealing a cover plate to the patterned fluidic substrate. Typically, channel collapse or substrate dissolution occurs during assembly making the device inoperable resulting in low process yield rates. In this work, we report a low temperature hybrid assembly approach for the generation of functional thermoplastic nanofluidic devices with high process yield rates (>90%) and with a short total assembly time (16 min). The approach involves thermally sealing a high T(g) (glass transition temperature) substrate containing the nanofluidic structures to a cover plate possessing a lower T(g). Nanofluidic devices with critical feature sizes ranging between 25-250 nm were fabricated in a thermoplastic substrate (T(g) = 104 °C) and sealed with a cover plate (T(g) = 75 °C) at a temperature significantly below the T(g) of the substrate. Results obtained from sealing tests revealed that the integrity of the nanochannels remained intact after assembly and devices were useful for fluorescence imaging at high signal-to-noise ratios. The functionality of the assembled devices was demonstrated by studying the stretching and translocation dynamics of dsDNA in the enclosed thermoplastic nanofluidic channels.

Homology modeling, molecular docking and molecular dynamics studies of the catalytic domain of chitin deacetylase from Cryptococcus laurentii strain RY1.

PubMed

Sarkar, Soumyadev; Gupta, Suchetana; Chakraborty, Writachit; Senapati, Sanjib; Gachhui, Ratan

2017-11-01

This study provides structural insights into chitin deacetylase, over-expressing under nitrogen limiting condition in Cryptococcus laurentii strain RY1. The enzyme converts chitin, the second most abundant natural biopolymer, to chitosan, which offers tremendous applications in diverse fields. To elucidate the structure-function relationship of this biologically and industrially important enzyme, a homology model of the catalytic domain was constructed. The stability of the structure was assessed by molecular dynamics simulation studies. Tryptophan 151 of the domain was identified to form hydrogen bond and stacking interaction with chitin upon docking. In Silico substitution of Tryptophan (W) to Alanine (A), Phenylalanine (F) and Aspartate (D) corroborated the importance of the Tryptophan residue in interaction with the substrate. This is the first report of unravelling the structural characteristics of chitin deacetylase from Cryptococcus and understanding the approach of the enzyme towards its substrate. Our results would be helpful to perform experimental validations and apply quantum mechanics/molecular mechanics techniques to determine the detailed catalytic mechanism and enhance the industrial potency of the enzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

Fungal Diversity Is Not Determined by Mineral and Chemical Differences in Serpentine Substrates

PubMed Central

Daghino, Stefania; Murat, Claude; Sizzano, Elisa; Girlanda, Mariangela; Perotto, Silvia

2012-01-01

The physico-chemical properties of serpentine soils lead to strong selection of plant species. Whereas many studies have described the serpentine flora, little information is available on the fungal communities dwelling in these sites. Asbestos minerals, often associated with serpentine rocks, can be weathered by serpentine-isolated fungi, suggesting an adaptation to this substrate. In this study, we have investigated whether serpentine substrates characterized by the presence of rocks with distinct mineral composition could select for different fungal communities. Both fungal isolation and 454 pyrosequencing of amplicons obtained from serpentine samples following direct DNA extraction revealed some fungal taxa shared by the four ophiolitic substrates, but also highlighted several substrate-specific taxa. Bootstrap analysis of 454 OTU abundances indicated weak clustering of fungal assemblages from the different substrates, which did not match substrate classification based on exchangeable macronutrients and metals. Intra-substrate variability, as assessed by DGGE profiles, was similar across the four serpentine substrates, and comparable to inter-substrate variability. These findings indicate the absence of a correlation between the substrate (mineral composition and available cations) and the diversity of the fungal community. Comparison of culture-based and culture-independent methods supports the higher taxonomic precision of the former, as complementation of the better performance of the latter. PMID:23028507

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships.

PubMed

Zeke, András; Misheva, Mariya; Reményi, Attila; Bogoyevitch, Marie A

2016-09-01

The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Structural Analysis of the Tobramycin and Gentamicin Clinical Resistome Reveals Limitations for Next-generation Aminoglycoside Design.

PubMed

Bassenden, Angelia V; Rodionov, Dmitry; Shi, Kun; Berghuis, Albert M

2016-05-20

Widespread use and misuse of antibiotics has allowed for the selection of resistant bacteria capable of avoiding the effects of antibiotics. The primary mechanism for resistance to aminoglycosides, a broad-spectrum class of antibiotics, is through covalent enzymatic modification of the drug, waning their bactericidal effect. Tobramycin and gentamicin are two medically important aminoglycosides targeted by several different resistance factors, including aminoglycoside 2″-nucleotidyltransferase [ANT(2″)], the primary cause of aminoglycoside resistance in North America. We describe here two crystal structures of ANT(2″), each in complex with AMPCPP, Mn(2+), and either tobramycin or gentamicin. Together these structures outline ANT(2″)'s specificity for clinically used substrates. Importantly, these structures complete our structural knowledge for the set of enzymes that most frequently confer clinically observed resistance to tobramycin and gentamicin. Comparison of tobramycin and gentamicin binding to enzymes in this resistome, as well as to the intended target, the bacterial ribosome, reveals surprising diversity in observed drug-target interactions. Analysis of the diverse binding modes informs that there are limited opportunities for developing aminoglycoside analogs capable of evading resistance.

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

Imker,H.; Fedorov, A.; Fedorov, E.

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the D-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ('enolization') of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-D-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-likemore » protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this 'enolase' is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and 'enolase'-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg{sup 2+}, we sought to establish structure-function relationships for the 'enolase' reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the 'enolases' from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from D-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the 'enolase'-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated 'enolase' from G. kaustophilus (carboxylated on Lys 173) liganded with Mg{sup 2+} and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal {alpha}+ {beta} domain instead of the C-terminal ({beta}/{alpha}){sub 8}-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all 'enolases', is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus 'enolase' is unable to catalyze the 'enolase' reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg{sup 2+}, but the proton abstraction is catalyzed by a different general base.« less

Assessment of benthic macroinvertebrates at Nile tilapia production using artificial substrate samplers.

PubMed

Moura E Silva, M S G; Graciano, T S; Losekann, M E; Luiz, A J B

2016-05-17

Biomonitoring is a cheap and effective tool for evaluation of water quality, and infer on the balance of aquatic ecosystems. The benthic macroinvertebrates are bioindicators sensitive to environmental changes, and can assist in detecting and preventing impacts such as organic enrichment and imbalance in the food chain. We compared the structure of benthic communities on artificial substrate samplers located in places near and far from net cages for production of Nile tilapia (Oreochromis niloticus). Samplers were manufactured with nylon net, using substrates such as crushed stone, gravel, loofah and cattail leaves. Samples were collected after 30 days of colonization, rinsed and then the specimens were identified and quantified. The following metrics were calculated: richness of Operational Taxonomic Units, Margalef richness, abundance of individuals, Shannon index and evenness index. The macrobenthic community structure was strongly modified according to the proximity of the net cages. Metrics showed significant differences (p < 0.05) between near and distant sites, for both periods (dry and rainy seasons). The position of the samplers significantly affected the structure of macroinvertebrate community, as near sites showed higher values for the community metrics, such as richness and diversity. Near sites presented a larger number of individuals, observed both in the dry and rainy seasons, with a predominance of Chironomidae (Diptera) in the dry season and Tubificidae (Oligochaeta) in the rainy season.

Phylogenetic and Functional Substrate Specificity for Endolithic Microbial Communities in Hyper-Arid Environments

PubMed Central

Crits-Christoph, Alexander; Robinson, Courtney K.; Ma, Bing; Ravel, Jacques; Wierzchos, Jacek; Ascaso, Carmen; Artieda, Octavio; Souza-Egipsy, Virginia; Casero, M. Cristina; DiRuggiero, Jocelyne

2016-01-01

Under extreme water deficit, endolithic (inside rock) microbial ecosystems are considered environmental refuges for life in cold and hot deserts, yet their diversity and functional adaptations remain vastly unexplored. The metagenomic analyses of the communities from two rock substrates, calcite and ignimbrite, revealed that they were dominated by Cyanobacteria, Actinobacteria, and Chloroflexi. The relative distribution of major phyla was significantly different between the two substrates and biodiversity estimates, from 16S rRNA gene sequences and from the metagenomic data, all pointed to a higher taxonomic diversity in the calcite community. While both endolithic communities showed adaptations to extreme aridity and to the rock habitat, their functional capabilities revealed significant differences. ABC transporters and pathways for osmoregulation were more diverse in the calcite chasmoendolithic community. In contrast, the ignimbrite cryptoendolithic community was enriched in pathways for secondary metabolites, such as non-ribosomal peptides (NRP) and polyketides (PK). Assemblies of the metagenome data produced population genomes for the major phyla found in both communities and revealed a greater diversity of Cyanobacteria population genomes for the calcite substrate. Draft genomes of the dominant Cyanobacteria in each community were constructed with more than 93% estimated completeness. The two annotated proteomes shared 64% amino acid identity and a significantly higher number of genes involved in iron update, and NRPS gene clusters, were found in the draft genomes from the ignimbrite. Both the community-wide and genome-specific differences may be related to higher water availability and the colonization of large fissures and cracks in the calcite in contrast to a harsh competition for colonization space and nutrient resources in the narrow pores of the ignimbrite. Together, these results indicated that the habitable architecture of both lithic substrates- chasmoendolithic versus cryptoendolithic – might be an essential element in determining the colonization and the diversity of the microbial communities in endolithic substrates at the dry limit for life. PMID:27014224

Large-scale impacts of herbivores on the structural diversity of African savannas

PubMed Central

Asner, Gregory P.; Levick, Shaun R.; Kennedy-Bowdoin, Ty; Knapp, David E.; Emerson, Ruth; Jacobson, James; Colgan, Matthew S.; Martin, Roberta E.

2009-01-01

African savannas are undergoing management intensification, and decision makers are increasingly challenged to balance the needs of large herbivore populations with the maintenance of vegetation and ecosystem diversity. Ensuring the sustainability of Africa's natural protected areas requires information on the efficacy of management decisions at large spatial scales, but often neither experimental treatments nor large-scale responses are available for analysis. Using a new airborne remote sensing system, we mapped the three-dimensional (3-D) structure of vegetation at a spatial resolution of 56 cm throughout 1640 ha of savanna after 6-, 22-, 35-, and 41-year exclusions of herbivores, as well as in unprotected areas, across Kruger National Park in South Africa. Areas in which herbivores were excluded over the short term (6 years) contained 38%–80% less bare ground compared with those that were exposed to mammalian herbivory. In the longer-term (> 22 years), the 3-D structure of woody vegetation differed significantly between protected and accessible landscapes, with up to 11-fold greater woody canopy cover in the areas without herbivores. Our maps revealed 2 scales of ecosystem response to herbivore consumption, one broadly mediated by geologic substrate and the other mediated by hillslope-scale variation in soil nutrient availability and moisture conditions. Our results are the first to quantitatively illustrate the extent to which herbivores can affect the 3-D structural diversity of vegetation across large savanna landscapes. PMID:19258457

Microsatellite analysis of Saccharomyces uvarum diversity.

PubMed

Masneuf-Pomarede, Isabelle; Salin, Franck; Börlin, Marine; Coton, Emmanuel; Coton, Monika; Jeune, Christine Le; Legras, Jean-Luc

2016-03-01

Considered as a sister species of Saccharomyces cerevisiae, S. uvarum is, to a lesser extent, an interesting species for fundamental and applied research studies. Despite its potential interest as a new gene pool for fermenting agents, the intraspecific molecular genetic diversity of this species is still poorly investigated. In this study, we report the use of nine microsatellite markers to describe S. uvarum genetic diversity and population structure among 108 isolates from various geographical and substrate origins (wine, cider and natural sources). Our combined microsatellite markers set allowed differentiating 89 genotypes. In contrast to S. cerevisiae genetic diversity, wild and human origin isolates were intertwined. A total of 75% of strains were proven to be homozygotes and estimated heterozygosity suggests a selfing rate above 0.95 for the different population tested here. From this point of view, the S. uvarum life cycle appears to be more closely related to S. paradoxus or S. cerevisiae of natural resources than S. cerevisiae wine isolates. Population structure could not be correlated to distinct geographic or technological origins, suggesting lower differentiation that may result from a large exchange between human and natural populations mediated by insects or human activities. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Carbon and nitrogen inputs affect soil microbial community structure and function

NASA Astrophysics Data System (ADS)

Liu, X. J. A.; Mau, R. L.; Hayer, M.; Finley, B. K.; Schwartz, E.; Dijkstra, P.; Hungate, B. A.

2016-12-01

Climate change has been projected to increase energy and nutrient inputs to soils, affecting soil organic matter (SOM) decomposition (priming effect) and microbial communities. However, many important questions remain: how do labile C and/or N inputs affect priming and microbial communities? What is the relationship between them? To address these questions, we applied N (NH4NO3 ; 100 µg N g-1 wk-1), C (13C glucose; 1000 µg C g-1 wk-1), C+N to four different soils for five weeks. We found: 1) N showed no effect, whereas C induced the greatest priming, and C+N had significantly lower priming than C. 2) C and C+N additions increased the relative abundance of actinobacteria, proteobacteria, and firmicutes, but reduced relative abundance of acidobacteria, chloroflexi, verrucomicrobia, planctomycetes, and gemmatimonadetes. 3) Actinobacteria and proteobacteria increased relative abundance over time, but most others decreased over time. 4) substrate additions (N, C, C+N) significantly reduced microbial alpha diversity, which also decreased over time. 5) For beta diversity, C and C+N formed significantly different communities compare to the control and N treatments. Overtime, microbial community structure significantly altered. Four soils have drastically different community structures. These results indicate amounts of substrate C were determinant factors in modulating the rate of SOM decomposition and microbial communities. Variable responses of different microbial communities to labile C and N inputs indicate that complex relationships between priming and microbial functions. In general, we demonstrate that energy inputs can quickly accelerate SOM decomposition whereas extra N input can slow this process, though both had similar microbial community responses.

Selective oxidation of aliphatic C-H bonds in alkylphenols by a chemomimetic biocatalytic system.

PubMed

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Feng, Yingang; Liu, Shuang-Jiang; Li, Shengying

2017-06-27

Selective oxidation of aliphatic C-H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C-H bonds of p - and m -alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity.

Selective oxidation of aliphatic C–H bonds in alkylphenols by a chemomimetic biocatalytic system

PubMed Central

Du, Lei; Dong, Sheng; Zhang, Xingwang; Jiang, Chengying; Chen, Jingfei; Yao, Lishan; Wang, Xiao; Wan, Xiaobo; Liu, Xi; Wang, Xinquan; Huang, Shaohua; Cui, Qiu; Liu, Shuang-Jiang; Li, Shengying

2017-01-01

Selective oxidation of aliphatic C–H bonds in alkylphenols serves significant roles not only in generation of functionalized intermediates that can be used to synthesize diverse downstream chemical products, but also in biological degradation of these environmentally hazardous compounds. Chemo-, regio-, and stereoselectivity; controllability; and environmental impact represent the major challenges for chemical oxidation of alkylphenols. Here, we report the development of a unique chemomimetic biocatalytic system originated from the Gram-positive bacterium Corynebacterium glutamicum. The system consisting of CreHI (for installation of a phosphate directing/anchoring group), CreJEF/CreG/CreC (for oxidation of alkylphenols), and CreD (for directing/anchoring group offloading) is able to selectively oxidize the aliphatic C–H bonds of p- and m-alkylated phenols in a controllable manner. Moreover, the crystal structures of the central P450 biocatalyst CreJ in complex with two representative substrates provide significant structural insights into its substrate flexibility and reaction selectivity. PMID:28607077

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.

Lysine-Tryptophan-Crosslinked Peptides Produced by Radical SAM Enzymes in Pathogenic Streptococci.

PubMed

Schramma, Kelsey R; Seyedsayamdost, Mohammad R

2017-04-21

Macrocycles represent a common structural framework in many naturally occurring peptides. Several strategies exist for macrocyclization, and the enzymes that incorporate them are of great interest, as they enhance our repertoire for creating complex molecules. We recently discovered a new peptide cyclization reaction involving a crosslink between the side chains of lysine and tryptophan that is installed by a radical SAM enzyme. Herein, we characterize relatives of this metalloenzyme from the pathogens Streptococcus agalactiae and Streptococcus suis. Our results show that the corresponding enzymes, which we call AgaB and SuiB, contain multiple [4Fe-4S] clusters and catalyze Lys-Trp crosslink formation in their respective substrates. Subsequent high-resolution-MS and 2D-NMR analyses located the site of macrocyclization. Moreover, we report that AgaB can accept modified substrates containing natural or unnatural amino acids. Aside from providing insights into the mechanism of this unusual modification, the substrate promiscuity of AgaB may be exploited to create diverse macrocyclic peptides.

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

DNA materials: bridging nanotechnology and biotechnology.

PubMed

Yang, Dayong; Hartman, Mark R; Derrien, Thomas L; Hamada, Shogo; An, Duo; Yancey, Kenneth G; Cheng, Ru; Ma, Minglin; Luo, Dan

2014-06-17

CONSPECTUS: In recent decades, DNA has taken on an assortment of diverse roles, not only as the central genetic molecule in biological systems but also as a generic material for nanoscale engineering. DNA possesses many exceptional properties, including its biological function, biocompatibility, molecular recognition ability, and nanoscale controllability. Taking advantage of these unique attributes, a variety of DNA materials have been created with properties derived both from the biological functions and from the structural characteristics of DNA molecules. These novel DNA materials provide a natural bridge between nanotechnology and biotechnology, leading to far-ranging real-world applications. In this Account, we describe our work on the design and construction of DNA materials. Based on the role of DNA in the construction, we categorize DNA materials into two classes: substrate and linker. As a substrate, DNA interfaces with enzymes in biochemical reactions, making use of molecular biology's "enzymatic toolkit". For example, employing DNA as a substrate, we utilized enzymatic ligation to prepare the first bulk hydrogel made entirely of DNA. Using this DNA hydrogel as a structural scaffold, we created a protein-producing DNA hydrogel via linking plasmid DNA onto the hydrogel matrix through enzymatic ligation. Furthermore, to fully make use of the advantages of both DNA materials and polymerase chain reaction (PCR), we prepared thermostable branched DNA that could remain intact even under denaturing conditions, allowing for their use as modular primers for PCR. Moreover, via enzymatic polymerization, we have recently constructed a physical DNA hydrogel with unique internal structure and mechanical properties. As a linker, we have used DNA to interface with other functional moieties, including gold nanoparticles, clay minerals, proteins, and lipids, allowing for hybrid materials with unique properties for desired applications. For example, we recently designed a DNA-protein conjugate as a universal adapter for protein detection. We further demonstrate a diverse assortment of applications for these DNA materials including diagnostics, protein production, controlled drug release systems, the exploration of life evolution, and plasmonics. Although DNA has shown great potential as both substrate and linker in the construction of DNA materials, it is still in the initial stages of becoming a well-established and widely used material. Important challenges include the ease of design and fabrication, scaling-up, and minimizing cost. We envision that DNA materials will continue to bridge the gap between nanotechnology and biotechnology and will ultimately be employed for many real-world applications.

Erasers of Histone Acetylation: The Histone Deacetylase Enzymes

PubMed Central

Seto, Edward; Yoshida, Minoru

2014-01-01

Histone deacetylases (HDACs) are enzymes that catalyze the removal of acetyl functional groups from the lysine residues of both histone and nonhistone proteins. In humans, there are 18 HDAC enzymes that use either zinc- or NAD+-dependent mechanisms to deacetylate acetyl lysine substrates. Although removal of histone acetyl epigenetic modification by HDACs regulates chromatin structure and transcription, deacetylation of nonhistones controls diverse cellular processes. HDAC inhibitors are already known potential anticancer agents and show promise for the treatment of many diseases. PMID:24691964

Distribution and Catabolic Diversity of 3-Chlorobenzoic Acid Degrading Bacteria Isolated from Geographically-Separated Pristine Soils

DTIC Science & Technology

1994-08-01

influence on the structure of 3-CBA degrading populations. This suggests that if substrate exposure is important, secondary metabolites produced during the...enrichments were treated with 50 gIg 3-CBAgodry-soil- 1 and secondary enrichments were performed with 50 p.g 3-CBA ml-1 d•tfined medium. Isolates were...not attempt to determine metabolites or if transformations were biologically mediated. As a result, it was not clear whether disappearance of a

Comparative Bioinformatic Analysis of Active Site Structures in Evolutionarily Remote Homologues of α,β-Hydrolase Superfamily Enzymes.

PubMed

Suplatov, D A; Arzhanik, V K; Svedas, V K

2011-01-01

Comparative bioinformatic analysis is the cornerstone of the study of enzymes' structure-function relationship. However, numerous enzymes that derive from a common ancestor and have undergone substantial functional alterations during natural selection appear not to have a sequence similarity acceptable for a statistically reliable comparative analysis. At the same time, their active site structures, in general, can be conserved, while other parts may largely differ. Therefore, it sounds both plausible and appealing to implement a comparative analysis of the most functionally important structural elements - the active site structures; that is, the amino acid residues involved in substrate binding and the catalytic mechanism. A computer algorithm has been developed to create a library of enzyme active site structures based on the use of the PDB database, together with programs of structural analysis and identification of functionally important amino acid residues and cavities in the enzyme structure. The proposed methodology has been used to compare some α,β-hydrolase superfamily enzymes. The insight has revealed a high structural similarity of catalytic site areas, including the conservative organization of a catalytic triad and oxyanion hole residues, despite the wide functional diversity among the remote homologues compared. The methodology can be used to compare the structural organization of the catalytic and substrate binding sites of various classes of enzymes, as well as study enzymes' evolution and to create of a databank of enzyme active site structures.

Molecular Architecture of Strictosidine Glucosidase: The Gateway to the Biosynthesis of the Monoterpenoid Indole Alkaloid Family[W

PubMed Central

Barleben, Leif; Panjikar, Santosh; Ruppert, Martin; Koepke, Juergen; Stöckigt, Joachim

2007-01-01

Strictosidine β-d-glucosidase (SG) follows strictosidine synthase (STR1) in the production of the reactive intermediate required for the formation of the large family of monoterpenoid indole alkaloids in plants. This family is composed of ∼2000 structurally diverse compounds. SG plays an important role in the plant cell by activating the glucoside strictosidine and allowing it to enter the multiple indole alkaloid pathways. Here, we report detailed three-dimensional information describing both native SG and the complex of its inactive mutant Glu207Gln with the substrate strictosidine, thus providing a structural characterization of substrate binding and identifying the amino acids that occupy the active site surface of the enzyme. Structural analysis and site-directed mutagenesis experiments demonstrate the essential role of Glu-207, Glu-416, His-161, and Trp-388 in catalysis. Comparison of the catalytic pocket of SG with that of other plant glucosidases demonstrates the structural importance of Trp-388. Compared with all other glucosidases of plant, bacterial, and archaeal origin, SG's residue Trp-388 is present in a unique structural conformation that is specific to the SG enzyme. In addition to STR1 and vinorine synthase, SG represents the third structural example of enzymes participating in the biosynthetic pathway of the Rauvolfia alkaloid ajmaline. The data presented here will contribute to deciphering the structure and reaction mechanism of other higher plant glucosidases. PMID:17890378

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.

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

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.

Substrate roughening improves swimming performance in two small-bodied riverine fishes: implications for culvert remediation and design

PubMed Central

Rodgers, Essie M.; Heaslip, Breeana M.; Cramp, Rebecca L.; Riches, Marcus; Gordos, Matthew A.

2017-01-01

Abstract Worldwide declines in riverine fish abundance and diversity have been linked to the fragmentation of aquatic habitats through the installation of instream structures (e.g. culverts, dams, weirs and barrages). Restoring riverine connectivity can be achieved by remediating structures impeding fish movements by, for example, replacing smooth substrates of pipe culverts with naturalistic substrates (i.e. river stones; culvert roughening). However, empirical evaluations of the efficacy of such remediation efforts are often lacking despite the high economic cost. We assessed the effectiveness of substrate roughening in improving fish swimming performance and linked this to estimates of upstream passage success. Critical swimming speeds (Ucrit) of two small-bodied fish, purple-spotted gudgeon (Mogurnda adspersa; 7.7–11.6 cm total length, BL) and crimson-spotted rainbowfish (Melanotaenia duboulayi; 4.2–8.7 cm BL) were examined. Swimming trials were conducted in a hydraulic flume fitted with either a smooth acrylic substrate (control) or a rough substrate with fixed river stones. Swimming performance was improved on the rough compared to the smooth substrate, with Mo. adspersa (Ucrit-smooth = 0.28 ± 0.0 m s−1, 2.89 ± 0.1 BL s−1, Ucrit-rough = 0.36 ± 0.02 m s−1, 3.66 ± 0.22 BL s−1, mean ± s.e) and Me. duboulayi (Ucrit-smooth = 0.46 ± 0.01 m s−1, 7.79 ± 0.33 BL s−1; Ucrit-rough = = 0.55 ± 0.03 m s−1, 9.83 ± 0.67 BL s−1, mean ± s.e.) both experiencing a 26% increase in relative Ucrit. Traversable water velocity models predicted maximum water speeds allowing successful upstream passage of both species to substantially increase following roughening remediation. Together these findings suggest culvert roughening may be a solution which allows hydraulic efficiency goals to be met, without compromising fish passage. PMID:28567285

Substrate roughening improves swimming performance in two small-bodied riverine fishes: implications for culvert remediation and design.

PubMed

Rodgers, Essie M; Heaslip, Breeana M; Cramp, Rebecca L; Riches, Marcus; Gordos, Matthew A; Franklin, Craig E

2017-01-01

Worldwide declines in riverine fish abundance and diversity have been linked to the fragmentation of aquatic habitats through the installation of instream structures (e.g. culverts, dams, weirs and barrages). Restoring riverine connectivity can be achieved by remediating structures impeding fish movements by, for example, replacing smooth substrates of pipe culverts with naturalistic substrates (i.e. river stones; culvert roughening). However, empirical evaluations of the efficacy of such remediation efforts are often lacking despite the high economic cost. We assessed the effectiveness of substrate roughening in improving fish swimming performance and linked this to estimates of upstream passage success. Critical swimming speeds ( U crit ) of two small-bodied fish, purple-spotted gudgeon ( Mogurnda adspersa ; 7.7-11.6 cm total length, BL) and crimson-spotted rainbowfish ( Melanotaenia duboulayi ; 4.2-8.7 cm BL) were examined. Swimming trials were conducted in a hydraulic flume fitted with either a smooth acrylic substrate (control) or a rough substrate with fixed river stones. Swimming performance was improved on the rough compared to the smooth substrate, with Mo. adspersa ( U crit-smooth = 0.28 ± 0.0 m s -1 , 2.89 ± 0.1 BL s -1 , U crit-rough = 0.36 ± 0.02 m s -1 , 3.66 ± 0.22 BL s -1 , mean ± s.e) and Me. duboulayi ( U crit-smooth = 0.46 ± 0.01 m s -1 , 7.79 ± 0.33 BL s -1 ; U crit-rough = = 0.55 ± 0.03 m s -1 , 9.83 ± 0.67 BL s -1 , mean ± s.e.) both experiencing a 26% increase in relative U crit . Traversable water velocity models predicted maximum water speeds allowing successful upstream passage of both species to substantially increase following roughening remediation. Together these findings suggest culvert roughening may be a solution which allows hydraulic efficiency goals to be met, without compromising fish passage.

Characterization of regioselective flavonoid O-methyltransferase from the Streptomyces sp. KCTC 0041BP.

PubMed

Darsandhari, Sumangala; Dhakal, Dipesh; Shrestha, Biplav; Parajuli, Prakash; Seo, Joo-Hyun; Kim, Tae-Su; Sohng, Jae Kyung

2018-06-01

A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-β-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4'-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C 2 and the C 3 and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented. Copyright © 2018 Elsevier Inc. 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

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.

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.

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

Structural, molecular motions, and free-energy landscape of Leishmania sterol-14α-demethylase wild type and drug resistant mutant: a comparative molecular dynamics study.

PubMed

Vijayakumar, Saravanan; Das, Pradeep

2018-04-18

Sterol-14α-demethylase (CYP51) is an ergosterol pathway enzyme crucial for the survival of infectious Leishmania parasite. Recent high-throughput metabolomics and whole genome sequencing study revealed amphotericin B resistance in Leishmania is indeed due to mutation in CYP51. The residue of mutation (asparagine 176) is conserved across the kinetoplastidae and not in yeast or humans, portraying its functional significance. In order to understand the possible cause for the resistance, knowledge of structural changes due to mutation is of high importance. To shed light on the structural changes of wild and mutant CYP51, we conducted comparative molecular dynamics simulation study. The active site, substrate biding cavity, substrate channel entrance (SCE), and cavity involving the mutated site were studied based on basic parameters and large concerted molecular motions derived from essential dynamics analyses of 100 ns simulation. Results indicated that mutant CYP51 is stable and less compact than the wild type. Correspondingly, the solvent accessible surface area (SASA) of the mutant was found to be increased, especially in active site and cavities not involving the mutation site. Free-energy landscape analysis disclosed mutant to have a rich conformational diversity than wild type, with various free-energy conformations of mutant having SASA greater than wild type with SCE open. More residues were found to interact with the mutant CYP51 upon docking of substrate to both the wild and mutant CYP51. These results indicate that, relative to wild type, the N176I mutation of CYP51 in Leishmania mexicana could possibly favor increased substrate binding efficiency.

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

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

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

DOE PAGES

Urbanus, Malene L.; Quaile, Andrew T.; Stogios, Peter J.; ...

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, tomore » query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila–translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Here, metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.« less

When lithography meets self-assembly: a review of recent advances in the directed assembly of complex metal nanostructures on planar and textured surfaces

NASA Astrophysics Data System (ADS)

Hughes, Robert A.; Menumerov, Eredzhep; Neretina, Svetlana

2017-07-01

One of the foremost challenges in nanofabrication is the establishment of a processing science that integrates wafer-based materials, techniques, and devices with the extraordinary physicochemical properties accessible when materials are reduced to nanoscale dimensions. Such a merger would allow for exacting controls on nanostructure positioning, promote cooperative phenomenon between adjacent nanostructures and/or substrate materials, and allow for electrical contact to individual or groups of nanostructures. With neither self-assembly nor top-down lithographic processes being able to adequately meet this challenge, advancements have often relied on a hybrid strategy that utilizes lithographically-defined features to direct the assembly of nanostructures into organized patterns. While these so-called directed assembly techniques have proven viable, much of this effort has focused on the assembly of periodic arrays of spherical or near-spherical nanostructures comprised of a single element. Work directed toward the fabrication of more complex nanostructures, while still at a nascent stage, has nevertheless demonstrated the possibility of forming arrays of nanocubes, nanorods, nanoprisms, nanoshells, nanocages, nanoframes, core-shell structures, Janus structures, and various alloys on the substrate surface. In this topical review, we describe the progress made in the directed assembly of periodic arrays of these complex metal nanostructures on planar and textured substrates. The review is divided into three broad strategies reliant on: (i) the deterministic positioning of colloidal structures, (ii) the reorganization of deposited metal films at elevated temperatures, and (iii) liquid-phase chemistry practiced directly on the substrate surface. These strategies collectively utilize a broad range of techniques including capillary assembly, microcontact printing, chemical surface modulation, templated dewetting, nanoimprint lithography, and dip-pen nanolithography and employ a wide scope of chemical processes including redox reactions, alloying, dealloying, phase separation, galvanic replacement, preferential etching, template-mediated reactions, and facet-selective capping agents. Taken together, they highlight the diverse toolset available when fabricating organized surfaces of substrate-supported nanostructures.

Conformal Robotic Stereolithography

PubMed Central

Stevens, Adam G.; Oliver, C. Ryan; Kirchmeyer, Matthieu; Wu, Jieyuan; Chin, Lillian; Polsen, Erik S.; Archer, Chad; Boyle, Casey; Garber, Jenna

2016-01-01

Abstract Additive manufacturing by layerwise photopolymerization, commonly called stereolithography (SLA), is attractive due to its high resolution and diversity of materials chemistry. However, traditional SLA methods are restricted to planar substrates and planar layers that are perpendicular to a single-axis build direction. Here, we present a robotic system that is capable of maskless layerwise photopolymerization on curved surfaces, enabling production of large-area conformal patterns and the construction of conformal freeform objects. The system comprises an industrial six-axis robot and a custom-built maskless projector end effector. Use of the system involves creating a mesh representation of the freeform substrate, generation of a triangulated toolpath with curved layers that represents the target object to be printed, precision mounting of the substrate in the robot workspace, and robotic photopatterning of the target object by coordinated motion of the robot and substrate. We demonstrate printing of conformal photopatterns on spheres of various sizes, and construction of miniature three-dimensional objects on spheres without requiring support features. Improvement of the motion accuracy and development of freeform toolpaths would enable construction of polymer objects that surpass the size and support structure constraints imparted by traditional SLA systems. PMID:29577062

Alternative modes of client binding enable functional plasticity of Hsp70

NASA Astrophysics Data System (ADS)

Mashaghi, Alireza; Bezrukavnikov, Sergey; Minde, David P.; Wentink, Anne S.; Kityk, Roman; Zachmann-Brand, Beate; Mayer, Matthias P.; Kramer, Günter; Bukau, Bernd; Tans, Sander J.

2016-11-01

The Hsp70 system is a central hub of chaperone activity in all domains of life. Hsp70 performs a plethora of tasks, including folding assistance, protection against aggregation, protein trafficking, and enzyme activity regulation, and interacts with non-folded chains, as well as near-native, misfolded, and aggregated proteins. Hsp70 is thought to achieve its many physiological roles by binding peptide segments that extend from these different protein conformers within a groove that can be covered by an ATP-driven helical lid. However, it has been difficult to test directly how Hsp70 interacts with protein substrates in different stages of folding and how it affects their structure. Moreover, recent indications of diverse lid conformations in Hsp70-substrate complexes raise the possibility of additional interaction mechanisms. Addressing these issues is technically challenging, given the conformational dynamics of both chaperone and client, the transient nature of their interaction, and the involvement of co-chaperones and the ATP hydrolysis cycle. Here, using optical tweezers, we show that the bacterial Hsp70 homologue (DnaK) binds and stabilizes not only extended peptide segments, but also partially folded and near-native protein structures. The Hsp70 lid and groove act synergistically when stabilizing folded structures: stabilization is abolished when the lid is truncated and less efficient when the groove is mutated. The diversity of binding modes has important consequences: Hsp70 can both stabilize and destabilize folded structures, in a nucleotide-regulated manner; like Hsp90 and GroEL, Hsp70 can affect the late stages of protein folding; and Hsp70 can suppress aggregation by protecting partially folded structures as well as unfolded protein chains. Overall, these findings in the DnaK system indicate an extension of the Hsp70 canonical model that potentially affects a wide range of physiological roles of the Hsp70 system.

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.

Borophene as a prototype for synthetic 2D materials development.

PubMed

Mannix, Andrew J; Zhang, Zhuhua; Guisinger, Nathan P; Yakobson, Boris I; Hersam, Mark C

2018-06-01

The synthesis of 2D materials with no analogous bulk layered allotropes promises a substantial breadth of physical and chemical properties through the diverse structural options afforded by substrate-dependent epitaxy. However, despite the joint theoretical and experimental efforts to guide materials discovery, successful demonstrations of synthetic 2D materials have been rare. The recent synthesis of 2D boron polymorphs (that is, borophene) provides a notable example of such success. In this Perspective, we discuss recent progress and future opportunities for borophene research. Borophene combines unique mechanical properties with anisotropic metallicity, which complements the canon of conventional 2D materials. The multi-centre characteristics of boron-boron bonding lead to the formation of configurationally varied, vacancy-mediated structural motifs, providing unprecedented diversity in a mono-elemental 2D system with potential for electronic applications, chemical functionalization, materials synthesis and complex heterostructures. With its foundations in computationally guided synthesis, borophene can serve as a prototype for ongoing efforts to discover and exploit synthetic 2D materials.

Phage phenomics: Physiological approaches to characterize novel viral proteins

ScienceCinema

Sanchez, Savannah E. [San Diego State Univ., San Diego, CA (United States); Cuevas, Daniel A. [San Diego State Univ., San Diego, CA (United States); Rostron, Jason E. [San Diego State Univ., San Diego, CA (United States); Liang, Tiffany Y. [San Diego State Univ., San Diego, CA (United States); Pivaroff, Cullen G. [San Diego State Univ., San Diego, CA (United States); Haynes, Matthew R. [San Diego State Univ., San Diego, CA (United States); Nulton, Jim [San Diego State Univ., San Diego, CA (United States); Felts, Ben [San Diego State Univ., San Diego, CA (United States); Bailey, Barbara A. [San Diego State Univ., San Diego, CA (United States); Salamon, Peter [San Diego State Univ., San Diego, CA (United States); Edwards, Robert A. [San Diego State Univ., San Diego, CA (United States); Argonne National Lab. (ANL), Argonne, IL (United States); Burgin, Alex B. [Broad Institute, Cambridge, MA (United States); Segall, Anca M. [San Diego State Univ., San Diego, CA (United States); Rohwer, Forest [San Diego State Univ., San Diego, CA (United States)

2018-06-21

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Borophene as a prototype for synthetic 2D materials development

NASA Astrophysics Data System (ADS)

Mannix, Andrew J.; Zhang, Zhuhua; Guisinger, Nathan P.; Yakobson, Boris I.; Hersam, Mark C.

2018-06-01

The synthesis of 2D materials with no analogous bulk layered allotropes promises a substantial breadth of physical and chemical properties through the diverse structural options afforded by substrate-dependent epitaxy. However, despite the joint theoretical and experimental efforts to guide materials discovery, successful demonstrations of synthetic 2D materials have been rare. The recent synthesis of 2D boron polymorphs (that is, borophene) provides a notable example of such success. In this Perspective, we discuss recent progress and future opportunities for borophene research. Borophene combines unique mechanical properties with anisotropic metallicity, which complements the canon of conventional 2D materials. The multi-centre characteristics of boron-boron bonding lead to the formation of configurationally varied, vacancy-mediated structural motifs, providing unprecedented diversity in a mono-elemental 2D system with potential for electronic applications, chemical functionalization, materials synthesis and complex heterostructures. With its foundations in computationally guided synthesis, borophene can serve as a prototype for ongoing efforts to discover and exploit synthetic 2D materials.

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

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

Insights into Hsp70 Chaperone Activity from a Crystal Structure of the Yeast Hsp110 Sse1

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

Liu,Q.; Hendrickson, W.

2007-01-01

Classic Hsp70 chaperones assist in diverse processes of protein folding and translocation, and Hsp110s had seemed by sequence to be distant relatives within an Hsp70 superfamily. The 2.4 Angstroms resolution structure of Sse1 with ATP shows that Hsp110s are indeed Hsp70 relatives, and it provides insight into allosteric coupling between sites for ATP and polypeptide-substrate binding in Hsp70s. Subdomain structures are similar in intact Sse1(ATP) and in the separate Hsp70 domains, but conformational dispositions are radically different. Interfaces between Sse1 domains are extensive, intimate, and conservative in sequence with Hsp70s. We propose that Sse1(ATP) may be an evolutionary vestige ofmore » the Hsp70(ATP) state, and an analysis of 64 mutant variants in Sse1 and three Hsp70 homologs supports this hypothesis. An atomic-level understanding of Hsp70 communication between ATP and substrate-binding domains follows. Requirements on Sse1 for yeast viability are in keeping with the distinct function of Hsp110s as nucleotide exchange factors.« less

DNA recognition by an RNA-guided bacterial Argonaute

PubMed Central

Doudna, Jennifer A.

2017-01-01

Argonaute (Ago) proteins are widespread in prokaryotes and eukaryotes and share a four-domain architecture capable of RNA- or DNA-guided nucleic acid recognition. Previous studies identified a prokaryotic Argonaute protein from the eubacterium Marinitoga piezophila (MpAgo), which binds preferentially to 5′-hydroxylated guide RNAs and cleaves single-stranded RNA (ssRNA) and DNA (ssDNA) targets. Here we present a 3.2 Å resolution crystal structure of MpAgo bound to a 21-nucleotide RNA guide and a complementary 21-nucleotide ssDNA substrate. Comparison of this ternary complex to other target-bound Argonaute structures reveals a unique orientation of the N-terminal domain, resulting in a straight helical axis of the entire RNA-DNA heteroduplex through the central cleft of the protein. Additionally, mismatches introduced into the heteroduplex reduce MpAgo cleavage efficiency with a symmetric profile centered around the middle of the helix. This pattern differs from the canonical mismatch tolerance of other Argonautes, which display decreased cleavage efficiency for substrates bearing sequence mismatches to the 5′ region of the guide strand. This structural analysis of MpAgo bound to a hybrid helix advances our understanding of the diversity of target recognition mechanisms by Argonaute proteins. PMID:28520746

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.

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.

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.

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

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

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance.

PubMed

Kern, Marcelo; McGeehan, John E; Streeter, Simon D; Martin, Richard N A; Besser, Katrin; Elias, Luisa; Eborall, Will; Malyon, Graham P; Payne, Christina M; Himmel, Michael E; Schnorr, Kirk; Beckham, Gregg T; Cragg, Simon M; Bruce, Neil C; McQueen-Mason, Simon J

2013-06-18

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

PubMed Central

Kern, Marcelo; McGeehan, John E.; Streeter, Simon D.; Martin, Richard N. A.; Besser, Katrin; Elias, Luisa; Eborall, Will; Malyon, Graham P.; Payne, Christina M.; Himmel, Michael E.; Schnorr, Kirk; Beckham, Gregg T.; Cragg, Simon M.; Bruce, Neil C.; McQueen-Mason, Simon J.

2013-01-01

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes. PMID:23733951

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

Crystal structures of catalytic complexes of the oxidative DNA/RNA repair enzyme AlkB

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

Yu,B.; Edstrom, W.; Benach, J.

2006-01-01

Nucleic acid damage by environmental and endogenous alkylation reagents creates lesions that are both mutagenic and cytotoxic, with the latter effect accounting for their widespread use in clinical cancer chemotherapy. Escherichia coliAlkB and the homologous human proteins ABH2 and ABH3 (refs 5, 7) promiscuously repair DNA and RNA bases damaged by SN2 alkylation reagents, which attach hydrocarbons to endocyclic ring nitrogen atoms (N1 of adenine and guanine and N3 of thymine and cytosine). Although the role of AlkB in DNA repair has long been established based on phenotypic studies, its exact biochemical activity was only elucidated recently after sequence profilemore » analysis revealed it to be a member of the Fe-oxoglutarate-dependent dioxygenase superfamily. These enzymes use an Fe(ii) cofactor and 2-oxoglutarate co-substrate to oxidize organic substrates. AlkB hydroxylates an alkylated nucleotide base to produce an unstable product that releases an aldehyde to regenerate the unmodified base. Here we have determined crystal structures of substrate and product complexes of E. coli AlkB at resolutions from 1.8 to 2.3 Angstroms. Whereas the Fe-2-oxoglutarate dioxygenase core matches that in other superfamily members, a unique subdomain holds a methylated trinucleotide substrate into the active site through contacts to the polynucleotide backbone. Amide hydrogen exchange studies and crystallographic analyses suggest that this substrate-binding 'lid' is conformationally flexible, which may enable docking of diverse alkylated nucleotide substrates in optimal catalytic geometry. Different crystal structures show open and closed states of a tunnel putatively gating O2 diffusion into the active site. Exposing crystals of the anaerobic Michaelis complex to air yields slow but substantial oxidation of 2-oxoglutarate that is inefficiently coupled to nucleotide oxidation. These observations suggest that protein dynamics modulate redox chemistry and that a hypothesized migration of the reactive oxy-ferryl ligand on the catalytic Fe ion may be impeded when the protein is constrained in the crystal lattice.« less

Subjects harboring presenilin familial Alzheimer’s disease mutations exhibit diverse white matter biochemistry alterations

PubMed Central

Roher, Alex E; Maarouf, Chera L; Malek-Ahmadi, Michael; Wilson, Jeffrey; Kokjohn, Tyler A; Daugs, Ian D; Whiteside, Charisse M; Kalback, Walter M; Macias, MiMi P; Jacobson, Sandra A; Sabbagh, Marwan N; Ghetti, Bernardino; Beach, Thomas G

2013-01-01

Alzheimer’s disease (AD) dementia impacts all facets of higher order cognitive function and is characterized by the presence of distinctive pathological lesions in the gray matter (GM). The profound alterations in GM structure and function have fostered the view that AD impacts are primarily a consequence of GM damage. However, the white matter (WM) represents about 50% of the cerebrum and this area of the brain is substantially atrophied and profoundly abnormal in both sporadic AD (SAD) and familial AD (FAD). We examined the WM biochemistry by ELISA and Western blot analyses of key proteins in 10 FAD cases harboring mutations in the presenilin genes PSEN1 and PSEN2 as well as in 4 non-demented control (NDC) individuals and 4 subjects with SAD. The molecules examined were direct substrates of PSEN1 such as Notch-1 and amyloid precursor protein (APP). In addition, apolipoproteins, axonal transport molecules, cytoskeletal and structural proteins, neurotrophic factors and synaptic proteins were examined. PSEN-FAD subjects had, on average, higher amounts of WM amyloid-beta (Aβ) peptides compared to SAD, which may play a role in the devastating dysfunction of the brain. However, the PSEN-FAD mutations we examined did not produce uniform increases in the relative proportions of Aβ42 and exhibited substantial variability in total Aβ levels. These observations suggest that neurodegeneration and dementia do not depend solely on enhanced Aβ42 levels. Our data revealed additional complexities in PSEN-FAD individuals. Some direct substrates of γ-secretase, such as Notch, N-cadherin, Erb-B4 and APP, deviated substantially from the NDC group baseline for some, but not all, mutation types. Proteins that were not direct γ-secretase substrates, but play key structural and functional roles in the WM, likewise exhibited varied concentrations in the distinct PSEN mutation backgrounds. Detailing the diverse biochemical pathology spectrum of PSEN mutations may offer valuable insights into dementia progression and the design of effective therapeutic interventions for both SAD and FAD. PMID:24093083

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.

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

Crystal structure of the Streptomyces coelicolor sortase E1 transpeptidase provides insight into the binding mode of the novel class E sorting signal

DOE PAGES

Kattke, Michele D.; Chan, Albert H.; Duong, Andrew; ...

2016-12-09

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

Diverse 2D structures obtained by adsorption of charged ABA triblock copolymer on different surfaces

NASA Astrophysics Data System (ADS)

Kontturi, Katri S.; Vesterinen, Arja-Helena; Seppälä, Jukka; Laine, Janne

2012-11-01

In the larger context of 2D polymeric structures, the morphologies obtained by adsorption and subsequent drying of charged, ABA type amphiphilic triblock copolymer of poly[2-(dimethylamino)ethyl metacrylate] (PDMAEMA) and poly(propylene oxide) (PPO) were investigated with atomic force microscopy and X-ray photoelectron spectroscopy as well as in situ adsorption analysis with quartz crystal microbalance with dissipation monitoring. Hydrophilic silica and hydrophobic polystyrene (PS) were used as substrates for adsorption. The structures emerging from the self-assembly of adsorbing polymer were profoundly influenced by composition of the aqueous solution and the choice of substrate. When adsorbed from dilute polymer solution where the concentration is so low that the polymer does not yet show surface-active behavior, the triblock copolymer unimers associated on hydrophilic silica surface forming large, irregular clustered aggregates, with sizes increasing with electrolyte concentration of the solution. On a hydrophobic PS substrate, on the other hand, unimers spread much more evenly, forming clear surface patterns. The roughness of these patterned structures was tuned with the electrolyte concentration of the solution. Adsorption from a more concentrated polymer solution, where the surface-activity of the polymer is perceptible, resulted in the formation of a smooth film with complete coverage over the hydrophilic silica substrate when the electrolyte concentration was high. On PS, on the other hand, nucleation of evenly scattered globular, disk-like micelles was induced. Besides the dry film morphology, the even distribution of the irreversibly adsorbed polymer over the PS surface was likely to serve as an optimal platform for the build-up of reversible hydrophobically bound multilayers at high electrolyte concentration. The multilayer formation was reversible because a decrease in the electrolyte concentration of the solution re-introduces strong electrostatic repulsion between the multilayered polymer coils which results in breakdown of the layer.

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...

A review of epiphyte community development: surface interactions and settlement on seagrass.

PubMed

Michael, Teena S; Shin, Hyun Woung; Hanna, Richard; Spafford, David C

2008-07-01

A focus of community ecology is the spatial distribution of species assemblages and the interactions among species and abiotic features of the environment. While the ubiquity of species associations is apparent, it is less clear if interactions within a community impart an organizational structure to the community. Do settlement processes in early stages of community development contribute to later community structure? What are the interfacial forces that lead to recruitment and colonization of diverse substrata? This review examines seagrasses as living substrates for epiphyte colonization and the surface interactions which may determine settlement success. These epiphytes include primary producers which contribute to biodiversity and are bioindicators of pollution/nutrient enrichment.

Compact multilayer film structure for angle insensitive color filtering.

PubMed

Yang, Chenying; Shen, Weidong; Zhang, Yueguang; Li, Kan; Fang, Xu; Zhang, Xing; Liu, Xu

2015-03-19

Here we report a compact multilayer film structure for angle robust color filtering, which is verified by theoretical calculations and experiment results. The introduction of the amorphous silicon in the proposed unsymmetrical resonant cavity greatly reduces the angular sensitivity of the filters, which is confirmed by the analysis of the phase shift within the structure. The temperature of the substrate during the deposition is expressly investigated to obtain the best optical performance with high peak reflectance and good angle insensitive color filtering by compromising the refractive index of dielectric layer and the surface roughness of the multilayer film. And the outlayer of the structure, worked as the anti-reflection layer, have an enormous impact on the filtering performance. This method, described in this paper, can have enormous potential for diverse applications in display, colorful decoration, anti-counterfeiting and so forth.

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*

PubMed Central

Meekins, David A.; Raththagala, Madushi; Auger, Kyle D.; Turner, Benjamin D.; Santelia, Diana; Kötting, Oliver; Gentry, Matthew S.; Vander Kooi, Craig W.

2015-01-01

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. PMID:26231210

A one-dimensional ice structure built from pentagons

NASA Astrophysics Data System (ADS)

Carrasco, Javier; Michaelides, Angelos; Forster, Matthew; Haq, Sam; Raval, Rasmita; Hodgson, Andrew

2009-05-01

Heterogeneous ice nucleation has a key role in fields as diverse as atmospheric chemistry and biology. Ice nucleation on metal surfaces affords an opportunity to watch this process unfold at the molecular scale on a well-defined, planar interface. A common feature of structural models for such films is that they are built from hexagonal arrangements of molecules. Here we show, through a combination of scanning tunnelling microscopy, infrared spectroscopy and density-functional theory, that about 1-nm-wide ice chains that nucleate on Cu(110) are not built from hexagons, but instead are built from a face-sharing arrangement of water pentagons. The pentagon structure is favoured over others because it maximizes the water-metal bonding while maintaining a strong hydrogen-bonding network. It reveals an unanticipated structural adaptability of water-ice films, demonstrating that the presence of the substrate can be sufficient to favour non-hexagonal structural units.

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.

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

Mechanism of endonuclease cleavage by the HigB toxin

PubMed Central

Schureck, Marc A.; Repack, Adrienne; Miles, Stacey J.; Marquez, Jhomar; Dunham, Christine M.

2016-01-01

Bacteria encode multiple type II toxin–antitoxin modules that cleave ribosome-bound mRNAs in response to stress. All ribosome-dependent toxin family members structurally characterized to date adopt similar microbial RNase architectures despite possessing low sequence identities. Therefore, determining which residues are catalytically important in this specialized RNase family has been a challenge in the field. Structural studies of RelE and YoeB toxins bound to the ribosome provided significant insights but biochemical experiments with RelE were required to clearly demonstrate which residues are critical for acid-base catalysis of mRNA cleavage. Here, we solved an X-ray crystal structure of the wild-type, ribosome-dependent toxin HigB bound to the ribosome revealing potential catalytic residues proximal to the mRNA substrate. Using cell-based and biochemical assays, we further determined that HigB residues His54, Asp90, Tyr91 and His92 are critical for activity in vivo, while HigB H54A and Y91A variants have the largest effect on mRNA cleavage in vitro. Comparison of X-ray crystal structures of two catalytically inactive HigB variants with 70S-HigB bound structures reveal that HigB active site residues undergo conformational rearrangements likely required for recognition of its mRNA substrate. These data support the emerging concept that ribosome-dependent toxins have diverse modes of mRNA recognition. PMID:27378776

Photo-excited multi-frequency terahertz switch based on a composite metamaterial structure

NASA Astrophysics Data System (ADS)

Ji, Hongyu; Zhang, Bo; Wang, Guocui; Wang, Wei; Shen, Jingling

2018-04-01

We propose a photo-excited tunable multi-frequency metamaterial (MM) switch that can be used in the terahertz region. This metamaterial switch is composed of a polyimide substrate and a hybrid metal-semiconductor square split-ring resonator (SRR) with two gaps, with various semiconductors placed in critical regions of the metallic resonator. By changing the incident pump power, we were able to tune the conductivity of the diverse semiconductors filling the gaps of the SRR, and by using an external exciting beam, we were able to modulate the resonant absorption properties of the composite metamaterial structure. We demonstrated the tunable multi-frequency metamaterial switch by irradiating the composite metamaterial structure with a pump laser. In addition, we proposed a tunable metamaterial switch based on a circular metallic split-ring resonator.

Modelling of the frictional behaviour of the snake skin covered by anisotropic surface nanostructures.

PubMed

Filippov, Alexander E; Gorb, Stanislav N

2016-03-23

Previous experimental data clearly revealed anisotropic friction on the ventral scale surface of snakes. However, it is known that frictional properties of the ventral surface of the snake skin range in a very broad range and the degree of anisotropy ranges as well to a quite strong extent. This might be due to the variety of species studied, diversity of approaches used for the friction characterization, and/or due to the variety of substrates used as a counterpart in the experiments. In order to understand the interactions between the nanostructure arrays of the ventral surface of the snake skin, this study was undertaken, which is aimed at numerical modeling of frictional properties of the structurally anisotropic surfaces in contact with various size of asperities. The model shows that frictional anisotropy appears on the snake skin only on the substrates with a characteristic range of roughness, which is less or comparable with dimensions of the skin microstructure. In other words, scale of the skin relief should reflect an adaptation to the particular range of surfaces asperities of the substrate.

Bioerosion structures in high-salinity marine environments: Evidence from the Al-Khafji coastline, Saudi Arabia

NASA Astrophysics Data System (ADS)

El-Sorogy, Abdelbaset S.; Alharbi, Talal; Richiano, Sebastián

2018-05-01

Salinity is one the major stress factors that controls the biotic activities in marine environments. In general, the mixture with fresh-water has been mention as a great stress factor, but the opposite, i.e. high-salinity conditions, is less developed in the ichnological literature. Along the Al-Khafji coastline, Saudi Arabia, hard substrates (constituted by gastropods, bivalves and coral skeletons) contain diverse and abundant bioerosion traces and associated encrusters. Field and laboratory observations allowed the recognition of eight ichnospecies belong to the ichnogenera Gastrochaenolites, Entobia, Oichnus, Caulostrepsis and Trypanites, which can be attributed to various activities produced by bivalves, sponges, gastropods and annelids. The borings demonstrate two notable ichnological boring assemblages, namely, Entobia-dominated and Gastrochaenolites-dominated assemblages. The highly diversified bioerosion and encrustation in the studied hard organic substrate indicate a long exposition period of organic substrate with slow to moderate rate of deposition in a restricted (high-salinity) marine environment. This bioerosion study shows that high-salinity, at least for the study area, is not an important controlling factor for ichnology.

Non-leaky modes and bandgaps of surface acoustic waves in wrinkled stiff-film/compliant-substrate bilayers

NASA Astrophysics Data System (ADS)

Li, Guo-Yang; Xu, Guoqiang; Zheng, Yang; Cao, Yanping

2018-03-01

Surface acoustic wave (SAW) devices have found a wide variety of technical applications, including SAW filters, SAW resonators, microfluidic actuators, biosensors, flow measurement devices, and seismic wave shields. Stretchable/flexible electronic devices, such as sensory skins for robotics, structural health monitors, and wearable communication devices, have received considerable attention across different disciplines. Flexible SAW devices are essential building blocks for these applications, wherein piezoelectric films may need to be integrated with the compliant substrates. When piezoelectric films are much stiffer than soft substrates, SAWs are usually leaky and the devices incorporating them suffer from acoustic losses. In this study, the propagation of SAWs in a wrinkled bilayer system is investigated, and our analysis shows that non-leaky modes can be achieved by engineering stress patterns through surface wrinkles in the system. Our analysis also uncovers intriguing bandgaps (BGs) related to the SAWs in a wrinkled bilayer system; these are caused by periodic deformation patterns, which indicate that diverse wrinkling patterns could be used as metasurfaces for controlling the propagation of SAWs.

Modelling of the frictional behaviour of the snake skin covered by anisotropic surface nanostructures

PubMed Central

Filippov, Alexander E.; Gorb, Stanislav N.

2016-01-01

Previous experimental data clearly revealed anisotropic friction on the ventral scale surface of snakes. However, it is known that frictional properties of the ventral surface of the snake skin range in a very broad range and the degree of anisotropy ranges as well to a quite strong extent. This might be due to the variety of species studied, diversity of approaches used for the friction characterization, and/or due to the variety of substrates used as a counterpart in the experiments. In order to understand the interactions between the nanostructure arrays of the ventral surface of the snake skin, this study was undertaken, which is aimed at numerical modeling of frictional properties of the structurally anisotropic surfaces in contact with various size of asperities. The model shows that frictional anisotropy appears on the snake skin only on the substrates with a characteristic range of roughness, which is less or comparable with dimensions of the skin microstructure. In other words, scale of the skin relief should reflect an adaptation to the particular range of surfaces asperities of the substrate. PMID:27005001

Modelling of the frictional behaviour of the snake skin covered by anisotropic surface nanostructures

NASA Astrophysics Data System (ADS)

Filippov, Alexander E.; Gorb, Stanislav N.

2016-03-01

Previous experimental data clearly revealed anisotropic friction on the ventral scale surface of snakes. However, it is known that frictional properties of the ventral surface of the snake skin range in a very broad range and the degree of anisotropy ranges as well to a quite strong extent. This might be due to the variety of species studied, diversity of approaches used for the friction characterization, and/or due to the variety of substrates used as a counterpart in the experiments. In order to understand the interactions between the nanostructure arrays of the ventral surface of the snake skin, this study was undertaken, which is aimed at numerical modeling of frictional properties of the structurally anisotropic surfaces in contact with various size of asperities. The model shows that frictional anisotropy appears on the snake skin only on the substrates with a characteristic range of roughness, which is less or comparable with dimensions of the skin microstructure. In other words, scale of the skin relief should reflect an adaptation to the particular range of surfaces asperities of the substrate.

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.

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.

Changes in microbial structure and functional communities at different soil depths during 13C labelled root litter degradation

NASA Astrophysics Data System (ADS)

Sanaullah, Muhammad; Baumann, Karen; Chabbi, Abad; Dignac, Marie-France; Maron, Pierre-Alain; Kuzyakov, Yakov; Rumpel, Cornelia

2014-05-01

Soil organic matter turnover depends on substrate quality and microbial activity in soil but little is known about how addition of freshly added organic material modifies the diversity of soil microbial communities with in a soil profile. We took advantage of a decomposition experiment, which was carried out at different soil depths under field conditions and sampled litterbags with 13C-labelled wheat roots, incubated in subsoil horizons at 30, 60 and 90 cm depth for up to 36 months. The effect of root litter addition on microbial community structure, diversity and activity was studied by determining total microbial biomass, PLFA signatures, molecular tools (DNA genotyping and pyrosequencing of 16S and 18S rDNAs) and extracellular enzyme activities. Automated ribosomal intergenic spacer analysis (ARISA) was also carried out to determine the differences in microbial community structure. We found that with the addition of root litter, total microbial biomass as well as microbial community composition and structure changed at different soil depths and change was significantly higher at top 30cm soil layer. Moreover, in the topsoil, population of both gram-positive and gram-negative bacteria increased with root litter addition over time, while subsoil horizons were relatively dominated by fungal community. Extra-cellular enzyme activities confirmed relatively higher fungal community at subsoil horizons compared with surface soil layer with bacteria dominant microbial population. Bacterial-ARISA profiling illustrated that the addition of root litter enhanced the abundance of Actinobacteria and Proteobacteria, at all three soil depths. These bacteria correspond to copiotrophic attributes, which can preferentially consume of labile soil organic C pools. While disappearance of oligotrophic Acidobacteria confirmed the shifting of microbial communities due to the addition of readily available substrate. We concluded that root litter mixing altered microbial community development which was soil horizon specific and its effects on soil microbial activity may impact on nutrient cycling.

Evolutionarily Conserved Linkage between Enzyme Fold, Flexibility, and Catalysis

PubMed Central

Ramanathan, Arvind; Agarwal, Pratul K.

2011-01-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme–substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme–substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design. PMID:22087074

Evolutionarily conserved linkage between enzyme fold, flexibility, and catalysis.

PubMed

Ramanathan, Arvind; Agarwal, Pratul K

2011-11-01

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function. Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 Å away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme-substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme-substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.

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

Ramanathan, Arvind; Agarwal, Pratul K

Proteins are intrinsically flexible molecules. The role of internal motions in a protein's designated function is widely debated. The role of protein structure in enzyme catalysis is well established, and conservation of structural features provides vital clues to their role in function. Recently, it has been proposed that the protein function may involve multiple conformations: the observed deviations are not random thermodynamic fluctuations; rather, flexibility may be closely linked to protein function, including enzyme catalysis. We hypothesize that the argument of conservation of important structural features can also be extended to identification of protein flexibility in interconnection with enzyme function.more » Three classes of enzymes (prolyl-peptidyl isomerase, oxidoreductase, and nuclease) that catalyze diverse chemical reactions have been examined using detailed computational modeling. For each class, the identification and characterization of the internal protein motions coupled to the chemical step in enzyme mechanisms in multiple species show identical enzyme conformational fluctuations. In addition to the active-site residues, motions of protein surface loop regions (>10 away) are observed to be identical across species, and networks of conserved interactions/residues connect these highly flexible surface regions to the active-site residues that make direct contact with substrates. More interestingly, examination of reaction-coupled motions in non-homologous enzyme systems (with no structural or sequence similarity) that catalyze the same biochemical reaction shows motions that induce remarkably similar changes in the enzyme substrate interactions during catalysis. The results indicate that the reaction-coupled flexibility is a conserved aspect of the enzyme molecular architecture. Protein motions in distal areas of homologous and non-homologous enzyme systems mediate similar changes in the active-site enzyme substrate interactions, thereby impacting the mechanism of catalyzed chemistry. These results have implications for understanding the mechanism of allostery, and for protein engineering and drug design.« less

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.

Substrate Sorting by a Supercharged Nanoreactor

PubMed Central

2017-01-01

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts. PMID:29278496

A non-canonical peptide synthetase adenylates 3-methyl-2-oxovaleric acid for auriculamide biosynthesis.

PubMed

Braga, Daniel; Hoffmeister, Dirk; Nett, Markus

2016-01-01

Auriculamide is the first natural product known from the predatory bacterium Herpetosiphon aurantiacus. It is composed of three unusual building blocks, including the non-proteinogenic amino acid 3-chloro-L-tyrosine, the α-hydroxy acid L-isoleucic acid, and a methylmalonyl-CoA-derived ethane unit. A candidate genetic locus for auriculamide biosynthesis was identified and encodes four enzymes. Among them, the non-canonical 199 kDa four-domain nonribosomal peptide synthetase, AulA, is extraordinary in that it features two consecutive adenylation domains. Here, we describe the functional characterization of the recombinantly produced AulA. The observed activation of 3-methyl-2-oxovaleric acid by the enzyme supports the hypothesis that it participates in the biosynthesis of auriculamide. An artificially truncated version of AulA that lacks the first adenylation domain activated this substrate like the full-length enzyme which shows that the first adenylation domain is dispensable. Additionally, we provide evidence that the enzyme tolerates structural variation of the substrate. α-Carbon substituents significantly affected the substrate turnover. While all tested aliphatic α-keto acids were accepted by the enzyme and minor differences in chain size and branches did not interfere with the enzymatic activity, molecules with methylene α-carbons led to low turnover. Such enzymatic plasticity is an important attribute to help in the perpetual search for novel molecules and to access a greater structural diversity by mutasynthesis.

Distribution of Chironomidae in a semiarid intermittent river of Brazil.

PubMed

Farias, R L; Carvalho, L K; Medeiros, E S F

2012-12-01

The effects of the intermittency of water flow on habitat structure and substrate composition have been reported to create a patch dynamics for the aquatic fauna, mostly for that associated with the substrate. This study aims to describe the spatial distribution of Chironomidae in an intermittent river of semiarid Brazil and to associate assemblage composition with environmental variables. Benthic invertebrates were sampled during the wet and dry seasons using a D-shaped net (40 cm wide and 250 μm mesh), and the Chironomidae were identified to genus level. The most abundant genera were Tanytarsus, Polypedilum, and Saetheria with important contributions of the genera Procladius, Aedokritus, and Dicrotendipes. Richness and density were not significantly different between the study sites, and multiple regression showed that the variation in richness and density explained by the environmental variables was significant only for substrate composition. The composition of genera showed significant spatial segregation across the study sites. Canonical Correspondence Analysis showed significant correspondence between Chironomidae composition and the environmental variables, with submerged vegetation, elevation, and leaf litter being important predictors of the Chironomidae fauna. This study showed that Chironomidae presented important spatial variation along the river and that this variation was substantially explained by environmental variables associated with the habitat structure and river hierarchy. We suggest that the observed spatial segregation in the fauna results in the high diversity of this group of organisms in intermittent streams.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

PubMed

Urbanus, Malene L; Quaile, Andrew T; Stogios, Peter J; Morar, Mariya; Rao, Chitong; Di Leo, Rosa; Evdokimova, Elena; Lam, Mandy; Oatway, Christina; Cuff, Marianne E; Osipiuk, Jerzy; Michalska, Karolina; Nocek, Boguslaw P; Taipale, Mikko; Savchenko, Alexei; Ensminger, Alexander W

2016-12-16

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Broad range tuning of structural and optical properties of Zn x Mg1-x O nanostructures grown by vapor transport method

NASA Astrophysics Data System (ADS)

Vanjaria, Jignesh V.; Azhar, Ebraheem Ali; Yu, Hongbin

2016-11-01

One-dimensional (1D) Zn x Mg1-x O nanomaterials have drawn global attention due to their remarkable chemical and physical properties, and their diverse current and future technological applications. In this work, 1D ZnMgO nanostructures with different magnesium concentrations and different morphologies were grown directly on zinc oxide-coated silicon substrates by thermal evaporation of zinc oxide, magnesium boride and graphite powders. Highly well-defined Mg-rich ZnMgO nanorods with a rock salt structure and Zn-rich ZnMgO nanostructures with a wurtzite structure have been deposited individually by careful optimization of the source mixture and process parameters. Structural and optical properties of the deposited products were studied by scanning electron microscopy, energy dispersive x-ray spectroscopy, x-ray diffraction, and Raman spectroscopy. Cathodoluminescence measurements demonstrate strong dominant peaks at 3.3 eV in Mg poor ZnMgO nanostructures and 4.8 eV in Mg rich nanostructures implying that the ZnMgO nanostructures can be used for the fabrication of deep UV optoelectronic devices. A mechanism for the formation and achieved diverse morphology of the ZnMgO nanostructures was proposed based on the characterization results.

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

Rhizosphere effect of colonizer plant species on the development of soil microbial community during primary succession on postmining sites

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

Elhottova, D.; Kristufek, V.; Maly, S.

2009-07-01

The impact of pioneer plant species Tussilago farfara on structural, functional, and growth characterization of microbial community colonizing the spoil colliery substrate was studied in a laboratory microcosm experiment. Microcosms consisting of spoil substrate (0.7 dm{sup 3} of tertiary alkaline clay sediment from Sokolov brown-coal mine area) from a pioneer site (without vegetation, 5 years after heaping) were cultivated in a greenhouse with one plant of this species. Plant roots substantially increased microbial diversity and biomass after one season (7 months) of cultivation. Roots influenced the microbial community and had nearly twice the size, higher growth, and metabolic potential inmore » comparison to the control. The development of microbial specialists improves the plant nutrient status. Bacterial nitrogen (N{sub 2}) fixators (Bradyrhizobium japonicum, Rhizobium radiobacter) and arbuscular mycorrhizal fungi were confirmed in the rhizosphere of Tussilago farfara.« less

Synthesis of an A'B' Precursor to Angelmicin B: Product Diversification in the Suárez Lactol Fragmentation.

PubMed

Li, Jialiang; Todaro, Louis; Mootoo, David R

2011-11-01

We describe a synthetic strategy for the angelimicin family of anthraquinoid natural products that involves converting a central highly oxygenated decalin intermediate to the AB and A'B' subunits. Herein, we report the synthesis of the bicyclic A'B' subunit that complements our earlier route to the tricyclic AB framework. The differentiating tact in the two syntheses focused on controlling the Suárez radical fragmentation of lactol precursors by modulating the substrate's structural rigidity. A more flexible lactol gave the tricyclic AB framework, whereas a more rigid substrate led to the bicyclic A'B' precursor, presumably through divergent pathways from the radical produced in the initial fragmentation step. These results establish a versatile advanced synthetic precursor for the angelimicins, and on a more general note, illustrate strategies for applying the Suárez fragmentation to diverse and complex molecular frameworks.

Biological substrates of schizophrenia.

PubMed

Kovelman, J A; Scheibel, A B

1986-01-01

Schizophrenia is increasingly believed to represent a group of organic disorders which primarily, although not exclusively, affect the central nervous system. Our purpose is to review a representative sample of twentieth-century literature which speaks to the biological substrates of the syndrome. Subjects reviewed include genetic and environmental contributions to the onset of illness, early and recent findings of gross structural anomalies, and apparent histopathological alterations in cerebral cortex, cerebellar vermis, limbic system, and brain stem, as well as problems of cerebral asymmetry. Data from a diverse group of electrophysiological studies reveal several promising correlates of these areas of investigation. Despite the inconsistent nature of the findings to date, several themes have begun to emerge, including patterns of hypofrontal/hyperparietal regional cerebral flow and glucose utilization, left hemispheric dysfunction, and deficits of interhemispheric information processing. The interpretation and significance of these emerging patterns remains unclear and must await more profound insights into the nature of normal and abnormal cerebral function.

Discovery of a Dual PRMT5-PRMT7 Inhibitor.

PubMed

Smil, David; Eram, Mohammad S; Li, Fengling; Kennedy, Steven; Szewczyk, Magdalena M; Brown, Peter J; Barsyte-Lovejoy, Dalia; Arrowsmith, Cheryl H; Vedadi, Masoud; Schapira, Matthieu

2015-04-09

The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5-PRMT7 axis as a target for therapy.

Discovery of a Dual PRMT5–PRMT7 Inhibitor

PubMed Central

2015-01-01

The protein arginine methyltransferases PRMT7 and PRMT5, respectively, monomethylate and symmetrically dimethylate arginine side-chains of proteins involved in diverse cellular mechanisms, including chromatin-mediated control of gene transcription, splicing, and the RAS to ERK transduction cascade. It is believed that PRMT5 and PRMT7 act in conjunction to methylate their substrates, and genetic deletions support the notion that these enzymes derepress cell proliferation and migration in cancer. Using available structures of PRMT5, we designed DS-437, a PRMT5 inhibitor with an IC50 value of 6 μM against both PRMT5 and PRMT7 that is inactive against 29 other human protein-, DNA-, and RNA-methyltransferases and inhibits symmetrical dimethylation of PRMT5 substrates in cells. This compound behaves as a cofactor competitor and represents a valid scaffold to interrogate the potential of the PRMT5–PRMT7 axis as a target for therapy. PMID:25893041

Microbial ecology of extreme environments: Antarctic dry valley yeasts and growth in substrate limited habitats

NASA Technical Reports Server (NTRS)

Vishniac, H. S.

1981-01-01

The multiple stresses temperature, moisture, and for chemoheterotrophs, sources of carbon and energy of the Dry Valley Antarctica soils allow at best depauperate communities, low in species diversity and population density. The nature of community structure, the operation of biogeochemical cycles, the evolution and mechanisms of adaptation to this habitat are of interest in informing speculations upon life on other planets as well as in modeling the limits of gene life. Yeasts of the Cryptococcus vishniacil complex (Basidiobiastomycetes) are investigated, as the only known indigenes of the most hostile, lichen free, parts of the Dry Valleys. Methods were developed for isolating these yeasts (methods which do not exclude the recovery of other microbiota). The definition of the complex was refined and the importance of nitrogen sources was established as well as substrate competition in fitness to the Dry Valley habitats.

Soil microbial community profiles and functional diversity in limestone cedar glades

USGS Publications Warehouse

Cartwright, Jennifer M.; Dzantor, E. Kudjo; Momen, Bahram

2016-01-01

Rock outcrop ecosystems, such as limestone cedar glades (LCGs), are known for their rare and endemic plant species adapted to high levels of abiotic stress. Soils in LCGs are thin (< 25 cm), soil-moisture conditions fluctuate seasonally between xeric and saturated, and summer soil temperatures commonly exceed 48 °C. The effects of these stressors on soil microbial communities (SMC) remain largely unstudied, despite the importance of SMC-plant interactions in regulating the structure and function of terrestrial ecosystems. SMC profiles and functional diversity were characterized in LCGs using community level physiological profiling (CLPP) and plate-dilution frequency assays (PDFA). Most-probable number (MPN) estimates and microbial substrate-utilization diversity (H) were positively related to soil thickness, soil organic matter (OM), soil water content, and vegetation density, and were diminished in alkaline soil relative to circumneutral soil. Soil nitrate showed no relationship to SMCs, suggesting lack of N-limitation. Canonical correlation analysis indicated strong correlations between microbial CLPP patterns and several physical and chemical properties of soil, primarily temperature at the ground surface and at 4-cm depth, and secondarily soil-water content, enabling differentiation by season. Thus, it was demonstrated that several well-described abiotic determinants of plant community structure in this ecosystem are also reflected in SMC profiles.

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...

Pharmacogenomics of CYP2C9: Functional and Clinical Considerations†

PubMed Central

Rettie, Allan E.; Fowler, Douglas M.; Miners, John O.

2017-01-01

CYP2C9 is the most abundant CYP2C subfamily enzyme in human liver and the most important contributor from this subfamily to drug metabolism. Polymorphisms resulting in decreased enzyme activity are common in the CYP2C9 gene and this, combined with narrow therapeutic indices for several key drug substrates, results in some important issues relating to drug safety and efficacy. CYP2C9 substrate selectivity is detailed and, based on crystal structures for the enzyme, we describe how CYP2C9 catalyzes these reactions. Factors relevant to clinical response to CYP2C9 substrates including inhibition, induction and genetic polymorphism are discussed in detail. In particular, we consider the issue of ethnic variation in pattern and frequency of genetic polymorphisms and clinical implications. Warfarin is the most well studied CYP2C9 substrate; recent work on use of dosing algorithms that include CYP2C9 genotype to improve patient safety during initiation of warfarin dosing are reviewed and prospects for their clinical implementation considered. Finally, we discuss a novel approach to cataloging the functional capabilities of rare ‘variants of uncertain significance’, which are increasingly detected as more exome and genome sequencing of diverse populations is conducted. PMID:29283396

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.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins.

PubMed

Stetz, Gabrielle; Verkhivker, Gennady M

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones.

Dancing through Life: Molecular Dynamics Simulations and Network-Centric Modeling of Allosteric Mechanisms in Hsp70 and Hsp110 Chaperone Proteins

PubMed Central

Stetz, Gabrielle; Verkhivker, Gennady M.

2015-01-01

Hsp70 and Hsp110 chaperones play an important role in regulating cellular processes that involve protein folding and stabilization, which are essential for the integrity of signaling networks. Although many aspects of allosteric regulatory mechanisms in Hsp70 and Hsp110 chaperones have been extensively studied and significantly advanced in recent experimental studies, the atomistic picture of signal propagation and energetics of dynamics-based communication still remain unresolved. In this work, we have combined molecular dynamics simulations and protein stability analysis of the chaperone structures with the network modeling of residue interaction networks to characterize molecular determinants of allosteric mechanisms. We have shown that allosteric mechanisms of Hsp70 and Hsp110 chaperones may be primarily determined by nucleotide-induced redistribution of local conformational ensembles in the inter-domain regions and the substrate binding domain. Conformational dynamics and energetics of the peptide substrate binding with the Hsp70 structures has been analyzed using free energy calculations, revealing allosteric hotspots that control negative cooperativity between regulatory sites. The results have indicated that cooperative interactions may promote a population-shift mechanism in Hsp70, in which functional residues are organized in a broad and robust allosteric network that can link the nucleotide-binding site and the substrate-binding regions. A smaller allosteric network in Hsp110 structures may elicit an entropy-driven allostery that occurs in the absence of global structural changes. We have found that global mediating residues with high network centrality may be organized in stable local communities that are indispensable for structural stability and efficient allosteric communications. The network-centric analysis of allosteric interactions has also established that centrality of functional residues could correlate with their sensitivity to mutations across diverse chaperone functions. This study reconciles a wide spectrum of structural and functional experiments by demonstrating how integration of molecular simulations and network-centric modeling may explain thermodynamic and mechanistic aspects of allosteric regulation in chaperones. PMID:26619280

Method of transferring strained semiconductor structure

DOEpatents

Nastasi, Michael A [Santa Fe, NM; Shao, Lin [College Station, TX

2009-12-29

The transfer of strained semiconductor layers from one substrate to another substrate involves depositing a multilayer structure on a substrate having surface contaminants. An interface that includes the contaminants is formed in between the deposited layer and the substrate. Hydrogen atoms are introduced into the structure and allowed to diffuse to the interface. Afterward, the deposited multilayer structure is bonded to a second substrate and is separated away at the interface, which results in transferring a multilayer structure from one substrate to the other substrate. The multilayer structure includes at least one strained semiconductor layer and at least one strain-induced seed layer. The strain-induced seed layer can be optionally etched away after the layer transfer.

Structural mechanisms of DNA binding and unwinding in bacterial RecQ helicases

DOE PAGES

Manthei, Kelly A.; Hill, Morgan C.; Burke, Jordan E.; ...

2015-03-23

RecQ helicases unwind remarkably diverse DNA structures as key components of many cellular processes. How RecQ enzymes accommodate different substrates in a unified mechanism that couples ATP hydrolysis to DNA unwinding is unknown. In this paper, the X-ray crystal structure of the Cronobacter sakazakii RecQ catalytic core domain bound to duplex DNA with a 3' single-stranded extension identifies two DNA-dependent conformational rearrangements: a winged-helix domain pivots ~90° to close onto duplex DNA, and a conserved aromatic-rich loop is remodeled to bind ssDNA. These changes coincide with a restructuring of the RecQ ATPase active site that positions catalytic residues for ATPmore » hydrolysis. Complex formation also induces a tight bend in the DNA and melts a portion of the duplex. Finally, this bending, coupled with translocation, could provide RecQ with a mechanism for unwinding duplex and other DNA structures.« less

A one-dimensional ice structure built from pentagons

NASA Astrophysics Data System (ADS)

Carrasco, Javier; Michaelides, Angelos

2010-03-01

Heterogeneous nucleation of water plays a key role in fields as diverse as atmospheric chemistry, astrophysics, and biology. Ice nucleation on metal surfaces offers an opportunity to watch this process unfold, providing a molecular-scale description at a well-defined, planar interface. We discuss a density-functional theory study on a metal surface specifically designed to understand such phenomena. Together with our colleges at the University of Liverpool, we found that the nanometer wide water-ice chains experimentally observed to nucleate and grow on Cu(110) are built from a face sharing arrangement of water pentagons [1]. The novel one-dimensional pentagon structure maximizes the water-metal bonding whilst simultaneously maintaining a strong hydrogen bonding network. These results reveal an unanticipated structural adaptability of water-ice films, demonstrating that the presence of the substrate can be sufficient to favor non-conventional structural units. [4pt] [1] J. Carrasco et al., Nature Mater. 8, 427 (2009).

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn 2+/Mn 2+ heterobinucleation

DOE PAGES

Ransey, Elizabeth; Paredes, Eduardo; Dey, Sourav K.; ...

2017-05-17

Here, the RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni 2+, Mn 2+, Mg 2+, Fe 2+, and Zn 2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn 2+ ion), recent structures determined a Zn 2+/Fe 2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn 2+/Mn 2+ occupancy.more » ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.« less

Metal hierarchical patterning by direct nanoimprint lithography

PubMed Central

Radha, Boya; Lim, Su Hui; Saifullah, Mohammad S. M.; Kulkarni, Giridhar U.

2013-01-01

Three-dimensional hierarchical patterning of metals is of paramount importance in diverse fields involving photonics, controlling surface wettability and wearable electronics. Conventionally, this type of structuring is tedious and usually involves layer-by-layer lithographic patterning. Here, we describe a simple process of direct nanoimprint lithography using palladium benzylthiolate, a versatile metal-organic ink, which not only leads to the formation of hierarchical patterns but also is amenable to layer-by-layer stacking of the metal over large areas. The key to achieving such multi-faceted patterning is hysteretic melting of ink, enabling its shaping. It undergoes transformation to metallic palladium under gentle thermal conditions without affecting the integrity of the hierarchical patterns on micro- as well as nanoscale. A metallic rice leaf structure showing anisotropic wetting behavior and woodpile-like structures were thus fabricated. Furthermore, this method is extendable for transferring imprinted structures to a flexible substrate to make them robust enough to sustain numerous bending cycles. PMID:23446801

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.

Crystal structure of the Entamoeba histolytica RNA lariat debranching enzyme EhDbr1 reveals a catalytic Zn 2+/Mn 2+ heterobinucleation

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

Ransey, Elizabeth; Paredes, Eduardo; Dey, Sourav K.

Here, the RNA lariat debranching enzyme, Dbr1, is a metallophosphoesterase that cleaves 2'-5' phosphodiester bonds within intronic lariats. Previous reports have indicated that Dbr1 enzymatic activity is supported by diverse metal ions including Ni 2+, Mn 2+, Mg 2+, Fe 2+, and Zn 2+. While in initial structures of the Entamoeba histolytica Dbr1 only one of the two catalytic metal-binding sites were observed to be occupied (with a Mn 2+ ion), recent structures determined a Zn 2+/Fe 2+ heterobinucleation. We solved a high-resolution X-ray crystal structure (1.8 Å) of the E. histolytica Dbr1 and determined a Zn 2+/Mn 2+ occupancy.more » ICP-AES corroborate this finding, and in vitro debranching assays with fluorescently labeled branched substrates confirm activity.« less

Soil Parameters Drive the Structure, Diversity and Metabolic Potentials of the Bacterial Communities Across Temperate Beech Forest Soil Sequences.

PubMed

Jeanbille, M; Buée, M; Bach, C; Cébron, A; Frey-Klett, P; Turpault, M P; Uroz, S

2016-02-01

Soil and climatic conditions as well as land cover and land management have been shown to strongly impact the structure and diversity of the soil bacterial communities. Here, we addressed under a same land cover the potential effect of the edaphic parameters on the soil bacterial communities, excluding potential confounding factors as climate. To do this, we characterized two natural soil sequences occurring in the Montiers experimental site. Spatially distant soil samples were collected below Fagus sylvatica tree stands to assess the effect of soil sequences on the edaphic parameters, as well as the structure and diversity of the bacterial communities. Soil analyses revealed that the two soil sequences were characterized by higher pH and calcium and magnesium contents in the lower plots. Metabolic assays based on Biolog Ecoplates highlighted higher intensity and richness in usable carbon substrates in the lower plots than in the middle and upper plots, although no significant differences occurred in the abundance of bacterial and fungal communities along the soil sequences as assessed using quantitative PCR. Pyrosequencing analysis of 16S ribosomal RNA (rRNA) gene amplicons revealed that Proteobacteria, Acidobacteria and Bacteroidetes were the most abundantly represented phyla. Acidobacteria, Proteobacteria and Chlamydiae were significantly enriched in the most acidic and nutrient-poor soils compared to the Bacteroidetes, which were significantly enriched in the soils presenting the higher pH and nutrient contents. Interestingly, aluminium, nitrogen, calcium, nutrient availability and pH appeared to be the best predictors of the bacterial community structures along the soil sequences.

The Type of Forage Substrate Preparation Included as Substrate in a RUSITEC System Affects the Ruminal Microbiota and Fermentation Characteristics

PubMed Central

Duarte, Andrea C.; Holman, Devin B.; Alexander, Trevor W.; Durmic, Zoey; Vercoe, Philip E.; Chaves, Alexandre V.

2017-01-01

In vitro fermentation systems such as the rumen simulation technique (RUSITEC) are frequently used to assess dietary manipulations in livestock, thereby limiting the use of live animals. Despite being in use for nearly 40 years, improvements are continually sought in these systems to better reflect and mimic natural processes in ruminants. The aim of this study was to evaluate the effect of forage preparation, i.e., frozen minced (FM) and freeze-dried and ground (FDG), on the ruminal microbiota and on fermentation characteristics when included as a substrate in a RUSITEC system. A completely randomized design experiment was performed over a 15-day period, with 7 days of adaptation and an 8-day experimental period. Fermentation parameters (total gas, CH4, and volatile fatty acid production) were analyzed on a daily basis over the experimental period and the archaeal and bacterial microbiota (liquid-associated microbes [LAM] and solid-associated microbes [SAM] was assessed at 0, 5, 10, and 15 days using high-throughput sequencing of the 16S rRNA gene. Results from this study suggested a tendency (P = 0.09) of FM treatment to increase daily CH4 (mg/d) production by 16.7% when compared with FDG treatment. Of the major volatile fatty acids (acetate, propionate, and butyrate), only butyrate production was greater (P = 0.01) with FM treatment compared with FDG substrate. The archaeal and bacterial diversity and richness did not differ between the forage preparations, although feed particle size of the forage had a significant effect on microbial community structure in the SAM and LAM samples. The Bacteroidetes phylum was more relatively abundant in the FM substrate treatment, while Proteobacteria was enriched in the FDG treatment. At the genus-level, Butyrivibrio, Prevotella, and Roseburia were enriched in the FM substrate treatment and Campylobacter and Lactobacillus in the FDG substrate treatment. Evidence from this study suggests that forage preparation affects CH4 production, butyrate production, and the structure of the rumen microbiota during in vitro fermentation. PMID:28473826

Matching the Diversity of Sulfated Biomolecules: Creation of a Classification Database for Sulfatases Reflecting Their Substrate Specificity

PubMed Central

Barbeyron, Tristan; Brillet-Guéguen, Loraine; Carré, Wilfrid; Carrière, Cathelène; Caron, Christophe; Czjzek, Mirjam; Hoebeke, Mark; Michel, Gurvan

2016-01-01

Sulfatases cleave sulfate groups from various molecules and constitute a biologically and industrially important group of enzymes. However, the number of sulfatases whose substrate has been characterized is limited in comparison to the huge diversity of sulfated compounds, yielding functional annotations of sulfatases particularly prone to flaws and misinterpretations. In the context of the explosion of genomic data, a classification system allowing a better prediction of substrate specificity and for setting the limit of functional annotations is urgently needed for sulfatases. Here, after an overview on the diversity of sulfated compounds and on the known sulfatases, we propose a classification database, SulfAtlas (http://abims.sb-roscoff.fr/sulfatlas/), based on sequence homology and composed of four families of sulfatases. The formylglycine-dependent sulfatases, which constitute the largest family, are also divided by phylogenetic approach into 73 subfamilies, each subfamily corresponding to either a known specificity or to an uncharacterized substrate. SulfAtlas summarizes information about the different families of sulfatases. Within a family a web page displays the list of its subfamilies (when they exist) and the list of EC numbers. The family or subfamily page shows some descriptors and a table with all the UniProt accession numbers linked to the databases UniProt, ExplorEnz, and PDB. PMID:27749924

Electrical characterization of anodic alumina substrate with via-in-pad structure

NASA Astrophysics Data System (ADS)

Kim, Moonjung

2013-10-01

An anodic alumina substrate has been developed as a package substrate for dynamic random access memory devices. Unlike the conventional package substrates commonly made by laminating an epoxy-based core and cladding with copper, this substrate is fabricated using aluminum anodization technology. The anodization process produces a thick aluminum oxide layer on the aluminum substrate to be used as a dielectric layer. Placing copper patterns on the anodic aluminum oxide layer forms a new substrate structure that consists of a layered structure of aluminum, anodic aluminum oxide, and copper. Using selective anodization in the fabrication process, a via structure connecting the top copper layer and bottom aluminum layer is demonstrated. Additionally, by putting vias directly in the bond and ball pads in the substrate design, the via-in-pad structure is applied in this work. These two-layer metal structures and via-in-pad arrangements make routing easier and thus provide more design flexibility. Additionally, this new package substrate has improved the power distribution network impedance given the characteristics of these structures.

[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.

Thought beyond language: neural dissociation of algebra and natural language.

PubMed

Monti, Martin M; Parsons, Lawrence M; Osherson, Daniel N

2012-08-01

A central question in cognitive science is whether natural language provides combinatorial operations that are essential to diverse domains of thought. In the study reported here, we addressed this issue by examining the role of linguistic mechanisms in forging the hierarchical structures of algebra. In a 3-T functional MRI experiment, we showed that processing of the syntax-like operations of algebra does not rely on the neural mechanisms of natural language. Our findings indicate that processing the syntax of language elicits the known substrate of linguistic competence, whereas algebraic operations recruit bilateral parietal brain regions previously implicated in the representation of magnitude. This double dissociation argues against the view that language provides the structure of thought across all cognitive domains.

Structural and Biochemical Characterization of Acinetobacter spp. Aminoglycoside Acetyltransferases Highlights Functional and Evolutionary Variation among Antibiotic Resistance Enzymes.

PubMed

Stogios, Peter J; Kuhn, Misty L; Evdokimova, Elena; Law, Melissa; Courvalin, Patrice; Savchenko, Alexei

2017-02-10

Modification of aminoglycosides by N-acetyltransferases (AACs) is one of the major mechanisms of resistance to these antibiotics in human bacterial pathogens. More than 50 enzymes belonging to the AAC(6') subfamily have been identified in Gram-negative and Gram-positive clinical isolates. Our understanding of the molecular function and evolutionary origin of these resistance enzymes remains incomplete. Here we report the structural and enzymatic characterization of AAC(6')-Ig and AAC(6')-Ih from Acinetobacter spp. The crystal structure of AAC(6')-Ig in complex with tobramycin revealed a large substrate-binding cleft remaining partially unoccupied by the substrate, which is in stark contrast with the previously characterized AAC(6')-Ib enzyme. Enzymatic analysis indicated that AAC(6')-Ig and -Ih possess a broad specificity against aminoglycosides but with significantly lower turnover rates as compared to other AAC(6') enzymes. Structure- and function-informed phylogenetic analysis of AAC(6') enzymes led to identification of at least three distinct subfamilies varying in oligomeric state, active site composition, and drug recognition mode. Our data support the concept of AAC(6') functionality originating through convergent evolution from diverse Gcn5-related-N-acetyltransferase (GNAT) ancestral enzymes, with AAC(6')-Ig and -Ih representing enzymes that may still retain ancestral nonresistance functions in the cell as provided by their particular active site properties.

Electrochemical Deposition of Conformal and Functional Layers on High Aspect Ratio Silicon Micro/Nanowires.

PubMed

Ozel, Tuncay; Zhang, Benjamin A; Gao, Ruixuan; Day, Robert W; Lieber, Charles M; Nocera, Daniel G

2017-07-12

Development of new synthetic methods for the modification of nanostructures has accelerated materials design advances to furnish complex architectures. Structures based on one-dimensional (1D) silicon (Si) structures synthesized using top-down and bottom-up methods are especially prominent for diverse applications in chemistry, physics, and medicine. Yet further elaboration of these structures with distinct metal-based and polymeric materials, which could open up new opportunities, has been difficult. We present a general electrochemical method for the deposition of conformal layers of various materials onto high aspect ratio Si micro- and nanowire arrays. The electrochemical deposition of a library of coaxial layers comprising metals, metal oxides, and organic/inorganic semiconductors demonstrate the materials generality of the synthesis technique. Depositions may be performed on wire arrays with varying diameter (70 nm to 4 μm), pitch (5 μ to 15 μ), aspect ratio (4:1 to 75:1), shape (cylindrical, conical, hourglass), resistivity (0.001-0.01 to 1-10 ohm/cm 2 ), and substrate orientation. Anisotropic physical etching of wires with one or more coaxial shells yields 1D structures with exposed tips that can be further site-specifically modified by an electrochemical deposition approach. The electrochemical deposition methodology described herein features a wafer-scale synthesis platform for the preparation of multifunctional nanoscale devices based on a 1D Si substrate.

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.

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

Phage phenomics: Physiological approaches to characterize novel viral proteins

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

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

Expanding the chemical diversity of natural esters by engineering a polyketide-derived pathway into Escherichia coli.

PubMed

Menendez-Bravo, Simón; Comba, Santiago; Sabatini, Martín; Arabolaza, Ana; Gramajo, Hugo

2014-07-01

Microbial fatty acid (FA)-derived molecules have emerged as promising alternatives to petroleum-based chemicals for reducing dependence on fossil hydrocarbons. However, native FA biosynthetic pathways often yield limited structural diversity, and therefore restricted physicochemical properties, of the end products by providing only a limited variety of usually linear hydrocarbons. Here we have engineered into Escherichia coli a mycocerosic polyketide synthase-based biosynthetic pathway from Mycobacterium tuberculosis and redefined its biological role towards the production of multi-methyl-branched-esters (MBEs) with novel chemical structures. Expression of FadD28, Mas and PapA5 enzymes enabled the biosynthesis of multi-methyl-branched-FA and their further esterification to an alcohol. The high substrate tolerance of these enzymes towards different FA and alcohol moieties resulted in the biosynthesis of a broad range of MBE. Further metabolic engineering of the MBE producer strain coupled this system to long-chain-alcohol biosynthetic pathways resulting in de novo production of branched wax esters following addition of only propionate. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Phage phenomics: Physiological approaches to characterize novel viral proteins

DOE PAGES

Sanchez, Savannah E.; Cuevas, Daniel A.; Rostron, Jason E.; ...

2015-06-11

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysismore » by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Thus, representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.« less

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.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

New World Bats Harbor Diverse Influenza A Viruses

PubMed Central

Tong, Suxiang; Zhu, Xueyong; Li, Yan; Shi, Mang; Zhang, Jing; Bourgeois, Melissa; Yang, Hua; Chen, Xianfeng; Recuenco, Sergio; Gomez, Jorge; Chen, Li-Mei; Johnson, Adam; Tao, Ying; Dreyfus, Cyrille; Yu, Wenli; McBride, Ryan; Carney, Paul J.; Gilbert, Amy T.; Chang, Jessie; Guo, Zhu; Davis, Charles T.; Paulson, James C.; Stevens, James; Rupprecht, Charles E.; Holmes, Edward C.; Wilson, Ian A.; Donis, Ruben O.

2013-01-01

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses. PMID:24130481

Recovery of Seamount Precious Coral Beds From Heavy Trawling Disturbance with Links to Carbonate Chemistry Changes

NASA Astrophysics Data System (ADS)

Roark, E. B.; Baco-Taylor, A.; Morgan, N. B.; Shamberger, K.; Miller, K.; Brooks, J.

2016-12-01

Increasing anthropogenic impacts in the deep sea make studies of resilience and recovery time critical, with deep-sea hard-substrate habitats and large-scale disturbances having received little attention. Seamount hard-substrate habitats in particular are thought to have low resilience due to the slow growth rates and recruitment limitations of key structure-forming taxa. Seamounts of the far Northwestern Hawaiian Islands and Emperor Chain have had some of the heaviest trawl impacts in the world, from both fish and precious coral fisheries, and include sites that are still trawled and recovering ones that have been protected since establishment of the EEZ in 1977. To test the hypothesis of low resilience we compare these impacted seamounts to untrawled sites. We used the AUV Sentry in 2014 and 2015 to image nine features (three per "treatment") and analyze for substrate and visible megafauna. Sites in the "still trawled" treatment were characterized by extensive areas of bare substrate with abundant trawl scars. Sites in the "recovering" and "never trawled" locations had abundant megafauna in hard substrate areas. Initial comparisons of transects at 700m depth for three sites indicate that Yuryaku in the "still trawled" treatment had lower diversity and abundance of megafauna compared to the "recovering" and "never trawled" locations with a dominance of sea urchins. The "recovering" and "never trawled" sites were dominated by cnidarians, fishes, and echinoderms, but differed in dominant species, diversity, abundances and occurrence of dead coral skeletons. These preliminary results suggest that the recovering sites have not returned to a pre-impact community type in the 38 years since they were trawled. The megafauna distribution, in particular that of deep-sea corals, was compared to environmental water column variables at the study sites across the Northwestern Hawaiian Islands. Deep-sea corals with calcium carbonate skeletons were found living below the aragonite saturation horizon (ASH; Ωarg=1), which ranges from 500-600 m depth and deepens moving northwest along the island chain. How deep-sea corals build and maintain their skeletons in undersaturated waters is poorly understood, but it is possible that saturation horizons may play a role in their recovery rates.

Glycoscience@Synchrotron: Synchrotron radiation applied to structural glycoscience

PubMed Central

de Sanctis, Daniele

2017-01-01

Synchrotron radiation is the most versatile way to explore biological materials in different states: monocrystalline, polycrystalline, solution, colloids and multiscale architectures. Steady improvements in instrumentation have made synchrotrons the most flexible intense X-ray source. The wide range of applications of synchrotron radiation is commensurate with the structural diversity and complexity of the molecules and macromolecules that form the collection of substrates investigated by glycoscience. The present review illustrates how synchrotron-based experiments have contributed to our understanding in the field of structural glycobiology. Structural characterization of protein–carbohydrate interactions of the families of most glycan-interacting proteins (including glycosyl transferases and hydrolases, lectins, antibodies and GAG-binding proteins) are presented. Examples concerned with glycolipids and colloids are also covered as well as some dealing with the structures and multiscale architectures of polysaccharides. Insights into the kinetics of catalytic events observed in the crystalline state are also presented as well as some aspects of structure determination of protein in solution. PMID:28684994

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

Influence of Vegetations' Metabolites on the Composition and Functioning of Soil Microbial Complex

NASA Astrophysics Data System (ADS)

Biryukov, Mikhail

2013-04-01

Microbiota is one of the major factors of soils fertility. It transforms organic substances in soil and, therefore, serves as the main component in the cycles of carbon and nitrogen. Microbial communities (MC) are characterized as highly diverse and extremely complex structures. This allows them to adapt to any affection and provide all the necessary biospheric functions. Hence, the study of their functional diversity and adaptivity of microbiota provides the key to the understanding of the ecosystems' functioning and their adaptivity to the human impact. The formation of MC at the initial stage is regulated by the fluxes of substrates and biologically active substances (BAS), which vary greatly in soils under different vegetations. These fluxes are presented by: low molecular weights organic substances (LMWOS), which can be directly included in metabolism of microbes; polymers, that can be decomposed to LMWOS by exoenzymes; and more complex compounds, having different "drug effects" (e.g. different types of phenolic acids) and regulating growth and enzymatic properties of microbiota. Therefore, the main hypothesis of the research was formulated as follows: penetration of different types of substrates and BAS into soil leads to the emergence of MC varying in enzymatic properties and structure. As a soil matrix we used the soil from the untreated variant of the lysimeter model experiment taking place in the faculty of Soil Science of the MSU for over the last 40 years. It was sieved with a 2mm sieves, humidified and incubated at 25C during one week. Subsequently, the samples were air-dried with occasional stirring for one more week. Thereafter, aliquots of the prepared soil were taken for the different experimental variants. The samples were rewetted with solutions of various substrates (glucose, cellulose, starch, etc.) and thoroughly mixed. The control variant was established with addition of deionised water. The samples were incubated at the 25C. During the incubation the rate of mineralisation of organic substances was assessed with CO2 measurements. In 5, 10 and 21 days of incubation the enzymatic properties of the formed MC were studied by the hydrolysis of fluorogenic substrates. The influence of BAS on enzymatic properties of MC were researched by addition of different concentrations of phenolic acids (e.g. salicylic, vanillic, benzoic, etc.) to the samples from various substrates treatments. The acute toxicity of BAS was studied with bacterial luminescent test. After the last measurement, the isolations of microorganisms on elective nutrient medias were made. The dominant microorganisms were collected to the library for further identification and physiological tests. MeOH-chloroform extraction of phospholipids were performed with the remaining samples. Finally, they were stored for subsequent FAME identifications. The obtained data prove that penetration of various substrates into the soil determines the formation of MC different in structure and properties. It was found, that EC50 of the most studied phenolic acids are similar to naturally occurring concentrations. This means that they can be the real drivers of forming endemical MC under various vegetations along with the plant-specific fluxes of nutrients.

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.

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

Spatial impacts of inorganic ligand availability and localized microbial community structure on mitigation of zinc laden mine water in sulfate-reducing bioreactors.

PubMed

Drennan, Dina M; Almstrand, Robert; Ladderud, Jeffrey; Lee, Ilsu; Landkamer, Lee; Figueroa, Linda; Sharp, Jonathan O

2017-05-15

Sulfate-reducing bioreactors (SRBRs) represent a passive, sustainable, and long-term option for mitigating mining influenced water (MIW) during release. Here we investigate spatial zinc precipitation profiles as influenced by substrate differentiation, inorganic ligand availability (inorganic carbon and sulfide), and microbial community structure in pilot-scale SRBR columns fed with sulfate and zinc-rich MIW. Through a combination of aqueous sampling, geochemical digests, electron microscopy and energy-dispersive x-ray spectroscopy, we were able to delineate zones of enhanced zinc removal, identify precipitates of varying stability, and discern the temporal and spatial evolution of zinc, sulfur, and calcium associations. These geochemical insights revealed spatially variable immobilization regimes between SRBR columns that could be further contrasted as a function of labile (alfalfa-dominated) versus recalcitrant (woodchip-dominated) solid-phase substrate content. Both column subsets exhibited initial zinc removal as carbonates; however precipitation in association with labile substrates was more pronounced and dominated by metal-sulfide formation in the upper portions of the down flow columns with micrographs visually suggestive of sphalerite (ZnS). In contrast, a more diffuse and lower mass of zinc precipitation in the presence of gypsum-like precipitates occurred within the more recalcitrant column systems. While removal and sulfide-associated precipitation were spatially variable, whole bacterial community structure (ANOSIM) and diversity estimates were comparatively homogeneous. However, two phyla exhibited a potentially selective relationship with a significant positive correlation between the ratio of Firmicutes to Bacteroidetes and sulfide-bound zinc. Collectively these biogeochemical insights indicate that depths of maximal zinc sulfide precipitation are temporally dynamic, influenced by substrate composition and broaden our understanding of bio-immobilized zinc species, microbial interactions and potential operational and monitoring tools in these types of passive bioreactors. Copyright © 2017 Elsevier Ltd. All rights reserved.

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.

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.

Species richness, distribution and genetic diversity of Caenorhabditis nematodes in a remote tropical rainforest

PubMed Central

2013-01-01

Background In stark contrast to the wealth of detail about C. elegans developmental biology and molecular genetics, biologists lack basic data for understanding the abundance and distribution of Caenorhabditis species in natural areas that are unperturbed by human influence. Methods Here we report the analysis of dense sampling from a small, remote site in the Amazonian rain forest of the Nouragues Natural Reserve in French Guiana. Results Sampling of rotting fruits and flowers revealed proliferating populations of Caenorhabditis, with up to three different species co-occurring within a single substrate sample, indicating remarkable overlap of local microhabitats. We isolated six species, representing the highest local species richness for Caenorhabditis encountered to date, including both tropically cosmopolitan and geographically restricted species not previously isolated elsewhere. We also documented the structure of within-species molecular diversity at multiple spatial scales, focusing on 57 C. briggsae isolates from French Guiana. Two distinct genetic subgroups co-occur even within a single fruit. However, the structure of C. briggsae population genetic diversity in French Guiana does not result from strong local patterning but instead presents a microcosm of global patterns of differentiation. We further integrate our observations with new data from nearly 50 additional recently collected C. briggsae isolates from both tropical and temperate regions of the world to re-evaluate local and global patterns of intraspecific diversity, providing the most comprehensive analysis to date for C. briggsae population structure across multiple spatial scales. Conclusions The abundance and species richness of Caenorhabditis nematodes is high in a Neotropical rainforest habitat that is subject to minimal human interference. Microhabitat preferences overlap for different local species, although global distributions include both cosmopolitan and geographically restricted groups. Local samples for the cosmopolitan C. briggsae mirror its pan-tropical patterns of intraspecific polymorphism. It remains an important challenge to decipher what drives Caenorhabditis distributions and diversity within and between species. PMID:23311925

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

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme coopted from flavonoid metabolism.

PubMed

Salim, Vonny; Jones, A Daniel; DellaPenna, Dean

2018-04-22

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of Class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMT in vivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Structural Characterization and Ligand/Inhibitor Identification Provide Functional Insights into the Mycobacterium tuberculosis Cytochrome P450 CYP126A1*

PubMed Central

Chenge, Jude T.; Duyet, Le Van; Swami, Shalini; McLean, Kirsty J.; Kavanagh, Madeline E.; Coyne, Anthony G.; Rigby, Stephen E. J.; Cheesman, Myles R.; Girvan, Hazel M.; Levy, Colin W.; Rupp, Bernd; von Kries, Jens P.; Abell, Chris; Leys, David; Munro, Andrew W.

2017-01-01

The Mycobacterium tuberculosis H37Rv genome encodes 20 cytochromes P450, including P450s crucial to infection and bacterial viability. Many M. tuberculosis P450s remain uncharacterized, suggesting that their further analysis may provide new insights into M. tuberculosis metabolic processes and new targets for drug discovery. CYP126A1 is representative of a P450 family widely distributed in mycobacteria and other bacteria. Here we explore the biochemical and structural properties of CYP126A1, including its interactions with new chemical ligands. A survey of azole antifungal drugs showed that CYP126A1 is inhibited strongly by azoles containing an imidazole ring but not by those tested containing a triazole ring. To further explore the molecular preferences of CYP126A1 and search for probes of enzyme function, we conducted a high throughput screen. Compounds containing three or more ring structures dominated the screening hits, including nitroaromatic compounds that induce substrate-like shifts in the heme spectrum of CYP126A1. Spectroelectrochemical measurements revealed a 155-mV increase in heme iron potential when bound to one of the newly identified nitroaromatic drugs. CYP126A1 dimers were observed in crystal structures of ligand-free CYP126A1 and for CYP126A1 bound to compounds discovered in the screen. However, ketoconazole binds in an orientation that disrupts the BC-loop regions at the P450 dimer interface and results in a CYP126A1 monomeric crystal form. Structural data also reveal that nitroaromatic ligands “moonlight” as substrates by displacing the CYP126A1 distal water but inhibit enzyme activity. The relatively polar active site of CYP126A1 distinguishes it from its most closely related sterol-binding P450s in M. tuberculosis, suggesting that further investigations will reveal its diverse substrate selectivity. PMID:27932461

Graphene-on-semiconductor substrates for analog electronics

DOEpatents

Lagally, Max G.; Cavallo, Francesca; Rojas-Delgado, Richard

2016-04-26

Electrically conductive material structures, analog electronic devices incorporating the structures and methods for making the structures are provided. The structures include a layer of graphene on a semiconductor substrate. The graphene layer and the substrate are separated by an interfacial region that promotes transfer of charge carriers from the surface of the substrate to the graphene.

Novel carboxy functionalized sol-gel precursors

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

Wolter, H.; Storch, W.; Gellermann, C.

1996-12-31

A novel family of inorganic-organic copolymers (ORMOCER`s) derived from urethane- and thioether(meth)acrylate alkoxysilanes has been successfully exploited for a variety of diverse applications. In order to widen the range of applications an additional functionality (carboxy group) has been incorporated int his silane type. Conventional sol-gel processing facilitates the formation of an inorganic Si-O-Si-network via hydrolysis and polycondensation reactions of alkoxysilyl moieties and in addition, the (meth)acrylate groups are available for radically induced polymerization to obtain a complementary organic polymer structure. The presence of a carboxy group would appear to have great potential for a range of diverse areas of application,more » such as an internal catalyst for the sol-gel process, complexation of elements such as Zr and Ti, increasing the adhesion to various substrates and modification of solubility. A number of novel silanes and their syntheses will be described in this paper.« less

Eukaryotic major facilitator superfamily transporter modeling based on the prokaryotic GlpT crystal structure.

PubMed

Lemieux, M Joanne

2007-01-01

The major facilitator superfamily (MFS) of transporters represents the largest family of secondary active transporters and has a diverse range of substrates. With structural information for four MFS transporters, we can see a strong structural commonality suggesting, as predicted, a common architecture for MFS transporters. The rate for crystal structure determination of MFS transporters is slow, making modeling of both prokaryotic and eukaryotic transporters more enticing. In this review, models of eukaryotic transporters Glut1, G6PT, OCT1, OCT2 and Pho84, based on the crystal structures of the prokaryotic GlpT, based on the crystal structure of LacY are discussed. The techniques used to generate the different models are compared. In addition, the validity of these models and the strategy of using prokaryotic crystal structures to model eukaryotic proteins are discussed. For comparison, E. coli GlpT was modeled based on the E. coli LacY structure and compared to the crystal structure of GlpT demonstrating that experimental evidence is essential for accurate modeling of membrane proteins.

STN1 OB Fold Mutation Alters DNA Binding and Affects Selective Aspects of CST Function

PubMed Central

Bhattacharjee, Anukana; Stewart, Jason; Chaiken, Mary; Price, Carolyn M.

2016-01-01

Mammalian CST (CTC1-STN1-TEN1) participates in multiple aspects of telomere replication and genome-wide recovery from replication stress. CST resembles Replication Protein A (RPA) in that it binds ssDNA and STN1 and TEN1 are structurally similar to RPA2 and RPA3. Conservation between CTC1 and RPA1 is less apparent. Currently the mechanism underlying CST action is largely unknown. Here we address CST mechanism by using a DNA-binding mutant, (STN1 OB-fold mutant, STN1-OBM) to examine the relationship between DNA binding and CST function. In vivo, STN1-OBM affects resolution of endogenous replication stress and telomere duplex replication but telomeric C-strand fill-in and new origin firing after exogenous replication stress are unaffected. These selective effects indicate mechanistic differences in CST action during resolution of different replication problems. In vitro binding studies show that STN1 directly engages both short and long ssDNA oligonucleotides, however STN1-OBM preferentially destabilizes binding to short substrates. The finding that STN1-OBM affects binding to only certain substrates starts to explain the in vivo separation of function observed in STN1-OBM expressing cells. CST is expected to engage DNA substrates of varied length and structure as it acts to resolve different replication problems. Since STN1-OBM will alter CST binding to only some of these substrates, the mutant should affect resolution of only a subset of replication problems, as was observed in the STN1-OBM cells. The in vitro studies also provide insight into CST binding mechanism. Like RPA, CST likely contacts DNA via multiple OB folds. However, the importance of STN1 for binding short substrates indicates differences in the architecture of CST and RPA DNA-protein complexes. Based on our results, we propose a dynamic DNA binding model that provides a general mechanism for CST action at diverse forms of replication stress. PMID:27690379

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

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.

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...

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase*S⃞

PubMed Central

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-01-01

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes. PMID:19158086

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

PubMed

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-04-03

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

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

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.

Microbial diversity and methanogenic activity of Antrim Shale formation waters from recently fractured wells

PubMed Central

Wuchter, Cornelia; Banning, Erin; Mincer, Tracy J.; Drenzek, Nicholas J.; Coolen, Marco J. L.

2013-01-01

The Antrim Shale in the Michigan Basin is one of the most productive shale gas formations in the U.S., but optimal resource recovery strategies must rely on a thorough understanding of the complex biogeochemical, microbial, and physical interdependencies in this and similar systems. We used Illumina MiSeq 16S rDNA sequencing to analyze the diversity and relative abundance of prokaryotic communities present in Antrim shale formation water of three closely spaced recently fractured gas-producing wells. In addition, the well waters were incubated with a suite of fermentative and methanogenic substrates in an effort to stimulate microbial methane generation. The three wells exhibited substantial differences in their community structure that may arise from their different drilling and fracturing histories. Bacterial sequences greatly outnumbered those of archaea and shared highest similarity to previously described cultures of mesophiles and moderate halophiles within the Firmicutes, Bacteroidetes, and δ- and ε-Proteobacteria. The majority of archaeal sequences shared highest sequence similarity to uncultured euryarchaeotal environmental clones. Some sequences closely related to cultured methylotrophic and hydrogenotrophic methanogens were also present in the initial well water. Incubation with methanol and trimethylamine stimulated methylotrophic methanogens and resulted in the largest increase in methane production in the formation waters, while fermentation triggered by the addition of yeast extract and formate indirectly stimulated hydrogenotrophic methanogens. The addition of sterile powdered shale as a complex natural substrate stimulated the rate of methane production without affecting total methane yields. Depletion of methane indicative of anaerobic methane oxidation (AMO) was observed over the course of incubation with some substrates. This process could constitute a substantial loss of methane in the shale formation. PMID:24367357

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

Increased Alignment in Carbon Nanotube Growth

NASA Technical Reports Server (NTRS)

Delzeit, Lance D. (Inventor)

2007-01-01

Method and system for fabricating an array of two or more carbon nanotube (CNT) structures on a coated substrate surface, the structures having substantially the same orientation with respect to a substrate surface. A single electrode, having an associated voltage source with a selected voltage, is connected to a substrate surface after the substrate is coated and before growth of the CNT structures, for a selected voltage application time interval. The CNT structures are then grown on a coated substrate surface with the desired orientation. Optionally, the electrode can be disconnected before the CNT structures are grown.

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

High Transparent and Conductive TiO2/Ag/TiO2 Multilayer Electrode Films Deposited on Sapphire Substrate

NASA Astrophysics Data System (ADS)

Loka, Chadrasekhar; Moon, Sung Whan; Choi, YiSik; Lee, Kee-Sun

2018-03-01

Transparent conducting oxides attract intense interests due to its diverse industrial applications. In this study, we report sapphire substrate-based TiO2/Ag/TiO2 (TAT) multilayer structure of indium-free transparent conductive multilayer coatings. The TAT thin films were deposited at room temperature on sapphire substrates and a rigorous analysis has been presented on the electrical and optical properties of the films as a function of Ag thickness. The optical and electrical properties were mainly controlled by the Ag mid-layer thickness of the TAT tri-layer. The TAT films showed high luminous transmittance 84% at 550 nm along with noteworthy low electrical resistance 3.65 × 10-5 Ω-cm and sheet resistance of 3.77 Ω/square, which is better are than those of amorphous ITO films and any sapphire-based dielectric/metal/dielectric multilayer stack. The carrier concentration of the films was increased with respect to Ag thickness. We obtained highest Hackke's figure of merit 43.97 × 10-3 Ω-1 from the TAT multilayer thin film with a 16 nm thick Ag mid-layer.

Repetitive Protein Unfolding by the trans Ring of the GroEL-GroES Chaperonin Complex Stimulates Folding*

PubMed Central

Lin, Zong; Puchalla, Jason; Shoup, Daniel; Rye, Hays S.

2013-01-01

A key constraint on the growth of most organisms is the slow and inefficient folding of many essential proteins. To deal with this problem, several diverse families of protein folding machines, known collectively as molecular chaperones, developed early in evolutionary history. The functional role and operational steps of these remarkably complex nanomachines remain subjects of active debate. Here we present evidence that, for the GroEL-GroES chaperonin system, the non-native substrate protein enters the folding cycle on the trans ring of the double-ring GroEL-ATP-GroES complex rather than the ADP-bound complex. The properties of this ATP complex are designed to ensure that non-native substrate protein binds first, followed by ATP and finally GroES. This binding order ensures efficient occupancy of the open GroEL ring and allows for disruption of misfolded structures through two phases of multiaxis unfolding. In this model, repeated cycles of partial unfolding, followed by confinement within the GroEL-GroES chamber, provide the most effective overall mechanism for facilitating the folding of the most stringently dependent GroEL substrate proteins. PMID:24022487

Nitrogen removal and its relationship with the nitrogen-cycle genes and microorganisms in the horizontal subsurface flow constructed wetlands with different design parameters.

PubMed

Chen, Jun; Ying, Guang-Guo; Liu, You-Sheng; Wei, Xiao-Dong; Liu, Shuang-Shuang; He, Liang-Ying; Yang, Yong-Qiang; Chen, Fan-Rong

2017-07-03

This study aims to investigate nitrogen removal and its relationship with the nitrogen-cycle genes and microorganisms in the horizontal subsurface flow constructed wetlands (CWs) with different design parameters. Twelve mesocosm-scale CWs with four substrates and three hydraulic loading rates were set up in the outdoor. The result showed the CWs with zeolite as substrate and HLR of 20 cm/d were selected as the best choice for the TN and NH 3 -N removal. It was found that the single-stage mesocosm-scale CWs were incapable to achieve high removals of TN and NH 3 -N due to inefficient nitrification process in the systems. This was demonstrated by the lower abundance of the nitrification genes (AOA and AOB) than the denitrification genes (nirK and nirS), and the less diverse nitrification microorganisms than the denitrification microorganisms in the CWs. The results also show that microorganism community structure including nitrogen-cycle microorganisms in the constructed wetland systems was affected by the design parameters especially the substrate type. These findings show that nitrification is a limiting factor for the nitrogen removal by CWs.

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

Substrate dependent hierarchical structures of RF sputtered ZnS films

NASA Astrophysics Data System (ADS)

Chalana, S. R.; Mahadevan Pillai, V. P.

2018-05-01

RF magnetron sputtering technique was employed to fabricate ZnS nanostructures with special emphasis given to study the effect of substrates (quartz, glass and quartz substrate pre-coated with Au, Ag, Cu and Pt) on the structure, surface evolution and optical properties. Type of substrate has a significant influence on the crystalline phase, film morphology, thickness and surface roughness. The present study elucidates the suitability of quartz substrate for the deposition of stable and highly crystalline ZnS films. We found that the role of metal layer on quartz substrate is substantial in the preparation of hierarchical ZnS structures and these structures are of great importance due to its high specific area and potential applications in various fields. A mechanism for morphological evolution of ZnS structures is also presented based on the roughness of substrates and primary nonlocal effects in sputtering. Furthermore, the findings suggest that a controlled growth of hierarchical ZnS structures may be achieved with an ordinary RF sputtering technique by changing the substrate type.

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

Aquatic macroinvertebrates and water quality of Sandia Canyon, Los Alamos National Laboratory, December 1992--October 1993. Status report

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

Cross, S.

1994-09-01

In the summer of 1990, an accidental spill from the TA-3 Power Plant Environment Tank released more than 3,785 liters of sulfuric acid into upper Sandia Canyon. The Biological Resource Evaluation Team (BRET) of EM-8 at Los Alamos National Laboratory (LANL) has collected aquatic samples from the stream within Sandia Canyon since then. These field studies gather water quality measurements and collect macroinvertebrates from permanent sampling sites. An earlier report by Bennett (1994) discusses previous BRET aquatic studies in Sandia Canyon. This report updates and expands Bennett`s initial findings. During 1993, BRET collected water quality data and aquatic macroinvertebrates atmore » five permanent stations within the canyon. The substrates of the upper three stations are largely sands and silts while the substrates of the two lower stations are largely rock and cobbles. The two upstream stations are located near outfalls that discharge industrial and sanitary waste effluent. The third station is within a natural cattail marsh, approximately 0.4 km (0.25 mi) downstream from Stations SC1 and SC2. Water quality parameters are slightly different at these first three stations from those expected of natural streams, suggesting slightly degraded water quality. Correspondingly, the macroinvertebrate communities at these stations are characterized by low diversities and poorly-developed community structures. The two downstream stations appear to be in a zone of recovery, where water quality parameters more closely resemble those found in natural streams of the area. Macroinvertebrate diversity increases and community structure becomes more complex at the two lower stations, which are further indications of improved water quality downstream.« less

A novel β-glucosidase isolated from the microbial metagenome of Lake Poraquê (Amazon, Brazil).

PubMed

Toyama, Danyelle; de Morais, Mariana Abrahão Bueno; Ramos, Felipe Cardoso; Zanphorlin, Letícia Maria; Tonoli, Celisa Caldana Costa; Balula, Augusto Furio; de Miranda, Fernando Pellon; Almeida, Vitor Medeiros; Marana, Sandro Roberto; Ruller, Roberto; Murakami, Mario Tyago; Henrique-Silva, Flavio

2018-04-01

The Amazon region holds most of the biological richness of Brazil. Despite their ecological and biotechnological importance, studies related to microorganisms from this region are limited. Metagenomics leads to exciting discoveries, mainly regarding non-cultivable microorganisms. Herein, we report the discovery of a novel β-glucosidase (glycoside hydrolase family 1) gene from a metagenome from Lake Poraquê in the Amazon region. The gene encodes a protein of 52.9 kDa, named AmBgl-LP, which was recombinantly expressed in Escherichia coli and biochemically and structurally characterized. Although AmBgl-LP hydrolyzed the synthetic substrate p-nitrophenyl-β-d-glucopyranoside (pNPβG) and the natural substrate cellobiose, it showed higher specificity for pNPβG (k cat /K m  = 6 s -1 ·mM -1 ) than cellobiose (k cat /K m  = 0.6 s -1 ·mM -1 ). AmBgl-LP showed maximum activity at 40 °C and pH 6.0 when pNPβG was used as the substrate. Glucose is a competitive inhibitor of AmBgl-LP, presenting a K i of 14 mM. X-ray crystallography and Small Angle X-ray Scattering were used to determine the AmBgl-LP three-dimensional structure and its oligomeric state. Interestingly, despite sharing similar active site architecture with other structurally characterized GH1 family members which are monomeric, AmBgl-LP forms stable dimers in solution. The identification of new GH1 members by metagenomics might extend our understanding of the molecular mechanisms and diversity of these enzymes, besides enabling us to survey their industrial applications. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

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

Environmental controls on denitrifying communities and denitrification rates--Insights from molecular methods

USGS Publications Warehouse

Wallenstein, Matthew D.; Myrold, David D.; Firestone, Mary; Voytek, Mary

2006-01-01

The advent of molecular techniques has improved our understanding of the microbial communities responsible for denitrification and is beginning to address their role in controlling denitrification processes. There is a large diversity of bacteria, archaea, and fungi capable of denitrification, and their community composition is structured by long-term environmental drivers. The range of temperature and moisture conditions, substrate availability, competition, and disturbances have long-lasting legacies on denitrifier community structure. These communities may differ in physiology, environmental tolerances to pH and O2, growth rate, and enzyme kinetics. Although factors such as O2, pH, C availability, and NO3− pools affect instantaneous rates, these drivers act through the biotic community. This review summarizes the results of molecular investigations of denitrifier communities in natural environments and provides a framework for developing future research for addressing connections between denitrifier community structure and function.

Structure and Biocatalytic Scope of Coclaurine N-Methyltransferase.

PubMed

Bennett, Matthew; Thompson, Mark; Shepherd, Sarah; Dunstan, Mark; Herbert, Abigail; Smith, Duncan; Cronin, Victoria; Menon, Binuraj; Levy, Colin; Micklefield, Jason

2018-05-23

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse family of plant secondary metabolites which have been exploited to develop analgesics, antibiotics, antitumor agents and other therapeutic agents. Biosynthesis of BIAs proceeds via a common pathway from tyrosine to (S)-reticulene at which point the pathway diverges. Coclaurine N-methyltransferase (CNMT) is a key enzyme in the pathway to (S)-reticulene, installing the N-methyl substituent that is essential for the bioactivity of many BIAs. In this paper, we describe the first crystal structure of CNMT which, along with mutagenesis studies, defines the enzymes active site architecture. The specificity of CNMT was also explored with a range of natural and synthetic substrates as well as co-factor analogues. Knowledge from this study could be used to generate improved CNMT variants required to produce BIAs or synthetic derivatives. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

Superconductive articles including cerium oxide layer

DOEpatents

Wu, X.D.; Muenchausen, R.E.

1993-11-16

A ceramic superconductor comprising a metal oxide substrate, a ceramic high temperature superconductive material, and a intermediate layer of a material having a cubic crystal structure, said layer situated between the substrate and the superconductive material is provided, and a structure for supporting a ceramic superconducting material is provided, said structure comprising a metal oxide substrate, and a layer situated over the surface of the substrate to substantially inhibit interdiffusion between the substrate and a ceramic superconducting material deposited upon said structure. 7 figures.

Superconductive articles including cerium oxide layer

DOEpatents

Wu, Xin D.; Muenchausen, Ross E.

1993-01-01

A ceramic superconductor comprising a metal oxide substrate, a ceramic high temperature superconductive material, and a intermediate layer of a material having a cubic crystal structure, said layer situated between the substrate and the superconductive material is provided, and a structure for supporting a ceramic superconducting material is provided, said structure comprising a metal oxide substrate, and a layer situated over the surface of the substrate to substantially inhibit interdiffusion between the substrate and a ceramic superconducting material deposited upon said structure.

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.

Network-Level Structure-Function Relationships in Human Neocortex

PubMed Central

Mišić, Bratislav; Betzel, Richard F.; de Reus, Marcel A.; van den Heuvel, Martijn P.; Berman, Marc G.; McIntosh, Anthony R.; Sporns, Olaf

2016-01-01

The dynamics of spontaneous fluctuations in neural activity are shaped by underlying patterns of anatomical connectivity. While numerous studies have demonstrated edge-wise correspondence between structural and functional connections, much less is known about how large-scale coherent functional network patterns emerge from the topology of structural networks. In the present study, we deploy a multivariate statistical technique, partial least squares, to investigate the association between spatially extended structural networks and functional networks. We find multiple statistically robust patterns, reflecting reliable combinations of structural and functional subnetworks that are optimally associated with one another. Importantly, these patterns generally do not show a one-to-one correspondence between structural and functional edges, but are instead distributed and heterogeneous, with many functional relationships arising from nonoverlapping sets of anatomical connections. We also find that structural connections between high-degree hubs are disproportionately represented, suggesting that these connections are particularly important in establishing coherent functional networks. Altogether, these results demonstrate that the network organization of the cerebral cortex supports the emergence of diverse functional network configurations that often diverge from the underlying anatomical substrate. PMID:27102654

Arginine Kinase. Joint Crystallographic & NMR RDC Analyses link Substrate-Associated Motions to Intrinsic Flexibility

PubMed Central

Niu, Xiaogang; Brüschweiler-Li, Lei; Davulcu, Omar; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

2010-01-01

The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a “high energy” phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and –bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection. PMID:21075117

Multi-Isotope Secondary Ion Mass Spectrometry Combining Heavy Water 2H with 15N Labeling As Complementary Tracers for Metabolic Heterogeneity at the Single-Cell Level

NASA Astrophysics Data System (ADS)

Kopf, S.; McGlynn, S.; Cowley, E.; Green, A.; Newman, D. K.; Orphan, V. J.

2014-12-01

Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system measurements of 2H/1H, 13C/12C and 15N/14N and apply it to study of microbial metabolic heterogeneity and nitrogen metabolism in a continuous culture case study. Our data provide insight into both the diversity of microbial activity rates, as well as patterns of ammonium utilization at the single cell level.

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

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

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

Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.

2008-06-23

Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target for tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 {angstrom}. The structure of the apoenzyme reveals that the protein is composed of five -helicesmore » with connecting loops and is a member of the {alpha}-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between {alpha}-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.« less

Oyster reef restoration in the Northern Gulf of Mexico: effect of artificial substrate and sge on nekton and benthic macroinvertebrate assemblage use

USGS Publications Warehouse

Brown, Laura A.; Furlong, Jessica N.; Brown, Kenneth M.; LaPeyre, Megan K.

2013-01-01

In the northern Gulf of Mexico (GOM), reefs built by eastern oysters, Crassostrea virginica, provide critical habitat within shallow estuaries, and recent efforts have focused on restoring reefs to benefit nekton and benthic macroinvertebrates. We compared nekton and benthic macroinvertebrate assemblages at historic, newly created (<5years) and old (>6years) shell and rock substrate reefs. Using crab traps, gill-nets, otter trawls, cast nets, and benthic macroinvertebrate collectors, 20 shallow reefs (<5m) in the northern GOM were sampled throughout the summer of 2011. We compared nekton and benthic assemblage abundance, diversity and composition across reef types. Except for benthic macroinvertebrate abundance, which was significantly higher on old rock reefs as compared to historic reefs, all reefs were similar to historic reefs, suggesting created reefs provide similar support of nekton and benthic assemblages as historic reefs. To determine refuge value of oyster structure for benthic macroinvertebrates compared to bare bottom, we tested preferences of juvenile crabs across depth and refuge complexity in the presence and absence of adult blue crabs (Callinectes sapidus). Juveniles were more likely to use deep water with predators present only when provided oyster structure. Provision of structural material to support and sustain development of benthic and mobile reef communities may be the most important factor in determining reef value to these assemblages, with biophysical characteristics related to reef location influencing assemblage patterns in areas with structure; if so, appropriately locating created reefs is critical.

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

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.

The Structure of Lombricine Kinase

PubMed Central

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S.

2011-01-01

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. PMID:21212263

Spun-wrapped aligned nanofiber (SWAN) lithography for fabrication of micro/nano-structures on 3D objects

NASA Astrophysics Data System (ADS)

Ye, Zhou; Nain, Amrinder S.; Behkam, Bahareh

2016-06-01

Fabrication of micro/nano-structures on irregularly shaped substrates and three-dimensional (3D) objects is of significant interest in diverse technological fields. However, it remains a formidable challenge thwarted by limited adaptability of the state-of-the-art nanolithography techniques for nanofabrication on non-planar surfaces. In this work, we introduce Spun-Wrapped Aligned Nanofiber (SWAN) lithography, a versatile, scalable, and cost-effective technique for fabrication of multiscale (nano to microscale) structures on 3D objects without restriction on substrate material and geometry. SWAN lithography combines precise deposition of polymeric nanofiber masks, in aligned single or multilayer configurations, with well-controlled solvent vapor treatment and etching processes to enable high throughput (>10-7 m2 s-1) and large-area fabrication of sub-50 nm to several micron features with high pattern fidelity. Using this technique, we demonstrate whole-surface nanopatterning of bulk and thin film surfaces of cubes, cylinders, and hyperbola-shaped objects that would be difficult, if not impossible to achieve with existing methods. We demonstrate that the fabricated feature size (b) scales with the fiber mask diameter (D) as b1.5 ~ D. This scaling law is in excellent agreement with theoretical predictions using the Johnson, Kendall, and Roberts (JKR) contact theory, thus providing a rational design framework for fabrication of systems and devices that require precisely designed multiscale features.Fabrication of micro/nano-structures on irregularly shaped substrates and three-dimensional (3D) objects is of significant interest in diverse technological fields. However, it remains a formidable challenge thwarted by limited adaptability of the state-of-the-art nanolithography techniques for nanofabrication on non-planar surfaces. In this work, we introduce Spun-Wrapped Aligned Nanofiber (SWAN) lithography, a versatile, scalable, and cost-effective technique for fabrication of multiscale (nano to microscale) structures on 3D objects without restriction on substrate material and geometry. SWAN lithography combines precise deposition of polymeric nanofiber masks, in aligned single or multilayer configurations, with well-controlled solvent vapor treatment and etching processes to enable high throughput (>10-7 m2 s-1) and large-area fabrication of sub-50 nm to several micron features with high pattern fidelity. Using this technique, we demonstrate whole-surface nanopatterning of bulk and thin film surfaces of cubes, cylinders, and hyperbola-shaped objects that would be difficult, if not impossible to achieve with existing methods. We demonstrate that the fabricated feature size (b) scales with the fiber mask diameter (D) as b1.5 ~ D. This scaling law is in excellent agreement with theoretical predictions using the Johnson, Kendall, and Roberts (JKR) contact theory, thus providing a rational design framework for fabrication of systems and devices that require precisely designed multiscale features. Electronic supplementary information (ESI) available: SWAN lithography on silicon; comparison of SWAN lithography and state-of-the-art nanopatterning methods; replica molding using SWAN lithography fabricated template; PDMS nanofluidic device, gold nanopattern characterization. See DOI: 10.1039/c6nr03323g

Microbial community diversity associated with moonmilk deposits in a karstic cave system in Ireland

NASA Astrophysics Data System (ADS)

Rooney, D.; Hutchens, E.; Clipson, Nick; McDermott, Frank

2009-04-01

Microbial ecology in subterranean systems has yet to be fully studied. Cave systems present highly unusual and extreme habitats, where microbial activity can potentially play a major role in nutrient cycling and possibly contribute to the formation of characteristic subaerial structures. How microorganisms actually function in cave systems, and what ecological roles they may perform, has yet to be widely addressed, although recent studies using molecular techniques combined with analytical geochemistry have begun to answer some questions surrounding subterranean microbial ecology (Northup et al., 2003). Moonmilk has a ‘cottage-cheese' like consistency, comprised of fine crystal aggregates of carbonate minerals, commonly calcite, hydromagnesite and gypsum, and is believed to be at least partially precipitated by microbial activity (Baskar et al., 2006). Microbial metabolic processes have been implicated in the formation of moonmilk, probably a result of biochemical corrosion of bedrock under high moisture conditions. Mineral weathering via bacterial activity has become accepted as a major influence on subsurface geochemistry and formation of belowground structures (Summers-Engel et al., 2004). While many studies focus on bacterial communities in subterranean systems, fungal community structure is also likely to be important in cave systems, given the important role fungi play in the transformations of organic and inorganic substrates (Gadd, 2004) and the significant role of fungi in mineral dissolution and secondary mineral formation (Burford et al., 2003). In general, it is agreed that both biotic and abiotic processes influence moonmilk formation, yet the diversity of the microbial community associated with moonmilk formations has not been characterised to date. Ballinamintra Cave (Waterford County, Ireland) is largely protected from human influence due to accessibility difficulties and thereby offers an opportunity to study microbial community structure that has been unaltered by human disturbance or practices. The aim of this study was to examine microbial community diversity associated with moonmilk deposits at Ballynamintra Cave, Ireland using automated ribosomal intergenic spacer analysis (ARISA). The results revealed considerable bacterial and fungal diversity associated with moonmilk in a karstic cave system, suggesting that the microbial community implicated in moonmilk formation may be more diverse than previously thought. These results suggest that microbes may have important functional roles in subterranean environments. Although the moonmilk in this study was largely comprised of calcite, microbial involvement in calcite precipitation could result in the bioavailability of a range of organic compounds for subsequent microbial metabolism. References: Baskar, S., Baskar, R., Mauclaire, L., and McKenzie, J.A. 2006. Microbially induced calcite precipitation in culture experiments: Possible origin for stalactites in Sahastradhara caves, Dehradun, India. Current Science 90: 58-64. Burford, E.P., Fomina, M., Gadd, G. 2003. Fungal involvement in bioweathering and biotrasformations of rocks and minerals. Min Mag 67(6):1172-1155. Engel, A.S., Stern, L.A., Bennett, P.C. 2004. Microbial contributions to cave formation: new insights into sulfuric acid speleogenesis. Geology 32(5): 369-372. Gadd, G.M. (2004). Mycotransformation of organic and inorganic substrates. Mycologist 18: 60-70. Northup, D., Barns, S.M., Yu, Laura, E., Spilde, M.N., Schelble, R.T., Dano, K.E., Crossey, L.J., Connolly, C.A., Boston, P.J., and Dahm, C.N. 2003. Diverse microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves. Environmental Microbiology 5(11): 1071-1086.

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.

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.

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

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

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase.

PubMed

Mitchell, Andrew J; Dunham, Noah P; Martinie, Ryan J; Bergman, Jonathan A; Pollock, Christopher J; Hu, Kai; Allen, Benjamin D; Chang, Wei-Chen; Silakov, Alexey; Bollinger, J Martin; Krebs, Carsten; Boal, Amie K

2017-10-04

Iron(II)- and 2-(oxo)-glutarate-dependent oxygenases catalyze diverse oxidative transformations that are often initiated by abstraction of hydrogen from carbon by iron(IV)-oxo (ferryl) complexes. Control of the relative orientation of the substrate C-H and ferryl Fe-O bonds, primarily by direction of the oxo group into one of two cis-related coordination sites (termed inline and offline), may be generally important for control of the reaction outcome. Neither the ferryl complexes nor their fleeting precursors have been crystallographically characterized, hindering direct experimental validation of the offline hypothesis and elucidation of the means by which the protein might dictate an alternative oxo position. Comparison of high-resolution X-ray crystal structures of the substrate complex, an Fe(II)-peroxysuccinate ferryl precursor, and a vanadium(IV)-oxo mimic of the ferryl intermediate in the l-arginine 3-hydroxylase, VioC, reveals coordinated motions of active site residues that appear to control the intermediate geometries to determine reaction outcome.

The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions

PubMed Central

Klemm, Bradley P.; Wu, Nancy; Chen, Yu; Liu, Xin; Kaitany, Kipchumba J.; Howard, Michael J.; Fierke, Carol A.

2016-01-01

Ribonuclease P (RNase P) is an essential endonuclease responsible for catalyzing 5’ end maturation in precursor transfer RNAs. Since its discovery in the 1970s, RNase P enzymes have been identified and studied throughout the three domains of life. Interestingly, RNase P is either RNA-based, with a catalytic RNA subunit, or a protein-only (PRORP) enzyme with differential evolutionary distribution. The available structural data, including the active site data, provides insight into catalysis and substrate recognition. The hydrolytic and kinetic mechanisms of the two forms of RNase P enzymes are similar, yet features unique to the RNA-based and PRORP enzymes are consistent with different evolutionary origins. The various RNase P enzymes, in addition to their primary role in tRNA 5’ maturation, catalyze cleavage of a variety of alternative substrates, indicating a diversification of RNase P function in vivo. The review concludes with a discussion of recent advances and interesting research directions in the field. PMID:27187488

Efficient and Highly Selective Solvent-Free Oxidation of Primary Alcohols to Aldehydes Using Bucky Nanodiamond.

PubMed

Lin, Yangming; Wu, Kuang-Hsu Tim; Yu, Linhui; Heumann, Saskia; Su, Dang Sheng

2017-09-11

Selective oxidation of alcohols to aldehydes is widely applicable to the synthesis of various green chemicals. The poor chemoselectivity for complicated primary aldehydes over state-of-the-art metal-free or metal-based catalysts represents a major obstacle for industrial application. Bucky nanodiamond is a potential green catalyst that exhibits excellent chemoselectivity and cycling stability for the selective oxidation of primary alcohols in diverse structures (22 examples, including aromatic, substituted aromatic, unsaturated, heterocyclic, and linear chain alcohols) to their corresponding aldehydes. The results are comparable to reported transition-metal catalysts including conventional Pt/C and Ru/C catalysts for certain substrates under solvent-free conditions. The possible activation process of the oxidant and substrates by the surface oxygen groups and defect species are revealed with model catalysts, ex situ electrochemical measurements, and ex situ attenuated total reflectance. The zigzag edges of sp 2 carbon planes are shown to play a key role in these reactions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hierarchical macroscopic fibrillar adhesives: in situ study of buckling and adhesion mechanisms on wavy substrates.

PubMed

Bauer, Christina T; Kroner, Elmar; Fleck, Norman A; Arzt, Eduard

2015-10-23

Nature uses hierarchical fibrillar structures to mediate temporary adhesion to arbitrary substrates. Such structures provide high compliance such that the flat fibril tips can be better positioned with respect to asperities of a wavy rough substrate. We investigated the buckling and adhesion of hierarchically structured adhesives in contact with flat smooth, flat rough and wavy rough substrates. A macroscopic model for the structural adhesive was fabricated by molding polydimethylsiloxane into pillars of diameter in the range of 0.3-4.8 mm, with up to three different hierarchy levels. Both flat-ended and mushroom-shaped hierarchical samples buckled at preloads one quarter that of the single level structures. We explain this behavior by a change in the buckling mode; buckling leads to a loss of contact and diminishes adhesion. Our results indicate that hierarchical structures can have a strong influence on the degree of adhesion on both flat and wavy substrates. Strategies are discussed that achieve highly compliant substrates which adhere to rough substrates.

The Crystal Structure of Streptococcus pyogenes Uridine Phosphorylase Reveals a Distinct Subfamily of Nucleoside Phosphorylases

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

Tran, Timothy H.; Christoffersen, S.; Allan, Paula W.

2011-09-20

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2'-deoxyuridine to uracil and ribose 1-phosphate or 2'-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 {angstrom} resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamermore » with an ?/? monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild-type SpUP showed that its substrate specificity is similar to that of EcUP, while EcUP is {approx}7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.« less

Structural insights into conserved L-arabinose metabolic enzymes reveal the substrate binding site of a thermophilic L-arabinose isomerase.

PubMed

Lee, Yong-Jik; Lee, Sang-Jae; Kim, Seong-Bo; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

2014-03-18

Structural genomics demonstrates that despite low levels of structural similarity of proteins comprising a metabolic pathway, their substrate binding regions are likely to be conserved. Herein based on the 3D-structures of the α/β-fold proteins involved in the ara operon, we attempted to predict the substrate binding residues of thermophilic Geobacillus stearothermophilus L-arabinose isomerase (GSAI) with no 3D-structure available. Comparison of the structures of L-arabinose catabolic enzymes revealed a conserved feature to form the substrate-binding modules, which can be extended to predict the substrate binding site of GSAI (i.e., D195, E261 and E333). Moreover, these data implicated that proteins in the l-arabinose metabolic pathway might retain their substrate binding niches as the modular structure through conserved molecular evolution even with totally different structural scaffolds. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Integrative Pericyclic Cascade: An Atom Economic, Multi C-C Bond-Forming Strategy for the Construction of Molecular Complexity.

PubMed

Tejedor, David; Delgado-Hernández, Samuel; Peyrac, Jesús; González-Platas, Javier; García-Tellado, Fernando

2017-07-26

An all-pericyclic manifold is developed for the construction of topologically diverse, structurally complex and natural product-like polycyclic chemotypes. The manifold uses readily accessible tertiary propargyl vinyl ethers as substrates and imidazole as a catalyst to form up to two new rings, three new C-C bonds, six stereogenic centers and one transannular oxo-bridge. The manifold is efficient, scalable and instrumentally simple to perform and entails a propargyl Claisen rearrangement-[1,3]H shift, an oxa-6π-electrocyclization, and an intramolecular Diels-Alder reaction. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Tunable thermal rectification in graphene/hexagonal boron nitride hybrid structures

NASA Astrophysics Data System (ADS)

Chen, Xue-Kun; Hu, Ji-Wen; Wu, Xi-Jun; Jia, Peng; Peng, Zhi-Hua; Chen, Ke-Qiu

2018-02-01

Using non-equilibrium molecular dynamics simulations, we investigate thermal rectification (TR) in graphene/hexagonal boron nitride (h-BN) hybrid structures. Two different structural models, partially substituting graphene into h-BN (CBN) and partially substituting h-BN into graphene (BNC), are considered. It is found that CBN has a significant TR effect while that of BNC is very weak. The observed TR phenomenon can be attributed to the resonance effect between out-of-plane phonons of graphene and h-BN domains in the low-frequency region under negative temperature bias. In addition, the influences of ambient temperature, system size, defect number and substrate interaction are also studied to obtain the optimum conditions for TR. More importantly, the TR ratio could be effectively tuned through chemical and structural diversity. A moderate C/BN ratio and parallel arrangement are found to enhance the TR ratio. Detailed phonon spectra analyses are conducted to understand the thermal transport behavior. This work extends hybrid engineering to 2D materials for achieving TR.

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...

AMP-forming acetyl-CoA synthetases in Archaea show unexpected diversity in substrate utilization

PubMed Central

Ingram-Smith, Cheryl; Smith, Kerry S.

2007-01-01

Adenosine monophosphate (AMP)-forming acetyl-CoA synthetase (ACS; acetate:CoA ligase (AMP-forming), EC 6.2.1.1) is a key enzyme for conversion of acetate to acetyl-CoA, an essential intermediate at the junction of anabolic and catabolic pathways. Phylogenetic analysis of putative short and medium chain acyl-CoA synthetase sequences indicates that the ACSs form a distinct clade from other acyl-CoA synthetases. Within this clade, the archaeal ACSs are not monophyletic and fall into three groups composed of both bacterial and archaeal sequences. Kinetic analysis of two archaeal enzymes, an ACS from Methanothermobacter thermautotrophicus (designated as MT-ACS1) and an ACS from Archaeoglobus fulgidus (designated as AF-ACS2), revealed that these enzymes have very different properties. MT-ACS1 has nearly 11-fold higher affinity and 14-fold higher catalytic efficiency with acetate than with propionate, a property shared by most ACSs. However, AF-ACS2 has only 2.3-fold higher affinity and catalytic efficiency with acetate than with propionate. This enzyme has an affinity for propionate that is almost identical to that of MT-ACS1 for acetate and nearly tenfold higher than the affinity of MT-ACS1 for propionate. Furthermore, MT-ACS1 is limited to acetate and propionate as acyl substrates, whereas AF-ACS2 can also utilize longer straight and branched chain acyl substrates. Phylogenetic analysis, sequence alignment and structural modeling suggest a molecular basis for the altered substrate preference and expanded substrate range of AF-ACS2 versus MT-ACS1. PMID:17350930

A novel cinnamyl alcohol dehydrogenase (CAD)-like reductase contributes to the structural diversity of monoterpenoid indole alkaloids in Rauvolfia.

PubMed

Geissler, Marcus; Burghard, Marie; Volk, Jascha; Staniek, Agata; Warzecha, Heribert

2016-03-01

Based on findings described herein, we contend that the reduction of vomilenine en route to antiarrhythmic ajmaline in planta might proceed via an alternative, novel sequence of biosynthetic steps. In the genus Rauvolfia, monoterpenoid indole alkaloids (MIAs) are formed via complex biosynthetic sequences. Despite the wealth of information about the biochemistry and molecular genetics underlying these processes, many reaction steps involving oxygenases and oxidoreductases are still elusive. Here, we describe molecular cloning and characterization of three cinnamyl alcohol dehydrogenase (CAD)-like reductases from Rauvolfia serpentina cell culture and R. tetraphylla roots. Functional analysis of the recombinant proteins, with a set of MIAs as potential substrates, led to identification of one of the enzymes as a CAD, putatively involved in lignin formation. The two remaining reductases comprise isoenzymes derived from orthologous genes of the investigated alternative Rauvolfia species. Their catalytic activity consists of specific conversion of vomilenine to 19,20-dihydrovomilenine, thus proving their exclusive involvement in MIA biosynthesis. The obtained data suggest the existence of a previously unknown bypass in the biosynthetic route to ajmaline further expanding structural diversity within the MIA family of specialized plant metabolites.

Improving protein delivery of fibroblast growth factor-2 from bacterial inclusion bodies used as cell culture substrates.

PubMed

Seras-Franzoso, Joaquin; Peebo, Karl; García-Fruitós, Elena; Vázquez, Esther; Rinas, Ursula; Villaverde, Antonio

2014-03-01

Bacterial inclusion bodies (IBs) have recently been used to generate biocompatible cell culture interfaces, with diverse effects on cultured cells such as cell adhesion enhancement, stimulation of cell growth or induction of mesenchymal stem cell differentiation. Additionally, novel applications of IBs as sustained protein delivery systems with potential applications in regenerative medicine have been successfully explored. In this scenario, with IBs gaining significance in the biomedical field, the fine tuning of this functional biomaterial is crucial. In this work, the effect of temperature on fibroblast growth factor-2 (FGF-2) IB production and performance has been evaluated. FGF-2 was overexpressed in Escherichia coli at 25 and 37 °C, producing IBs with differences in size, particle structure and biological activity. Cell culture topographies made with FGF-2 IBs biofabricated at 25 °C showed higher levels of biological activity as well as a looser supramolecular structure, enabling a higher protein release from the particles. In addition, the controlled use of FGF-2 protein particles enabled the generation of functional topographies with multiple biological activities being effective on diverse cell types. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Analysis on the Spatial Difference of Bacterial Community Structure in Micro-pressure Air-lift Loop Reactor

NASA Astrophysics Data System (ADS)

Wan, L. G.; Lin, Q.; Bian, D. J.; Ren, Q. K.; Xiao, Y. B.; Lu, W. X.

2018-02-01

In order to reveal the spatial difference of the bacterial community structure in the Micro-pressure Air-lift Loop Reactor, the activated sludge bacterial at five different representative sites in the reactor were studied by denaturing gradient gel electrophoresis (DGGE). The results of DGGE showed that the difference of environmental conditions (such as substrate concentration, dissolved oxygen and PH, etc.) resulted in different diversity and similarity of microbial flora in different spatial locations. The Shannon-Wiener diversity index of the total bacterial samples from five sludge samples varied from 0.92 to 1.28, the biodiversity index was the smallest at point 5, and the biodiversity index was the highest at point 2. The similarity of the flora between the point 2, 3 and 4 was 80% or more, respectively. The similarity of the flora between the point 5 and the other samples was below 70%, and the similarity of point 2 was only 59.2%. Due to the different contribution of different strains to the removal of pollutants, it can give full play to the synergistic effect of bacterial degradation of pollutants, and further improve the efficiency of sewage treatment.

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

Laminated rare earth structure and method of making

DOEpatents

Senor, David J [West Richland, WA; Johnson, Roger N [Richland, WA; Reid, Bruce D [Pasco, WA; Larson, Sandra [Richland, WA

2002-07-30

A laminated structure having two or more layers, wherein at least one layer is a metal substrate and at least one other layer is a coating comprising at least one rare earth element. For structures having more than two layers, the coating and metal substrate layers alternate. In one embodiment of the invention, the structure is a two-layer laminate having a rare earth coating electrospark deposited onto a metal substrate. In another embodiment of the invention, the structure is a three-layer laminate having the rare earth coating electrospark deposited onto a first metal substrate and the coating subsequently abonded to a second metal substrate. The bonding of the coating to the second metal substrate may be accomplished by hot pressing, hot rolling, high deformation rate processing, or combinations thereof. The laminated structure may be used in nuclear components where reactivity control or neutron absorption is desired and in non-nuclear applications such as magnetic and superconducting films.

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.

The interdigitating loop of the enolase superfamily as a specificity binding determinant or 'flying buttress'.

PubMed

Bearne, Stephen L

2017-05-01

Enzymes of the enolase superfamily (ENS) are mechanistically diverse, yet share a common partial reaction (abstraction of the α-proton from a carboxylate substrate). While the catalytic machinery responsible for the deprotonation reaction has been conserved, divergent evolution has led to numerous ENS members that catalyze different overall reactions. This rich functional diversity has made the ENS an excellent model system for developing the approaches necessary to validate enzyme function. However, enzymes of the ENS also share a common bidomain structure ((β/α) 7 β-barrel domain and α+β capping domain) which makes validation of function from structural information challenging. This review presents a comparative survey of the structural data obtained over the past decade for enzymes from all seven subgroups that comprise the ENS. Of the seven ENS subgroups (enolase, mandelate racemase (MR), muconate lactonizing enzyme, β-methylaspartate ammonia lyase, d-glucarate dehydratase, d-mannonate dehydratase (ManD), and galactarate dehydratase 2), only enzymes of the MR and ManD subgroups exhibit an additional feature of structural complexity-an interdigitating loop. This loop emanates from one protomer of a homodimeric pair and penetrates into the adjacent, symmetry-related protomer to either contribute a binding determinant to the active site of the adjacent protomer, or act as a 'flying buttress' to support residues of the active site. The analysis presented in this review suggests that the interdigitating loop is the only gross structural element that permits functional distinction between ENS subgroups at the tertiary level of protein structure. Copyright © 2017 Elsevier B.V. All rights reserved.

Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes

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

Jin, Lei; Wei, Wentao; Jiang, Yaobin

2009-11-04

SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into themore » conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.« less

The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

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

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.

2012-05-29

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR ofmore » arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.« less

Methods and systems for positioning micro elements

DOEpatents

Stalford, Harold L

2015-04-28

A micro device may comprise a substrate, a first micro structure coupled to the substrate, a second micro structure coupled to the substrate, and port configured to receive an input. The first micro structure is configured to move into engagement with the second micro structure in response to the input.

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...

Optothermal Manipulations of Colloidal Particles and Living Cells.

PubMed

Lin, Linhan; Hill, Eric H; Peng, Xiaolei; Zheng, Yuebing

2018-05-25

Optical manipulation techniques are important in many fields. For instance, they enable bottom-up assembly of nanomaterials and high-resolution and in situ analysis of biological cells and molecules, providing opportunities for discovery of new materials, medical diagnostics, and nanomedicines. Traditional optical tweezers have their applications limited due to the use of rigorous optics and high optical power. New strategies have been established for low-power optical manipulation techniques. Optothermal manipulation, which exploits photon-phonon conversion and matter migration under a light-controlled temperature gradient, is one such emerging technique. Elucidation of the underlying physics of optothermo-matter interaction and rational engineering of optical environments are required to realize diverse optothermal manipulation functionalities. This Account covers the working principles, design concepts, and applications of a series of newly developed optothermal manipulation techniques, including bubble-pen lithography, opto-thermophoretic tweezers, opto-thermoelectric tweezers, optothermal assembly, and opto-thermoelectric printing. In bubble-pen lithography, optical heating of a plasmonic substrate generates microbubbles at the solid-liquid interface to print diverse colloidal particles on the substrates. Programmable bubble printing of semiconductor quantum dots on different substrates and haptic control of printing have also been achieved. The key to optothermal tweezers is the ability to deliver colloidal particles from cold to hot regions of a temperature gradient or a negative Soret effect. We explore different driving forces for the two types of optothermal tweezers. Opto-thermophoretic tweezers rely on an abnormal permittivity gradient built by structured solvent molecules in the electric double layer of colloidal particles and living cells in response to heat-induced entropy, and opto-thermoelectric tweezers exploit a thermophoresis-induced thermoelectric field for the low-power manipulation of small nanoparticles with minimum diameter around 20 nm. Furthermore, by incorporating depletion attraction into the optothermal tweezers system as particle-particle or particle-substrate binding force, we have achieved bottom-up assembly and reconfigurable optical printing of artificial colloidal matter. Beyond optothermal manipulation techniques in liquid environments, we also review recent progress of gas-phase optothermal manipulation based on photophoresis. Photophoretic trapping and transport of light-absorbing materials have been achieved through optical engineering to tune particle-molecule interactions during optical heating, and a novel optical trap display has been demonstrated. An improved understanding of the colloidal response to temperature gradients will surely facilitate further innovations in optothermal manipulation. With their low-power operation, simple optics, and diverse functionalities, optothermal manipulation techniques will find a wide range of applications in life sciences, colloidal science, materials science, and nanoscience, as well as in the developments of colloidal functional devices and nanomedicine.

Structure-based Analysis to Hu-DNA Binding

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

Swinger,K.; Rice, P.

2007-01-01

HU and IHF are prokaryotic proteins that induce very large bends in DNA. They are present in high concentrations in the bacterial nucleoid and aid in chromosomal compaction. They also function as regulatory cofactors in many processes, such as site-specific recombination and the initiation of replication and transcription. HU and IHF have become paradigms for understanding DNA bending and indirect readout of sequence. While IHF shows significant sequence specificity, HU binds preferentially to certain damaged or distorted DNAs. However, none of the structurally diverse HU substrates previously studied in vitro is identical with the distorted substrates in the recently publishedmore » Anabaena HU(AHU)-DNA cocrystal structures. Here, we report binding affinities for AHU and the DNA in the cocrystal structures. The binding free energies for formation of these AHU-DNA complexes range from 10-14.5 kcal/mol, representing K{sub d} values in the nanomolar to low picomolar range, and a maximum stabilization of at least 6.3 kcal/mol relative to complexes with undistorted, non-specific DNA. We investigated IHF binding and found that appropriate structural distortions can greatly enhance its affinity. On the basis of the coupling of structural and relevant binding data, we estimate the amount of conformational strain in an IHF-mediated DNA kink that is relieved by a nick (at least 0.76 kcal/mol) and pinpoint the location of the strain. We show that AHU has a sequence preference for an A+T-rich region in the center of its DNA-binding site, correlating with an unusually narrow minor groove. This is similar to sequence preferences shown by the eukaryotic nucleosome.« less

The 1.25 Å resolution structure of phosphoribosyl-ATP pyrophosphohydrolase from Mycobacterium tuberculosis

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

Javid-Majd, Farah; Yang, Dong; Ioerger, Thomas R.

2008-06-01

The crystal structure of M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase, the second enzyme in the histidine-biosynthetic pathway, is presented. The structural and inferred functional relationships between M. tuberculosis phosphoribosyl-ATP pyrophosphohydrolase and other members of the nucleoside-triphosphate pyrophosphatase-fold family are described. Phosphoribosyl-ATP pyrophosphohydrolase is the second enzyme in the histidine-biosynthetic pathway, irreversibly hydrolyzing phosphoribosyl-ATP to phosphoribosyl-AMP and pyrophosphate. It is encoded by the hisE gene, which is present as a separate gene in many bacteria and archaea but is fused to hisI in other bacteria, fungi and plants. Because of its essentiality for growth in vitro, HisE is a potential drug target formore » tuberculosis. The crystal structures of two native (uncomplexed) forms of HisE from Mycobacterium tuberculosis have been determined to resolutions of 1.25 and 1.79 Å. The structure of the apoenzyme reveals that the protein is composed of five α-helices with connecting loops and is a member of the α-helical nucleoside-triphosphate pyrophosphatase superfamily. The biological unit of the protein is a homodimer, with an active site on each subunit composed of residues exclusively from that subunit. A comparison with the Campylobacter jejuni dUTPase active site allowed the identification of putative metal- and substrate-binding sites in HisE, including four conserved glutamate and glutamine residues in the sequence that are consistent with a motif for pyrophosphohydrolase activity. However, significant differences between family members are observed in the loop region between α-helices H1 and H3. The crystal structure of M. tuberculosis HisE provides insights into possible mechanisms of substrate binding and the diversity of the nucleoside-triphosphate pyrophosphatase superfamily.« less

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

One-step assembly of coordination complexes for versatile film and particle engineering.

PubMed

Ejima, Hirotaka; Richardson, Joseph J; Liang, Kang; Best, James P; van Koeverden, Martin P; Such, Georgina K; Cui, Jiwei; Caruso, Frank

2013-07-12

The development of facile and versatile strategies for thin-film and particle engineering is of immense scientific interest. However, few methods can conformally coat substrates of different composition, size, shape, and structure. We report the one-step coating of various interfaces using coordination complexes of natural polyphenols and Fe(III) ions. Film formation is initiated by the adsorption of the polyphenol and directed by pH-dependent, multivalent coordination bonding. Aqueous deposition is performed on a range of planar as well as inorganic, organic, and biological particle templates, demonstrating an extremely rapid technique for producing structurally diverse, thin films and capsules that can disassemble. The ease, low cost, and scalability of the assembly process, combined with pH responsiveness and negligible cytotoxicity, makes these films potential candidates for biomedical and environmental applications.

Material-Independent Nanotransfer onto a Flexible Substrate Using Mechanical-Interlocking Structure.

PubMed

Seo, Min-Ho; Choi, Seon-Jin; Park, Sang Hyun; Yoo, Jae-Young; Lim, Sung Kyu; Lee, Jae-Shin; Choi, Kwang-Wook; Jo, Min-Seung; Kim, Il-Doo; Yoon, Jun-Bo

2018-05-22

Nanowire-transfer technology has received much attention thanks to its capability to fabricate high-performance flexible nanodevices with high simplicity and throughput. However, it is still challenging to extend the conventional nanowire-transfer method to the fabrication of a wide range of devices since a chemical-adhesion-based nanowire-transfer mechanism is complex and time-consuming, hindering successful transfer of diverse nanowires made of various materials. Here, we introduce a material-independent mechanical-interlocking-based nanowire-transfer (MINT) method, fabricating ultralong and fully aligned nanowires on a large flexible substrate (2.5 × 2 cm 2 ) in a highly robust manner. For the material-independent nanotransfer, we developed a mechanics-based nanotransfer method, which employs a dry-removable amorphous carbon (a-C) sacrificial layer between a vacuum-deposited nanowire and the underlying master mold. The controlled etching of the sacrificial layer enables the formation of a mechanical-interlocking structure under the nanowire, facilitating peeling off of the nanowire from the master mold robustly and reliably. Using the developed MINT method, we successfully fabricated various metallic and semiconductor nanowire arrays on flexible substrates. We further demonstrated that the developed method is well suited to the reliable fabrication of highly flexible and high-performance nanoelectronic devices. As examples, a fully aligned gold (Au) microheater array exhibited high bending stability (10 6 cycling) and ultrafast (∼220 ms) heating operation up to ∼100 °C. An ultralong Au heater-embedded cuprous-oxide (Cu 2 O) nanowire chemical gas sensor showed significantly improved reversible reaction kinetics toward NO 2 with 10-fold enhancement in sensitivity at 100 °C.

Substrate dependence of TM-polarized light emission characteristics of BAlGaN/AlN quantum wells

NASA Astrophysics Data System (ADS)

Park, Seoung-Hwan; Ahn, Doyeol

2018-06-01

To study the substrate dependence of light emission characteristics of transverse-magnetic (TM)-polarized light emitted from BAlGaN/AlN quantum wells (QWs) grown on GaN and AlN substrates were investigated theoretically. It is found that the topmost valence subband for QW structures grown on AlN substrate, is heavy hole state (HH1) while that for QW structures grown on GaN substrate is crystal-field split off light hole state (CL1), irrespective of the boron content. Since TM-polarized light emission is associated with the light hole state, the TM-polarized emission peak of BAlGaN/AlN QW structures grown on GaN substrate is expected to be much larger than that of the QW structure grown on AlN substrate. Also, both QW structures show that the spontaneous emission peak of BAlGaN/AlN QW structures would be improved with the inclusion of the boron. However, it rapidly begins to decrease when the boron content exceeds a critical value.

Effects of SPORL and dilute acid pretreatment on substrate morphology, cell physical and chemical wall structures, and subsequent enzymatic hydrolysis of lodgepole pine

Treesearch

Xinping Li; Xiaolin Luo; Kecheng Li; J.Y. Zhu; J. Dennis Fougere; Kimberley Clarke

2012-01-01

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL...

"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.

Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue.

PubMed

Tseng, Hubert; Daquinag, Alexes C; Souza, Glauco R; Kolonin, Mikhail G

2018-01-01

White adipose tissue (WAT) has attracted interest for tissue engineering and cell-based therapies as an abundant source of adipose stem/stromal cells (ASC). However, technical challenges in WAT cell culture have limited its applications in regenerative medicine. Traditional two-dimensional (2D) cell culture models, which are essentially monolayers of cells on glass or plastic substrates, inadequately represent tissue architecture, biochemical concentration gradients, substrate stiffness, and most importantly for WAT research, cell phenotypic heterogeneity. Physiological cell culture platforms for WAT modeling must recapitulate the native diversity of cell types and their coordination within the organ. For this purpose, we developed a three-dimensional (3D) model using magnetic levitation. Here, we describe our protocol that we successfully employed to build adipose tissue organoids (adipospheres) that preserve the heterogeneity of the constituent cell types in vitro. We demonstrate the capacity of assembling adipospheres from multiple cell types, including ASCs, endohtelial cells, and leukocytes that recreate tissue organization. These adipospheres mimicked WAT organogenesis in that they enabled the formation of vessel-like endothelial structures with lumens and differentiation of unilocular adipocytes. Altogether, magnetic levitation is a cell culture platform that recreates tissue structure, function, and heterogeneity in vitro, and serves as a foundation for high-throughput WAT tissue culture and analysis.

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.

Mechanosensitivity in axon growth and guidance

NASA Astrophysics Data System (ADS)

Urbach, Jeff

2013-03-01

In the developing nervous system, axons respond to a diverse array of cues to generate the intricate connection network required for proper function. The growth cone, a highly motile structure at the tip of a growing axon, integrates information about the local environment and modulates outgrowth and guidance, but little is known about effects of external mechanical cues and internal mechanical forces on growth cone behavior. We have investigated axon outgrowth and force generation on soft elastic substrates for dorsal root ganglion (DRG) neurons (from the peripheral nervous system) and hippocampal neurons (from the central) to see how the mechanics of the microenvironment affect different populations. We find that force generation and stiffness-dependent outgrowth are strongly dependent on cell type. We also observe very different internal dynamics and substrate coupling in the two populations, suggesting that the difference in force generation is due to stronger adhesions and therefore stronger substrate engagement in the peripheral nervous system neurons. We will discuss the biological origins of these differences, and recent analyses of the dynamic aspects of growth cone force generation and the implications for the role of mechanosensitivity in axon guidance. In collaboration with D. Koch, W. Rosoff, and H. M. Geller. Supported by NINDS grant 1R01NS064250-01 (J.S.U.) and the NHLBI Intramural Research Program (H.M.G.).

Direct hydrothermal growth of GDC nanorods for low temperature solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Hong, Soonwook; Lee, Dohaeng; Yang, Hwichul; Kim, Young-Beom

2018-06-01

We report a novel synthesis technique of gadolinia-doped ceria (GDC) nano-rod (NRs) via direct hydrothermal process to enhance performance of low temperature solid oxide fuel cell by increasing active reaction area and ionic conductivity at interface between cathode and electrolyte. The cerium nitrate hexahydrate, gadolinium nitrate hexahydrate and urea were used to synthesis GDC NRs for growth on diverse substrate. The directly grown GDC NRs on substrate had a width from 819 to 490 nm and height about 2200 nm with a varied urea concentration. Under the optimized urea concentration of 40 mMol, we confirmed that GDC NRs able to fully cover the substrate by enlarging active reaction area. To maximize ionic conductivity of GDC NRs, we synthesis varied GDC NRs with different ratio of gadolinium and cerium precursor. Electrochemical analysis revealed a significant enhanced performance of fuel cells applying synthesized GDC NRs with a ratio of 2:8 gadolinium and cerium precursor by reducing polarization resistance, which was chiefly attributed to the enlarged active reaction area and enhanced ionic conductivity of GDC NRs. This method of direct hydrothermal growth of GDC NRs enhancing fuel cell performance was considered to apply other types of catalyzing application using nano-structure such as gas sensing and electrolysis fields.

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

Thermal Design and Characterization of Heterogeneously Integrated InGaP/GaAs HBTs

DOE PAGES

Choi, Sukwon; Peake, Gregory M.; Keeler, Gordon A.; ...

2016-04-21

Flip-chip heterogeneously integrated n-p-n InGaP/GaAs heterojunction bipolar transistors (HBTs) with integrated thermal management on wide-bandgap AlN substrates followed by GaAs substrate removal are demonstrated. Without thermal management, substrate removal after integration significantly aggravates self-heating effects, causing poor I–V characteristics due to excessive device self-heating. An electrothermal codesign scheme is demonstrated that involves simulation (design), thermal characterization, fabrication, and evaluation. Thermoreflectance thermal imaging, electrical-temperature sensitive parameter-based thermometry, and infrared thermography were utilized to assess the junction temperature rise in HBTs under diverse configurations. In order to reduce the thermal resistance of integrated devices, passive cooling schemes assisted by structural modification, i.e.,more » positioning indium bump heat sinks between the devices and the carrier, were employed. By implementing thermal heat sinks in close proximity to the active region of flip-chip integrated HBTs, the junction-to-baseplate thermal resistance was reduced over a factor of two, as revealed by junction temperature measurements and improvement of electrical performance. In conclusion, the suggested heterogeneous integration method accounts for not only electrical but also thermal requirements providing insight into realization of advanced and robust III–V/Si heterogeneously integrated electronics.« less

The AAA+ ATPase p97, a cellular multitool

PubMed Central

Stach, Lasse

2017-01-01

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy. PMID:28819009

Characteristics of Various Photodiode Structures in CMOS Technology with Monolithic Signal Processing Electronics

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

Mukhopadhyay, Sourav; Chandratre, V. B.; Sukhwani, Menka

2011-10-20

Monolithic optical sensor with readout electronics are needed in optical communication, medical imaging and scintillator based gamma spectroscopy system. This paper presents the design of three different CMOS photodiode test structures and two readout channels in a commercial CMOS technology catering to the need of nuclear instrumentation. The three photodiode structures each of 1 mm{sup 2} with readout electronics are fabricated in 0.35 um, 4 metal, double poly, N-well CMOS process. These photodiode structures are based on available P-N junction of standard CMOS process i.e. N-well/P-substrate, P+/N-well/P-substrate and inter-digitized P+/N-well/P-substrate. The comparisons of typical characteristics among three fabricated photo sensorsmore » are reported in terms of spectral sensitivity, dark current and junction capacitance. Among the three photodiode structures N-well/P-substrate photodiode shows higher spectral sensitivity compared to the other two photodiode structures. The inter-digitized P+/N-well/P-substrate structure has enhanced blue response compared to N-well/P-substrate and P+/N-well/P-substrate photodiode. Design and test results of monolithic readout electronics, for three different CMOS photodiode structures for application related to nuclear instrumentation, are also reported.« less

Ecotoxicological assessment of pesticides and their combination on rhizospheric microbial community structure and function of Vigna radiata.

PubMed

Walvekar, Varsha Ashok; Bajaj, Swati; Singh, Dileep K; Sharma, Shilpi

2017-07-01

India is one of the leading countries in production and indiscriminate consumption of pesticides. Owing to their xenobiotic nature, pesticides affect soil microorganisms that serve as mediators in plant growth promotion. Our study aimed to deliver a comprehensive picture, by comparing the effects of synthetic pesticides (chlorpyriphos, cypermethrin, and a combination of both) with a biopesticide (azadirachtin) at their recommended field application level (L), and three times the recommended dosage (H) on structure and function of microbial community in rhizosphere of Vigna radiata. Effect on culturable fraction was assessed by enumeration on selective media, while PCR-denaturing gradient gel electrophoresis (DGGE) was employed to capture total bacterial community diversity. This was followed by a metabolic sketch using community-level physiological profiling (CLPP), to obtain a broader picture of the non-target effects on rhizospheric microbial community. Although plant parameters were not significantly affected by pesticide application, the microbial community structure experienced an undesirable impact as compared to control devoid of pesticide treatment. Examination of DGGE banding patterns through cluster analysis revealed that microbial community structure of pesticide-treated soils had only 70% resemblance to control rhizospheric soil even at 45 days post application. Drastic changes in the metabolic profiles of pesticide-treated soils were also detected in terms of substrate utilization, rhizospheric diversity, and evenness. It is noteworthy that the effects exacerbated by biopesticide were comparable to that of synthetic pesticides, thus emphasizing the significance of ecotoxicological assessments before tagging biopesticides as "safe alternatives."

Influence of substrate structure on turnover of the organic cation/H+ exchanger of the renal luminal membrane.

PubMed

Wright, S H; Wunz, T M

1998-08-01

We examined the influence of organic cation (OC) structure on the rate of turnover of the OC/H+ exchanger in rabbit renal brush-border membrane vesicles (BBMV). The rate of efflux of [14C]tetraethylammonium ([14C]TEA) from BBMV, measured in the presence of an inwardly directed chemical gradient for test agent, provided an indirect measure of activity of the OC/H+(OC) exchanger. The trans-stimulation of [14C]TEA efflux from BBMV was a saturable function of increasing extravesicular concentration of both unlabeled TEA and tetramethylammonium (TMA), with an apparent Michaelis constant (Kt) for the interaction of these compounds with the OC/H+(OC) exchanger of 25 microM and 1 mM, respectively. The effect on [14C]TEA efflux of saturating extravesicular concentrations of a series of n-tetraalkylammonium compounds was examined. Whereas the short-chain compounds TMA and TEA markedly stimulated [14C]TEA efflux (by 830% and 690%, respectively), the long-chain compounds tetrapropylammonium and tetrabutylammonium were less effective, increasing efflux by only 40% and 120%, respectively. When the exchanger was saturated with tetrapentylammonium, mediated efflux of [14C]TEA was reduced. Increasing alkyl chain length was also correlated with an increase in the inhibitory effect (as measured by the apparent inhibition constant, Ki, or the IC50 value) that these compounds had against transport of [14C]TEA by the OC/H+(OC) exchanger; i.e., there was a correlation between decreasing IC50 and decreasing turnover of the OC/H+(OC) exchanger. This same correlation was observed for a broader set of test agents of diverse molecular structure, including a series of n-tetraalkylammonium and -phosphonium compounds and the OCs, choline, N1-methyl nicotinamide, 1-methyl-4-phenylpyridinium, and amiloride. Because high affinity of substrates for the OC/H+(OC) exchanger is correlated with increasing substrate hydrophobicity, we conclude that the interaction of hydrophobic OCs with the renal OC/H+(OC) exchanger results in the formation of a substrate-exchanger complex that has a comparatively low rate of turnover.

Molecular Dynamics of CYP2D6 Polymorphisms in the Absence and Presence of a Mechanism-Based Inactivator Reveals Changes in Local Flexibility and Dominant Substrate Access Channels

PubMed Central

de Waal, Parker W.; Sunden, Kyle F.; Furge, Laura Lowe

2014-01-01

Cytochrome P450 enzymes (CYPs) represent an important enzyme superfamily involved in metabolism of many endogenous and exogenous small molecules. CYP2D6 is responsible for ∼15% of CYP-mediated drug metabolism and exhibits large phenotypic diversity within CYPs with over 100 different allelic variants. Many of these variants lead to functional changes in enzyme activity and substrate selectivity. Herein, a molecular dynamics comparative analysis of four different variants of CYP2D6 was performed. The comparative analysis included simulations with and without SCH 66712, a ligand that is also a mechanism-based inactivator, in order to investigate the possible structural basis of CYP2D6 inactivation. Analysis of protein stability highlighted significantly altered flexibility in both proximal and distal residues from the variant residues. In the absence of SCH 66712, *34, *17-2, and *17-3 displayed more flexibility than *1, and *53 displayed more rigidity. SCH 66712 binding reversed flexibility in *17-2 and *17-3, through *53 remained largely rigid. Throughout simulations with docked SCH 66712, ligand orientation within the heme-binding pocket was consistent with previously identified sites of metabolism and measured binding energies. Subsequent tunnel analysis of substrate access, egress, and solvent channels displayed varied bottle-neck radii. Taken together, our results indicate that SCH 66712 should inactivate these allelic variants, although varied flexibility and substrate binding-pocket accessibility may alter its interaction abilities. PMID:25286176

Silencing of tryptamine biosynthesis for production of nonnatural alkaloids in plant culture.

PubMed

Runguphan, Weerawat; Maresh, Justin J; O'Connor, Sarah E

2009-08-18

Natural products have long served as both a source and inspiration for pharmaceuticals. Modifying the structure of a natural product often improves the biological activity of the compound. Metabolic engineering strategies to ferment "unnatural" products have been enormously successful in microbial organisms. However, despite the importance of plant derived natural products, metabolic engineering strategies to yield unnatural products from complex, lengthy plant pathways have not been widely explored. Here, we show that RNA mediated suppression of tryptamine biosynthesis in Catharanthus roseus hairy root culture eliminates all production of monoterpene indole alkaloids, a class of natural products derived from two starting substrates, tryptamine and secologanin. To exploit this chemically silent background, we introduced an unnatural tryptamine analog to the production media and demonstrated that the silenced plant culture could produce a variety of novel products derived from this unnatural starting substrate. The novel alkaloids were not contaminated by the presence of the natural alkaloids normally present in C. roseus. Suppression of tryptamine biosynthesis therefore did not appear to adversely affect expression of downstream biosynthetic enzymes. Targeted suppression of substrate biosynthesis therefore appears to be a viable strategy for programming a plant alkaloid pathway to more effectively produce desirable unnatural products. Moreover, although tryptamine is widely found among plants, this silenced line demonstrates that tryptamine does not play an essential role in growth or development in C. roseus root culture. Silencing the biosynthesis of an early starting substrate enhances our ability to harness the rich diversity of plant based natural products.

Shifts of microbial communities of wheat (Triticum aestivum L.) cultivation in a closed artificial ecosystem.

PubMed

Qin, Youcai; Fu, Yuming; Dong, Chen; Jia, Nannan; Liu, Hong

2016-05-01

The microbial communities of plant ecosystems are in relation to plant growing environment, but the alteration in biodiversity of rhizosphere and phyllosphere microbial communities in closed and controlled environments is unknown. The purpose of this study is to analyze the change regularity of microbial communities with wheat plants dependent-cultivated in a closed artificial ecosystem. The microbial community structures in closed-environment treatment plants were investigated by a culture-dependent approach, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), and Illumina Miseq high-throughput sequencing. The results indicated that the number of microbes decreased along with time, and the magnitude of bacteria, fungi, and actinomycetes were 10(7)-10(8), 10(5), and 10(3)-10(4) CFU/g (dry weight), respectively. The analysis of PCR-DGGE and Illumina Miseq revealed that the wheat leaf surface and near-root substrate had different microbial communities at different periods of wheat ecosystem development and showed that the relative highest diversity of microbial communities appeared at late and middle periods of the plant ecosystem, respectively. The results also indicated that the wheat leaf and substrate had different microbial community compositions, and the wheat substrate had higher richness of microbial community than the leaf. Flavobacterium, Pseudomonas, Paenibacillus, Enterobacter, Penicillium, Rhodotorula, Acremonium, and Alternaria were dominant in the wheat leaf samples, and Pedobacter, Flavobacterium, Halomonas, Marinobacter, Salinimicrobium, Lysobacter, Pseudomonas, Halobacillus, Xanthomonas, Acremonium, Monographella, and Penicillium were dominant populations in the wheat near-root substrate samples.

Integrated chemical and multi-scale structural analyses for the processes of acid pretreatment and enzymatic hydrolysis of corn stover.

PubMed

Chen, Longjian; Li, Junbao; Lu, Minsheng; Guo, Xiaomiao; Zhang, Haiyan; Han, Lujia

2016-05-05

Corn stover was pretreated with acid under moderate conditions (1.5%, w/w, 121°C, 60min), and kinetic enzymolysis experiments were performed on the pretreated substrate using a mixture of Celluclast 1.5L (20FPU/g dry substrate) and Novozyme 188 (40CBU/g dry substrate). Integrated chemical and multi-scale structural methods were then used to characterize both processes. Chemical analysis showed that acid pretreatment removed considerable hemicellulose (from 19.7% in native substrate to 9.28% in acid-pretreated substrate) and achieved a reasonably high conversion efficiency (58.63% of glucose yield) in the subsequent enzymatic hydrolysis. Multi-scale structural analysis indicated that acid pretreatment caused structural changes via cleaving acetyl linkages, solubilizing hemicellulose, relocating cell wall surfaces and enlarging substrate porosity (pore volume increased from 0.0067cm(3)/g in native substrate to 0.019cm(3)/g in acid-pretreated substrate), thereby improving the polysaccharide digestibility. Copyright © 2016 Elsevier Ltd. All rights reserved.

A trait based dynamic energy budget approach to explore emergent microalgal community structure

NASA Astrophysics Data System (ADS)

Cheng, Y.; Bouskill, N.; Karaoz, U.; Geng, H.; Lane, T.; Pett-Ridge, J.; Mayali, X.; Brodie, E.

2015-12-01

Microalgae play important roles in the global carbon budget. Phytoplankton, including microalgae, are responsible for around 50% of global primary production, and also hold promise as a viable renewable biofuel source. Research has been underway for decades to realize the full potential of algal biofuels at the commercial scale, however, uni-algal ponds are typically threatened by collapse due to microalgal grazing and parasite invasions. Recently, it has been proposed that functionally diverse microalgal-bacterial communities can achieve high biomass and/or lipid yields, and are more stable (less susceptible to invasion) than a monoculture. Similar positive diversity-productivity relationships have been observed in a wide range of ecosystem studies, but the purposeful maintenance of a diverse microbiome is less common in managed systems. In our work, a trait based dynamic energy budget model was developed to explore emergent microalgal community structure under various environmental (e.g. light, temperature, nutrient availability) conditions. The complex algal community can be reduced into functional groups (guilds). Each guild (algae or bacteria) is characterized by distinct physiological traits (e.g. nutrient requirement, growth rate, substrate affinity, lipid production) constrained by biochemical trade-offs. These trait values are derived from literature and information encoded in genomic data. Metabolism of the algae and the bacterial species (symbiotic or non-symbiotic) are described within a dynamic energy budget framework. The model offers a mechanistic framework to predict the optimal microalgal community assemblage towards high productivity and resistance to invasion under prevailing environmental conditions.

Playing RNase P evolution: swapping the RNA catalyst for a protein reveals functional uniformity of highly divergent enzyme forms.

PubMed

Weber, Christoph; Hartig, Andreas; Hartmann, Roland K; Rossmanith, Walter

2014-08-01

The RNase P family is a diverse group of endonucleases responsible for the removal of 5' extensions from tRNA precursors. The diversity of enzyme forms finds its extremes in the eukaryal nucleus where RNA-based catalysis by complex ribonucleoproteins in some organisms contrasts with single-polypeptide enzymes in others. Such structural contrast suggests associated functional differences, and the complexity of the ribonucleoprotein was indeed proposed to broaden the enzyme's functionality beyond tRNA processing. To explore functional overlap and differences between most divergent forms of RNase P, we replaced the nuclear RNase P of Saccharomyces cerevisiae, a 10-subunit ribonucleoprotein, with Arabidopsis thaliana PRORP3, a single monomeric protein. Surprisingly, the RNase P-swapped yeast strains were viable, displayed essentially unimpaired growth under a wide variety of conditions, and, in a certain genetic background, their fitness even slightly exceeded that of the wild type. The molecular analysis of the RNase P-swapped strains showed a minor disturbance in tRNA metabolism, but did not point to any RNase P substrates or functions beyond that. Altogether, these results indicate the full functional exchangeability of the highly dissimilar enzymes. Our study thereby establishes the RNase P family, with its combination of structural diversity and functional uniformity, as an extreme case of convergent evolution. It moreover suggests that the apparently gratuitous complexity of some RNase P forms is the result of constructive neutral evolution rather than reflecting increased functional versatility.

Sacrificial plastic mold with electroplatable base

DOEpatents

Domeier, Linda A.; Hruby, Jill M.; Morales, Alfredo M.

2002-01-01

A sacrificial plastic mold having an electroplatable backing is provided. One embodiment consists of the infusion of a softened or molten thermoplastic through a porous metal substrate (sheet, screen, mesh or foam) and into the features of a micro-scale molding tool contacting the porous metal substrate. Upon demolding, the porous metal substrate will be embedded within the thermoplastic and will project a plastic structure with features determined by the mold tool. This plastic structure, in turn, provides a sacrificial plastic mold mechanically bonded to the porous metal substrate which provides a conducting support suitable for electroplating either contiguous or non-contiguous metal replicates. After electroplating and lapping, the sacrificial plastic can be dissolved to leave the desired metal structure bonded to the porous metal substrate. Optionally, the electroplated structures may be debonded from the porous substrate by selective dissolution of the porous substrate or a coating thereon.

Sacrificial Plastic Mold With Electroplatable Base

DOEpatents

Domeier, Linda A.; Hruby, Jill M.; Morales, Alfredo M.

2005-08-16

A sacrificial plastic mold having an electroplatable backing is provided. One embodiment consists of the infusion of a softened or molten thermoplastic through a porous metal substrate (sheet, screen, mesh or foam) and into the features of a micro-scale molding tool contacting the porous metal substrate. Upon demolding, the porous metal substrate will be embedded within the thermoplastic and will project a plastic structure with features determined by the mold tool. This plastic structure, in turn, provides a sacrificial plastic mold mechanically bonded to the porous metal substrate which provides a conducting support suitable for electroplating either contiguous or non-contiguous metal replicates. After electroplating and lapping, the sacrificial plastic can be dissolved to leave the desired metal structure bonded to the porous metal substrate. Optionally, the electroplated structures may be debonded from the porous substrate by selective dissolution of the porous substrate or a coating thereon.

Squalene Synthase As a Target for Chagas Disease Therapeutics

PubMed Central

Chan, Hsiu-Chien; Li, Jikun; Zheng, Yingying; Huang, Chun-Hsiang; Ren, Feifei; Chen, Chun-Chi; Zhu, Zhen; Galizzi, Melina; Li, Zhu-Hong; Rodrigues-Poveda, Carlos A.; Gonzalez-Pacanowska, Dolores; Veiga-Santos, Phercyles; de Carvalho, Tecia Maria Ulisses; de Souza, Wanderley; Urbina, Julio A.; Wang, Andrew H.-J.; Docampo, Roberto; Li, Kai; Liu, Yi-Liang; Oldfield, Eric; Guo, Rey-Ting

2014-01-01

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease. PMID:24789335

Enzyme-adenylate structure of a bacterial ATP-dependent DNA ligase with a minimized DNA-binding surface.

PubMed

Williamson, Adele; Rothweiler, Ulli; Leiros, Hanna Kirsti Schrøder

2014-11-01

DNA ligases are a structurally diverse class of enzymes which share a common catalytic core and seal breaks in the phosphodiester backbone of double-stranded DNA via an adenylated intermediate. Here, the structure and activity of a recombinantly produced ATP-dependent DNA ligase from the bacterium Psychromonas sp. strain SP041 is described. This minimal-type ligase, like its close homologues, is able to ligate singly nicked double-stranded DNA with high efficiency and to join cohesive-ended and blunt-ended substrates to a more limited extent. The 1.65 Å resolution crystal structure of the enzyme-adenylate complex reveals no unstructured loops or segments, and suggests that this enzyme binds the DNA without requiring full encirclement of the DNA duplex. This is in contrast to previously characterized minimal DNA ligases from viruses, which use flexible loop regions for DNA interaction. The Psychromonas sp. enzyme is the first structure available for the minimal type of bacterial DNA ligases and is the smallest DNA ligase to be crystallized to date.

Insights Into the Bifunctional Aphidicolan-16-ß-ol Synthase Through Rapid Biomolecular Modeling Approaches.

PubMed

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B

2018-01-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modeling techniques offer an alternative route to study the enzyme's reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modeling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modeling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789, and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modeling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme's active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modeling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.

Insights into the bifunctional Aphidicolan-16-ß-ol synthase through rapid biomolecular modelling approaches

NASA Astrophysics Data System (ADS)

Hirte, Max; Meese, Nicolas; Mertz, Michael; Fuchs, Monika; Brück, Thomas B.

2018-04-01

Diterpene synthases catalyze complex, multi-step C-C coupling reactions thereby converting the universal, aliphatic precursor geranylgeranyl diphosphate into diverse olefinic macrocylces that form the basis for the structural diversity of the diterpene natural product family. Since catalytically relevant crystal structures of diterpene synthases are scarce, homology based biomolecular modelling techniques offer an alternative route to study the enzyme’s reaction mechanism. However, precise identification of catalytically relevant amino acids is challenging since these models require careful preparation and refinement techniques prior to substrate docking studies. Targeted amino acid substitutions in this protein class can initiate premature quenching of the carbocation centered reaction cascade. The structural characterization of those alternative cyclization products allows for elucidation of the cyclization reaction cascade and provides a new source for complex macrocyclic synthons. In this study, new insights into structure and function of the fungal, bifunctional Aphidicolan-16-ß-ol synthase were achieved using a simplified biomolecular modelling strategy. The applied refinement methodologies could rapidly generate a reliable protein-ligand complex, which provides for an accurate in silico identification of catalytically relevant amino acids. Guided by our modelling data, ACS mutations lead to the identification of the catalytically relevant ACS amino acid network I626, T657, Y658, A786, F789 and Y923. Moreover, the ACS amino acid substitutions Y658L and D661A resulted in a premature termination of the cyclization reaction cascade en-route from syn-copalyl diphosphate to Aphidicolan-16-ß-ol. Both ACS mutants generated the diterpene macrocycle syn-copalol and a minor, non-hydroxylated labdane related diterpene, respectively. Our biomolecular modelling and mutational studies suggest that the ACS substrate cyclization occurs in a spatially restricted location of the enzyme’s active site and that the geranylgeranyl diphosphate derived pyrophosphate moiety remains in the ACS active site thereby directing the cyclization process. Our cumulative data confirm that amino acids constituting the G-loop of diterpene synthases are involved in the open to the closed, catalytically active enzyme conformation. This study demonstrates that a simple and rapid biomolecular modelling procedure can predict catalytically relevant amino acids. The approach reduces computational and experimental screening efforts for diterpene synthase structure-function analyses.

Increasing the Size of the Microbial Biomass Altered Bacterial Community Structure which Enhances Plant Phosphorus Uptake

PubMed Central

Shen, Pu; Murphy, Daniel Vaughan; George, Suman J.; Lapis-Gaza, Hazel; Xu, Minggang

2016-01-01

Agricultural production can be limited by low phosphorus (P) availability, with soil P being constrained by sorption and precipitation reactions making it less available for plant uptake. There are strong links between carbon (C) and nitrogen (N) availability and P cycling within soil P pools, with microorganisms being an integral component of soil P cycling mediating the availability of P to plants. Here we tested a conceptual model that proposes (i) the addition of readily-available organic substrates would increase the size of the microbial biomass thus exhausting the pool of easily-available P and (ii) this would cause the microbial biomass to access P from more recalcitrant pools. In this model it is hypothesised that the size of the microbial population is regulating access to less available P rather than the diversity of organisms contained within this biomass. To test this hypothesis we added mixtures of simple organic compounds that reflect typical root exudates at different C:N ratios to a soil microcosm experiment and assessed changes in soil P pools, microbial biomass and bacterial diversity measures. We report that low C:N ratio (C:N = 12.5:1) artificial root exudates increased the size of the microbial biomass while high C:N ratio (C:N = 50:1) artificial root exudates did not result in a similar increase in microbial biomass. Interestingly, addition of the root exudates did not alter bacterial diversity (measured via univariate diversity indices) but did alter bacterial community structure. Where C, N and P supply was sufficient to support plant growth the increase observed in microbial biomass occurred with a concurrent increase in plant yield. PMID:27893833

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

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

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families

DOE PAGES

Popovic, Ana; Hai, Tran; Tchigvintsev, Anatoly; ...

2017-03-08

Metagenomics has made accessible an enormous reserve of global biochemical diversity. In order to tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. Here, we validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalyticmore » residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.« less

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...

Buckling analysis of stiff thin films suspended on a substrate with tripod surface relief structure

NASA Astrophysics Data System (ADS)

Yu, Qingmin; Chen, Furong; Li, Ming; Cheng, Huanyu

2017-09-01

A wavy configuration is a simple yet powerful structural design strategy, which has been widely used in flexible and stretchable electronics. A buckled structure created from a prestretch-contact-release process represents an early effort. Substrates with engineered surface relief structures (e.g., rectangular islands or tripod structure) have enabled stretchability to the devices without sacrificing their electric performance (e.g., high areal coverage for LEDs/photovoltaics/batteries/supercapacitors). In particular, the substrate with a tripod surface relief structure allows wrinkled devices to be suspended on a soft tripod substrate. This minimizes the contact area between devices and the deformed substrate, which contributes to a significantly reduced interfacial stress/strain. To uncover the underlying mechanism of such a design, we exploit the energy method to analytically investigate the buckling and postbuckling behaviors of stiff films suspended on a stretchable polymeric substrate with a tripod surface relief structure. Validated by finite element analysis, the predications from such an analytical study elucidate the deformed profile and maximum strain in the buckled and postbuckled stiff thin device films, providing a useful toolkit for future experimental designs.

Association mapping of starch chain length distribution and amylose content in pea (Pisum sativum L.) using carbohydrate metabolism candidate genes.

PubMed

Carpenter, Margaret A; Shaw, Martin; Cooper, Rebecca D; Frew, Tonya J; Butler, Ruth C; Murray, Sarah R; Moya, Leire; Coyne, Clarice J; Timmerman-Vaughan, Gail M

2017-08-01

Although starch consists of large macromolecules composed of glucose units linked by α-1,4-glycosidic linkages with α-1,6-glycosidic branchpoints, variation in starch structural and functional properties is found both within and between species. Interest in starch genetics is based on the importance of starch in food and industrial processes, with the potential of genetics to provide novel starches. The starch metabolic pathway is complex but has been characterized in diverse plant species, including pea. To understand how allelic variation in the pea starch metabolic pathway affects starch structure and percent amylose, partial sequences of 25 candidate genes were characterized for polymorphisms using a panel of 92 diverse pea lines. Variation in the percent amylose composition of extracted seed starch and (amylopectin) chain length distribution, one measure of starch structure, were characterized for these lines. Association mapping was undertaken to identify polymorphisms associated with the variation in starch chain length distribution and percent amylose, using a mixed linear model that incorporated population structure and kinship. Associations were found for polymorphisms in seven candidate genes plus Mendel's r locus (which conditions the round versus wrinkled seed phenotype). The genes with associated polymorphisms are involved in the substrate supply, chain elongation and branching stages of the pea carbohydrate and starch metabolic pathways. The association of polymorphisms in carbohydrate and starch metabolic genes with variation in amylopectin chain length distribution and percent amylose may help to guide manipulation of pea seed starch structural and functional properties through plant breeding.

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.

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.

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.

Effect of tacticity on the structure and glass transition temperature of polystyrene adsorbed onto solid surfaces

NASA Astrophysics Data System (ADS)

Negash, Solomon; Tatek, Yergou B.; Tsige, Mesfin

2018-04-01

We have carried out atomistic (all-atom) molecular dynamics simulations to investigate the effect of tacticity on the structure and glass transition temperature (Tg) of polystyrene (PS) thin films adsorbed on two distinct types of solid substrates. The systems consist of thin films made of atactic, isotactic, and syndiotactic PS chains supported by graphite or hydroxylated α-quartz substrates, which are known to be atomically flat but chemically and structurally different. We have observed a marked dependence of the film structure on substrate type as well as on tacticity. For instance, rings' orientation near substrate surfaces depends on substrate type for atactic PS and isotactic PS films, while no such dependence is observed for syndiotactic PS films whose interfacial structure seems to result from their propensity to adopt the trans conformation rather than their specific interaction with the substrates. Moreover, our results indicate that glass transition temperatures of substrate supported polystyrene films are higher compared to those of the corresponding free-standing films. More specifically, PS films on graphite exhibit larger Tg values than those on α-quartz, and we have noticed that syndiotactic PS has the largest Tg irrespective of the substrate type. Furthermore, the local Tg in the region of the film in contact with the substrates shows a strong tacticity and substrate dependence, whereas no dependencies were found for the local Tg in the middle of the film. Substrate-film interaction energy and chains' dynamics near substrate-film interfaces were subsequently investigated in order to substantiate the obtained Tgs, and it was found that films with higher Tgs are strongly adsorbed on the substrates and/or exhibit smaller interfacial chains' dynamics essentially due to steric hindrance.

Detection of electromagnetic radiation using nonlinear materials

DOEpatents

Hwang, Harold Y.; Liu, Mengkun; Averitt, Richard D.; Nelson, Keith A.; Sternbach, Aaron; Fan, Kebin

2016-06-14

An apparatus for detecting electromagnetic radiation within a target frequency range is provided. The apparatus includes a substrate and one or more resonator structures disposed on the substrate. The substrate can be a dielectric or semiconductor material. Each of the one or more resonator structures has at least one dimension that is less than the wavelength of target electromagnetic radiation within the target frequency range, and each of the resonator structures includes at least two conductive structures separated by a spacing. Charge carriers are induced in the substrate near the spacing when the resonator structures are exposed to the target electromagnetic radiation. A measure of the change in conductivity of the substrate due to the induced charge carriers provides an indication of the presence of the target electromagnetic radiation.

Variable waveband infrared imager

DOEpatents

Hunter, Scott R.

2013-06-11

A waveband imager includes an imaging pixel that utilizes photon tunneling with a thermally actuated bimorph structure to convert infrared radiation to visible radiation. Infrared radiation passes through a transparent substrate and is absorbed by a bimorph structure formed with a pixel plate. The absorption generates heat which deflects the bimorph structure and pixel plate towards the substrate and into an evanescent electric field generated by light propagating through the substrate. Penetration of the bimorph structure and pixel plate into the evanescent electric field allows a portion of the visible wavelengths propagating through the substrate to tunnel through the substrate, bimorph structure, and/or pixel plate as visible radiation that is proportional to the intensity of the incident infrared radiation. This converted visible radiation may be superimposed over visible wavelengths passed through the imaging pixel.

Solution structural ensembles of substrate-free cytochrome P450(cam).

PubMed

Asciutto, Eliana K; Young, Matthew J; Madura, Jeffry; Pochapsky, Susan Sondej; Pochapsky, Thomas C

2012-04-24

Removal of substrate (+)-camphor from the active site of cytochrome P450(cam) (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The (1)H-(15)N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for (15)N-(1)H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. Nuclear magnetic resonance-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information about relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of the substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.

Influence of nanostructural environment and fluid flow on osteoblast-like cell behavior: a model for cell-mechanics studies.

PubMed

Prodanov, L; Semeins, C M; van Loon, J J W A; te Riet, J; Jansen, J A; Klein-Nulend, J; Walboomers, X F

2013-05-01

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Unprecedented access of phenolic substrates to the heme active site of a catalase: substrate binding and peroxidase-like reactivity of Bacillus pumilus catalase monitored by X-ray crystallography and EPR spectroscopy.

PubMed

Loewen, Peter C; Villanueva, Jacylyn; Switala, Jacek; Donald, Lynda J; Ivancich, Anabella

2015-05-01

Heme-containing catalases and catalase-peroxidases catalyze the dismutation of hydrogen peroxide as their predominant catalytic activity, but in addition, individual enzymes support low levels of peroxidase and oxidase activities, produce superoxide, and activate isoniazid as an antitubercular drug. The recent report of a heme enzyme with catalase, peroxidase and penicillin oxidase activities in Bacillus pumilus and its categorization as an unusual catalase-peroxidase led us to investigate the enzyme for comparison with other catalase-peroxidases, catalases, and peroxidases. Characterization revealed a typical homotetrameric catalase with one pentacoordinated heme b per subunit (Tyr340 being the axial ligand), albeit in two orientations, and a very fast catalatic turnover rate (kcat  = 339,000 s(-1) ). In addition, the enzyme supported a much slower (kcat  = 20 s(-1) ) peroxidatic activity utilizing substrates as diverse as ABTS and polyphenols, but no oxidase activity. Two binding sites, one in the main access channel and the other on the protein surface, accommodating pyrogallol, catechol, resorcinol, guaiacol, hydroquinone, and 2-chlorophenol were identified in crystal structures at 1.65-1.95 Å. A third site, in the heme distal side, accommodating only pyrogallol and catechol, interacting with the heme iron and the catalytic His and Arg residues, was also identified. This site was confirmed in solution by EPR spectroscopy characterization, which also showed that the phenolic oxygen was not directly coordinated to the heme iron (no low-spin conversion of the Fe(III) high-spin EPR signal upon substrate binding). This is the first demonstration of phenolic substrates directly accessing the heme distal side of a catalase. © 2015 Wiley Periodicals, 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.

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

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

Aripirala, Srinivas; Gonzalez-Pacanowska, Dolores; Oldfield, Eric

Structural insights into L. major farnesyl diphosphate synthase, a key enzyme in the mevalonate pathway, are described. Farnesyl diphosphate synthase (FPPS) is an essential enzyme involved in the biosynthesis of sterols (cholesterol in humans and ergosterol in yeasts, fungi and trypanosomatid parasites) as well as in protein prenylation. It is inhibited by bisphosphonates, a class of drugs used in humans to treat diverse bone-related diseases. The development of bisphosphonates as antiparasitic compounds targeting ergosterol biosynthesis has become an important route for therapeutic intervention. Here, the X-ray crystallographic structures of complexes of FPPS from Leishmania major (the causative agent of cutaneousmore » leishmaniasis) with three bisphosphonates determined at resolutions of 1.8, 1.9 and 2.3 Å are reported. Two of the inhibitors, 1-(2-hydroxy-2,2-diphosphonoethyl)-3-phenylpyridinium (300B) and 3-butyl-1-(2,2-diphosphonoethyl)pyridinium (476A), co-crystallize with the homoallylic substrate isopentenyl diphosphate (IPP) and three Ca{sup 2+} ions. A third inhibitor, 3-fluoro-1-(2-hydroxy-2,2-diphosphonoethyl)pyridinium (46I), was found to bind two Mg{sup 2+} ions but not IPP. Calorimetric studies showed that binding of the inhibitors is entropically driven. Comparison of the structures of L. major FPPS (LmFPPS) and human FPPS provides new information for the design of bisphosphonates that will be more specific for inhibition of LmFPPS. The asymmetric structure of the LmFPPS–46I homodimer indicates that binding of the allylic substrate to both monomers of the dimer results in an asymmetric dimer with one open and one closed homoallylic site. It is proposed that IPP first binds to the open site, which then closes, opening the site on the other monomer, which closes after binding the second IPP, leading to the symmetric fully occupied FPPS dimer observed in other structures.« less

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.

Derivation of a 3D pharmacophore model for the angiotensin-II site one receptor

NASA Astrophysics Data System (ADS)

Prendergast, Kristine; Adams, Kym; Greenlee, William J.; Nachbar, Robert B.; Patchett, Arthur A.; Underwood, Dennis J.

1994-10-01

A systematic search has been used to derive a hypothesis for the receptor-bound conformation of A-II antagonists at the AT1 receptor. The validity of the pharmacophore hypothesis has been tested using CoMFA, which included 50 diverse A-II antagonists, spanning four orders of magnitude in activity. The resulting cross-validated R2 of 0.64 (conventional R2 of 0.76) is indicative of a good predictive model of activity, and has been used to estimate potency for a variety of non-peptidyl antagonists. The structural model for the non-peptide has been compared with respect to the natural substrate, A-II, by generating peptide to non-peptide overlays.

Optimized structural designs for stretchable silicon integrated circuits.

PubMed

Kim, Dae-Hyeong; Liu, Zhuangjian; Kim, Yun-Soung; Wu, Jian; Song, Jizhou; Kim, Hoon-Sik; Huang, Yonggang; Hwang, Keh-Chih; Zhang, Yongwei; Rogers, John A

2009-12-01

Materials and design strategies for stretchable silicon integrated circuits that use non-coplanar mesh layouts and elastomeric substrates are presented. Detailed experimental and theoretical studies reveal many of the key underlying aspects of these systems. The results shpw, as an example, optimized mechanics and materials for circuits that exhibit maximum principal strains less than 0.2% even for applied strains of up to approximately 90%. Simple circuits, including complementary metal-oxide-semiconductor inverters and n-type metal-oxide-semiconductor differential amplifiers, validate these designs. The results suggest practical routes to high-performance electronics with linear elastic responses to large strain deformations, suitable for diverse applications that are not readily addressed with conventional wafer-based technologies.

Stabilization of different types of transition states in a single enzyme active site: QM/MM analysis of enzymes in the alkaline phosphatase superfamily.

PubMed

Hou, Guanhua; Cui, Qiang

2013-07-17

The first step for the hydrolysis of a phosphate monoester (pNPP(2-)) in enzymes of the alkaline phosphatase (AP) superfamily, R166S AP and wild-type NPP, is studied using QM/MM simulations based on an approximate density functional theory (SCC-DFTBPR) and a recently introduced QM/MM interaction Hamiltonian. The calculations suggest that similar loose transition states are involved in both enzymes, despite the fact that phosphate monoesters are the cognate substrates for AP but promiscuous substrates for NPP. The computed loose transition states are clearly different from the more synchronous ones previously calculated for diester reactions in the same AP enzymes. Therefore, our results explicitly support the proposal that AP enzymes are able to recognize and stabilize different types of transition states in a single active site. Analysis of the structural features of computed transition states indicates that the plastic nature of the bimetallic site plays a minor role in accommodating multiple types of transition states and that the high degree of solvent accessibility of the AP active site also contributes to its ability to stabilize diverse transition-state structures without the need of causing large structural distortions of the bimetallic motif. The binding mode of the leaving group in the transition state highlights that vanadate may not always be an ideal transition state analog for loose phosphoryl transfer transition states.

Spreading properties of cosmetic emollients: Use of synthetic skin surface to elucidate structural effect.

PubMed

Douguet, Marine; Picard, Céline; Savary, Géraldine; Merlaud, Fabien; Loubat-Bouleuc, Nathalie; Grisel, Michel

2017-06-01

The study focuses on the impact of structural and physicochemical properties of emollients on their spreadability. Fifty-three emollients, among which esters, silicones, vegetable and mineral oils, have been characterized. Their viscosity, surface tension, density and spreadability have been measured. Vitro-skin ® , an artificial skin substitute, was used as an artificial porous substrate to measure spreadability. Two different methods have been selected to characterize spreadability, namely contact angle and spreading value. Dynamic contact angle measurements showed that emollient spreadability is first governed by spontaneous spreading and that, in a second phase, absorption and migration into the porous substrate becomes the driver of the extension of the spreading area. Statistical analysis of physicochemical and spreading value data revealed that viscosity has a major impact on the spreading behavior of emollients whatever their chemical type. A special emphasis was placed on the ester family in which chemical diversity is very wide. The results highlighted a difference between "high viscosity esters" for which viscosity is the main factor impacting spreadability and "low viscosity esters" for which structural variations (mono/diester, saturated/unsaturated chain, linear/branched chain) have to be considered in addition to viscosity. Linear regressions were used to express spreading value as a function of viscosity for each of the four emollient families tested (esters, silicones, vegetable and mineral oils). These regressions allowed the development of reliable predictive models as a powerful tool for formulators to forecast spreadability of emollients. Copyright © 2017 Elsevier B.V. All rights reserved.

Castable plastic mold with electroplatable base

DOEpatents

Domeier, Linda A.; Morales, Alfredo M.; Gonzales, Marcela G.; Keifer, Patrick M.

2004-01-20

A sacrificial plastic mold having an electroplatable backing is provided as are methods of making such a mold via the infusion of a castable liquid formulation through a porous metal substrate (sheet, screen, mesh or foam) and into the features of a micro-scale master mold. Upon casting and demolding, the porous metal substrate is embedded within the cast formulation and projects a plastic structure with features determined by the mold tool. The plastic structure provides a sacrificial plastic mold mechanically bonded to the porous metal substrate, which provides a conducting support suitable for electroplating either contiguous or non-contiguous metal replicates. After electroplating and lapping, the sacrificial plastic can be dissolved, leaving the desired metal structure bonded to the porous metal substrate. Optionally, the electroplated structures may be debonded from the porous substrate by selective dissolution of the porous substrate or a coating thereon.

Structures and fabrication techniques for solid state electrochemical devices

DOEpatents

Visco, Steven J.; Jacobson, Craig P.; DeJonghe, Lutgard C.

2006-10-10

Low-cost, mechanically strong, highly electronically conductive porous substrates and associated structures for solid-state electrochemical devices, techniques for forming these structures, and devices incorporating the structures provide solid state electrochemical device substrates of novel composition and techniques for forming thin electrode/membrane/electrolyte coatings on the novel or more conventional substrates. In particular, in one aspect the invention provides techniques for co-firing of device substrate (often an electrode) with an electrolyte or membrane layer to form densified electrolyte/membrane films 5 to 20 microns thick. In another aspect, densified electrolyte/membrane films 5 to 20 microns thick may be formed on a pre-sintered substrate by a constrained sintering process. In some cases, the substrate may be a porous metal, alloy, or non-nickel cermet incorporating one or more of the transition metals Cr, Fe and Cu, or alloys thereof.

Available nitrogen is the key factor influencing soil microbial functional gene diversity in tropical rainforest.

PubMed

Cong, Jing; Liu, Xueduan; Lu, Hui; Xu, Han; Li, Yide; Deng, Ye; Li, Diqiang; Zhang, Yuguang

2015-08-20

Tropical rainforests cover over 50% of all known plant and animal species and provide a variety of key resources and ecosystem services to humans, largely mediated by metabolic activities of soil microbial communities. A deep analysis of soil microbial communities and their roles in ecological processes would improve our understanding on biogeochemical elemental cycles. However, soil microbial functional gene diversity in tropical rainforests and causative factors remain unclear. GeoChip, contained almost all of the key functional genes related to biogeochemical cycles, could be used as a specific and sensitive tool for studying microbial gene diversity and metabolic potential. In this study, soil microbial functional gene diversity in tropical rainforest was analyzed by using GeoChip technology. Gene categories detected in the tropical rainforest soils were related to different biogeochemical processes, such as carbon (C), nitrogen (N) and phosphorus (P) cycling. The relative abundance of genes related to C and P cycling detected mostly derived from the cultured bacteria. C degradation gene categories for substrates ranging from labile C to recalcitrant C were all detected, and gene abundances involved in many recalcitrant C degradation gene categories were significantly (P < 0.05) different among three sampling sites. The relative abundance of genes related to N cycling detected was significantly (P < 0.05) different, mostly derived from the uncultured bacteria. The gene categories related to ammonification had a high relative abundance. Both canonical correspondence analysis and multivariate regression tree analysis showed that soil available N was the most correlated with soil microbial functional gene structure. Overall high microbial functional gene diversity and different soil microbial metabolic potential for different biogeochemical processes were considered to exist in tropical rainforest. Soil available N could be the key factor in shaping the soil microbial functional gene structure and metabolic potential.

Thermal stability of Pt nanoclusters interacting to carbon sublattice

NASA Astrophysics Data System (ADS)

Baidyshev, V. S.; Gafner, Yu. Ya.; Gafner, S. L.; Redel, L. V.

2017-12-01

The catalytic activity of Pt clusters is dependent not only on the nanoparticle size and its composition, but also on its internal structure. To determine the real structure of the nanoparticles used in catalysis, the boundaries of the thermal structure stability of Pt clusters to 8.0 nm in diameter interacting with carbon substrates of two types: a fixed α-graphite plane and a mobile substrate with the diamond structure. The effect of a substrate on the processes melting of Pt nanoclusters is estimated. The role of the cooling rate in the formation of the internal structure of Pt clusters during crystallization is studied. The regularities obtained in the case of "free" Pt clusters and Pt clusters on a substrate are compared. It is concluded that platinum nanoparticles with diameter D ≤ 4.0 nm disposed on a carbon substrate conserve the initial fcc structure during cooling.

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

DOE PAGES

Park, Kiyoung; Li, Ning; Kwak, Yeonju; ...

2017-05-01

Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reactionmore » shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.« less

Structural basis for an inositol pyrophosphate kinase surmounting phosphate crowding

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

Wang, Huanchen; Falck, J.R.; Hall, Traci M. Tanaka

2012-01-11

Inositol pyrophosphates (such as IP7 and IP8) are multifunctional signaling molecules that regulate diverse cellular activities. Inositol pyrophosphates have 'high-energy' phosphoanhydride bonds, so their enzymatic synthesis requires that a substantial energy barrier to the transition state be overcome. Additionally, inositol pyrophosphate kinases can show stringent ligand specificity, despite the need to accommodate the steric bulk and intense electronegativity of nature's most concentrated three-dimensional array of phosphate groups. Here we examine how these catalytic challenges are met by describing the structure and reaction cycle of an inositol pyrophosphate kinase at the atomic level. We obtained crystal structures of the kinase domainmore » of human PPIP5K2 complexed with nucleotide cofactors and either substrates, product or a MgF{sub 3}{sup -} transition-state mimic. We describe the enzyme's conformational dynamics, its unprecedented topological presentation of nucleotide and inositol phosphate, and the charge balance that facilitates partly associative in-line phosphoryl transfer.« less

[Effect of landscape change on the structure of the sting-less bee community (Hymenoptera: Apidae) in Meta, Colombia].

PubMed

Nates-Parra, Guiomar; Palacios, Eliana; Parra-H, Alejandro

2008-09-01

Stingless bees represent one of the most diversified components of the natural Apoidea fauna of pollinators in the tropics. They use diverse kinds of substrates and inhabit varied habitats. Some species are typical for some natural either artificial place. The landscape alteration were this group of bees nests, has and important impact on the natural composition of its community structure, fact which is reflected in the nest density. We analyzed the structure composition of the stingless bees' community in three environments in the Colombian Ilanos piedmont, an important region that represents the transition between Andean ecosystems and a savannah that is seriously threatened by cattle practices. We made systematic samples in secondary forest, agro-ecosystems and urban areas, recording the presence of 204 nests from 11 genera (24 species). The nest density per landscape was heterogeneous and never higher than 16 nests/Ha. We observed two nesting patterns and an effect of sampling criterion on the measured biodiversity.

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.

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

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

Park, Kiyoung; Li, Ning; Kwak, Yeonju

Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reactionmore » shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.« less

Erosion rates as a potential bottom-up control of forest structural characteristics in the Sierra Nevada Mountains.

PubMed

Milodowski, David T; Mudd, Simon M; Mitchard, Edward T A

2015-01-01

The physical characteristics of landscapes place fundamental constraints on vegetation growth and ecosystem function. In actively eroding landscapes, many of these characteristics are controlled by long-term erosion rates: increased erosion rates generate steeper topography and reduce the depth and extent of weathering, limiting moisture storage capacity and impacting nutrient availability. Despite the potentially important bottom-up control that erosion rates place on substrate characteristics, the relationship between the two is largely unexplored. We investigate spatial variations in aboveground biomass (AGB) across a structurally diverse mixed coniferous/deciduous forest with an order of magnitude erosion-rate gradient in the Northern Californian Sierra Nevada, USA, using high resolution LiDAR data and field plots. Mean basin slope, a proxy for erosion rate, accounts for 32% of variance in AGB within our field area (P < 0.001), considerably outweighing the effects of mean annual precipitation, temperature, and bedrock lithology. This highlights erosion rate as a potentially important, but hitherto unappreciated, control on AGB and forest structure.

New faces of porous Prussian blue: interfacial assembly of integrated hetero-structures for sensing applications.

PubMed

Kong, Biao; Selomulya, Cordelia; Zheng, Gengfeng; Zhao, Dongyuan

2015-11-21

Prussian blue (PB), the oldest synthetic coordination compound, is a classic and fascinating transition metal coordination material. Prussian blue is based on a three-dimensional (3-D) cubic polymeric porous network consisting of alternating ferric and ferrous ions, which provides facile assembly as well as precise interaction with active sites at functional interfaces. A fundamental understanding of the assembly mechanism of PB hetero-interfaces is essential to enable the full potential applications of PB crystals, including chemical sensing, catalysis, gas storage, drug delivery and electronic displays. Developing controlled assembly methods towards functionally integrated hetero-interfaces with adjustable sizes and morphology of PB crystals is necessary. A key point in the functional interface and device integration of PB nanocrystals is the fabrication of hetero-interfaces in a well-defined and oriented fashion on given substrates. This review will bring together these key aspects of the hetero-interfaces of PB nanocrystals, ranging from structure and properties, interfacial assembly strategies, to integrated hetero-structures for diverse sensing.

Divergent Synthesis of Heparan Sulfate Oligosaccharides

PubMed Central

2015-01-01

Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis. PMID:26574650

Structural Basis for Nicotinamide Inhibition and Base Exchange in Sir2 Enzymes

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

Sanders, B.; Zhao, K; Slama, J

2007-01-01

The Sir2 family of proteins consists of broadly conserved NAD+-dependent deacetylases that are implicated in diverse biological processes, including DNA regulation, metabolism, and longevity. Sir2 proteins are regulated in part by the cellular concentrations of a noncompetitive inhibitor, nicotinamide, that reacts with a Sir2 reaction intermediate via a base-exchange reaction to reform NAD+ at the expense of deacetylation. To gain a mechanistic understanding of nicotinamide inhibition in Sir2 enzymes, we captured the structure of nicotinamide bound to a Sir2 homolog, yeast Hst2, in complex with its acetyl-lysine 16 histone H4 substrate and a reaction intermediate analog, ADP-HPD. Together with relatedmore » biochemical studies and structures, we identify a nicotinamide inhibition and base-exchange site that is distinct from the so-called 'C pocket' binding site for the nicotinamide group of NAD+. These results provide insights into the Sir2 mechanism of nicotinamide inhibition and have important implications for the development of Sir2-specific effectors.« less

Substrate structures for InP-based devices

DOEpatents

Wanlass, Mark W.; Sheldon, Peter

1990-01-01

A substrate structure for an InP-based semiconductor device having an InP based film is disclosed. The substrate structure includes a substrate region having a lightweight bulk substrate and an upper GaAs layer. An interconnecting region is disposed between the substrate region and the InP-based device. The interconnecting region includes a compositionally graded intermediate layer substantially lattice-matched at one end to the GaAs layer and substantially lattice-matched at the opposite end to the InP-based film. The interconnecting region further includes a dislocation mechanism disposed between the GaAs layer and the InP-based film in cooperation with the graded intermediate layer, the buffer mechanism blocking and inhibiting propagation of threading dislocations between the substrate region, and the InP-based device.

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

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

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.