Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Choline and N,N-dimethylethanolamine as direct substrates for methanogens.

PubMed

Watkins, Andrew J; Roussel, Erwan G; Webster, Gordon; Parkes, R John; Sass, Henrik

2012-12-01

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

Choline and N,N-Dimethylethanolamine as Direct Substrates for Methanogens

PubMed Central

Watkins, Andrew J.; Roussel, Erwan G.; Webster, Gordon; Parkes, R. John

2012-01-01

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply. PMID:23001649

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

NASA Astrophysics Data System (ADS)

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

PubMed Central

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

2011-01-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. PMID:21974603

A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

PubMed

Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

2011-09-01

Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics

Properties of Acetate Kinase Isozymes and a Branched-Chain Fatty Acid Kinase from a Spirochete

PubMed Central

Harwood, Caroline S.; Canale-Parola, Ercole

1982-01-01

Spirochete MA-2, which is anaerobic, ferments glucose, forming acetate as a major product. The spirochete also ferments (but does not utilize as growth substrates) small amounts of l-leucine, l-isoleucine, and l-valine, forming the branched-chain fatty acids isovalerate, 2-methylbutyrate, and isobutyrate, respectively, as end products. Energy generated through the fermentation of these amino acids is utilized to prolong cell survival under conditions of growth substrate starvation. A branched-chain fatty acid kinase and two acetate kinase isozymes were resolved from spirochete MA-2 cell extracts. Kinase activity was followed by measuring the formation of acyl phosphate from fatty acid and ATP. The branched-chain fatty acid kinase was active with isobutyrate, 2-methylbutyrate, isovalerate, butyrate, valerate, or propionate as a substrate but not with acetate as a substrate. The acetate kinase isozymes were active with acetate and propionate as substrates but not with longer-chain fatty acids as substrates. The acetate kinase isozymes and the branched-chain fatty acid kinase differed in nucleoside triphosphate and cation specificities. Each acetate kinase isozyme had an apparent molecular weight of approximately 125,000, whereas the branched-chain fatty acid kinase had a molecular weight of approximately 76,000. These results show that spirochete MA-2 synthesizes a branched-chain fatty acid kinase specific for leucine, isoleucine, and valine fermentation. It is likely that a phosphate branched-chain amino acids is also synthesized by spirochete MA-2. Thus, in spirochete MA-2, physiological mechanisms have evolved which serve specifically to generate maintenance energy from branched-chain amino acids. PMID:6288660

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease.

PubMed

Rockwell, N C; Fuller, R S

2001-10-19

Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.

Influence of hormonal status on substrate utilization at rest and during exercise in the female population.

PubMed

Isacco, Laurie; Duché, Pascale; Boisseau, Nathalie

2012-04-01

During exercise, substrate utilization plays a major role in performance and disease prevention. The contribution of fat and carbohydrates to energy expenditure during exercise is modulated by several factors, including intensity and duration of exercise, age, training and diet, but also gender. Because sex hormone levels change throughout a woman's lifetime (in connection with puberty, the menstrual cycle, use of oral contraceptives and menopause), the female population has to be considered specifically in terms of substrate utilization, and metabolic and hormonal responses to exercise. Before puberty, there is no difference between males and females when it comes to substrate oxidation during exercise. This is not the case during adulthood, since women are known to rely more on fat than men for the same relative intensity of exercise. Among adult women, the menstrual cycle and use of oral contraceptives may influence substrate oxidation. While some authors have noted that the luteal phase of the menstrual cycle is connected with greater lipid oxidation, compared with the follicular stage, other authors have found no difference. Among oral contraceptive users, fat oxidation is sometimes increased during prolonged exercise with a concomitant rise in lipolytic hormones, as well as growth hormone. If this result is not always observed, the type of oral contraceptive (monophasic vs triphasic) and hormone doses may be implicated. Menopause represents a hormonal transition in a woman's life, leading to a decline in ovarian hormone production. A decrease in fat oxidation is consequently observed, and some studies have demonstrated a similar respiratory exchange ratio during prolonged exercise in postmenopausal women and in men. As is the case during puberty, no sex difference should thus appear after menopause in the absence of hormonal replacement therapy (HRT). Results concerning women who take HRT remain conflicting. HRT may act on fat loss by increasing lipid metabolism, but this depends on how the treatment is administered (orally vs transdermally). To better understand the role of ovarian hormones in substrate oxidation, studies have made use of animal protocols to investigate cellular mechanisms. Estradiol and progesterone seem to have opposite effects, with greater lipid oxidation when estradiol is used alone. However, the concentrations used (physiological levels or pharmacological doses) may considerably modify fuel selection. In cases where conflicting data are observed in studies of substrate utilization and prolonged exercise in women, methodological reasons must be called into question. Too many parameters, which oftentimes are not specified, may modulate substrate utilization and metabolic and hormonal responses to prolonged exercise. Although information is generally provided about the type of exercise, its duration and the subjects' training level, detailed information is not always given about the subjects' nutritional state and, more specifically, the hormonal status of female subjects. The primary purpose of this review was to identify the impact of hormonal status on substrate oxidation among female subjects at rest and during exercise. A second aim was to describe gender differences in substrate utilization during exercise.

Direct growth of single-crystalline III–V semiconductors on amorphous substrates

DOE PAGES

Chen, Kevin; Kapadia, Rehan; Harker, Audrey; ...

2016-01-27

The III–V compound semiconductors exhibit superb electronic and optoelectronic properties. Traditionally, closely lattice-matched epitaxial substrates have been required for the growth of high-quality single-crystal III–V thin films and patterned microstructures. To remove this materials constraint, here we introduce a growth mode that enables direct writing of single-crystalline III–V’s on amorphous substrates, thus further expanding their utility for various applications. The process utilizes templated liquid-phase crystal growth that results in user-tunable, patterned micro and nanostructures of single-crystalline III–V’s of up to tens of micrometres in lateral dimensions. InP is chosen as a model material system owing to its technological importance. Themore » patterned InP single crystals are configured as high-performance transistors and photodetectors directly on amorphous SiO 2 growth substrates, with performance matching state-of-the-art epitaxially grown devices. In conclusion, the work presents an important advance towards universal integration of III–V’s on application-specific substrates by direct growth.« less

Direct growth of single-crystalline III–V semiconductors on amorphous substrates

PubMed Central

Chen, Kevin; Kapadia, Rehan; Harker, Audrey; Desai, Sujay; Seuk Kang, Jeong; Chuang, Steven; Tosun, Mahmut; Sutter-Fella, Carolin M.; Tsang, Michael; Zeng, Yuping; Kiriya, Daisuke; Hazra, Jubin; Madhvapathy, Surabhi Rao; Hettick, Mark; Chen, Yu-Ze; Mastandrea, James; Amani, Matin; Cabrini, Stefano; Chueh, Yu-Lun; Ager III, Joel W.; Chrzan, Daryl C.; Javey, Ali

2016-01-01

The III–V compound semiconductors exhibit superb electronic and optoelectronic properties. Traditionally, closely lattice-matched epitaxial substrates have been required for the growth of high-quality single-crystal III–V thin films and patterned microstructures. To remove this materials constraint, here we introduce a growth mode that enables direct writing of single-crystalline III–V's on amorphous substrates, thus further expanding their utility for various applications. The process utilizes templated liquid-phase crystal growth that results in user-tunable, patterned micro and nanostructures of single-crystalline III–V's of up to tens of micrometres in lateral dimensions. InP is chosen as a model material system owing to its technological importance. The patterned InP single crystals are configured as high-performance transistors and photodetectors directly on amorphous SiO2 growth substrates, with performance matching state-of-the-art epitaxially grown devices. The work presents an important advance towards universal integration of III–V's on application-specific substrates by direct growth. PMID:26813257

A model framework to describe growth-linked biodegradation of trace-level pollutants in the presence of coincidental carbon substrates and microbes.

PubMed

Liu, Li; Helbling, Damian E; Kohler, Hans-Peter E; Smets, Barth F

2014-11-18

Pollutants such as pesticides and their degradation products occur ubiquitously in natural aquatic environments at trace concentrations (μg L(-1) and lower). Microbial biodegradation processes have long been known to contribute to the attenuation of pesticides in contaminated environments. However, challenges remain in developing engineered remediation strategies for pesticide-contaminated environments because the fundamental processes that regulate growth-linked biodegradation of pesticides in natural environments remain poorly understood. In this research, we developed a model framework to describe growth-linked biodegradation of pesticides at trace concentrations. We used experimental data reported in the literature or novel simulations to explore three fundamental kinetic processes in isolation. We then combine these kinetic processes into a unified model framework. The three kinetic processes described were: the growth-linked biodegradation of micropollutant at environmentally relevant concentrations; the effect of coincidental assimilable organic carbon substrates; and the effect of coincidental microbes that compete for assimilable organic carbon substrates. We used Monod kinetic models to describe substrate utilization and microbial growth rates for specific pesticide and degrader pairs. We then extended the model to include terms for utilization of assimilable organic carbon substrates by the specific degrader and coincidental microbes, growth on assimilable organic carbon substrates by the specific degrader and coincidental microbes, and endogenous metabolism. The proposed model framework enables interpretation and description of a range of experimental observations on micropollutant biodegradation. The model provides a useful tool to identify environmental conditions with respect to the occurrence of assimilable organic carbon and coincidental microbes that may result in enhanced or reduced micropollutant biodegradation.

Mixing of water masses caused by a drifting iceberg affects bacterial activity, community composition and substrate utilization capability in the Southern Ocean.

PubMed

Dinasquet, Julie; Richert, Inga; Logares, Ramiro; Yager, Patricia; Bertilsson, Stefan; Riemann, Lasse

2017-06-01

The number of icebergs produced from ice-shelf disintegration has increased over the past decade in Antarctica. These drifting icebergs mix the water column, influence stratification and nutrient condition, and can affect local productivity and food web composition. Data on whether icebergs affect bacterioplankton function and composition are scarce, however. We assessed the influence of iceberg drift on bacterial community composition and on their ability to exploit carbon substrates during summer in the coastal Southern Ocean. An elevated bacterial production and a different community composition were observed in iceberg-influenced waters relative to the undisturbed water column nearby. These major differences were confirmed in short-term incubations with bromodeoxyuridine followed by CARD-FISH. Furthermore, one-week bottle incubations amended with inorganic nutrients and carbon substrates (a mix of substrates, glutamine, N-acetylglucosamine, or pyruvate) revealed contrasting capacity of bacterioplankton to utilize specific carbon substrates in the iceberg-influenced waters compared with the undisturbed site. Our study demonstrates that the hydrographical perturbations introduced by a drifting iceberg can affect activity, composition, and substrate utilization capability of marine bacterioplankton. Consequently, in a context of global warming, increased frequency of drifting icebergs in polar regions holds the potential to affect carbon and nutrient biogeochemistry at local and possibly regional scales. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Chaperone substrate provides missing link for cancer drug discovery.

PubMed

Byrd, Katherine M; Blagg, Brian S J

2018-02-16

Both Hsp70 and Hsp90 chaperones are overexpressed in cancer, making them relevant targets for the development of cancer chemotherapeutics, but a lack of biomolecular readouts for Hsp70 inhibition has limited the pursuit of specific inhibitors for this enzyme. A new study from Cesa et al. identifies two inhibitors of apoptosis proteins (IAPs) as specific client substrates of Hsp70. These results establish biomarkers that can be utilized to monitor Hsp70 inhibition and provide a framework for future efforts to deconvolute chaperone networks. © 2018 Byrd and Blagg.

Gender Differences in Skeletal Muscle Substrate Metabolism – Molecular Mechanisms and Insulin Sensitivity

PubMed Central

Lundsgaard, Anne-Marie; Kiens, Bente

2014-01-01

It has become increasingly apparent that substrate metabolism is subject to gender-specific regulation, and the aim of this review is to outline the available evidence of molecular gender differences in glucose and lipid metabolism of skeletal muscle. Female sex has been suggested to have a favorable effect on glucose homeostasis, and the available evidence from hyperinsulinemic–euglycemic clamp studies is summarized to delineate whether there is a gender difference in whole-body insulin sensitivity and in particular insulin-stimulated glucose uptake of skeletal muscle. Whether an eventual higher insulin sensitivity of female skeletal muscle can be related to gender-specific regulation of molecular metabolism will be topic for discussion. Gender differences in muscle fiber type distribution and substrate availability to and in skeletal muscle are highly relevant for substrate metabolism in men and women. In particular, the molecular machinery for glucose and fatty acid oxidative and storage capacities in skeletal muscle and its implications for substrate utilization during metabolic situations of daily living are discussed, emphasizing their relevance for substrate choice in the fed and fasted state, and during periods of physical activity and recovery. Together, handling of carbohydrate and lipids and regulation of their utilization in skeletal muscle have implications for whole-body glucose homeostasis in men and women. 17-β estradiol is the most important female sex hormone, and the identification of estradiol receptors in skeletal muscle has opened for a role in regulation of substrate metabolism. Also, higher levels of circulating adipokines as adiponectin and leptin in women and their implications for muscle metabolism will be considered. PMID:25431568

Phylogenetic Affiliation of Soil Bacteria That Degrade Aliphatic Polyesters Available Commercially as Biodegradable Plastics

PubMed Central

Suyama, Tetsushi; Tokiwa, Yutaka; Ouichanpagdee, Pornpimol; Kanagawa, Takahiro; Kamagata, Yoichi

1998-01-01

Thirty-nine morphologically different soil bacteria capable of degrading poly(β-hydroxyalkanoate), poly(ɛ-caprolactone), poly(hexamethylene carbonate), or poly(tetramethylene succinate) were isolated. Their phylogenetic positions were determined by 16S ribosomal DNA sequencing, and all of them fell into the classes Firmicutes and Proteobacteria. Determinations of substrate utilization revealed characteristic patterns of substrate specificities. PMID:9835597

Purification of SUMO conjugating enzymes and kinetic analysis of substrate conjugation

PubMed Central

Yunus, Ali A.; Lima, Christopher D.

2009-01-01

SUMO conjugation to protein substrates requires the concerted action of a dedicated E2 ubiquitin conjugation enzyme (Ubc9) and associated E3 ligases. Although Ubc9 can directly recognize and modify substrate lysine residues that occur within a consensus site for SUMO modification, E3 ligases can redirect specificity and enhance conjugation rates during SUMO conjugation in vitro and in vivo. In this chapter, we will describe methods utilized to purify SUMO conjugating enzymes and model substrates which can be used for analysis of SUMO conjugation in vitro. We will also describe methods to extract kinetic parameters during E3-dependent or E3-independent substrate conjugation. PMID:19107417

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

[Structure and function of the bacterial flagellar type III protein export system in Salmonella
].

PubMed

Minamino, Tohru

2015-01-01

The bacterial flagellum is a filamentous organelle that propels the bacterial cell body in liquid media. For construction of the bacterial flagellum beyond the cytoplasmic membrane, flagellar component proteins are transported by its specific protein export apparatus from the cytoplasm to the distal end of the growing flagellar structure. The flagellar export apparatus consists of a transmembrane export gate complex and a cytoplasmic ATPase ring complex. Flagellar substrate-specific chaperones bind to their cognate substrates in the cytoplasm and escort the substrates to the docking platform of the export gate. The export apparatus utilizes ATP and proton motive force across the cytoplasmic membrane as the energy sources to drive protein export and coordinates protein export with assembly by ordered export of substrates to parallel with their order of assembly. In this review, we summarize our current understanding of the structure and function of the flagellar protein export system in Salmonella enterica serovar Typhimurium.

Role of catabolite regulatory mechanisms in control of carbohydrate utilization by the rumen anaerobic fungus Neocallimastix frontalis.

PubMed Central

Mountfort, D O; Asher, R A

1983-01-01

Neocallimastix frontalis PN-1 utilized the soluble sugars D-glucose, D-cellobiose, D-fructose, maltose, sucrose, and D-xylose for growth. L-Arabinose, D-galactose, D-mannose, and D-xylitol did not support growth of the fungus. Paired substrate test systems were used to determine whether any two sugars were utilized simultaneously or sequentially. Of the paired monosaccharides tested, glucose was found to be preferentially utilized compared with fructose and xylose. The disaccharides cellobiose and sucrose were preferentially utilized compared with fructose and glucose, respectively, an cellobiose was also the preferred substrate compared with xylose. Xylose was the preferred substrate compared with maltose. In further incubations, the fungus was grown on the substrate utilized last in the two-substrate tests. After moderate growth was attained, the preferred substrate was added to the culture medium. Inhibition of nonpreferred substrate utilization by the addition of the preferred substrate was taken as evidence of catabolite regulation. For the various combinations of substrates tested, fructose and xylose utilization was found to be inhibited in the presence of glucose, indicating that catabolite regulation was involved. No clear-cut inhibition was observed with any of the other substrate combinations tested. The significance of these findings in relation to rumen microbial interactions and competitions is discussed. PMID:6660873

Functional characterization of LePGT1, a membrane-bound prenyltransferase involved in the geranylation of p-hydroxybenzoic acid.

PubMed

Ohara, Kazuaki; Muroya, Ayumu; Fukushima, Nobuhiro; Yazaki, Kazufumi

2009-06-26

The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane alpha-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.

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.

Characterization of Hybrid Toluate and Benzoate Dioxygenases

PubMed Central

Ge, Yong; Eltis, Lindsay D.

2003-01-01

Toluate dioxygenase of Pseudomonas putida mt-2 (TADOmt2) and benzoate dioxygenase of Acinetobacter calcoaceticus ADP1 (BADOADP1) catalyze the 1,2-dihydroxylation of different ranges of benzoates. The catalytic component of these enzymes is an oxygenase consisting of two subunits. To investigate the structural determinants of substrate specificity in these ring-hydroxylating dioxygenases, hybrid oxygenases consisting of the α subunit of one enzyme and the β subunit of the other were prepared, and their respective specificities were compared to those of the parent enzymes. Reconstituted BADOADP1 utilized four of the seven tested benzoates in the following order of apparent specificity: benzoate > 3-methylbenzoate > 3-chlorobenzoate > 2-methylbenzoate. This is a significantly narrower apparent specificity than for TADOmt2 (3-methylbenzoate > benzoate ∼ 3-chlorobenzoate > 4-methylbenzoate ∼ 4-chlorobenzoate ≫ 2-methylbenzoate ∼ 2-chlorobenzoate [Y. Ge, F. H. Vaillancourt, N. Y. Agar, and L. D. Eltis, J. Bacteriol. 184:4096-4103, 2002]). The apparent substrate specificity of the αBβT hybrid oxygenase for these benzoates corresponded to that of BADOADP1, the parent from which the α subunit originated. In contrast, the apparent substrate specificity of the αTβB hybrid oxygenase differed slightly from that of TADOmt2 (3-chlorobenzoate > 3-methylbenzoate > benzoate ∼ 4-methylbenzoate > 4-chlorobenzoate > 2-methylbenzoate > 2-chlorobenzoate). Moreover, the αTβB hybrid catalyzed the 1,6-dihydroxylation of 2-methylbenzoate, not the 1,2-dihydroxylation catalyzed by the TADOmt2 parent. Finally, the turnover of this ortho-substituted benzoate was much better coupled to O2 utilization in the hybrid than in the parent. Overall, these results support the notion that the α subunit harbors the principal determinants of specificity in ring-hydroxylating dioxygenases. However, they also demonstrate that the β subunit contributes significantly to the enzyme's function. PMID:12949084

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

PubMed Central

Thygesen, Uffe H.; Riemann, Lasse; Stedmon, Colin A.

2015-01-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. PMID:26253668

Detection of alkaline phosphatase in canine cells previously stained with Wright-Giemsa and its utility in differentiating osteosarcoma from other mesenchymal tumors.

PubMed

Ryseff, Julia K; Bohn, Andrea A

2012-09-01

Osteosarcoma (OSA) is a common primary bone tumor in dogs. Demonstration of alkaline phosphatase (ALP) reactivity by tumor cells on unstained slides is useful in differentiating osteosarcoma from other types of sarcoma. However, unstained slides are not always available. The objectives of this study were to evaluate the diagnostic utility of detecting ALP expression in differentiating osteosarcoma from other sarcomas in dogs using cytologic material previously stained with Wright-Giemsa stain and to assess the sensitivity and specificity of ALP expression for diagnosing osteosarcoma using a specific protocol. Archived aspirates of histologically confirmed sarcomas in dogs that had been previously stained with Wright-Giemsa stain were treated with 5-bromo, 4-chloro, 3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) as a substrate for ALP. Cells were evaluated for expression of ALP after incubation with BCIP/NBT for 1 hour. Sensitivity and specificity of ALP expression for diagnosis of OSA were calculated. In samples from 83 dogs, cells from 15/17 OSAs and from 4/66 tumors other than OSA (amelanotic melanoma, gastrointestinal stromal tumor, collision tumor, and anaplastic sarcoma) expressed ALP. Sensitivity and specificity of ALP expression detected using BCIP/NBT substrate applied to cells previously stained with Wright-Giemsa stain for OSA were 88 and 94%, respectively. ALP expression detected using BCIP/NBT substrate applied to previously stained cells is useful in differentiating canine OSA from other mesenchymal neoplasms. © 2012 American Society for Veterinary Clinical Pathology.

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

PubMed Central

Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

2008-01-01

The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

Characterization of a Salmonella sugar kinase essential for the utilization of fructose-asparagine.

PubMed

Biswas, Pradip K; Behrman, Edward J; Gopalan, Venkat

2017-04-01

Salmonella can utilize fructose-asparagine (F-Asn), a naturally occurring Amadori product, as its sole carbon and nitrogen source. Conversion of F-Asn to the common intermediates glucose-6-phosphate, aspartate, and ammonia was predicted to involve the sequential action of an asparaginase, a kinase, and a deglycase. Mutants lacking the deglycase are highly attenuated in mouse models of intestinal inflammation owing to the toxic build-up of the deglycase substrate. The limited distribution of this metabolic pathway in the animal gut microbiome raises the prospects for antibacterial discovery. We report the biochemical characterization of the kinase that was expected to transform fructose-aspartate to 6-phosphofructose-aspartate during F-Asn utilization. In addition to confirming its anticipated function, we determined through studies of fructose-aspartate analogues that this kinase exhibits a substrate-specificity with greater tolerance to changes to the amino acid (including the d-isomer of aspartate) than to the sugar.

Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Lin, S. Jack; Kirchhofer, Daniel; Salvesen, Guy S.; Drag, Marcin

2015-01-01

Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs. PMID:26172376

Biostability analysis for drinking water distribution systems.

PubMed

Srinivasan, Soumya; Harrington, Gregory W

2007-05-01

The ability to limit regrowth in drinking water is referred to as biological stability and depends on the concentration of disinfectant residual and on the concentration of substrate required for the growth of microorganisms. The biostability curve, based on this fundamental concept of biological stability, is a graphical approach to study the two competing effects that determine bacterial regrowth in a distribution system: inactivation due to the presence of a disinfectant, and growth due to the presence of a substrate. Biostability curves are a practical, system specific approach for addressing the problem of bacterial regrowth in distribution systems. This paper presents a standardized algorithm for generating biostability curves and this will enable water utilities to incorporate this approach for their site-specific needs. Using data from pilot scale studies, it was found that this algorithm was applicable to control regrowth of HPC in chlorinated systems where AOC is the growth limiting substrate, and growth of AOB in chloraminated systems, where ammonia is the growth limiting substrate.

Peptidomics methods for the identification of peptidase-substrate interactions

PubMed Central

Lone, Anna Mari; Kim, Yun-Gon; Saghatelian, Alan

2013-01-01

Peptidases have important roles in controlling physiological signaling through their regulation of bioactive peptides. Understanding and controlling bioactive peptide regulation is of great biomedical interest and approaches that elucidate the interplay between peptidases and their substrates are vital for achieving this goal. Here, we highlight the utility of recent peptidomics approaches in identifying endogenous substrates of peptidases. These approaches reveal bioactive substrates and help characterize the biochemical functions of the enzyme. Most recently, peptidomics approaches have been applied to address the challenging question of identifying the peptidases responsible for regulating specific bioactive peptides. Since peptidases are of great biomedical interest, these approaches will begin to impact our ability to identify new drug targets that regulate important bioactive peptides. PMID:23332665

Portable pathogen detection system

DOEpatents

Colston, Billy W.; Everett, Matthew; Milanovich, Fred P.; Brown, Steve B.; Vendateswaran, Kodumudi; Simon, Jonathan N.

2005-06-14

A portable pathogen detection system that accomplishes on-site multiplex detection of targets in biological samples. The system includes: microbead specific reagents, incubation/mixing chambers, a disposable microbead capture substrate, and an optical measurement and decoding arrangement. The basis of this system is a highly flexible Liquid Array that utilizes optically encoded microbeads as the templates for biological assays. Target biological samples are optically labeled and captured on the microbeads, which are in turn captured on an ordered array or disordered array disposable capture substrate and then optically read.

Therapeutic drug monitoring of flucytosine in serum using a SERS-active membrane system

NASA Astrophysics Data System (ADS)

Berger, Adam G.; White, Ian M.

2017-02-01

A need exists for near real-time therapeutic drug monitoring (TDM), in particular for antibiotics and antifungals in patient samples at the point-of-care. To truly fit the point-of-care need, techniques must be rapid and easy to use. Here we report a membrane system utilizing inkjet-fabricated surface enhanced Raman spectroscopy (SERS) sensors that allows sensitive and specific analysis despite the elimination of sophisticated chromatography equipment, expensive analytical instruments, and other systems relegated to the central lab. We utilize inkjet-fabricated paper SERS sensors as substrates for 5FC detection; the use of paper-based SERS substrates leverages the natural wicking ability and filtering properties of microporous membranes. We investigate the use of microporous membranes in the vertical flow assay to allow separation of the flucytosine from whole blood. The passive vertical flow assay serves as a valuable method for physical separation of target analytes from complex biological matrices. This work further establishes a platform for easy, sensitive, and specific TDM of 5FC from whole blood.

Metabolic behavior and enzymatic aspects of denitrifying EBPR sludge in a continuous-flow anaerobic-anoxic system.

PubMed

Zafiriadis, Ilias; Ntougias, Spyridon; Kapagiannidis, Anastasios G; Aivasidis, Alexander

2013-10-01

The metabolic aspects of enhanced biological phosphorus removal (EBPR) were investigated for the first time in a continuous-flow anaerobic-anoxic plant fed with acetate, propionate, or substrates which are involved in the tricarboxylic acid and/or glyoxylate cycle, i.e., fumarate, malate, or oxaloacetate, as the sole carbon source. Although the polyphosphate-accumulating organisms (PAOs) population remained stable with any carbon source examined, no typical EBPR metabolism was observed during fumarate, malate, or oxaloacetate utilization. Specific enzymatic activities related to EBPR were determined in activated sludge homogenates and directly correlated with the nutrient metabolic rates. The experimental results indicated the direct involvement of alkaline phosphatase, pyrophosphatase, and exopolyphosphatase in the denitrifying EBPR process. Metabolic aspects of glyoxylate cycle enzymes are discussed with regard to the biomass anaerobic and anoxic activity. Process performance was highly influenced by the kind of substrate utilized, indicating that specific metabolic pathways should be followed to favor efficient EBPR.

Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.

PubMed

Wang, Yanan; Song, Jiangning; Marquez-Lago, Tatiana T; Leier, André; Li, Chen; Lithgow, Trevor; Webb, Geoffrey I; Shen, Hong-Bin

2017-07-18

Matrix Metalloproteases (MMPs) are an important family of proteases that play crucial roles in key cellular and disease processes. Therefore, MMPs constitute important targets for drug design, development and delivery. Advanced proteomic technologies have identified type-specific target substrates; however, the complete repertoire of MMP substrates remains uncharacterized. Indeed, computational prediction of substrate-cleavage sites associated with MMPs is a challenging problem. This holds especially true when considering MMPs with few experimentally verified cleavage sites, such as for MMP-2, -3, -7, and -8. To fill this gap, we propose a new knowledge-transfer computational framework which effectively utilizes the hidden shared knowledge from some MMP types to enhance predictions of other, distinct target substrate-cleavage sites. Our computational framework uses support vector machines combined with transfer machine learning and feature selection. To demonstrate the value of the model, we extracted a variety of substrate sequence-derived features and compared the performance of our method using both 5-fold cross-validation and independent tests. The results show that our transfer-learning-based method provides a robust performance, which is at least comparable to traditional feature-selection methods for prediction of MMP-2, -3, -7, -8, -9 and -12 substrate-cleavage sites on independent tests. The results also demonstrate that our proposed computational framework provides a useful alternative for the characterization of sequence-level determinants of MMP-substrate specificity.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A model of extracellular enzymes in free-living microbes: which strategy pays off?

PubMed

Traving, Sachia J; Thygesen, Uffe H; Riemann, Lasse; Stedmon, Colin A

2015-11-01

An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A SENSITIVE FLUORESCENCE-BASED ASSAY FOR MONITORING GM2 GANGLIOSIDE HYDROLYSIS IN LIVE PATIENT CELLS AND THEIR LYSATES

PubMed Central

Tropak, Michael B.; Bukovac, Scott W.; Rigat, Brigitte A.; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J.

2010-01-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is anattractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are alsoinhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Herewe demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the β-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant β-hexosaminidase A and substrate-hydrolysis as compared to mock treated cells. PMID:19917668

A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

PubMed

Tropak, Michael B; Bukovac, Scott W; Rigat, Brigitte A; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J

2010-03-01

Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

PubMed

Park, Sun-Ha; Lee, Chang Woo; Cho, Sung Mi; Lee, Hyoungseok; Park, Hyun; Lee, Jungeun; Lee, Jun Hyuck

2018-01-01

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions.

Crystal structure and enzymatic properties of chalcone isomerase from the Antarctic vascular plant Deschampsia antarctica Desv.

PubMed Central

Lee, Hyoungseok; Park, Hyun; Lee, Jungeun

2018-01-01

Chalcone isomerase (CHI) is an important enzyme for flavonoid biosynthesis that catalyzes the intramolecular cyclization of chalcones into (S)-flavanones. CHIs have been classified into two types based on their substrate specificity. Type I CHIs use naringenin chalcone as a substrate and are found in most of plants besides legumes, whereas type II CHIs in leguminous plants can also utilize isoliquiritigenin. In this study, we found that the CHI from the Antarctic plant Deschampsia antarctica (DaCHI1) is of type I based on sequence homology but can use type II CHI substrates. To clarify the enzymatic mechanism of DaCHI1 at the molecular level, the crystal structures of unliganded DaCHI1 and isoliquiritigenin-bound DaCHI1 were determined at 2.7 and 2.1 Å resolutions, respectively. The structures revealed that isoliquiritigenin binds to the active site of DaCHI1 and induces conformational changes. Additionally, the activity assay showed that while DaCHI1 exhibits substrate preference for naringenin chalcone, it can also utilize isoliquiritigenin although the catalytic activity was relatively low. Based on these results, we propose that DaCHI1 uses various substrates to produce antioxidant flavonoids as an adaptation to oxidative stresses associated with harsh environmental conditions. PMID:29394293

Engineering peptide ligase specificity by proteomic identification of ligation sites.

PubMed

Weeks, Amy M; Wells, James A

2018-01-01

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme-substrate specificity limits its utility. We developed an approach for comprehensively characterizing peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme-substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins, including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. Our methods provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Identification of Putative Mek1 Substrates during Meiosis in Saccharomyces cerevisiae Using Quantitative Phosphoproteomics

PubMed Central

Suhandynata, Raymond T.; Wan, Lihong; Zhou, Huilin; Hollingsworth, Nancy M.

2016-01-01

Meiotic recombination plays a key role in sexual reproduction as it generates crossovers that, in combination with sister chromatid cohesion, physically connect homologous chromosomes, thereby promoting their proper segregation at the first meiotic division. Meiotic recombination is initiated by programmed double strand breaks (DSBs) catalyzed by the evolutionarily conserved, topoisomerase-like protein Spo11. Repair of these DSBs is highly regulated to create crossovers between homologs that are distributed throughout the genome. This repair requires the presence of the mitotic recombinase, Rad51, as well as the strand exchange activity of the meiosis-specific recombinase, Dmc1. A key regulator of meiotic DSB repair in Saccharomyces cerevisiae is the meiosis-specific kinase Mek1, which promotes interhomolog strand invasion and is required for the meiotic recombination checkpoint and the crossover/noncrossover decision. Understanding how Mek1 regulates meiotic recombination requires the identification of its substrates. Towards that end, an unbiased phosphoproteomic approach utilizing Stable Isotope Labeling by Amino Acids in Cells (SILAC) was utilized to generate a list of potential Mek1 substrates, as well as proteins containing consensus phosphorylation sites for cyclin-dependent kinase, the checkpoint kinases, Mec1/Tel1, and the polo-like kinase, Cdc5. These experiments represent the first global phosphoproteomic dataset for proteins in meiotic budding yeast. PMID:27214570

A microarray of ubiquitylated proteins for profiling deubiquitylase activity reveals the critical roles of both chain and substrate.

PubMed

Loch, Christian M; Strickler, James E

2012-11-01

Substrate ubiquitylation is a reversible process critical to cellular homeostasis that is often dysregulated in many human pathologies including cancer and neurodegeneration. Elucidating the mechanistic details of this pathway could unlock a large store of information useful to the design of diagnostic and therapeutic interventions. Proteomic approaches to the questions at hand have generally utilized mass spectrometry (MS), which has been successful in identifying both ubiquitylation substrates and profiling pan-cellular chain linkages, but is generally unable to connect the two. Interacting partners of the deubiquitylating enzymes (DUBs) have also been reported by MS, although substrates of catalytically competent DUBs generally cannot be. Where they have been used towards the study of ubiquitylation, protein microarrays have usually functioned as platforms for the identification of substrates for specific E3 ubiquitin ligases. Here, we report on the first use of protein microarrays to identify substrates of DUBs, and in so doing demonstrate the first example of microarray proteomics involving multiple (i.e., distinct, sequential and opposing) enzymatic activities. This technique demonstrates the selectivity of DUBs for both substrate and type (mono- versus poly-) of ubiquitylation. This work shows that the vast majority of DUBs are monoubiquitylated in vitro, and are incapable of removing this modification from themselves. This work also underscores the critical role of utilizing both ubiquitin chains and substrates when attempting to characterize DUBs. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. Copyright © 2012 Elsevier B.V. All rights reserved.

The three principles of action: a Pavlovian-instrumental transfer hypothesis

PubMed Central

Cartoni, Emilio; Puglisi-Allegra, Stefano; Baldassarre, Gianluca

2013-01-01

Pavlovian conditioned stimuli can influence instrumental responding, an effect called Pavlovian-instrumental transfer (PIT). During the last decade, PIT has been subdivided into two types: specific PIT and general PIT, each having its own neural substrates. Specific PIT happens when a conditioned stimulus (CS) associated with a reward enhances an instrumental response directed to the same reward. Under general PIT, instead, the CS enhances a response directed to a different reward. While important progress has been made into identifying the neural substrates, the function of specific and general PIT and how they interact with instrumental responses are still not clear. In the experimental paradigm that distinguishes specific and general PIT an effect of PIT inhibition has also been observed and is waiting for an explanation. Here we propose an hypothesis that links these three PIT effects (specific PIT, general PIT and PIT inhibition) to three aspects of action evaluation. These three aspects, which we call “principles of action”, are: context, efficacy, and utility. In goal-directed behavior, an agent has to evaluate if the context is suitable to accomplish the goal, the efficacy of his action in getting the goal, and the utility of the goal itself: we suggest that each of the three PIT effects is related to one of these aspects of action evaluation. In particular, we link specific PIT with the estimation of efficacy, general PIT with the evaluation of utility, and PIT inhibition with the adequacy of context. We also provide a latent cause Bayesian computational model that exemplifies this hypothesis. This hypothesis and the model provide a new framework and new predictions to advance knowledge about PIT functioning and its role in animal adaptation. PMID:24312025

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

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

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

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

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

PubMed

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

2017-09-01

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

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

PubMed Central

Malyavantham, Kishore S.; Suresh, Lakshmanan

2018-01-01

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

Quinone-based stable isotope probing for assessment of 13C substrate-utilizing bacteria

NASA Astrophysics Data System (ADS)

Kunihiro, Tadao; Katayama, Arata; Demachi, Toyoko; Veuger, Bart; Boschker, Henricus T. S.; van Oevelen, Dick

2015-04-01

In this study, we attempted to establish quinone-stable-isotope probing (SIP) technique to link substrate-utilizing bacterial group to chemotaxonomic group in bacterial community. To identify metabolically active bacterial group in various environments, SIP techniques combined with biomarkers have been widely utilized as an attractive method for environmental study. Quantitative approaches of the SIP technique have unique advantage to assess substrate-incorporation into bacteria. As a most major quantitative approach, SIP technique based on phospholipid-derived fatty acids (PLFA) have been applied to simultaneously assess substrate-incorporation rate into bacteria and microbial community structure. This approach is powerful to estimate the incorporation rate because of the high sensitivity due to the detection by a gas chromatograph-combustion interface-isotope ratio mass spectrometer (GC-c-IRMS). However, its phylogenetic resolution is limited by specificity of a compound-specific marker. We focused on respiratory quinone as a biomarker. Our previous study found a good correlation between concentrations of bacteria-specific PLFAs and quinones over several orders of magnitude in various marine sediments, and the quinone method has a higher resolution (bacterial phylum level) for resolving differences in bacterial community composition more than that of bacterial PLFA. Therefore, respiratory quinones are potentially good biomarkers for quantitative approaches of the SIP technique. The LC-APCI-MS method as molecular-mass based detection method for quinone was developed and provides useful structural information for identifying quinone molecular species in environmental samples. LC-MS/MS on hybrid triple quadrupole/linear ion trap, which enables to simultaneously identify and quantify compounds in a single analysis, can detect high molecular compounds with their isotope ions. Use of LC-MS/MS allows us to develop quinone-SIP based on molecular mass differences due to 13C abundance in the quinone. In this study, we verified carbon stable isotope of quinone compared with bulk carbon stable isotope of bacterial culture. Results indicated a good correlation between carbon stable isotope of quinone compared with bulk carbon stable isotope. However, our measurement conditions for detection of quinone isotope-ions incurred underestimation of 13C abundance in the quinone. The quinone-SIP technique needs further optimization for measurement conditions of LC-MS/MS.

Roles of Ala-149 in the catalytic activity of diadenosine tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.

PubMed

Mori, Shigetarou; Kim, Hyun; Rimbara, Emiko; Arakawa, Yoshichika; Shibayama, Keigo

2015-01-01

Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) phosphorylase from Mycobacterium tuberculosis H37Rv (MtAPA) belongs to the histidine triad motif (HIT) superfamily, but is the only member with an alanine residue at position 149 (Ala-149). Enzymatic analysis revealed that the Ala-149 deletion mutant displayed substrate specificity for diadenosine 5',5'''-P(1),P(5)-pentaphosphate and was inactive on Ap4A and other substrates that are utilized by the wild-type enzyme.

Splicing fidelity: DEAD/H-box ATPases as molecular clocks.

PubMed

Koodathingal, Prakash; Staley, Jonathan P

2013-07-01

The spliceosome discriminates against suboptimal substrates, both during assembly and catalysis, thereby enhancing specificity during pre-mRNA splicing. Central to such fidelity mechanisms are a conserved subset of the DEAD- and DEAH-box ATPases, which belong to a superfamily of proteins that mediate RNP rearrangements in almost all RNA-dependent processes in the cell. Through an investigation of the mechanisms contributing to the specificity of 5' splice site cleavage, two related reports, one from our lab and the other from the Cheng lab, have provided insights into fidelity mechanisms utilized by the spliceosome. In our work, we found evidence for a kinetic proofreading mechanism in splicing in which the DEAH-box ATPase Prp16 discriminates against substrates undergoing slow 5' splice site cleavage. Additionally, our study revealed that discriminated substrates are discarded through a general spliceosome disassembly pathway, mediated by another DEAH-box ATPase Prp43. In their work, Tseng et al. described the underlying molecular events through which Prp16 discriminates against a splicing substrate during 5' splice site cleavage. Here, we present a synthesis of these two studies and, additionally, provide the first biochemical evidence for discrimination of a suboptimal splicing substrate just prior to 5' splice site cleavage. Together, these findings support a general mechanism for a ubiquitous superfamily of ATPases in enhancing specificity during RNA-dependent processes in the cell.

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

PubMed Central

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

2014-01-01

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

Biochemical evaluation of a parsley tyrosine decarboxylase results in a novel 4-hydroxyphenylacetaldehyde synthase enzyme.

PubMed

Torrens-Spence, Michael P; Gillaspy, Glenda; Zhao, Bingyu; Harich, Kim; White, Robert H; Li, Jianyong

2012-02-10

Plant aromatic amino acid decarboxylases (AAADs) are effectively indistinguishable from plant aromatic acetaldehyde syntheses (AASs) through primary sequence comparison. Spectroscopic analyses of several characterized AASs and AAADs were performed to look for absorbance spectral identifiers. Although this limited survey proved inconclusive, the resulting work enabled the reevaluation of several characterized plant AAS and AAAD enzymes. Upon completion, a previously reported parsley AAAD protein was demonstrated to have AAS activity. Substrate specificity tests demonstrate that this novel AAS enzyme has a unique substrate specificity towards tyrosine (km 0.46mM) and dopa (km 1.40mM). Metabolite analysis established the abundance of tyrosine and absence of dopa in parsley extracts. Such analysis indicates that tyrosine is likely to be the sole physiological substrate. The resulting information suggests that this gene is responsible for the in vivo production of 4-hydroxyphenylacetaldehyde (4-HPAA). This is the first reported case of an AAS enzyme utilizing tyrosine as a primary substrate and the first report of a single enzyme capable of producing 4-HPAA from tyrosine. Copyright © 2012 Elsevier Inc. All rights reserved.

Characterization of a new oligoalginate lyase from marine bacterium Vibrio sp.

PubMed

Yu, Zuochen; Zhu, Benwei; Wang, Wenxia; Tan, Haidong; Yin, Heng

2018-06-01

A new oligoalginate lyase encoding gene, designed oal17A, was cloned from marine bacterium Vibrio sp. W13, and then expressed in Escherichia coli. The recombinant Oal17A was purified by NTA-Ni resin with maximal activity at 30°C and pH7.0. Oal17A exhibited broad substrate specificity, and preferred to degrade alginate than polyM or polyG into monosaccharide acid. The specific activity of Oal17A toward alginate, polyM and polyG was 21.14U/mg, 12.31U/mg and 7.43U/mg, respectively. With features of high-level expression and broad substrate specificity, Oal17A would be a potential tool for alginate monomer production process of alginate utilizing for biofuels and bioethanol production. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of energy substrate utilization and hepatic insulin sensitivity by phosphatidylcholine transfer protein/StarD2.

PubMed

Scapa, Erez F; Pocai, Alessandro; Wu, Michele K; Gutierrez-Juarez, Roger; Glenz, Lauren; Kanno, Keishi; Li, Hua; Biddinger, Sudha; Jelicks, Linda A; Rossetti, Luciano; Cohen, David E

2008-07-01

Phosphatidylcholine transfer protein (PC-TP, also known as StarD2) is a highly specific intracellular lipid binding protein with accentuated expression in oxidative tissues. Here we show that decreased plasma concentrations of glucose and free fatty acids in fasting PC-TP-deficient (Pctp(-/-)) mice are attributable to increased hepatic insulin sensitivity. In hyperinsulinemic-euglycemic clamp studies, Pctp(-/-) mice exhibited profound reductions in hepatic glucose production, gluconeogenesis, glycogenolysis, and glucose cycling. These changes were explained in part by the lack of PC-TP expression in liver per se and in part by marked alterations in body fat composition. Reduced respiratory quotients in Pctp(-/-) mice were indicative of preferential fatty acid utilization for energy production in oxidative tissues. In the setting of decreased hepatic fatty acid synthesis, increased clearance rates of dietary triglycerides and increased hepatic triglyceride production rates reflected higher turnover in Pctp(-/-) mice. Collectively, these data support a key biological role for PC-TP in the regulation of energy substrate utilization.

Plant-microbe interactions driven by exometabolite preferences of rhizosphere bacteria

NASA Astrophysics Data System (ADS)

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

2016-12-01

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

Changing appetites: The adaptive advantages of fuel choice

PubMed Central

Stanley, Illana A.; Ribeiro, Sofia M.; Giménez-Cassina, Alfredo; Norberg, Erik; Danial, Nika N.

2013-01-01

Cells are capable of metabolizing a variety of carbon substrates, including glucose, fatty acids, ketone bodies, and amino acids. Cellular fuel choice not only fulfills specific biosynthetic needs, but also enables programmatic adaptations to stress conditions beyond compensating for changes in nutrient availability. Emerging evidence indicates that specific switches from utilization of one substrate to another can have protective or permissive roles in disease pathogenesis. Understanding the molecular determinants of cellular fuel preference may provide insights into the homeostatic control of stress responses, and unveil therapeutic targets. Here, we highlight overarching themes encompassing cellular fuel choice, its link to cell fate and function, its advantages in stress protection, and its contribution to metabolic dependencies and maladaptations in pathologic conditions. PMID:24018218

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

PubMed Central

Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

2016-01-01

The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

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

Laser surface treatment of porous ceramic substrate for application in solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Mahmod, D. S. A.; Khan, A. A.; Munot, M. A.; Glandut, N.; Labbe, J. C.

2016-08-01

Laser has offered a large number of benefits for surface treatment of ceramics due to possibility of localized heating, very high heating/cooling rates and possibility of growth of structural configurations only produced under non-equilibrium high temperature conditions. The present work investigates oxidation of porous ZrB2-SiC sintered ceramic substrates through treatment by a 1072 ± 10 nm ytterbium fiber laser. A multi-layer structure is hence produced showing successively oxygen rich distinct layers. The porous bulk beneath these layers remained unaffected as this laser-formed oxide scale and protected the substrate from oxidation. A glassy SiO2 structure thus obtained on the surface of the substrate becomes subject of interest for further research, specifically for its utilization as solid protonic conductor in Solid Oxide Fuel Cells (SOFCs).

Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus

PubMed Central

Daou, Marianne; Piumi, François; Cullen, Daniel; Record, Eric

2016-01-01

ABSTRACT The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium. The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry. PMID:27260365

Specificity in transition state binding: the Pauling model revisited.

PubMed

Amyes, Tina L; Richard, John P

2013-03-26

Linus Pauling proposed that the large rate accelerations for enzymes are caused by the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogues of the transition states for enzymatic reactions often act as tight-binding inhibitors provided early support for this simple and elegant proposal. We review experimental results that support the proposal that Pauling's model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high-energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies that aimed to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-coenzyme A:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of coenzyme A (CoA) are responsible for a rate increase of 3 × 10(12)-fold, which is close to the estimated total 5 × 10(13)-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide an ~12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5'-monophosphate decarboxylase, and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6-8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with that of the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form E(O) to an active closed form E(C), by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis.

Specificity in Transition State Binding: The Pauling Model Revisited

PubMed Central

Amyes, Tina L.; Richard, John P.

2013-01-01

Linus Pauling proposed that the large rate accelerations for enzymes are due to the high specificity of the protein catalyst for binding the reaction transition state. The observation that stable analogs of the transition states for enzymatic reactions often act as tight-binding binding inhibitors provided early support for this simple and elegant proposal. We review experimental results which support the proposal that Pauling’s model provides a satisfactory explanation for the rate accelerations for many heterolytic enzymatic reactions through high energy reaction intermediates, such as proton transfer and decarboxylation. Specificity in transition state binding is obtained when the total intrinsic binding energy of the substrate is significantly larger than the binding energy observed at the Michaelis complex. The results of recent studies to characterize the specificity in binding of the enolate oxygen at the transition state for the 1,3-isomerization reaction catalyzed by ketosteroid isomerase are reviewed. Interactions between pig heart succinyl-CoA:3-oxoacid coenzyme A transferase (SCOT) and the nonreacting portions of CoA are responsible for a rate increase of 3 × 1012-fold, which is close to the estimated total 5 × 1013-fold enzymatic rate acceleration. Studies that partition the interactions between SCOT and CoA into their contributing parts are reviewed. Interactions of the protein with the substrate phosphodianion group provide a ca. 12 kcal/mol stabilization of the transition state for the reactions catalyzed by triosephosphate isomerase, orotidine 5′-monophosphate decarboxylase and α-glycerol phosphate dehydrogenase. The interactions of these enzymes with the substrate piece phosphite dianion provide a 6 – 8 kcal/mol stabilization of the transition state for reaction of the appropriate truncated substrate. Enzyme activation by phosphite dianion reflects the higher dianion affinity for binding to the enzyme-transition state complex compared with the free enzyme. Evidence is presented that supports a model in which the binding energy of the phosphite dianion piece, or the phosphodianion group of the whole substrate, is utilized to drive an enzyme conformational change from an inactive open form EO to an active closed form EC, by closure of a phosphodianion gripper loop. Members of the enolase and haloalkanoic acid dehalogenase superfamilies use variable capping domains to interact with nonreacting portions of the substrate and sequester the substrate from interaction with bulk solvent. Interactions of this capping domain with the phenyl group of mandelate have been shown to activate mandelate racemase for catalysis of deprotonation of α-carbonyl carbon. We propose that an important function of these capping domains is to utilize the binding interactions with nonreacting portions of the substrate to activate the enzyme for catalysis. PMID:23327224

C-Myc Induced Compensated Cardiac Hypertrophy Increases Free Fatty Acid Utilization for the Citric Acid Cycle

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

Olson, Aaron; Ledee, Dolena; Iwamoto, Kate

The protooncogene C-Myc (Myc) regulates cardiac hypertrophy. Myc promotes compensated cardiac function, suggesting that the operative mechanisms differ from those leading to heart failure. Myc regulation of substrate metabolism is a reasonable target, as Myc alters metabolism in other tissues. We hypothesize that Myc-induced shifts in substrate utilization signal and promote compensated hypertrophy. We used cardiac specific Myc-inducible C57/BL6 male mice between 4-6 months old that develop hypertrophy with tamoxifen (tam). Isolated working hearts and 13Carbon (13C )-NMR were used to measure function and fractional contributions (Fc) to the citric acid cycle by using perfusate containing 13C-labeled free fatty acids,more » acetoacetate, lactate, unlabeled glucose and insulin. Studies were performed at pre-hypertrophy (3-days tam, 3dMyc), established hypertrophy (7-days tam, 7dMyc) or vehicle control (cont). Non-transgenic siblings (NTG) received 7-days tam or vehicle to assess drug effect. Hypertrophy was confirmed by echocardiograms and heart weights. Western blots were performed on key metabolic enzymes. Hypertrophy occurred in 7dMyc only. Cardiac function did not differ between groups. Tam alone did not affect substrate contribution in NTG. Substrate utilization was not significantly altered in 3dMyc versus cont. The free fatty acid FC was significantly greater in 7dMyc vs cont with decreased unlabeled Fc, which is predominately exogenous glucose. Free fatty acid flux to the citric acid cycle increased while lactate flux was diminished in 7dMyc compared to cont. Total protein levels of a panel of key metabolic enzymes were unchanged; however total protein O-GlcNAcylation was increased in 7dMyc. Substrate utilization changes did not precede hypertrophy; therefore they are not the primary signal for cardiac growth in this model. Free fatty acid utilization and oxidation increase at established hypertrophy. Understanding the mechanisms whereby this change maintained compensated function could provide useful information for developing metabolic therapies to treat heart failure. The molecular signaling for this metabolic change may occur through O-GlcNAcylation.« less

Sialidases from gut bacteria: a mini-review.

PubMed

Juge, Nathalie; Tailford, Louise; Owen, C David

2016-02-01

Sialidases are a large group of enzymes, the majority of which catalyses the cleavage of terminal sialic acids from complex carbohydrates on glycoproteins or glycolipids. In the gastrointestinal (GI) tract, sialic acid residues are mostly found in terminal location of mucins via α2-3/6 glycosidic linkages. Many enteric commensal and pathogenic bacteria can utilize sialic acids as a nutrient source, but not all express the sialidases that are required to release free sialic acid. Sialidases encoded by gut bacteria vary in terms of their substrate specificity and their enzymatic reaction. Most are hydrolytic sialidases, which release free sialic acid from sialylated substrates. However, there are also examples with transglycosylation activities. Recently, a third class of sialidases, intramolecular trans-sialidase (IT-sialidase), has been discovered in gut microbiota, releasing (2,7-anhydro-Neu5Ac) 2,7-anydro-N-acetylneuraminic acid instead of sialic acid. Reaction specificity varies, with hydrolytic sialidases demonstrating broad activity against α2,3-, α2,6- and α2,8-linked substrates, whereas IT-sialidases tend to be specific for α2,3-linked substrates. In this mini-review, we summarize the current knowledge on the structural and biochemical properties of sialidases involved in the interaction between gut bacteria and epithelial surfaces. © 2016 Authors.

Tetraterpene Synthase Substrate and Product Specificity in the Green Microalga Botryococcus braunii Race L.

PubMed

Thapa, Hem R; Tang, Su; Sacchettini, James C; Devarenne, Timothy P

2017-09-15

Recently, the biosynthetic pathway for lycopadiene, a C 40 tetraterpenoid hydrocarbon, was deciphered from the L race of Botryococcus braunii, an alga that produces hydrocarbon oils capable of being converted into combustible fuels. The lycopadiene pathway is initiated by the squalene synthase (SS)-like enzyme lycopaoctaene synthase (LOS), which catalyzes the head-to-head condensation of two C 20 geranylgeranyl diphosphate (GGPP) molecules to produce C 40 lycopaoctaene. LOS shows unusual substrate promiscuity for SS or SS-like enzymes by utilizing C 15 farnesyl diphosphate (FPP) and C 20 phytyl diphosphate in addition to GGPP as substrates. These three substrates can be combined by LOS individually or in combinations to produce six different hydrocarbons of C 30 , C 35 , and C 40 chain lengths. To understand LOS substrate and product specificity, rational mutagenesis experiments were conducted based on sequence alignment with several SS proteins as well as a structural comparison with the human SS (HSS) crystal structure. Characterization of the LOS mutants in vitro identified Ser276 and Ala288 in the LOS active site as key amino acids responsible for controlling substrate binding, and thus the promiscuity of this enzyme. Mutating these residues to those found in HSS largely converted LOS from lycopaoctaene production to C 30 squalene production. Furthermore, these studies were confirmed in vivo by expressing LOS in E. coli cells metabolically engineered to produce high FPP and GGPP levels. These studies also offer insights into tetraterpene hydrocarbon metabolism in B. braunii and provide a foundation for engineering LOS for robust production of specific hydrocarbons of a desired chain length.

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

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

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

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

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

DOE PAGES

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

2017-07-01

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

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

PubMed

Kottom, Theodore J; Limper, Andrew H

2011-10-01

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

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

PubMed

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

2016-11-01

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

Conformational Changes Allow Processing of Bulky Substrates by a Haloalkane Dehalogenase with a Small and Buried Active Site.

PubMed

Kokkonen, Piia; Bednar, David; Dockalova, Veronika; Prokop, Zbynek; Damborsky, Jiri

2018-06-01

Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and which is likely the route for bulky substrates. These results provide insights into the structure-dynamics-function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to DCE introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed Central

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

2016-01-01

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

Laminin α5 substrates promote survival, network formation and functional development of human pluripotent stem cell-derived neurons in vitro.

PubMed

Hyysalo, Anu; Ristola, Mervi; Mäkinen, Meeri E-L; Häyrynen, Sergei; Nykter, Matti; Narkilahti, Susanna

2017-10-01

Laminins are one of the major protein groups in the extracellular matrix (ECM) and specific laminin isoforms are crucial for neuronal functions in the central nervous system in vivo. In the present study, we compared recombinant human laminin isoforms (LN211, LN332, LN411, LN511, and LN521) and laminin isoform fragment (LN511-E8) in in vitro cultures of human pluripotent stem cell (hPSC)-derived neurons. We showed that laminin substrates containing the α5-chain are important for neuronal attachment, viability and network formation, as detected by phase contrast imaging, viability staining, and immunocytochemistry. Gene expression analysis showed that the molecular mechanisms involved in the preference of hPSC-derived neurons for specific laminin isoforms could be related to ECM remodeling and cell adhesion. Importantly, the microelectrode array analysis revealed the widest distribution of electrophysiologically active neurons on laminin α5 substrates, indicating most efficient development of neuronal network functionality. This study shows that specific laminin α5 substrates provide a controlled in vitro culture environment for hPSC-derived neurons. These substrates can be utilized not only to enhance the production of functional hPSC-derived neurons for in vitro applications like disease modeling, toxicological studies, and drug discovery, but also for the production of clinical grade hPSC-derived cells for regenerative medicine applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme contributions to NAD+ biosynthesis.

PubMed

Sorci, Leonardo; Cimadamore, Flavio; Scotti, Stefania; Petrelli, Riccardo; Cappellacci, Loredana; Franchetti, Palmarisa; Orsomando, Giuseppe; Magni, Giulio

2007-04-24

Initial-rate and product inhibition studies revealed distinctive ordered ternary complex kinetic mechanisms, substrate specificities, and metal ion preferences for the three isozymes of human nicotinamide mononucleotide adenylyl-transferase (NMNAT, EC 2.7.7.1). ATP binds before NMN with nuclear isozyme NMNAT1 and Golgi apparatus NMNAT2, but the opposite order is observed with the mitochondrial isozyme NMNAT3. Only the latter utilizes ITP efficiently in place of ATP, and while NMNH conversion to NADH by NMNAT1 and NMNAT3 occurs at similar rates, conversion by NMNAT2 is much slower. These isozymes can also be discriminated by their action on tiazofurin monophosphate (TrMP), a metabolite of the antineoplastic prodrug tiazofurin. Our finding that TrMP is only a substrate with NMNAT1 and NMNAT3 reveals for the first time an organelle selectivity in the metabolism of this important drug. In search of additional ways to discriminate these isozymes, we synthesized and tested the P1-(nicotinamide/nicotinate-riboside-5')-Pn-(adenosine-5') dinucleotides Np3AD, Np4AD, and Nap4AD. In addition to being highly effective inhibitors, these multisubstrate geometric inhibitors gave inhibition patterns that are consistent with the aforementioned isozyme differences in substrate binding order. Distinctive differences in their substrate specificity and metal ion selectivity also permitted us to quantify individual isozyme contributions to NAD+ formation in human cell extracts.

Enzyme activation through the utilization of intrinsic dianion binding energy.

PubMed

Amyes, T L; Malabanan, M M; Zhai, X; Reyes, A C; Richard, J P

2017-03-01

We consider 'the proposition that the intrinsic binding energy that results from the noncovalent interaction of a specific substrate with the active site of the enzyme is considerably larger than is generally believed. An important part of this binding energy may be utilized to provide the driving force for catalysis, so that the observed binding energy represents only what is left over after this utilization' [Jencks,W.P. (1975) Adv. Enzymol. Relat. Areas. Mol. Biol. , , 219-410]. The large ~12 kcal/mol intrinsic substrate phosphodianion binding energy for reactions catalyzed by triosephosphate isomerase (TIM), orotidine 5'-monophosphate decarboxylase and glycerol-3-phosphate dehydrogenase is divided into 4-6 kcal/mol binding energy that is expressed on the formation of the Michaelis complex in anchoring substrates to the respective enzyme, and 6-8 kcal/mol binding energy that is specifically expressed at the transition state in activating the respective enzymes for catalysis. A structure-based mechanism is described where the dianion binding energy drives a conformational change that activates these enzymes for catalysis. Phosphite dianion plays the active role of holding TIM in a high-energy closed active form, but acts as passive spectator in showing no effect on transition-state structure. The result of studies on mutant enzymes is presented, which support the proposal that the dianion-driven enzyme conformational change plays a role in enhancing the basicity of side chain of E167, the catalytic base, by clamping the base between a pair of hydrophobic side chains. The insight these results provide into the architecture of enzyme active sites and the development of strategies for the de novo design of protein catalysts is discussed. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

DOE PAGES

Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; ...

2014-10-21

Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

Aerobic microbial mineralization of dichloroethene as sole carbon substrate

USGS Publications Warehouse

Bradley, P.M.; Chapelle, F.H.

2000-01-01

Microorganisms indigenous to the bed sediments of a black- water stream utilized 1,2-dichloroethene (1,2-DCE) as a sole carbon substrate for aerobic metabolism. Although no evidence of growth was observed in the minimal salts culture media used in this study, efficient aerobic microbial mineralization of 1,2-DCE as sole carbon substrate was maintained through three sequential transfers (107 final dilution) of the original environmental innoculum. These results indicate that 1,2-DCE can be utilized as a primary substrate to support microbial metabolism under aerobic conditions.Microorganisms indigenous to the bed sediments of a black-water stream utilized 1,2-dichloroethene (1,2-DCE) as a sole carbon substrate for aerobic metabolism. Although no evidence of growth was observed in the minimal salts culture media used in this study, efficient aerobic microbial mineralization of 1,2-DCE as sole carbon substrate was maintained through three sequential transfers (107 final dilution) of the original environmental innoculum. These results indicate that 1,2-DCE can be utilized as a primary substrate to support microbial metabolism under aerobic conditions.

Selective-area growth and controlled substrate coupling of transition metal dichalcogenides

NASA Astrophysics Data System (ADS)

Bersch, Brian M.; Eichfeld, Sarah M.; Lin, Yu-Chuan; Zhang, Kehao; Bhimanapati, Ganesh R.; Piasecki, Aleksander F.; Labella, Michael, III; Robinson, Joshua A.

2017-06-01

Developing a means for true bottom-up, selective-area growth of two-dimensional (2D) materials on device-ready substrates will enable synthesis in regions only where they are needed. Here, we demonstrate seed-free, site-specific nucleation of transition metal dichalcogenides (TMDs) with precise control over lateral growth by utilizing an ultra-thin polymeric surface functionalization capable of precluding nucleation and growth. This polymer functional layer (PFL) is derived from conventional photoresists and lithographic processing, and is compatible with multiple growth techniques, precursors (metal organics, solid-source) and TMDs. Additionally, we demonstrate that the substrate can play a major role in TMD transport properties. With proper TMD/substrate decoupling, top-gated field-effect transistors (FETs) fabricated with selectively-grown monolayer MoS2 channels are competitive with current reported MoS2 FETs. The work presented here demonstrates that substrate surface engineering is key to realizing precisely located and geometrically-defined 2D layers via unseeded chemical vapor deposition techniques.

Selection and deposition of nanoparticles using CO.sub.2-expanded liquids

DOEpatents

Roberts, Christopher B [Auburn, AL; McLeod, Marshall Chandler [Hillsboro, OR; Anand, Madhu [Auburn, AL

2008-06-10

A method for size selection of nanostructures comprising utilizing a gas-expanded liquids (GEL) and controlled pressure to precipitate desired size populations of nanostructures, e.g., monodisperse. The GEL can comprise CO.sub.2 antisolvent and an organic solvent. The method can be carried out in an apparatus comprising a first open vessel configured to allow movement of a liquid/particle solution to specific desired locations within the vessel, a second pressure vessel, a location controller for controlling location of the particles and solution within the first vessel, a inlet for addition of antisolvent to the first vessel, and a device for measuring the amount of antisolvent added. Also disclosed is a method for forming nanoparticle thin films comprising utilizing a GEL containing a substrate, pressurizing the solution to precipitate and deposit nanoparticles onto the substrate, removing the solvent thereby leaving a thin nanoparticle film, removing the solvent and antisolvent, and drying the film.

Selection of nanoparticles using CO.sub.2-expanded liquids

DOEpatents

Roberts, Christopher B; McLeod, Marshall Chandler; Anand, Madhu

2013-02-19

A method for size selection of nanostructures comprising utilizing a gas-expanded liquids (GEL) and controlled pressure to precipitate desired size populations of nanostructures, e.g., monodisperse. The GEL can comprise CO.sub.2 antisolvent and an organic solvent. The method can be carried out in an apparatus comprising a first open vessel configured to allow movement of a liquid/particle solution to specific desired locations within the vessel, a second pressure vessel, a location controller for controlling location of the particles and solution within the first vessel, a inlet for addition of antisolvent to the first vessel, and a device for measuring the amount of antisolvent added. Also disclosed is a method for forming nanoparticle thin films comprising utilizing a GEL containing a substrate, pressurizing the solution to precipitate and deposit nanoparticles onto the substrate, removing the solvent thereby leaving a thin nanoparticle film, removing the solvent and antisolvent, and drying the film.

The role of Monosaccharide Transport Proteins in carbohydrate assimilation, distribution, metabolism and homeostasis

PubMed Central

Cura, Anthony J.; Carruthers, Anthony

2012-01-01

The facilitated diffusion of glucose, galactose, fructose, urate, myoinositol and dehydroascorbic acid in mammals is catalyzed by a family of 14 monosaccharide transport proteins called GLUTs. These transporters may be divided into 3 classes according to sequence similarity and function/substrate specificity. GLUT1 appears to be highly expressed in glycolytically active cells and has been co-opted in vitamin C auxotrophs to maintain the redox state of the blood through transport of dehydroascorbate. Several GLUTs are definitive glucose/galactose transporters, GLUT2 and GLUT5 are physiologically important fructose transporters, GLUT9 appears to be a urate transporter while GLUT13 (HMIT1) is a proton/myoinositol co-transporter. The physiologic substrates of some GLUTs remain to be established. The GLUTs are expressed in a tissue specific manner where affinity, specificity and capacity for substrate transport are paramount for tissue function. Although great strides have been made in characterizing GLUT-catalyzed monosaccharide transport and mapping GLUT membrane topography and determinants of substrate specificity, a unifying model for GLUT structure and function remains elusive. The GLUTs play a major role in carbohydrate homeostasis and the redistribution of sugar-derived carbons among the various organ systems. This is accomplished through a multiplicity of GLUT-dependent glucose sensing and effector mechanisms that regulate monosaccharide ingestion, absorption, distribution, cellular transport and metabolism and recovery/retention. Glucose transport and metabolism have co-evolved in mammals to support cerebral glucose utilization. PMID:22943001

Impact of Lactic Acid and Hydrogen Ion on the Simultaneous Fermentation of Glucose and Xylose by the Carbon Catabolite Derepressed Lactobacillus brevis ATCC 14869.

PubMed

Jeong, Kyung Hun; Israr, Beenish; Shoemaker, Sharon P; Mills, David A; Kim, Jaehan

2016-07-28

Lactobacillus brevis ATCC 14869 exhibited a carbon catabolite de-repressed (CCR) phenotype which has ability to consume fermentable sugar simultaneously with glucose. To evaluate this unusual phenotype under harsh conditions during fermentation, the effect of lactic acid and hydrogen ion concentrations on L. brevis ATCC 14869 were examined. Kinetic equations describing the relationship between specific cell growth rate and lactic acid or hydrogen ion concentration has been reduced. The change of substrate utilization and product formation according to lactic acid and hydrogen ion concentration in the media were quantitatively described. Moreover; utilization of other compounds were also observed along with hydrogen ion and lactic acid concentration simultaneously. It has been found that substrate preference changes significantly regarding to utilization of compounds in media. That could result into formation of two-carbon products. In particular, acetic acid present in the media as sodium acetate were consumed by L. brevis ATCC 14869 under extreme pH of both acid and alkaline conditions.

Evolution of substrate specificity for the bile salt transporter ASBT (SLC10A2)[S

PubMed Central

Lionarons, Daniël A.; Boyer, James L.; Cai, Shi-Ying

2012-01-01

The apical Na+-dependent bile salt transporter (ASBT/SLC10A2) is essential for maintaining the enterohepatic circulation of bile salts. It is not known when Slc10a2 evolved as a bile salt transporter or how it adapted to substantial changes in bile salt structure during evolution. We characterized ASBT orthologs from two primitive vertebrates, the lamprey that utilizes early 5α-bile alcohols and the skate that utilizes structurally different 5β-bile alcohols, and compared substrate specificity with ASBT from humans who utilize modern 5β-bile acids. Everted gut sacs of skate but not the more primitive lamprey transported 3H-taurocholic acid (TCA), a modern 5β-bile acid. However, molecular cloning identified ASBT orthologs from both species. Cell-based assays using recombinant ASBT/Asbt's indicate that lamprey Asbt has high affinity for 5α-bile alcohols, low affinity for 5β-bile alcohols, and lacks affinity for TCA, whereas skate Asbt showed high affinity for 5α- and 5β-bile alcohols but low affinity for TCA. In contrast, human ASBT demonstrated high affinity for all three bile salt types. These findings suggest that ASBT evolved from the earliest vertebrates by gaining affinity for modern bile salts while retaining affinity for older bile salts. Also, our results indicate that the bile salt enterohepatic circulation is conserved throughout vertebrate evolution. PMID:22669917

Acidotolerant Bacteria and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil

PubMed Central

Morawe, Mareen; Hoeke, Henrike; Wissenbach, Dirk K.; Lentendu, Guillaume; Wubet, Tesfaye; Kröber, Eileen; Kolb, Steffen

2017-01-01

Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi. PMID:28790984

Kinetics and yields of pesticide biodegradation at low substrate concentrations and under conditions restricting assimilable organic carbon.

PubMed

Helbling, Damian E; Hammes, Frederik; Egli, Thomas; Kohler, Hans-Peter E

2014-02-01

The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter(-1). We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

PubMed Central

Ge, Yong; Vaillancourt, Frédéric H.; Agar, Nathalie Y. R.; Eltis, Lindsay D.

2002-01-01

Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, kcat = 3.9 ± 0.2 s−1, and KmO2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where KmO2 represents the Km for O2 and KmA represents the Km for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent Km of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates. PMID:12107126

Application of acoustic surface wave filter-beam lead component technology to deep space multimission hardware design

NASA Technical Reports Server (NTRS)

Kermode, A. W.; Boreham, J. F.

1974-01-01

This paper discusses the utilization of acoustic surface wave filters, beam lead components, and thin film metallized ceramic substrate technology as applied to the design of deep space, long-life, multimission transponder. The specific design to be presented is for a second mixer local oscillator module, operating at frequencies as high as 249 MHz.

Global Microarray Analysis of Carbohydrate Use in Alkaliphilic Hemicellulolytic Bacterium Bacillus sp. N16-5

PubMed Central

Song, Yajian; Xue, Yanfen; Ma, Yanhe

2013-01-01

The alkaliphilic hemicellulolytic bacterium Bacillus sp. N16-5 has a broad substrate spectrum and exhibits the capacity to utilize complex carbohydrates such as galactomannan, xylan, and pectin. In the monosaccharide mixture, sequential utilization by Bacillus sp. N16-5 was observed. Glucose appeared to be its preferential monosaccharide, followed by fructose, mannose, arabinose, xylose, and galactose. Global transcription profiles of the strain were determined separately for growth on six monosaccharides (glucose, fructose, mannose, galactose, arabinose, and xylose) and four polysaccharides (galactomannan, xylan, pectin, and sodium carboxymethylcellulose) using one-color microarrays. Numerous genes potentially related to polysaccharide degradation, sugar transport, and monosaccharide metabolism were found to respond to a specific substrate. Putative gene clusters for different carbohydrates were identified according to transcriptional patterns and genome annotation. Identification and analysis of these gene clusters contributed to pathway reconstruction for carbohydrate utilization in Bacillus sp. N16-5. Several genes encoding putative sugar transporters were highly expressed during growth on specific sugars, suggesting their functional roles. Two phosphoenolpyruvate-dependent phosphotransferase systems were identified as candidate transporters for mannose and fructose, and a major facilitator superfamily transporter was identified as a candidate transporter for arabinose and xylose. Five carbohydrate uptake transporter 1 family ATP-binding cassette transporters were predicted to participate in the uptake of hemicellulose and pectin degradation products. Collectively, microarray data improved the pathway reconstruction involved in carbohydrate utilization of Bacillus sp. N16-5 and revealed that the organism precisely regulates gene transcription in response to fluctuations in energy resources. PMID:23326578

Classification of bladder cancer cell lines using Raman spectroscopy: a comparison of excitation wavelength, sample substrate and statistical algorithms

NASA Astrophysics Data System (ADS)

Kerr, Laura T.; Adams, Aine; O'Dea, Shirley; Domijan, Katarina; Cullen, Ivor; Hennelly, Bryan M.

2014-05-01

Raman microspectroscopy can be applied to the urinary bladder for highly accurate classification and diagnosis of bladder cancer. This technique can be applied in vitro to bladder epithelial cells obtained from urine cytology or in vivo as an optical biopsy" to provide results in real-time with higher sensitivity and specificity than current clinical methods. However, there exists a high degree of variability across experimental parameters which need to be standardised before this technique can be utilized in an everyday clinical environment. In this study, we investigate different laser wavelengths (473 nm and 532 nm), sample substrates (glass, fused silica and calcium fluoride) and multivariate statistical methods in order to gain insight into how these various experimental parameters impact on the sensitivity and specificity of Raman cytology.

Recognition of Salmonella typhimurium by immobilized phage P22 monolayers

NASA Astrophysics Data System (ADS)

Handa, Hitesh; Gurczynski, Stephen; Jackson, Matthew P.; Auner, Gregory; Walker, Jeremy; Mao, Guangzhao

2008-04-01

Phages are promising alternatives to antibodies as the biorecognition element in a variety of biosensing applications. In this study, a monolayer of bacteriophage P22 whose tailspike proteins specifically recognize Salmonella serotypes was covalently bound to glass substrates through a bifunctional cross linker 3-aminopropyltrimethoxysilane. The specific binding of Salmonella typhimurium to the phage monolayer was studied by enzyme-linked immunosorbent assay and atomic force microscopy. Escherichia coli and a Gram-positive bacterium Listeria monocytogenes were also studied as control bacteria. The P22 particles show strong binding affinity to S. typhimurium. In addition, the dried P22 monolayer maintained 50% binding capacity to S. typhimurium after a one-week storage time. This is a promising method to prepare phage monolayer coatings on surface plasmon resonance and acoustic biosensor substrates in order to utilize the nascent phage display technology.

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09

PubMed Central

Rosberg, Anna Karin; Windstam, Sofia T.; Karlsson, Maria E.; Bergstrand, Karl-Johan; Khalil, Sammar; Wohanka, Walter

2017-01-01

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350–990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5–09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5–09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity. PMID:29267321

Light spectrum modifies the utilization pattern of energy sources in Pseudomonas sp. DR 5-09.

PubMed

Gharaie, Samareh; Vaas, Lea A I; Rosberg, Anna Karin; Windstam, Sofia T; Karlsson, Maria E; Bergstrand, Karl-Johan; Khalil, Sammar; Wohanka, Walter; Alsanius, Beatrix W

2017-01-01

Despite the overruling impact of light in the phyllosphere, little is known regarding the influence of light spectra on non-phototrophic bacteria colonizing the leaf surface. We developed an in vitro method to study phenotypic profile responses of bacterial pure cultures to different bands of the visible light spectrum using monochromatic (blue: 460 nm; red: 660 nm) and polychromatic (white: 350-990 nm) LEDs, by modification and optimization of a protocol for the Phenotype MicroArray™ technique (Biolog Inc., CA, USA). The new protocol revealed high reproducibility of substrate utilization under all conditions tested. Challenging the non-phototrophic bacterium Pseudomonas sp. DR 5-09 with white, blue, and red light demonstrated that all light treatments affected the respiratory profile differently, with blue LED having the most decisive impact on substrate utilization by impairing respiration of 140 substrates. The respiratory activity was decreased on 23 and 42 substrates under red and white LEDs, respectively, while utilization of one, 16, and 20 substrates increased in the presence of red, blue, and white LEDs, respectively. Interestingly, on four substrates contrasting utilization patterns were found when the bacterium was exposed to different light spectra. Although non-phototrophic bacteria do not rely directly on light as an energy source, Pseudomonas sp. DR 5-09 changed its respiratory activity on various substrates differently when exposed to different lights. Thus, ability to sense and distinguish between different wavelengths even within the visible light spectrum must exist, and leads to differential regulation of substrate usage. With these results, we hypothesize that different light spectra might be a hitherto neglected key stimulus for changes in microbial lifestyle and habits of substrate usage by non-phototrophic phyllospheric microbiota, and thus might essentially stratify leaf microbiota composition and diversity.

Biodegradation during contaminant transport in porous media: 1. mathematical analysis of controlling factors

NASA Astrophysics Data System (ADS)

Brusseau, Mark L.; Xie, Lily H.; Li, Li

1999-04-01

Interest in coupled biodegradation and transport of organic contaminants has expanded greatly in the past several years. In a system in which biodegradation is coupled with solute transport, the magnitude and rate of biodegradation is influenced not only by properties of the microbial population and the substrate, but also by hydrodynamic properties (e.g., residence time, dispersivity). By nondimensionalizing the coupled-process equations for transport and nonlinear biodegradation, we show that transport behavior is controlled by three characteristic parameters: the effective maximum specific growth rate, the relative half-saturation constant, and the relative substrate-utilization coefficient. The impact on biodegradation and transport of these parameters, which constitute various combinations of factors reflecting the influences of biotic and hydraulic properties of the system, are examined numerically. A type-curve diagram based on the three characteristic parameters is constructed to illustrate the conditions under which steady and non-steady transport is observed, and the conditions for which the linear, first-order approximation is valid for representing biodegradation. The influence of constraints to microbial growth and substrate utilization on contaminant transport is also briefly discussed. Additionally, the impact of biodegradation, with and without biomass growth, on spatial solute distribution and moments is examined.

Growth of the extremophilic Deinococcus geothermalis DSM 11302 using co-substrate fed-batch culture.

PubMed

Bornot, Julie; Molina-Jouve, Carole; Uribelarrea, Jean-Louis; Gorret, Nathalie

2014-02-01

Deinococcus geothermalis metabolism has been scarcely studied to date, although new developments on its utilization for bioremediation have been carried out. So, large-scale production of this strain and a better understanding of its physiology are required. A fed-batch experiment was conducted to achieve a high cell density non-limiting culture of D. geothermalis DSM 11302. A co-substrate nutritional strategy using glucose and yeast extract was carried out in a 20-L bioreactor in order to maintain a non-limited growth at a maximal growth rate of 1 h(-1) at 45 °C. Substrate supplies were adjusted by monitoring online culture parameters and physiological data (dissolved oxygen, gas analyses, respiratory quotient, biomass concentration). The results showed that yeast extract could serve as both carbon and nitrogen sources, although glucose and ammonia were consumed too. Yeast extract carbon-specific uptake rate reached a value 4.5 times higher than glucose carbon-specific uptake rate. Cell concentration of 9.6 g L(-1) dry cell weight corresponding to 99 g of biomass was obtained using glucose and yeast extract as carbon and nitrogen sources.

Regulation of ATP production: dependence on calcium concentration and respiratory state.

PubMed

Fink, Brian D; Bai, Fan; Yu, Liping; Sivitz, William I

2017-08-01

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca 2+ ] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H 2 O 2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca 2+ ], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca 2+ ] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca 2+ ] was positively and continuously associated with H 2 O 2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.

Key Issues Concerning Biolog Use for Aerobic and Anaerobic Freshwater Bacterial Community-Level Physiological Profiling

NASA Astrophysics Data System (ADS)

Christian, Bradley W.; Lind, Owen T.

2006-06-01

Bacterial heterotrophy in aquatic ecosystems is important in the overall carbon cycle. Biolog MicroPlates provide information into the metabolic potential of bacteria involved in carbon cycling. Specifically, Biolog EcoPlatesTM were developed with ecologically relevant carbon substrates to allow investigators to measure carbon substrate utilization patterns and develop community-level physiological profiles from natural bacterial assemblages. However, understanding of the functionality of these plates in freshwater research is limited. We explored several issues of EcoPlate use for freshwater bacterial assemblages including inoculum density, incubation temperature, non-bacterial color development, and substrate selectivity. Each of these has various effects on plate interpretation. We offer suggestions and techniques to resolve these interpretation issues. Lastly we propose a technique to allow EcoPlate use in anaerobic freshwater bacterial studies.

Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay.

PubMed

Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C

2016-01-01

Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.

Substrate specific effects of calcium on metabolism of rat heart mitochondria.

PubMed

Panov, A V; Scaduto, R C

1996-04-01

Oxidative metabolism in the heart is tightly coupled to mechanical work. Because this coupling process is believed to involve Ca2+, the roles of mitochondrial Ca2+ in the regulation of oxidative phosphorylation was studied in isolated rat heart mitochondria. The electrical component of the mitochondrial membrane potential (delta psi) and the redox state of the pyridine nucleotides were determined during the oxidation of various substrates under different metabolic states. In the absence of added adenine nucleotides, the NADP+ redox couple was almost completely reduced, regardless of the specific substrate and the presence of Ca2+, whereas NAD+ couple redox state was highly dependent on the substrate type and the presence of Ca2+. Titration of respiration with ADP, in the presence of excess hexokinase and glucose, showed that both respiration and NAD(P)+ reduction were very sensitive to ADP. The maximal enzyme reaction rate of ADP-stimulated respiration Michaelis constants (Km) for ADP were dependent on the particular substrate employed. delta psi was much less sensitive to ADP. With either alpha-ketoglutarate or glutamate as substrate, Ca2+ significantly increased reduction of NAD(P)+.Ca2+ did not influence NAD(P)+ reduction with either acetylcarnitine or pyruvate as substrate. In the presence of ADP, delta psi was increased by Ca2+ at all metabolic states with glutamate plus malate, 0.5 mM alpha-ketoglutarate plus malate, or pyruvate plus malate as substrates. The data presented support the hypothesis that cardiac respiration is controlled by the availability of both Ca2+ and ADP to mitochondria. The data indicate that an increase in substrate supply to mitochondria can increase mitochondrial respiration at given level of ADP. This effect can be produced by Ca2+ with substrates such as glutamate, which utilize alpha-ketoglutarate dehydrogenase activity for oxidation. Increases in respiration by Ca2+ may mitigate an increase in ADP during periods of increased cardiac work.

Efficient synthesis of supercoiled M13 DNA molecule containing a site specifically placed psoralen adduct and its use as a substrate for DNA replication

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

Kodadek, T.; Gamper, H.

The authors report a simple method for the in vitro synthesis of large quantities of site specifically modified DNA. The protocol involves extension of an oligonucleotide primer annealed to M13 single-stranded DNA using part of the T4 DNA polymerase holoenzyme. The resulting nicked double-stranded circles are ligated and supercoiled in the same tube, producing good yields of form I DNA. When the oligonucleotide primer is chemically modified, the resultant product contains a site-specific lesion. In this study, they report the synthesis of an M13 mp19 form I DNA which contains a psoralen monoadduct or cross-link at the KpnI site. Theymore » demonstrate the utility of these modified substrates by assessing the ability of the bacteriophage T4 DNA replication complex to bypass the damage and show that the psoralen monoadduct poses a severe block to the holoenzyme when attached to the template strand.« less

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

PubMed

Mayali, Xavier; Weber, Peter K

2018-05-01

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

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

Son, Ora; Kim, Sunghan; Hur, Yoon-Sun

TOR (target of rapamycin) kinase signaling plays central role as a regulator of growth and proliferation in all eukaryotic cells and its key signaling components and effectors are also conserved in plants. Unlike the mammalian and yeast counterparts, however, we found through yeast two-hybrid analysis that multiple regions of the Arabidopsis Raptor (regulatory associated protein of TOR) are required for binding to its substrate. We also identified that a 44-amino acid region at the N-terminal end of Arabidopsis ribosomal S6 kinase 1 (AtS6K1) specifically interacted with AtRaptor1, indicating that this region may contain a functional equivalent of the TOS (TOR-Signaling)more » motif present in the mammalian TOR substrates. Transient over-expression of this 44-amino acid fragment in Arabidopsis protoplasts resulted in significant decrease in rDNA transcription, demonstrating a feasibility of developing a new plant-specific TOR signaling inhibitor based upon perturbation of the Raptor-substrate interaction. - Highlights: • Multiple regions on the Arabidopsis Raptor protein were found to be involved in substrate binding. • N-terminal end of the Arabidopsis ribosomal S6 kinase 1 (AtS6K1) was responsible for interacting with AtRaptor1. • The Raptor-interacting fragment of AtS6K1 could be utilized as an effective inhibitor of plant TOR signaling.« less

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

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence

PubMed Central

Tuncil, Yunus E.; Xiao, Yao; Porter, Nathan T.; Reuhs, Bradley L.

2017-01-01

ABSTRACT When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. PMID:29018117

Direct Substrate Identification with an Analog Sensitive (AS) Viral Cyclin-Dependent Kinase (v-Cdk).

PubMed

Umaña, Angie C; Iwahori, Satoko; Kalejta, Robert F

2018-01-19

Viral cyclin-dependent kinases (v-Cdks) functionally emulate their cellular Cdk counterparts. Such viral mimicry is an established phenomenon that we extend here through chemical genetics. Kinases contain gatekeeper residues that limit the size of molecules that can be accommodated within the enzyme active site. Mutating gatekeeper residues to smaller amino acids allows larger molecules access to the active site. Such mutants can utilize bio-orthoganol ATPs for phosphate transfer and are inhibited by compounds ineffective against the wild type protein, and thus are referred to as analog-sensitive (AS) kinases. We identified the gatekeeper residues of the v-Cdks encoded by Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) and mutated them to generate AS kinases. The AS-v-Cdks are functional and utilize different ATP derivatives with a specificity closely matching their cellular ortholog, AS-Cdk2. The AS derivative of the EBV v-Cdk was used to transfer a thiolated phosphate group to targeted proteins which were then purified through covalent capture and identified by mass spectrometry. Pathway analysis of these newly identified direct substrates of the EBV v-Cdk extends the potential influence of this kinase into all stages of gene expression (transcription, splicing, mRNA export, and translation). Our work demonstrates the biochemical similarity of the cellular and viral Cdks, as well as the utility of AS v-Cdks for substrate identification to increase our understanding of both viral infections and Cdk biology.

Synthesis of FAEEs from glycerol in engineered Saccharomyces cerevisiae using endogenously produced ethanol by heterologous expression of an unspecific bacterial acyltransferase.

PubMed

Yu, Kyung Ok; Jung, Ju; Kim, Seung Wook; Park, Chul Hwan; Han, Sung Ok

2012-01-01

The high price of petroleum-based diesel fuel has led to the development of alternative fuels, such as ethanol. Saccharomyces cerevisiae was metabolically engineered to utilize glycerol as a substrate for ethanol production. For the synthesis of fatty acid ethyl esters (FAEEs) by engineered S. cerevisiae that utilize glycerol as substrate, heterologous expression of an unspecific acyltransferase from Acinetobacter baylyi with glycerol utilizing genes was established. As a result, the engineered YPH499 (pGcyaDak, pGupWs-DgaTCas) strain produced 0.24 g/L FAEEs using endogenous ethanol produced from glycerol. And this study also demonstrated the possibility of increasing FAEE production by enhancing ethanol production by minimizing the synthesis of glycerol. The overall FAEE production in strain YPH499 fps1Δ gpd2Δ (pGcyaDak, pGupWs-DgaTCas) was 2.1-fold more than in YPH499 (pGcyaDak, pGupWs-DgaTCas), with approximately 0.52 g/L FAEEs produced, while nearly 17 g/L of glycerol was consumed. These results clearly indicated that FAEEs were synthesized in engineered S. cerevisiae by esterifying exogenous fatty acids with endogenously produced ethanol from glycerol. This microbial system acts as a platform in applying metabolic engineering that allows the production of FAEEs from cheap and abundant substrates specifically glycerol through the use of endogenous bioethanol. Copyright © 2011 Wiley Periodicals, Inc.

Effect of high loading on substrate utilization kinetics and microbial community structure in super fast submerged membrane bioreactor.

PubMed

Sözen, S; Çokgör, E U; Başaran, S Teksoy; Aysel, M; Akarsubaşı, A; Ergal, I; Kurt, H; Pala-Ozkok, I; Orhon, D

2014-05-01

The study investigated the effect of high substrate loading on substrate utilization kinetics, and changes inflicted on the composition of the microbial community in a superfast submerged membrane bioreactor. Submerged MBR was sequentially fed with a substrate mixture and acetate; its performance was monitored at steady-state, at extremely low sludge age values of 2.0, 1.0 and 0.5d, all adjusted to a single hydraulic retention time of 8.0 h. Each MBR run was repeated when substrate feeding was increased from 200 mg COD/L to 1000 mg COD/L. Substrate utilization kinetics was altered to significantly lower levels when the MBR was adjusted to higher substrate loadings. Molecular analysis of the biomass revealed that variable process kinetics could be correlated with parallel changes in the composition of the microbial community, mainly by a replacement mechanism, where newer species, better adapted to the new growth conditions, substituted others that are washed out from the system. Copyright © 2014 Elsevier Ltd. All rights reserved.

New class of radioenzymatic assay for the quantification of p-tyramine and phenylethylamine

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

Henry, D.P.; Van Huysse, J.W.; Bowsher, R.R.

Radioenzymatic assays are widely used for the quantification of a number of biogenic amines. All previous procedures have utilized methyltransferases derived from mammalian tissues. In this assay for the quantification of the trace aralkylamines, p-tyramine (p-tym) and phenylethylamine (PEA), an enzyme, tyramine N-methyltransferase isolated from sprouted barley roots was used. The enzyme was specific for phenylethylamines. Of 26 structurally-related compounds, only p-tym, PEA, m-tym and amphetamine were substrates in vitro. Theoretic maximal methylation of substrates occurred at 10-20/sup 0/C. When TLC was used to separate the radiolabeled reaction products, a specific method was developed for p-tym and PEA. The assaymore » had a sensitivity of 0.8 and 2.8 pg/tube with a C.V. < 5% and was applicable to human plasma and urine. Assay throughput is similar to that of other TLC based radioenzymatic assays.« less

Crystal structure and mutational analysis of aminoacylhistidine dipeptidase from Vibrio alginolyticus reveal a new architecture of M20 metallopeptidases.

PubMed

Chang, Chin-Yuan; Hsieh, Yin-Cheng; Wang, Ting-Yi; Chen, Yi-Chin; Wang, Yu-Kuo; Chiang, Ting-Wei; Chen, Yi-Ju; Chang, Cheng-Hsiang; Chen, Chun-Jung; Wu, Tung-Kung

2010-12-10

Aminoacylhistidine dipeptidases (PepD, EC 3.4.13.3) belong to the family of M20 metallopeptidases from the metallopeptidase H clan that catalyze a broad range of dipeptide and tripeptide substrates, including L-carnosine and L-homocarnosine. Homocarnosine has been suggested as a precursor for the neurotransmitter γ-aminobutyric acid (GABA) and may mediate the antiseizure effects of GABAergic therapies. Here, we report the crystal structure of PepD from Vibrio alginolyticus and the results of mutational analysis of substrate-binding residues in the C-terminal as well as substrate specificity of the PepD catalytic domain-alone truncated protein PepD(CAT). The structure of PepD was found to exist as a homodimer, in which each monomer comprises a catalytic domain containing two zinc ions at the active site center for its hydrolytic function and a lid domain utilizing hydrogen bonds between helices to form the dimer interface. Although the PepD is structurally similar to PepV, which exists as a monomer, putative substrate-binding residues reside in different topological regions of the polypeptide chain. In addition, the lid domain of the PepD contains an "extra" domain not observed in related M20 family metallopeptidases with a dimeric structure. Mutational assays confirmed both the putative di-zinc allocations and the architecture of substrate recognition. In addition, the catalytic domain-alone truncated PepD(CAT) exhibited substrate specificity to l-homocarnosine compared with that of the wild-type PepD, indicating a potential value in applications of PepD(CAT) for GABAergic therapies or neuroprotection.

Carbon Nanotube Patterning on a Metal Substrate

NASA Technical Reports Server (NTRS)

Nguyen, Cattien V. (Inventor)

2016-01-01

A CNT electron source, a method of manufacturing a CNT electron source, and a solar cell utilizing a CNT patterned sculptured substrate are disclosed. Embodiments utilize a metal substrate which enables CNTs to be grown directly from the substrate. An inhibitor may be applied to the metal substrate to inhibit growth of CNTs from the metal substrate. The inhibitor may be precisely applied to the metal substrate in any pattern, thereby enabling the positioning of the CNT groupings to be more precisely controlled. The surface roughness of the metal substrate may be varied to control the density of the CNTs within each CNT grouping. Further, an absorber layer and an acceptor layer may be applied to the CNT electron source to form a solar cell, where a voltage potential may be generated between the acceptor layer and the metal substrate in response to sunlight exposure.

Alterations in Skeletal Muscle With Disuse Atrophy: The Effects of Countermeasures

NASA Technical Reports Server (NTRS)

Fitts, Robert H.

1996-01-01

The specific aims of this project concerned three general areas: (1) studies on the contractile function of single skinned fibers designed to determine the time course and cellular basis of the Hindlimb Suspension (HS) induced increase in fiber Vo (maximal shortening velocity), and the decrease in peak tension (Po); (2) studies designed to understand the effect of HS on single fiber substrate utilization during contractile activity, and how if at all such changes contribute to the increased muscle fatigue associated with HS; and (3) studies evaluating the effectiveness of standing and ladder climbing as countermeasures to the deleterious effects of HS. We have constructed all of the necessary equipment, and are currently conducting preliminary studies on T-tubular charge movement. A list of publications from this contract is included at the end of this report. The three objectives are (1) Functional Studies on the Single Skinned Fiber; (2) Fiber Substrate Utilization and Muscle Fatugue with Contracting Activity and (3) Exercise Countermeasures.

Nicotinamide riboside kinase structures reveal new pathways to NAD+.

PubMed

Tempel, Wolfram; Rabeh, Wael M; Bogan, Katrina L; Belenky, Peter; Wojcik, Marzena; Seidle, Heather F; Nedyalkova, Lyudmila; Yang, Tianle; Sauve, Anthony A; Park, Hee-Won; Brenner, Charles

2007-10-02

The eukaryotic nicotinamide riboside kinase (Nrk) pathway, which is induced in response to nerve damage and promotes replicative life span in yeast, converts nicotinamide riboside to nicotinamide adenine dinucleotide (NAD+) by phosphorylation and adenylylation. Crystal structures of human Nrk1 bound to nucleoside and nucleotide substrates and products revealed an enzyme structurally similar to Rossmann fold metabolite kinases and allowed the identification of active site residues, which were shown to be essential for human Nrk1 and Nrk2 activity in vivo. Although the structures account for the 500-fold discrimination between nicotinamide riboside and pyrimidine nucleosides, no enzyme feature was identified to recognize the distinctive carboxamide group of nicotinamide riboside. Indeed, nicotinic acid riboside is a specific substrate of human Nrk enzymes and is utilized in yeast in a novel biosynthetic pathway that depends on Nrk and NAD+ synthetase. Additionally, nicotinic acid riboside is utilized in vivo by Urh1, Pnp1, and Preiss-Handler salvage. Thus, crystal structures of Nrk1 led to the identification of new pathways to NAD+.

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier ProteinTethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus

DOE PAGES

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan; ...

2016-08-25

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

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.

Effect of Warm-Up on Plasma Free Fatty Acid Response and Substrate Utilization During Submaximal Exercise.

ERIC Educational Resources Information Center

Hetzler, Ronald K.; And Others

1986-01-01

This study examined the effect of preliminary walking on free fatty acid responses and substrate utilization during a 40-minute treadmill run by experienced male distance runners. Conclusions are presented. (Author/MT)

Determination of human DNA polymerase utilization for the repair of a model ionizing radiation-induced DNA strand break lesion in a defined vector substrate

NASA Technical Reports Server (NTRS)

Winters, T. A.; Russell, P. S.; Kohli, M.; Dar, M. E.; Neumann, R. D.; Jorgensen, T. J.

1999-01-01

Human DNA polymerase and DNA ligase utilization for the repair of a major class of ionizing radiation-induced DNA lesion [DNA single-strand breaks containing 3'-phosphoglycolate (3'-PG)] was examined using a novel, chemically defined vector substrate containing a single, site-specific 3'-PG single-strand break lesion. In addition, the major human AP endonuclease, HAP1 (also known as APE1, APEX, Ref-1), was tested to determine if it was involved in initiating repair of 3'-PG-containing single-strand break lesions. DNA polymerase beta was found to be the primary polymerase responsible for nucleotide incorporation at the lesion site following excision of the 3'-PG blocking group. However, DNA polymerase delta/straightepsilon was also capable of nucleotide incorporation at the lesion site following 3'-PG excision. In addition, repair reactions catalyzed by DNA polymerase beta were found to be most effective in the presence of DNA ligase III, while those catalyzed by DNA polymerase delta/straightepsilon appeared to be more effective in the presence of DNA ligase I. Also, it was demonstrated that the repair initiating 3'-PG excision reaction was not dependent upon HAP1 activity, as judged by inhibition of HAP1 with neutralizing HAP1-specific polyclonal antibody.

AAV Vectorization of DSB-mediated Gene Editing Technologies.

PubMed

Moser, Rachel J; Hirsch, Matthew L

2016-01-01

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

Production of DagA and ethanol by sequential utilization of sugars in a mixed-sugar medium simulating microalgal hydrolysate.

PubMed

Park, Juyi; Hong, Soon-Kwang; Chang, Yong Keun

2015-09-01

A novel two-step fermentation process using a mixed-sugar medium mimicking microalgal hydrolysate has been proposed to avoid glucose repression and thus to maximize substrate utilization efficiency. When DagA, a β-agarase was produced in one step in the mixed-sugar medium by using a recombinant Streptomyces lividans, glucose was found to have negative effects on the consumption of the other sugars and DagA biosynthesis causing low substrate utilization efficiency and low DagA productivity. To overcome such difficulties, a new strategy of sequential substrate utilization was developed. In the first step, glucose was consumed by Saccharomyces cerevisiae together with galactose and mannose producing ethanol, after which DagA was produced from the remaining sugars of xylose, rhamnose and ribose. Fucose was not consumed. By adopting this two-step process, the overall substrate utilization efficiency was increased approximately 3-fold with a nearly 2-fold improvement of DagA production, let alone the additional benefit of ethanol production. Copyright © 2015 Elsevier Ltd. All rights reserved.

Chemiluminescence assay for the detection of biological warfare agents

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

Langry, K; Horn, J

A chemiluminescent homogeneous immunoassay and a hand-size multiassay reader are described that could be used for detecting biological materials. The special feature of the assay is that it employs two different antibodies that each bind to a unique epitope on the same antigen. Each group of epitope-specific antibodies has linked to it an enzyme of a proximal-enzyme pair. One enzyme of the pair utilizes a substrate in high concentration to produce a second substrate required by the second enzyme. This new substrate enables the second enzyme to function. The reaction of the second enzyme is configured to produce light. Thismore » chemiluminescence is detected with a charge-coupled device (CCD) camera. The proximal pair enzymes must be in close proximity to one another to allow the second enzyme to react with the product of the first enzyme. This only occurs when the enzyme-linked antibodies are attached to the antigen, whether antigen is a single protein with multiple epitopes or the surface of a cell with a variety of different antigens. As a result of their juxtaposition, the enzymes produce light only in the presence of the biological material. A brief description is given as to how this assay could be utilized in a personal bio-agent detector system.« less

Alteration of Substrate and Inhibitor Specificity of Feline Immunodeficiency Virus Protease

PubMed Central

Lin, Ying-Chuan; Beck, Zachary; Lee, Taekyu; Le, Van-Duc; Morris, Garrett M.; Olson, Arthur J.; Wong, Chi-Huey; Elder, John H.

2000-01-01

Feline immunodeficiency virus (FIV) protease is structurally very similar to human immunodeficiency virus (HIV) protease but exhibits distinct substrate and inhibitor specificities. We performed mutagenesis of subsite residues of FIV protease in order to define interactions that dictate this specificity. The I37V, N55M, M56I, V59I, and Q99V mutants yielded full activity. The I37V, N55M, V59I, and Q99V mutants showed a significant increase in activity against the HIV-1 reverse transcriptase/integrase and P2/nucleocapsid junction peptides compared with wild-type (wt) FIV protease. The I37V, V59I, and Q99V mutants also showed an increase in activity against two rapidly cleaved peptides selected by cleavage of a phage display library with HIV-1 protease. Mutations at Q54K, I98P, and L101I dramatically reduced activity. Mutants containing a I35D or I57G substitution showed no activity against either FIV or HIV substrates. FIV proteases all failed to cut HIV-1 matrix/capsid, P1/P6, P6/protease, and protease/reverse transcriptase junctions, indicating that none of the substitutions were sufficient to change the specificity completely. The I37V, N55M, M56I, V59I, and Q99V mutants, compared with wt FIV protease, all showed inhibitor specificity more similar to that of HIV-1 protease. The data also suggest that FIV protease prefers a hydrophobic P2/P2′ residue like Val over Asn or Glu, which are utilized by HIV-1 protease, and that S2/S2′ might play a critical role in distinguishing FIV and HIV-1 protease by specificity. The findings extend our observations regarding the interactions involved in substrate binding and aid in the development of broad-based inhibitors. PMID:10775609

Construction of an alternative glycerol-utilization pathway for improved β-carotene production in Escherichia coli.

PubMed

Guo, Jin-Ying; Hu, Kun-Le; Bi, Chang-Hao; Li, Qing-Yan; Zhang, Xue-Li

2018-05-11

Glycerol, which is an inevitable by-product of biodiesel production, is an ideal carbon source for the production of carotenoids due to its low price, good availability and chemically reduced status, which results in a low requirement for additional reducing equivalents. In this study, an alternative carbon-utilization pathway was constructed in Escherichia coli to enable more efficient β-carotene production from glycerol. An aldehyde reductase gene (alrd) and an aldehyde dehydrogenase gene (aldH) from Ralstonia eutropha H16 were integrated into the E. coli chromosome to form a novel glycerol-utilization pathway. The β-carotene specific production value was increased by 50% after the introduction of alrd and aldH. It was found that the glycerol kinase gene (garK), alrd and aldH were the bottleneck of the alternative glycerol metabolic pathway, and modulation of garK gene with an mRS library further increased the β-carotene specific production value by 13%. Finally, co-modulation of genes in the introduced aldH-alrd operon led to 86% more of β-carotene specific production value than that of the strain without the alternative glycerol-utilization pathway and the glycerol-utilization rate was also increased. In this work, β-carotene production of E. coli was significantly improved by constructing and optimizing an alternative glycerol-utilization pathway. This strategy can potentially be used to improve the production of other isoprenoids using glycerol as a cheap and abundant substrate, and therefore has industrial relevance.

Graphene sheets stabilized on genetically engineered M13 viral templates as conducting frameworks for hybrid energy-storage materials.

PubMed

Oh, Dahyun; Dang, Xiangnan; Yi, Hyunjung; Allen, Mark A; Xu, Kang; Lee, Yun Jung; Belcher, Angela M

2012-04-10

Utilization of the material-specific peptide-substrate interactions of M13 virus broadens colloidal stability window of graphene. The homogeneous distribution of graphene is maintained in weak acids and increased ionic strengths by complexing with virus. This graphene/virus conducting template is utilized in the synthesis of energy-storage materials to increase the conductivity of the composite electrode. Successful formation of the hybrid biological template is demonstrated by the mineralization of bismuth oxyfluoride as a cathode material for lithium-ion batteries, with increased loading and improved electronic conductivity. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Inorganic Photovoltaics Materials and Devices: Past, Present, and Future

NASA Technical Reports Server (NTRS)

Hepp, Aloysius F.; Bailey, Sheila G.; Rafaelle, Ryne P.

2005-01-01

This report describes recent aspects of advanced inorganic materials for photovoltaics or solar cell applications. Specific materials examined will be high-efficiency silicon, gallium arsenide and related materials, and thin-film materials, particularly amorphous silicon and (polycrystalline) copper indium selenide. Some of the advanced concepts discussed include multi-junction III-V (and thin-film) devices, utilization of nanotechnology, specifically quantum dots, low-temperature chemical processing, polymer substrates for lightweight and low-cost solar arrays, concentrator cells, and integrated power devices. While many of these technologies will eventually be used for utility and consumer applications, their genesis can be traced back to challenging problems related to power generation for aerospace and defense. Because this overview of inorganic materials is included in a monogram focused on organic photovoltaics, fundamental issues and metrics common to all solar cell devices (and arrays) will be addressed.

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

PubMed

Russell, Thomas R; Demeler, Borries; Tu, Shiao-Chun

2004-02-17

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

Comparison Actin- and Glass-Supported Phospholipid Bilayer Diffusion Coefficients

PubMed Central

Sterling, Sarah M.; Dawes, Ryan; Allgeyer, Edward S.; Ashworth, Sharon L.; Neivandt, David J.

2015-01-01

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20–44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. PMID:25902434

A co-culture device with a tunable stiffness to understand combinatorial cell-cell and cell-matrix interactions.

PubMed

Rao, Nikhil; Grover, Gregory N; Vincent, Ludovic G; Evans, Samantha C; Choi, Yu Suk; Spencer, Katrina H; Hui, Elliot E; Engler, Adam J; Christman, Karen L

2013-11-01

Cell behavior on 2-D in vitro cultures is continually being improved to better mimic in vivo physiological conditions by combining niche cues including multiple cell types and substrate stiffness, which are well known to impact cell phenotype. However, no system exists in which a user can systematically examine cell behavior on a substrate with a specific stiffness (elastic modulus) in culture with a different cell type, while maintaining distinct cell populations. We demonstrate the modification of a silicon reconfigurable co-culture system with a covalently linked hydrogel of user-defined stiffness. This device allows the user to control whether two separate cell populations are in contact with each other or only experience paracrine interactions on substrates of controllable stiffness. To illustrate the utility of this device, we examined the role of substrate stiffness combined with myoblast co-culture on adipose derived stem cell (ASC) differentiation and found that the presence of myoblasts and a 10 kPa substrate stiffness increased ASC myogenesis versus co-culture on stiff substrates. As this example highlights, this technology better controls the in vitro microenvironment, allowing the user to develop a more thorough understanding of the combined effects of cell-cell and cell-matrix interactions.

Carbon catabolite repression and cell dispersal affect degradation of the xenobiotic compound 3,4-dichloroaniline in Comamonas testosteroni WDL7 biofilms.

PubMed

Horemans, Benjamin; Breugelmans, Philip; Hofkens, Johan; Springael, Dirk

2017-03-01

Organic pollutant degrading biofilms in natural ecosystems and water treatment systems are often exposed to other carbon sources in addition to the pollutant. The availability of auxiliary carbon sources can lead to surplus biomass growth, changes in biofilm structure and carbon catabolite repression (CCR) which together will affect pollutant degradation rate and efficiency of the system. To understand the interplay between these processes, continuous biofilms of the 3,4-dichloroaniline (3,4-DCA) degrading Comamonas testosteroni WDL7-RFP were grown in single- and dual-substrate conditions with 3,4-DCA and/or citrate and reciprocal effects on 3,4-DCA/citrate degradation, biofilm biomass and biofilm structure were examined. The main mechanism affecting 3,4-DCA degradation in biofilms in dual-substrate conditions was citrate-mediated CCR as reflected by a decrease in specific 3,4-DCA degrading activity. Growth on citrate partially compensated for the lowered specific 3,4-DCA degradation activity under dual substrate conditions but not to the extent expected from growth observed under single-substrate conditions with citrate. This was explained by higher residual 3,4-DCA concentrations in the presence of citrate that increased cell dispersal in the biofilms. Our results show hampered pollutant removal in biofilms due to a complex interplay of auxiliary organic C source utilization for growth affecting the specific pollutant degradation rate and changes in cell physiology due to increased exposure to the pollutant as a result of lowered pollutant degradation rates. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Processing and fractional utilization of lignocellulosic substrates by "pure" and "natural and defined mixed" culture C.TM1, C.SA IV and rumen mixed culture consortia CD.

PubMed

Sankar, M; Chandra, T S

2003-01-01

A detailed analysis was made of chemical fractions of common agro-residues before and after pretreatment (alkali and hydrogen peroxide), and the selective utilization of components such as WSS, EBS, TSS, lignin, cellulose and hemicellulose by pure and mixed cultures of cellulolytic and xylanolytic Clostridia was monitored and correlated with the organisms' enzyme activity. For all cultures pretreatment gave higher utilization of hemicellulose and cellulose fractions; hydrogen peroxide pretreatment was more effective than NaOH treatment. Lignin utilization was not very significant even on pretreatment. C.TM1 and C.SA IV utilized hemicellulose and cellulose better than mixed cultures in selected substrates. These results help to determine the substrate composition, pretreatment conditions and enzyme system of the organism needed when designing an inoculum for agricultural waste treatment processes such as composting or biogas generation.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase.

PubMed

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa; Sakuraba, Haruhiko

2017-06-01

A stable NADP + -dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P) + -dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. Copyright © 2017 American Society for Microbiology.

Structure-Based Engineering of an Artificially Generated NADP+-Dependent d-Amino Acid Dehydrogenase

PubMed Central

Hayashi, Junji; Seto, Tomonari; Akita, Hironaga; Watanabe, Masahiro; Hoshino, Tamotsu; Yoneda, Kazunari; Ohshima, Toshihisa

2017-01-01

ABSTRACT A stable NADP+-dependent d-amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso-diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d-phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d-leucine, d-norleucine, d-methionine, d-isoleucine, and d-tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP+ and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d-amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d-amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d-amino acids, but all include tedious steps. The use of NAD(P)+-dependent d-amino acid dehydrogenase (DAADH) makes single-step production of d-amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d-amino acids, such as d-leucine and d-isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d-amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs. PMID:28363957

Gain-of-function mutations identify amino acids within transmembrane domains of the yeast vacuolar transporter Zrc1 that determine metal specificity

PubMed Central

Lin, Huilan; Burton, Damali; Li, Liangtao; Warner, David E.; Phillips, John D.; Ward, Diane McVEY; Kaplan, Jerry

2015-01-01

Cation diffusion facilitator transporters are found in all three Kingdoms of life and are involved in transporting transition metals out of the cytosol. The metals they transport include Zn2+, Co2+, Fe2+, Cd2+, Ni2+ and Mn2+; however, no single transporter transports all metals. Previously we showed that a single amino acid mutation in the yeast vacuolar zinc transporter Zrc1 changed its substrate specificity from Zn2+ to Fe2+ and Mn2+ [Lin, Kumanovics, Nelson, Warner, Ward and Kaplan (2008) J. Biol. Chem. 283, 33865–33873]. Mutant Zrc1 that gained iron transport activity could protect cells with a deletion in the vacuolar iron transporter (CCC1) from high iron toxicity. Utilizing suppression of high iron toxicity and PCR mutagenesis of ZRC1, we identified other amino acid substitutions within ZRC1 that changed its metal specificity. All Zrc1 mutants that transported Fe2+ could also transport Mn2+. Some Zrc1 mutants lost the ability to transport Zn2+, but others retained the ability to transport Zn2+. All of the amino acid substitutions that resulted in a gain in Fe2+ transport activity were found in transmembrane domains. In addition to alteration of residues adjacent to the putative metal-binding site in two transmembrane domains, alteration of residues distant from the binding site affected substrate specificity. These results suggest that substrate selection involves co-operativity between transmembrane domains. PMID:19538181

Molecular Basis of Prodrug Activation by Human Valacyclovirase, an [alpha]-Amino Acid Ester Hydrolase

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

Lai, Longsheng; Xu, Zhaohui; Zhou, Jiahai

2008-07-08

Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for amino acid esters. Biochemical data demonstrate that the enzyme hydrolyzes esters of {alpha}-amino acids exclusively and displays a broad specificity spectrum for the aminoacyl moiety similar tomore » tricorn-interacting aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific {alpha}-amino acid ester hydrolase. Valacyclovirase is a valuable target for amino acid ester prodrug-based oral drug delivery enhancement strategies.« less

A Two-Layer Gene Circuit for Decoupling Cell Growth from Metabolite Production.

PubMed

Lo, Tat-Ming; Chng, Si Hui; Teo, Wei Suong; Cho, Han-Saem; Chang, Matthew Wook

2016-08-01

We present a synthetic gene circuit for decoupling cell growth from metabolite production through autonomous regulation of enzymatic pathways by integrated modules that sense nutrient and substrate. The two-layer circuit allows Escherichia coli to selectively utilize target substrates in a mixed pool; channel metabolic resources to growth by delaying enzymatic conversion until nutrient depletion; and activate, terminate, and re-activate conversion upon substrate availability. We developed two versions of controller, both of which have glucose nutrient sensors but differ in their substrate-sensing modules. One controller is specific for hydroxycinnamic acid and the other for oleic acid. Our hydroxycinnamic acid controller lowered metabolic stress 2-fold and increased the growth rate 2-fold and productivity 5-fold, whereas our oleic acid controller lowered metabolic stress 2-fold and increased the growth rate 1.3-fold and productivity 2.4-fold. These results demonstrate the potential for engineering strategies that decouple growth and production to make bio-based production more economical and sustainable. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Formation of extracellular polymeric substances from acidogenic sludge in H2-producing process.

PubMed

Sheng, Guo-Ping; Yu, Han-Qing

2007-02-01

In this study, the formation of extracellular polymeric substances (EPS) and surface characteristics of an acidogenic sludge in anaerobic H(2)-producing process was investigated. Results show that carbohydrates, proteins, and humic substances were the dominant components in bound EPS (BEPS), while in soluble EPS (SEPS), carbohydrates were the main component. The total content of BEPS initially increased but then kept almost unchanged during fermentation from 25 to 35 h; after that, it slightly decreased. The total content of SEPS increased to 172.5 +/- 0.05 mg C g(-1) volatile suspended solid with the time that increased to 23.5 h, and then rapidly decreased until 43 h; thereafter, it kept almost unchanged. The SEPS had good correlations with the specific H(2) production rate, substrate degradation rate, and specific aqueous products formation rate, but the BEPS seemed to have no such correlations with these specific rates. Results also confirm that part of EPS could be utilized by the H(2)-producing sludge. As the substrate was in short supply, the EPS would be hydrolyzed to sever as carbon and energy source.

Receptor-Selective Agonists Induce Emesis and Fos Expression in the Brain and Enteric Nervous System of the Least Shrew (Cryptotis parva)

PubMed Central

Ray, Andrew P.; Chebolu, Seetha; Darmani, Nissar A.

2009-01-01

Research on the mechanisms of emesis has implicated multiple neurotransmitters via both central (dorsal vagal complex) and peripheral (enteric neurons and enterochromaffin cells) anatomical substrates. Taking advantage of advances in receptor-specific agonists, and utilizing Fos expression as a functional activity marker, this study demonstrates a strong, but incomplete, overlap in anatomical substrates for a variety of emetogens. We used cisplatin and specific agonists to 5-HT3 serotonergic, D2/D3 dopaminergic, and NK1 tachykininergic receptors to induce vomiting in the least shrew (Cryptotis parva), and quantified the resulting Fos expression. The least shrew is a small mammal whose responses to emetic challenges are very similar to its human counterparts. In all cases, the enteric nervous system, nucleus of the solitary tract, and dorsal motor nucleus of the vagus demonstrated significantly increased Fos immunoreactivity (Fos-IR). However, Fos-IR induction was notably absent from the area postrema following the dopaminergic and NK1 receptor-specific agents. Two brain nuclei not usually discussed regarding emesis, the dorsal raphe nucleus and paraventricular thalamic nucleus, also demonstrated increased emesis-related Fos-IR. Taken together, these data suggest the dorsal vagal complex is part of a common pathway for a variety of distinct emetogens, but there are central emetic substrates, both medullary and diencephalic, that can be accessed without directly stimulating the area postrema. PMID:19699757

Prolonged Maltose-Limited Cultivation of Saccharomyces cerevisiae Selects for Cells with Improved Maltose Affinity and Hypersensitivity

PubMed Central

Jansen, Mickel L. A.; Daran-Lapujade, Pascale; de Winde, Johannes H.; Piper, Matthew D. W.; Pronk, Jack T.

2004-01-01

Prolonged cultivation (>25 generations) of Saccharomyces cerevisiae in aerobic, maltose-limited chemostat cultures led to profound physiological changes. Maltose hypersensitivity was observed when cells from prolonged cultivations were suddenly exposed to excess maltose. This substrate hypersensitivity was evident from massive cell lysis and loss of viability. During prolonged cultivation at a fixed specific growth rate, the affinity for the growth-limiting nutrient (i.e., maltose) increased, as evident from a decreasing residual maltose concentration. Furthermore, the capacity of maltose-dependent proton uptake increased up to 2.5-fold during prolonged cultivation. Genome-wide transcriptome analysis showed that the increased maltose transport capacity was not primarily due to increased transcript levels of maltose-permease genes upon prolonged cultivation. We propose that selection for improved substrate affinity (ratio of maximum substrate consumption rate and substrate saturation constant) in maltose-limited cultures leads to selection for cells with an increased capacity for maltose uptake. At the same time, the accumulative nature of maltose-proton symport in S. cerevisiae leads to unrestricted uptake when maltose-adapted cells are exposed to a substrate excess. These changes were retained after isolation of individual cell lines from the chemostat cultures and nonselective cultivation, indicating that mutations were involved. The observed trade-off between substrate affinity and substrate tolerance may be relevant for metabolic engineering and strain selection for utilization of substrates that are taken up by proton symport. PMID:15066785

A Land-Plant-Specific Glycerol-3-Phosphate Acyltransferase Family in Arabidopsis: Substrate Specificity, sn-2 Preference, and Evolution1[W][OA

PubMed Central

Yang, Weili; Simpson, Jeffrey P.; Li-Beisson, Yonghua; Beisson, Fred; Pollard, Mike; Ohlrogge, John B.

2012-01-01

Arabidopsis (Arabidopsis thaliana) has eight glycerol-3-phosphate acyltransferase (GPAT) genes that are members of a plant-specific family with three distinct clades. Several of these GPATs are required for the synthesis of cutin or suberin. Unlike GPATs with sn-1 regiospecificity involved in membrane or storage lipid synthesis, GPAT4 and -6 are unique bifunctional enzymes with both sn-2 acyltransferase and phosphatase activity resulting in 2-monoacylglycerol products. We present enzymology, pathway organization, and evolutionary analysis of this GPAT family. Within the cutin-associated clade, GPAT8 is demonstrated as a bifunctional sn-2 acyltransferase/phosphatase. GPAT4, -6, and -8 strongly prefer C16:0 and C18:1 ω-oxidized acyl-coenzyme As (CoAs) over unmodified or longer acyl chain substrates. In contrast, suberin-associated GPAT5 can accommodate a broad chain length range of ω-oxidized and unsubstituted acyl-CoAs. These substrate specificities (1) strongly support polyester biosynthetic pathways in which acyl transfer to glycerol occurs after oxidation of the acyl group, (2) implicate GPAT specificities as one major determinant of cutin and suberin composition, and (3) argue against a role of sn-2-GPATs (Enzyme Commission 2.3.1.198) in membrane/storage lipid synthesis. Evidence is presented that GPAT7 is induced by wounding, produces suberin-like monomers when overexpressed, and likely functions in suberin biosynthesis. Within the third clade, we demonstrate that GPAT1 possesses sn-2 acyltransferase but not phosphatase activity and can utilize dicarboxylic acyl-CoA substrates. Thus, sn-2 acyltransferase activity extends to all subbranches of the Arabidopsis GPAT family. Phylogenetic analyses of this family indicate that GPAT4/6/8 arose early in land-plant evolution (bryophytes), whereas the phosphatase-minus GPAT1 to -3 and GPAT5/7 clades diverged later with the appearance of tracheophytes. PMID:22864585

Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase▿ †

PubMed Central

Toyota, Cory G.; Berthold, Catrine L.; Gruez, Arnaud; Jónsson, Stefán; Lindqvist, Ylva; Cambillau, Christian; Richards, Nigel G. J.

2008-01-01

The yfdXWUVE operon appears to encode proteins that enhance the ability of Escherichia coli MG1655 to survive under acidic conditions. Although the molecular mechanisms underlying this phenotypic behavior remain to be elucidated, findings from structural genomic studies have shown that the structure of YfdW, the protein encoded by the yfdW gene, is homologous to that of the enzyme that mediates oxalate catabolism in the obligate anaerobe Oxalobacter formigenes, O. formigenes formyl coenzyme A transferase (FRC). We now report the first detailed examination of the steady-state kinetic behavior and substrate specificity of recombinant, wild-type YfdW. Our studies confirm that YfdW is a formyl coenzyme A (formyl-CoA) transferase, and YfdW appears to be more stringent than the corresponding enzyme (FRC) in Oxalobacter in employing formyl-CoA and oxalate as substrates. We also report the effects of replacing Trp-48 in the FRC active site with the glutamine residue that occupies an equivalent position in the E. coli protein. The results of these experiments show that Trp-48 precludes oxalate binding to a site that mediates substrate inhibition for YfdW. In addition, the replacement of Trp-48 by Gln-48 yields an FRC variant for which oxalate-dependent substrate inhibition is modified to resemble that seen for YfdW. Our findings illustrate the utility of structural homology in assigning enzyme function and raise the question of whether oxalate catabolism takes place in E. coli upon the up-regulation of the yfdXWUVE operon under acidic conditions. PMID:18245280

Hydrogen in thin Pd-based layers deposited on reticulated vitreous carbon-A new system for electrochemical capacitors

NASA Astrophysics Data System (ADS)

Łukaszewski, M.; Żurowski, A.; Czerwiński, A.

Reticulated vitreous carbon (RVC) has been used as a matrix for electrodeposition of thin layers of Pd and Pd-rich Pd-Rh alloys. It was found that RVC substrate does not affect qualitatively hydrogen absorption behavior of Pd-based deposits. Similarly to thin Pd or Pd alloy layers deposited on Au wires, the α-β phase transition controls the overall rate of hydrogen absorption and desorption into/from Pd-based/RVC electrodes. The possibility of the application of these materials as phase charging-discharging systems was investigated. The values of specific pseudocapacitance, specific power and specific energy were comparable with those for supercapacitors utilizing various redox reactions.

Enhancing substrate utilization and power production of a microbial fuel cell with nitrogen-doped carbon aerogel as cathode catalyst.

PubMed

Tardy, Gábor Márk; Lóránt, Bálint; Lóka, Máté; Nagy, Balázs; László, Krisztina

2017-07-01

Catalytic efficiency of a nitrogen-doped, mesoporous carbon aerogel cathode catalyst was investigated in a two-chambered microbial fuel cell (MFC) applying graphite felt as base material for cathode and anode, utilizing peptone as carbon source. This mesoporous carbon aerogel containing catalyst layer on the cathode increased the maximum power density normalized to the anode volume to 2.7 times higher compared to the maximum power density obtained applying graphite felt cathode without the catalyst layer. At high (2 and 3) cathode/anode volume ratios, maximum power density exceeded 40 W m -3 . At the same time, current density and specific substrate utilization rate increased by 58% resulting in 31.9 A m -3 and 18.8 g COD m -3  h -1 , respectively (normalized to anode volume). Besides the increase of the power and the rate of biodegradation, the investigated catalyst decreased the internal resistance from the range of 450-600 to 350-370 Ω. Although Pt/C catalyst proved to be more efficient, a considerable decrease in the material costs might be achieved by substituting it with nitrogen-doped carbon aerogel in MFCs. Such cathode still displays enhanced catalytic effect.

A Stilbenoid-Specific Prenyltransferase Utilizes Dimethylallyl Pyrophosphate from the Plastidic Terpenoid Pathway1[OPEN

PubMed Central

2016-01-01

Prenylated stilbenoids synthesized in some legumes exhibit plant pathogen defense properties and pharmacological activities with potential benefits to human health. Despite their importance, the biosynthetic pathways of these compounds remain to be elucidated. Peanut (Arachis hypogaea) hairy root cultures produce a diverse array of prenylated stilbenoids upon treatment with elicitors. Using metabolic inhibitors of the plastidic and cytosolic isoprenoid biosynthetic pathways, we demonstrated that the prenyl moiety on the prenylated stilbenoids derives from a plastidic pathway. We further characterized, to our knowledge for the first time, a membrane-bound stilbenoid-specific prenyltransferase activity from the microsomal fraction of peanut hairy roots. This microsomal fraction-derived resveratrol 4-dimethylallyl transferase utilizes 3,3-dimethylallyl pyrophosphate as a prenyl donor and prenylates resveratrol to form arachidin-2. It also prenylates pinosylvin to chiricanine A and piceatannol to arachidin-5, a prenylated stilbenoid identified, to our knowledge, for the first time in this study. This prenyltransferase exhibits strict substrate specificity for stilbenoids and does not prenylate flavanone, flavone, or isoflavone backbones, even though it shares several common features with flavonoid-specific prenyltransferases. PMID:27356974

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.

Modified telomeric repeat amplification protocol: a quantitative radioactive assay for telomerase without using electrophoresis.

PubMed

Szatmari, I; Tókés, S; Dunn, C B; Bardos, T J; Aradi, J

2000-06-15

A polymerase chain reaction (PCR)-based radioactive telomerase assay was developed in our laboratory which is quantitative and does not require electrophoretic evaluation (designated as TP-TRAP; it utilizes two reverse primers). The main steps of the assay include (1) extension of a 20-mer oligonucleotide substrate (MTS) by telomerase, (2) amplification of the telomerase products in the presence of [(3)H]dTTP using the substrate oligonucleotide and two reverse primers (RPC3, 38 mer; RP, 20 mer), (3) isolation of the amplified radioactive dsDNA by precipitation and filtration, (4) determination of the radioactivity of the acid-insoluble DNA. The length of the telomerase products does not increase on amplification. This valuable feature of the assay is achieved by utilization of the two reverse primers and a highly specific PCR protocol. The assay is linear, accurate, and suitable for cell-biological studies where slight quantitative differences in telomerase activity must be detected. The assay is also suitable for screening and characterization of telomerase inhibitors, as shown with a chemically modified oligonucleotide reverse transcriptase inhibitor [(s(4)dU)(35)]. Copyright 2000 Academic Press.

Laforin, a dual specificity phosphatase that dephosphorylates complex carbohydrates.

PubMed

Worby, Carolyn A; Gentry, Matthew S; Dixon, Jack E

2006-10-13

Laforin is the only phosphatase in the animal kingdom that contains a carbohydrate-binding module. Mutations in the gene encoding laforin result in Lafora disease, a fatal autosomal recessive neurodegenerative disorder, which is diagnosed by the presence of intracellular deposits of insoluble complex carbohydrates known as Lafora bodies. We demonstrate that laforin interacts with proteins known to be involved in glycogen metabolism and rule out several of these proteins as potential substrates. Surprisingly, we find that laforin displays robust phosphatase activity against a phosphorylated complex carbohydrate. Furthermore, this activity is unique to laforin, since several other phosphatases are unable to dephosphorylate polysaccharides. Finally, fusing the carbohydrate-binding module of laforin to the dual specific phosphatase VHR does not result in the ability of this phosphatase to dephosphorylate polysaccharides. Therefore, we hypothesize that laforin is unique in its ability to utilize a phosphorylated complex carbohydrate as a substrate and that this function may be necessary for the maintenance of normal cellular glycogen.

Breath carbon stable isotope ratios identify changes in energy balance and substrate utilization in humans

USDA-ARS?s Scientific Manuscript database

Rapid detection of shifts in substrate utilization and energy balance would provide a compelling biofeedback tool to enable individuals to lose weight. In a pilot study, we tested whether the natural abundance of exhaled carbon stable isotope ratios (breath d13C values) reflects shifts between negat...

Metagenome Sequencing of a Coastal Marine Microbial Community from Monterey Bay, California

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

Mueller, Ryan S.; Bryson, Sam; Kieft, Brandon

Heterotrophic microbes are critical components of aquatic food webs. Linkages between populations and the substrates they utilize are not well defined. Here we present the metagenome of microbial communities from the coastal Pacific Ocean exposed to various nutrient additions in order to better understand substrate utilization and partitioning in this environment.

Metagenome Sequencing of a Coastal Marine Microbial Community from Monterey Bay, California

DOE PAGES

Mueller, Ryan S.; Bryson, Sam; Kieft, Brandon; ...

2015-04-30

Heterotrophic microbes are critical components of aquatic food webs. Linkages between populations and the substrates they utilize are not well defined. Here we present the metagenome of microbial communities from the coastal Pacific Ocean exposed to various nutrient additions in order to better understand substrate utilization and partitioning in this environment.

Polysaccharides and Proteins Added to Flowing Drinking Water at Microgram-per-Liter Levels Promote the Formation of Biofilms Predominated by Bacteroidetes and Proteobacteria

PubMed Central

Sack, Eveline L. W.; van der Kooij, Dick

2014-01-01

Biopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 μg C liter−1 in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 μg C liter−1 per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm−2 day−1), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm−2 day−1). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells. Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew during polysaccharide addition, and Alpha-, Beta-, and Gammaproteobacteria, Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might impair the biological stability of distributed drinking water. PMID:24487544

Polysaccharides and proteins added to flowing drinking water at microgram-per-liter levels promote the formation of biofilms predominated by bacteroidetes and proteobacteria.

PubMed

Sack, Eveline L W; van der Wielen, Paul W J J; van der Kooij, Dick

2014-04-01

Biopolymers are important substrates for heterotrophic bacteria in (ultra)oligotrophic freshwater environments, but information about their utilization at microgram-per-liter levels by attached freshwater bacteria is lacking. This study aimed at characterizing biopolymer utilization in drinking-water-related biofilms by exposing such biofilms to added carbohydrates or proteins at 10 μg C liter(-1) in flowing tap water for up to 3 months. Individually added amylopectin was not utilized by the biofilms, whereas laminarin, gelatin, and caseinate were. Amylopectin was utilized during steady-state biofilm growth with simultaneously added maltose but not with simultaneously added acetate. Biofilm formation rates (BFR) at 10 μg C liter(-1) per substrate were ranked as follows, from lowest to highest: blank or amylopectin (≤6 pg ATP cm(-2) day(-1)), gelatin or caseinate, laminarin, maltose, acetate alone or acetate plus amylopectin, and maltose plus amylopectin (980 pg ATP cm(-2) day(-1)). Terminal restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene sequence analyses revealed that the predominant maltose-utilizing bacteria also dominated subsequent amylopectin utilization, indicating catabolic repression and (extracellular) enzyme induction. The accelerated BFR with amylopectin in the presence of maltose probably resulted from efficient amylopectin binding to and hydrolysis by inductive enzymes attached to the bacterial cells. Cytophagia, Flavobacteriia, Gammaproteobacteria, and Sphingobacteriia grew during polysaccharide addition, and Alpha-, Beta-, and Gammaproteobacteria, Cytophagia, Flavobacteriia, and Sphingobacteriia grew during protein addition. The succession of bacterial populations in the biofilms coincided with the decrease in the specific growth rate during biofilm formation. Biopolymers can clearly promote biofilm formation at microgram-per-liter levels in drinking water distribution systems and, depending on their concentrations, might impair the biological stability of distributed drinking water.

Three dehalogenases and physiological restraints in the biodegradation of haloalkanes by Arthrobacter sp. strain HA1.

PubMed Central

Scholtz, R; Messi, F; Leisinger, T; Cook, A M

1988-01-01

Arthrobacter sp. strain HA1 utilizes 18 C2-to-C8 1-haloalkanes for growth and synthesizes an inducible 1-bromoalkane debrominase of unknown physiological function (R. Scholtz, T. Leisinger, F. Suter, and A.M. Cook, J. Bacteriol. 169:5016-5021, 1987) in addition to an inducible 1-chlorohexane halidohydrolase which dehalogenates some 50 substrates, including alpha, omega-dihaloalkanes. alpha, omega-Dihaloalkanes were utilized by cultures of strain HA1 under certain conditions only. C9 and C8 homologs prevented growth. At suitable concentrations, C7-to-C5 homologs could serve as sole sources of carbon and energy for growth. C4 and C3 homologs could be utilized only in the presence of a second substrate (e.g., butanol), and the C2 homolog was not degraded. Kinetics of growth and substrate utilization indicated that cells of strain HA1 growing in butanol-salts medium could be used to test whether compounds induced the 1-chlorohexane halidohydrolase. No gratuitous induction of synthesis of the enzyme was observed. Many enzyme substrates (e.g., bromobenzene) did not induce synthesis of the enzyme, though the enzyme sequence to degrade the product (phenol) was present. Some inducers (e.g., bromomethane) were enzyme substrates but not growth substrates. In an attempt to find a physiological role for the 1-bromoalkane debrominase, we observed that several long-chain haloaliphatic compounds (greater than C9; e.g., 1-bromohexadecane and 1-chlorohexadecane) were utilized for growth and that induced cells could dehalogenate several 1-haloalkanes (at least C4 to C16). The dehalogenation of the long-chain compounds could not be assayed in the cell extract, so we presume that a third haloalkane dehalogenase was present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3223767

An introduction to the Astro Edge solar array

NASA Technical Reports Server (NTRS)

Spence, B. R.; Marks, G. W.

1994-01-01

The Astro Edge solar array is a new and innovative low concentrator power generating system which has been developed for applications requiring high specific power, high stiffness, low risk, light modular construction which utilizes conventional materials and technology, and standard photovoltaic solar cells and laydown processes. Mechanisms, restraint/release devices, wiring harnesses, substrates, and support structures are designed to be simple, functional, lightweight, and modular. A brief overview of the Astro Edge solar array is discussed.

Extracellular lignase: a key to enhanced cellulose utilization

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

Hira, A.; Barnett, S.M.; Shieh, C.H.

1978-01-01

An alternate approach to the conventional chemical processing of lignin, a potential renewable resource, is enzymic conversion. Biodegradation of wood, a lignin-cellulose complex, is accomplished naturally by various enzymes of microbial origin. Extracellular lignases have been isolated from pure cultures of Polyporus versicolor, Phanerochaete chrysosporium, and Pleurotus ostreatus. The isolated enzyme systems from these organisms have shown substrate specificity for guaiacol and hydroquinone and yielded a positive syringaldazine test. A commercial lignin was degraded by the enzyme system.

Photo-ionization and modification of nanoparticles on transparent substrates by ultrashort laser pulses

NASA Astrophysics Data System (ADS)

Gruzdev, Vitaly; Komolov, Vladimir; Li, Hao; Yu, Qingsong; Przhibel'skii, Sergey; Smirnov, Dmitry

2011-02-01

The objective of this combined experimental and theoretical research is to study the dynamics and mechanisms of nanoparticle interaction with ultrashort laser pulses and related modifications of substrate surface. For the experimental effort, metal (gold), dielectric (SiO2) and dielectric with metal coating (about 30 nm thick) spherical nanoparticles deposited on glass substrate are utilized. Size of the particles varies from 20 to 200 nm. Density of the particles varies from low (mean inter-particle distance 100 nm) to high (mean inter-particle distance less than 1 nm). The nanoparticle assemblies and the corresponding empty substrate surfaces are irradiated with single 130-fs laser pulses at wavelength 775 nm and different levels of laser fluence. Large diameter of laser spot (0.5-2 mm) provides gradient variations of laser intensity over the spot and allows observing different laser-nanoparticle interactions. The interactions vary from total removal of the nanoparticles in the center of laser spot to gentle modification of their size and shape and totally non-destructive interaction. The removed particles frequently form specific sub-micrometer-size pits on the substrate surface at their locations. The experimental effort is supported by simulations of the nanoparticle interactions with high-intensity ultrashort laser pulse. The simulation employs specific modification of the molecular dynamics approach applied to model the processes of non-thermal particle ablation following laser-induced electron emission. This technique delivers various characteristics of the ablation plume from a single nanoparticle including energy and speed distribution of emitted ions, variations of particle size and overall dynamics of its ablation. The considered geometry includes single isolated particle as well a single particle on a flat substrate that corresponds to the experimental conditions. The simulations confirm existence of the different regimes of laser-nanoparticle interactions depending on laser intensity and wavelength. In particular, implantation of ions departing from the nanoparticles towards the substrate is predicted.

Influence of four antimicrobials on methane-producing archaea and sulfate-reducing bacteria in anaerobic granular sludge.

PubMed

Du, Jingru; Hu, Yong; Qi, Weikang; Zhang, Yanlong; Jing, Zhaoqian; Norton, Michael; Li, Yu-You

2015-12-01

The influence of Cephalexin (CLX), Tetracycline (TC), Erythromycin (ERY) and Sulfathiazole (ST) on methane-producing archaea (MPA) and sulfate-reducing bacteria (SRB) in anaerobic sludge was investigated using acetate or ethanol as substrate. With antimicrobial concentrations below 400mgL(-1), the relative specific methanogenic activity (SMA) was above 50%, so that the antimicrobials exerted slight effects on archaea. However ERY and ST at 400mgL(-1) caused a 74.5% and 57.6% inhibition to specific sulfidogenic activity (SSA) when the sludge granules were disrupted and ethanol used as substrate. After disruption, microbial tolerance to antimicrobials decreased, but the rate at which MPA utilized acetate and ethanol increased from 0.95gCOD·(gVSS⋅d)(-1) to 1.45gCOD·(gVSS⋅d)(-1) and 0.90gCOD·(gVSS⋅d)(-1) to 1.15gCOD·(gVSS⋅d)(-1) respectively. The ethanol utilization rate for SRB also increased after disruption from 0.35gCOD·(gVSS⋅d)(-1) to 0.46gCOD·(gVSS⋅d)(-1). Removal rates for CLX approaching 20.0% and 25.0% were obtained used acetate and ethanol respectively. The disintegration of granules improved the CLX removal rate to 65% and 78%, but ST was not removed during this process. Copyright © 2014 Elsevier Ltd. All rights reserved.

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level.

PubMed

Prielhofer, Roland; Cartwright, Stephanie P; Graf, Alexandra B; Valli, Minoska; Bill, Roslyn M; Mattanovich, Diethard; Gasser, Brigitte

2015-03-11

The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

NASA Astrophysics Data System (ADS)

Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2014-05-01

Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

Glycine Betaine as a Direct Substrate for Methanogens (Methanococcoides spp.)

PubMed Central

Watkins, Andrew J.; Roussel, Erwan G.; Parkes, R. John

2014-01-01

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners. PMID:24162571

The Structure and Specificity of the Type III Secretion System Effector NleC Suggest a DNA Mimicry Mechanism of Substrate Recognition

PubMed Central

2015-01-01

Many pathogenic bacteria utilize the type III secretion system (T3SS) to translocate effector proteins directly into host cells, facilitating colonization. In enterohemmorhagic Escherichia coli (EHEC), a subset of T3SS effectors is essential for suppression of the inflammatory response in hosts, including humans. Identified as a zinc protease that cleaves NF-κB transcription factors, NleC is one such effector. Here, we investigate NleC substrate specificity, showing that four residues around the cleavage site in the DNA-binding loop of the NF-κB subunit RelA strongly influence the cleavage rate. Class I NF-κB subunit p50 is cleaved at a reduced rate consistent with conservation of only three of these four residues. However, peptides containing 10 residues on each side of the scissile bond were not efficiently cleaved by NleC, indicating that elements distal from the cleavage site are also important for substrate recognition. We present the crystal structure of NleC and show that it mimics DNA structurally and electrostatically. Consistent with this model, mutation of phosphate-mimicking residues in NleC reduces the level of RelA cleavage. We propose that global recognition of NF-κB subunits by DNA mimicry combined with a high sequence selectivity for the cleavage site results in exquisite NleC substrate specificity. The structure also shows that despite undetectable similarity of its sequence to those of other Zn2+ proteases beyond its conserved HExxH Zn2+-binding motif, NleC is a member of the Zincin protease superfamily, albeit divergent from its structural homologues. In particular, NleC displays a modified Ψ-loop motif that may be important for folding and refolding requirements implicit in T3SS translocation. PMID:25040221

ZnO deposition on metal substrates: Relating fabrication, morphology, and wettability

NASA Astrophysics Data System (ADS)

Beaini, Sara S.; Kronawitter, Coleman X.; Carey, Van P.; Mao, Samuel S.

2013-05-01

It is not common practice to deposit thin films on metal substrates, especially copper, which is a common heat exchanger metal and practical engineering material known for its heat transfer properties. While single crystal substrates offer ideal surfaces with uniform structure for compatibility with oxide deposition, metallic surfaces needed for industrial applications exhibit non-idealities that complicate the fabrication of oxide nanostructure arrays. The following study explored different ZnO fabrication techniques to deposit a (super)hydrophobic thin film of ZnO on a metal substrate, specifically copper, in order to explore its feasibility as an enhanced condensing surface. ZnO was selected for its non-toxicity, ability to be made (super)hydrophobic with hierarchical roughness, and its photoinduced hydrophilicity characteristic, which could be utilized to pattern it to have both hydrophobic-hydrophilic regions. We investigated the variation of ZnO's morphology and wetting state, using SEMs and sessile drop contact angle measurements, as a function of different fabrication techniques: sputtering, pulsed laser deposition (PLD), electrodeposition and annealing Zn. We successfully fabricated (super)hydrophobic ZnO on a mirror finish, commercially available copper substrate using the scalable electrodeposition technique. PLD for ZnO deposition did not prove viable, as the ZnO samples on metal substrates were hydrophilic and the process does not lend itself to scalability. The annealed Zn sheets did not exhibit consistent wetting state results.

Multi-substrate biodegradation interaction of 1, 4-dioxane and BTEX mixtures by Acinetobacter baumannii DD1.

PubMed

Zhou, YuYang; Huang, Huanlin; Shen, Dongsheng

2016-02-01

This study evaluated substrate interactions during the aerobic biodegradation of 1, 4-dioxane and BTEX mixtures by a pure culture, Acinetobacter baumannii DD1, which is capable of utilizing 1, 4-dioxane for growth. A. baumannii DD1 could utilize BTEX as a sole carbon source, but could not utilize m-xylene and p-xylene. In binary mixtures, there was a lag of about 14 h before the degradation of BTE, and 1, 4-dioxane only started to be utilized when BTE was completely degraded by 1, 4-dioxane-grown DD1. Furthermore, the biodegradation rate of 1, 4-dioxane decreased from 73.33 to 40.74 mg/(h g dry weight) after the biodegradation of benzene. 1, 4-dioxane could not be degraded after the biodegradation of o-xylene in 80 h. DD1 could also not degrade m-xylene and p-xylene coexisting with 1, 4-dioxane. The ability of DD1 to degrade BTEX occurred in the following order: benzene > ethylbenzene > toluene > o-xylene > m-xylene = p-xylene. The biodegradation of 1, 4-dioxane was not activated in the mixture with o-xylene, primarily because of the accumulation of the specific toxic intermediate, 2, 3-dimethylphenol. The lag in BTE degradation was presumably because of the induction of enzymes necessary for BTE degradation. Additionally, SDS-PAGE analysis demonstrated that there were different proteins during the degradation of benzene and 1, 4-dioxane.

Recognition of Nucleoside Monophosphate Substrates by Haemophilus influenzae Class C Acid Phosphatase

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

Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.

2010-12-08

The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD{sup +} utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5{prime},3{prime}-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5{prime}-AMP, 3{prime}-AMP, and 2{prime}-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pocketsmore » that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5{prime}-nucleotides and 3{prime}-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5{prime} substrates in an anti conformation and 3{prime} substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition.« less

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

PubMed Central

Ruiz-Moyano, Santiago; Totten, Sarah M.; Garrido, Daniel A.; Smilowitz, Jennifer T.; German, J. Bruce; Lebrilla, Carlito B.

2013-01-01

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota. PMID:23892749

Variation in consumption of human milk oligosaccharides by infant gut-associated strains of Bifidobacterium breve.

PubMed

Ruiz-Moyano, Santiago; Totten, Sarah M; Garrido, Daniel A; Smilowitz, Jennifer T; German, J Bruce; Lebrilla, Carlito B; Mills, David A

2013-10-01

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.

Mechanisms of pseudosubstrate inhibition of the anaphase promoting complex by Acm1

PubMed Central

Burton, Janet L; Xiong, Yong; Solomon, Mark J

2011-01-01

The anaphase promoting complex (APC) is a ubiquitin ligase that promotes the degradation of cell-cycle regulators by the 26S proteasome. Cdc20 and Cdh1 are WD40-containing APC co-activators that bind destruction boxes (DB) and KEN boxes within substrates to recruit them to the APC for ubiquitination. Acm1 is an APCCdh1 inhibitor that utilizes a DB and a KEN box to bind Cdh1 and prevent substrate binding, although Acm1 itself is not a substrate. We investigated what differentiates an APC substrate from an inhibitor. We identified the Acm1 A-motif that interacts with Cdh1 and together with the DB and KEN box is required for APCCdh1 inhibition. A genetic screen identified Cdh1 WD40 domain residues important for Acm1 A-motif interaction and inhibition that appears to reside near Cdh1 residues important for DB recognition. Specific lysine insertion mutations within Acm1 promoted its ubiquitination by APCCdh1 whereas lysine removal from the APC substrate Hsl1 converted it into a potent APCCdh1 inhibitor. These findings suggest that tight Cdh1 binding combined with the inaccessibility of ubiquitinatable lysines contributes to pseudosubstrate inhibition of APCCdh1. PMID:21460798

Comparison of [corrected] actin- and glass-supported phospholipid bilayer diffusion coefficients.

PubMed

Sterling, Sarah M; Dawes, Ryan; Allgeyer, Edward S; Ashworth, Sharon L; Neivandt, David J

2015-04-21

The formation of biomimetic lipid membranes has the potential to provide insights into cellular lipid membrane dynamics. The construction of such membranes necessitates not only the utilization of appropriate lipids, but also physiologically relevant substrate/support materials. The substrate materials employed have been shown to have demonstrable effects on the behavior of the overlying lipid membrane, and thus must be studied before use as a model cushion support. To our knowledge, we report the formation and investigation of a novel actin protein-supported lipid membrane. Specifically, inner leaflet lateral mobility of globular actin-supported DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers, deposited via the Langmuir-Blodgett/Langmuir Schaefer methodology, was investigated by z-scan fluorescence correlation spectroscopy across a temperature range of 20-44°C. The actin substrate was found to decrease the diffusion coefficient when compared to an identical membrane supported on glass. The depression of the diffusion coefficient occurred across all measured temperatures. These results indicated that the actin substrate exerted a direct effect on the fluidity of the lipid membrane and highlighted the fact that the choice of substrate/support is critical in studies of model lipid membranes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Single Source Precursors for Thin Film Solar Cells

NASA Technical Reports Server (NTRS)

Banger, Kulbinder K.; Hollingsworth, Jennifer A.; Harris, Jerry D.; Cowen, Jonathan; Buhro, William E.; Hepp, Aloysius F.

2002-01-01

The development of thin film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. At NASA GRC we have focused on the development of new single source precursors (SSP) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD (chemical vapor deposition) process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV (photovoltaic) devices.

Photovoltaic options for solar electric propulsion

NASA Technical Reports Server (NTRS)

Stella, Paul M.; Flood, Dennis J.

1990-01-01

During the past decade, a number of advances have occurred in solar cell and array technology. These advances have lead to performance improvement for both conventional space arrays and for advanced technology arrays. Performance enhancements have occurred in power density, specific power, and environmental capability. Both state-of-the-art and advanced development cells and array technology are discussed. Present technology will include rigid, rollout, and foldout flexible substrate designs, with silicon and GaAs solar cells. The use of concentrator array systems is also discussed based on both DOD and NASA efforts. The benefits of advanced lightweight array technology, for both near term and far term utilization, and of advanced high efficiency, thin, radiation resistant cells is examined. This includes gallium arsenide on germaniun substrates, indium phosphide, and thin film devices such as copper indium diselenide.

Highly flexible binder-free core-shell nanofibrous electrode for lightweight electrochemical energy storage using recycled water bottles

NASA Astrophysics Data System (ADS)

Shi, HaoTian H.; Naguib, Hani E.

2016-08-01

The creation of a novel flexible nanocomposite fiber with conductive polymer polyaniline (PAni) coating on a polyethylene terephthalate (PET) substrate allowed for increased electrochemical performance while retaining ideal mechanical properties such as very high flexibility. Binder-free PAni-wrapped PET (PAni@PET) fiber with a core-shell structure was successfully fabricated through a novel technique. The PET nanofiber substrate was fabricated through an optimized electrospinning method, while the PAni shell was chemically polymerized onto the surface of the nanofibers. The PET substrate can be made directly from recycled PETE1 grade plastic water bottles. The resulting nanofiber with an average diameter of 121 nm ± 39 nm, with a specific surface area of 83.72 m2 g-1, led to better ionic interactions at the electrode/electrolyte interface. The PAni active layer coating was found to be 69 nm in average thickness. The specific capacitance was found to have increased dramatically from pure PAni with carbon binders. The specific capacitance was found to be 347 F g-1 at a relatively high scan rate of 10 mV s-1. The PAni/PET fiber also experienced very little degradation (4.4%) in capacitance after 1500 galvanostatic charge/discharge cycles at a specific current of 1.2 A g-1. The mesoporous structure of the PAni@PET fibrous mat also allowed for tunable capacitance by controlling the pore sizes. This novel fabrication method offers insights for the utilization of recycled PETE1 based bottles as a high performance, low cost, highly flexible supercapacitor device.

Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids.

PubMed

Isikhuemhen, Omoanghe S; Mikiashvilli, Nona A

2009-11-01

Solid waste from anaerobic digestion of litter from the commercial production of broiler chickens has limited use as fertilizer. Its disposal is a major problem for digester operators who are seeking alternative use for anaerobic digester solids, also referred to as solid waste (SW). The use of SW as substrates for the cultivation of Pleurotus ostreatus strain MBFBL400 was investigated. Lignocellulolytic enzymes activity, substrate utilization, and mushroom yield were evaluated in ten different substrate combinations (SCs) containing varying amounts of solid waste, wheat straw, and millet. Nutritional content of mushrooms produced on the different substrates was also determined. Substrates containing 70-80% wheat straw, 10-20% SW, and 10-20% millet were found to produce the highest mushroom yield (874.8-958.3 g/kg). Loss of organic matter in all SCs tested varied from 45.8% to 56.2%, which had positive correlation with the biological efficiency. Laccase, peroxidase, and carboxymethylcellulase (CMCase) activities were higher before fruiting, whereas xylanase showed higher activities after mushroom fruiting. SW increased the nutritional content in mushrooms harvested, and the combination of wheat straw and SW with millet significantly improved mushroom yield. Our findings demonstrated the possibility of utilizing anaerobic digester solids in mushroom cultivation. The application of SW as such could improve the financial gains in the overall economy of anaerobic digester plants.

Composite perfluorohydrocarbon membranes, their preparation and use

DOEpatents

Ding, Yong; Bikson, Benjamin

2017-04-04

Composite porous hydrophobic membranes are prepared by forming a perfluorohydrocarbon layer on the surface of a preformed porous polymeric substrate. The substrate can be formed from poly (aryl ether ketone) and a perfluorohydrocarbon layer can be chemically grafted to the surface of the substrate. The membranes can be utilized for a broad range of fluid separations, such as microfiltration, nanofiltration, ultrafiltration as membrane contactors for membrane distillation and for degassing and dewatering of fluids. The membranes can further contain a dense ultra-thin perfluorohydrocarbon layer superimposed on the porous poly (aryl ether ketone) substrate and can be utilized as membrane contactors or as gas separation. membranes for natural gas treatment and gas dehydration.

Epitaxial growth of silicon for layer transfer

DOEpatents

Teplin, Charles; Branz, Howard M

2015-03-24

Methods of preparing a thin crystalline silicon film for transfer and devices utilizing a transferred crystalline silicon film are disclosed. The methods include preparing a silicon growth substrate which has an interface defining substance associated with an exterior surface. The methods further include depositing an epitaxial layer of silicon on the silicon growth substrate at the surface and separating the epitaxial layer from the substrate substantially along the plane or other surface defined by the interface defining substance. The epitaxial layer may be utilized as a thin film of crystalline silicon in any type of semiconductor device which requires a crystalline silicon layer. In use, the epitaxial transfer layer may be associated with a secondary substrate.

Versatile gold based SERS substrates fabricated by ultrafast laser ablation for sensing picric acid and ammonium nitrate

NASA Astrophysics Data System (ADS)

Byram, Chandu; Moram, Sree Sathya Bharathi; Shaik, Abdul Kalam; Soma, Venugopal Rao

2017-10-01

We demonstrate the detection of picric acid (PA) and ammonium nitrate (AN) at μM concentrations by utilizing gold (Au) nanostructures (NSs) as surface enhanced Raman scattering (SERS) substrates fabricated using the technique of ultrafast laser ablation in liquids. Au NPs and NSs were also utilized for detecting Rhodamine 6G (Rh6G) and methylene blue (MB). Detection of all the molecules using the same substrates (NPs and NSs) demonstrated their versatility. Detection limits of 10-5, 10-6, 10-7, 10-8 M were achieved for AN, PA, Rh6G and MB, respectively. Reproducibility of the SERS intensity using NSs and NPs as substrates demonstrate their efficacy.

Crystal structure of Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH: implications for substrate specificity and catalysis.

PubMed

Ud-Din, Abu I; Liu, Yu C; Roujeinikova, Anna

2015-01-01

Helicobacter pylori infection is the common cause of gastroduodenal diseases linked to a higher risk of the development of gastric cancer. Persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. It belongs to the GCN5-related N-acetyltransferase (GNAT) superfamily. The crystal structure of the PseH complex with cofactor acetyl-CoA has been determined at 2.3 Å resolution. This is the first crystal structure of the GNAT superfamily member with specificity to UDP-4-amino-4,6-dideoxy-β-L-AltNAc. PseH is a homodimer in the crystal, each subunit of which has a central twisted β-sheet flanked by five α-helices and is structurally homologous to those of other GNAT superfamily enzymes. Interestingly, PseH is more similar to the GNAT enzymes that utilize amino acid sulfamoyl adenosine or protein as a substrate than a different GNAT-superfamily bacterial nucleotide-sugar N-acetyltransferase of the known structure, WecD. Analysis of the complex of PseH with acetyl-CoA revealed the location of the cofactor-binding site between the splayed strands β4 and β5. The structure of PseH, together with the conservation of the active-site general acid among GNAT superfamily transferases, are consistent with a common catalytic mechanism for this enzyme that involves direct acetyl transfer from AcCoA without an acetylated enzyme intermediate. Based on structural homology with microcin C7 acetyltransferase MccE and WecD, the Michaelis complex can be modeled. The model suggests that the nucleotide- and 4-amino-4,6-dideoxy-β-L-AltNAc-binding pockets form extensive interactions with the substrate and are thus the most significant determinants of substrate specificity. A hydrophobic pocket accommodating the 6'-methyl group of the altrose dictates preference to the methyl over the hydroxyl group and thus to contributes to substrate specificity of PseH.

MDD-carb: a combinatorial model for the identification of protein carbonylation sites with substrate motifs.

PubMed

Kao, Hui-Ju; Weng, Shun-Long; Huang, Kai-Yao; Kaunang, Fergie Joanda; Hsu, Justin Bo-Kai; Huang, Chien-Hsun; Lee, Tzong-Yi

2017-12-21

Carbonylation, which takes place through oxidation of reactive oxygen species (ROS) on specific residues, is an irreversibly oxidative modification of proteins. It has been reported that the carbonylation is related to a number of metabolic or aging diseases including diabetes, chronic lung disease, Parkinson's disease, and Alzheimer's disease. Due to the lack of computational methods dedicated to exploring motif signatures of protein carbonylation sites, we were motivated to exploit an iterative statistical method to characterize and identify carbonylated sites with motif signatures. By manually curating experimental data from research articles, we obtained 332, 144, 135, and 140 verified substrate sites for K (lysine), R (arginine), T (threonine), and P (proline) residues, respectively, from 241 carbonylated proteins. In order to examine the informative attributes for classifying between carbonylated and non-carbonylated sites, multifarious features including composition of twenty amino acids (AAC), composition of amino acid pairs (AAPC), position-specific scoring matrix (PSSM), and positional weighted matrix (PWM) were investigated in this study. Additionally, in an attempt to explore the motif signatures of carbonylation sites, an iterative statistical method was adopted to detect statistically significant dependencies of amino acid compositions between specific positions around substrate sites. Profile hidden Markov model (HMM) was then utilized to train a predictive model from each motif signature. Moreover, based on the method of support vector machine (SVM), we adopted it to construct an integrative model by combining the values of bit scores obtained from profile HMMs. The combinatorial model could provide an enhanced performance with evenly predictive sensitivity and specificity in the evaluation of cross-validation and independent testing. This study provides a new scheme for exploring potential motif signatures at substrate sites of protein carbonylation. The usefulness of the revealed motifs in the identification of carbonylated sites is demonstrated by their effective performance in cross-validation and independent testing. Finally, these substrate motifs were adopted to build an available online resource (MDD-Carb, http://csb.cse.yzu.edu.tw/MDDCarb/ ) and are also anticipated to facilitate the study of large-scale carbonylated proteomes.

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

Diggin’ on U(biquitin): A Novel Method for the Identification of Physiological E3 Ubiquitin Ligase Substrates

PubMed Central

Rubel, Carrie E.; Schisler, Jonathan C.; Hamlett, Eric D.; DeKroon, Robert M.; Gautel, Mathias; Alzate, Oscar; Patterson, Cam

2013-01-01

The ubiquitin-proteasome system (UPS) plays a central role in maintaining protein homeostasis, emphasized by a myriad of diseases that are associated with altered UPS function such as cancer, muscle-wasting, and neurodegeneration. Protein ubiquitination plays a central role in both the promotion of proteasomal degradation as well as cellular signaling through regulation of the stability of transcription factors and other signaling molecules. Substrate specificity is a critical regulatory step of ubiquitination and is mediated by ubiquitin ligases. Recent studies implicate ubiquitin ligases in multiple models of cardiac diseases such as cardiac hypertrophy, atrophy, and ischemia/reperfusion injury, both in a cardioprotective and maladaptive role. Therefore, identifying physiological substrates of cardiac ubiquitin ligases provides both mechanistic insights into heart disease as well as possible therapeutic targets. Current methods identifying substrates for ubiquitin ligases rely heavily upon non-physiologic in vitro methods, impeding the unbiased discovery of physiological substrates in relevant model systems. Here we describe a novel method for identifying ubiquitin ligase substrates utilizing Tandem Ubiquitin Binding Entities (TUBE) technology, two-dimensional differential in gel electrophoresis (2-D DIGE), and mass spectrometry, validated by the identification of both known and novel physiological substrates of the ubiquitin ligase MuRF1 in primary cardiomyocytes. This method can be applied to any ubiquitin ligase, both in normal and disease model systems, in order to identify relevant physiological substrates under various biological conditions, opening the door to a clearer mechanistic understanding of ubiquitin ligase function and broadening their potential as therapeutic targets. PMID:23695782

Mechanistic and Structural Analysis of a Drosophila melanogaster Enzyme, Arylalkylamine N-Acetyltransferase Like 7, an Enzyme That Catalyzes the Formation of N-Acetylarylalkylamides and N-Acetylhistamine.

PubMed

Dempsey, Daniel R; Jeffries, Kristen A; Handa, Sumit; Carpenter, Anne-Marie; Rodriguez-Ospina, Santiago; Breydo, Leonid; Merkler, David J

2015-04-28

Arylalkylamine N-acetyltransferase like 7 (AANATL7) catalyzes the formation of N-acetylarylalkylamides and N-acetylhistamine from acetyl-CoA and the corresponding amine substrate. AANATL7 is a member of the GNAT superfamily of >10000 GCN5-related N-acetyltransferases, many members being linked to important roles in both human metabolism and disease. Drosophila melanogaster utilizes the N-acetylation of biogenic amines for the inactivation of neurotransmitters, the biosynthesis of melatonin, and the sclerotization of the cuticle. We have expressed and purified D. melanogaster AANATL7 in Escherichia coli and used the purified enzyme to define the substrate specificity for acyl-CoA and amine substrates. Information about the substrate specificity provides insight into the potential contribution made by AANATL7 to fatty acid amide biosynthesis because D. melanogaster has emerged as an important model system contributing to our understanding of fatty acid amide metabolism. Characterization of the kinetic mechanism of AANATL7 identified an ordered sequential mechanism, with acetyl-CoA binding first followed by histamine to generate an AANATL7·acetyl-CoA·histamine ternary complex prior to catalysis. Successive pH-activity profiling and site-directed mutagenesis experiments identified two ionizable groups: one with a pKa of 7.1 that is assigned to Glu-26 as a general base and a second pKa of 9.5 that is assigned to the protonation of the thiolate of the coenzyme A product. Using the data generated herein, we propose a chemical mechanism for AANATL7 and define functions for other important amino acid residues involved in substrate binding and regulation of catalysis.

Short-term starvation is a strategy to unravel the cellular capacity of oxidizing specific exogenous/endogenous substrates in mitochondria.

PubMed

Zeidler, Julianna D; Fernandes-Siqueira, Lorena O; Carvalho, Ana S; Cararo-Lopes, Eduardo; Dias, Matheus H; Ketzer, Luisa A; Galina, Antonio; Da Poian, Andrea T

2017-08-25

Mitochondrial oxidation of nutrients is tightly regulated in response to the cellular environment and changes in energy demands. In vitro studies evaluating the mitochondrial capacity of oxidizing different substrates are important for understanding metabolic shifts in physiological adaptations and pathological conditions, but may be influenced by the nutrients present in the culture medium or by the utilization of endogenous stores. One such influence is exemplified by the Crabtree effect (the glucose-mediated inhibition of mitochondrial respiration) as most in vitro experiments are performed in glucose-containing media. Here, using high-resolution respirometry, we evaluated the oxidation of endogenous or exogenous substrates by cell lines harboring different metabolic profiles. We found that a 1-h deprivation of the main energetic nutrients is an appropriate strategy to abolish interference of endogenous or undesirable exogenous substrates with the cellular capacity of oxidizing specific substrates, namely glutamine, pyruvate, glucose, or palmitate, in mitochondria. This approach primed mitochondria to immediately increase their oxygen consumption after the addition of the exogenous nutrients. All starved cells could oxidize exogenous glutamine, whereas the capacity for oxidizing palmitate was limited to human hepatocarcinoma Huh7 cells and to C2C12 mouse myoblasts that differentiated into myotubes. In the presence of exogenous glucose, starvation decreased the Crabtree effect in Huh7 and C2C12 cells and abrogated it in mouse neuroblastoma N2A cells. Interestingly, the fact that the Crabtree effect was observed only for mitochondrial basal respiration but not for the maximum respiratory capacity suggests it is not caused by a direct effect on the electron transport system. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Refurbishment of SRB aluminum components by walnut hull blast removal of protective coatings

NASA Technical Reports Server (NTRS)

Colberg, W. R.; Gordon, G. H.; Jackson, C. H.

1982-01-01

A test program was conducted to develop, optimize, and scale up an abrasive blasting procedure was developed for refurbishment of specific SRB components: aft skirt, forward skirt, frustrum, and painted piece parts. Test specimens utilizing 2219 T87 aluminum substrate of varying thicknesses were prepared and blasted at progressively increasing pressures with selected abrasives. Specimens were analyzed for material response. The optimum blasting parameters were determined on panel specimens and verified on a large cylindrical integrated test bed.

Nuclear Imaging for Assessment of Prostate Cancer Gene Therapy

DTIC Science & Technology

2007-03-01

thymidine kinase transfected EL4 cells . Further exploration of Tc-99m conjugated potential HSV1-TK substrates is still undergoing in our laboratory...prostate cancer cells , has been demonstrated the utility for tissue-specific toxic gene therapy for prostate cancer[10, 11]. Therefore, an adenovirus...BJ5183 together with pAdeasy-1, the viral DNA plasmid. The pAdeasy-1 is E1 and E3 deleted, its E1 function can be complemented in 293A cells . The

Effects of long-term microgravitation exposure on cell respiration of the rat musculus soleus fibers.

PubMed

Veselova, O M; Ogneva, I V; Larina, I M

2011-07-01

Cell respiration of the m. soleus fibers was studied in Wistar rats treated with succinic acid and exposed to microgravitation for 35 days. The results indicated that respiration rates during utilization of endogenous and exogenous substrates and the maximum respiration rate decreased in animals subjected to microgravitation without succinate treatment. The respiration rate during utilization of exogenous substrate did not increase in comparison with that on endogenous substrates. Succinic acid prevented the decrease in respiration rate on endogenous substrates and the maximum respiration rate. On the other hand, the respiration rate on exogenous substrates was reduced in vivarium control rats receiving succinate in comparison with intact control group. That could indicate changed efficiency of complex I of the respiratory chain due to reciprocal regulation of the tricarbonic acid cycle.

Enhancement of phonon backscattering due to confinement of ballistic phonon pathways in silicon as studied with a microfabricated phonon spectrometer

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

Otelaja, O. O.; Robinson, R. D., E-mail: rdr82@cornell.edu

2015-10-26

In this work, the mechanism for enhanced phonon backscattering in silicon is investigated. An understanding of phonon propagation through substrates has implications for engineering heat flow at the nanoscale, for understanding sources of decoherence in quantum systems, and for realizing efficient phonon-mediated particle detectors. In these systems, phonons that backscatter from the bottom of substrates, within the crystal or from interfaces, often contribute to the overall detector signal. We utilize a microscale phonon spectrometer, comprising superconducting tunnel junction emitters and detectors, to specifically probe phonon backscattering in silicon substrates (∼500 μm thick). By etching phonon “enhancers” or deep trenches (∼90 μm) aroundmore » the detectors, we show that the backscattered signal level increases by a factor of ∼2 for two enhancers versus one enhancer. Using a geometric analysis of the phonon pathways, we show that the mechanism of the backscattered phonon enhancement is due to confinement of the ballistic phonon pathways and increased scattering off the enhancer walls. Our result is applicable to the geometric design and patterning of substrates that are employed in phonon-mediated detection devices.« less

Agricultural waste from the tequila industry as substrate for the production of commercially important enzymes.

PubMed

Huitron, C; Perez, R; Sanchez, A E; Lappe, P; Rocha Zavaleta, L

2008-01-01

Approximately 1 million tons of Agave tequilana plants are processed annually by the Mexican Tequila industry generating vast amounts of agricultural waste. The aim of this study was to investigate the potential use of Agave tequilana waste as substrate for the production of commercially important enzymes. Two strains of Aspergillus niger (CH-A-2010 and CH-A-2016), isolated from agave fields, were found to grow and propagate in submerged cultures using Agave tequilana waste as substrate. Isolates showed simultaneous extracellular inulinase, xylanase, pectinase, and cellulase activities. Aspergillus CH-A-2010 showed the highest production of inulinase activity (1.48 U/ml), whereas Aspergillus niger CH-A-2016 produced the highest xylanase (1.52 U/ml) and endo-pectinase (2.7U/ml) activities. In both cases production of enzyme activities was significantly higher on Agave tequilana waste than that observed on lemon peel and specific polymeric carbohydrates. Enzymatic hydrolysis of raw A. tequilana stems and leaves, by enzymes secreted by the isolates yielded maximum concentrations of reducing sugars of 28.2 g/l, and 9.9 g/l respectively. In conclusion, Agave tequilana waste can be utilized as substrate for the production of important biotechnological enzymes.

Bioinformatics analysis identifies several intrinsically disordered human E3 ubiquitin-protein ligases.

PubMed

Boomsma, Wouter; Nielsen, Sofie V; Lindorff-Larsen, Kresten; Hartmann-Petersen, Rasmus; Ellgaard, Lars

2016-01-01

The ubiquitin-proteasome system targets misfolded proteins for degradation. Since the accumulation of such proteins is potentially harmful for the cell, their prompt removal is important. E3 ubiquitin-protein ligases mediate substrate ubiquitination by bringing together the substrate with an E2 ubiquitin-conjugating enzyme, which transfers ubiquitin to the substrate. For misfolded proteins, substrate recognition is generally delegated to molecular chaperones that subsequently interact with specific E3 ligases. An important exception is San1, a yeast E3 ligase. San1 harbors extensive regions of intrinsic disorder, which provide both conformational flexibility and sites for direct recognition of misfolded targets of vastly different conformations. So far, no mammalian ortholog of San1 is known, nor is it clear whether other E3 ligases utilize disordered regions for substrate recognition. Here, we conduct a bioinformatics analysis to examine >600 human and S. cerevisiae E3 ligases to identify enzymes that are similar to San1 in terms of function and/or mechanism of substrate recognition. An initial sequence-based database search was found to detect candidates primarily based on the homology of their ordered regions, and did not capture the unique disorder patterns that encode the functional mechanism of San1. However, by searching specifically for key features of the San1 sequence, such as long regions of intrinsic disorder embedded with short stretches predicted to be suitable for substrate interaction, we identified several E3 ligases with these characteristics. Our initial analysis revealed that another remarkable trait of San1 is shared with several candidate E3 ligases: long stretches of complete lysine suppression, which in San1 limits auto-ubiquitination. We encode these characteristic features into a San1 similarity-score, and present a set of proteins that are plausible candidates as San1 counterparts in humans. In conclusion, our work indicates that San1 is not a unique case, and that several other yeast and human E3 ligases have sequence properties that may allow them to recognize substrates by a similar mechanism as San1.

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

Performance and Reliability of Bonded Interfaces for High-Temperature Packaging. Annual Report

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

DeVoto, Douglas

2016-04-01

Current generation automotive power electronics packages utilize silicon devices and lead-free solder alloys. To meet stringent technical targets for 2020 and beyond (for cost, power density, specific power, efficiency and reliability), wide-bandgap devices are being considered since they offer advantages such as operation at higher frequencies, voltages, and temperatures. Traditional power electronics packages must be redesigned to utilize the full potential of wide-bandgap devices, and the die- and substrate-attach layers are key areas where new material development and validation is required. Present solder alloys do not meet the performance requirements for these new package designs while also meeting cost andmore » hazardous substance restrictions. Sintered silver (Ag) promises to meet the needs for die- and substrate-attach interfaces but synthesis optimization and reliability evaluation must be completed. Sintered Ag material was proposed as an alternative solution in power electronics packages almost 20 years back. However, synthesis pressure requirements up 40 MPa caused a higher complexity in the production process and more stringent flatness specifications for the substrates. Recently, several manufacturers have developed sintered Ag materials that require lower (3-5 MPa) or even no bonding pressures. Degradation mechanisms for these sintered Ag materials are not well known and need to be addressed. We are addressing these aspects to some extent in this project. We are developing generalized (i.e., independent of geometry) stress intensity factor versus cycles-to-failure relations for sintered Ag. Because sintered Ag is a relatively new material for automotive power electronics, the industry currently does not have a good understanding of recommended synthesis parameters or expected reliability under prescribed conditions. It is an important deliverable of this project to transfer findings to industry to eliminate barriers to using sintered Ag as a viable and commercialized die- and substrate-attach material. Only a few manufacturers produce sintered Ag pastes and may consider some processing conditions as proprietary. It is the goal of this project to openly explore and define best practices in order to impact the maximum number of power electronics module manufacturers and suppliers.« less

Genomic, proteomic, and biochemical analyses of oleaginous Mucor circinelloides: evaluating its capability in utilizing cellulolytic substrates for lipid production.

PubMed

Wei, Hui; Wang, Wei; Yarbrough, John M; Baker, John O; Laurens, Lieve; Van Wychen, Stefanie; Chen, Xiaowen; Taylor, Larry E; Xu, Qi; Himmel, Michael E; Zhang, Min

2013-01-01

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

An artificial transport metabolon facilitates improved substrate utilization in yeast.

PubMed

Thomik, Thomas; Wittig, Ilka; Choe, Jun-Yong; Boles, Eckhard; Oreb, Mislav

2017-11-01

Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

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

PubMed Central

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

2002-01-01

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

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

PubMed Central

Bakrania, Bhavisha; Granger, Joey P.; Harmancey, Romain

2016-01-01

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product. PMID:27768055

Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo.

PubMed

Harlow, Brittany E; Lawrence, Laurie M; Harris, Patricia A; Aiken, Glen E; Flythe, Michael D

2017-01-01

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition.

Exogenous lactobacilli mitigate microbial changes associated with grain fermentation (corn, oats, and wheat) by equine fecal microflora ex vivo

PubMed Central

Harlow, Brittany E.; Lawrence, Laurie M.; Harris, Patricia A.; Aiken, Glen E.

2017-01-01

Cereal grains are often included in equine diets. When starch intake exceeds foregut digestion starch will reach the hindgut, impacting microbial ecology. Probiotics (e.g., lactobacilli) are reported to mitigate GI dysbioses in other species. This study was conducted to determine the effect of exogenous lactobacilli on pH and the growth of amylolytic and lactate-utilizing bacteria. Feces were collected from 3 mature geldings fed grass hay with access to pasture. Fecal microbes were harvested by differential centrifugation, washed, and re-suspended in anaerobic media containing ground corn, wheat, or oats at 1.6% (w/v) starch and one of five treatments: Control (substrate only), L. acidophilus, L. buchneri, L. reuteri, or an equal mixture of all three (107 cells/mL, final concentration). After 24 h of incubation (37°C, 160 rpm), samples were collected for pH and enumerations of total amylolytics, Group D Gram-positive cocci (GPC; Enterococci, Streptococci), lactobacilli, and lactate-utilizing bacteria. Enumeration data were log transformed prior to ANOVA (SAS, v. 9.3). Lactobacilli inhibited pH decline in corn and wheat fermentations (P < 0.0001). Specifically, addition of either L. reuteri or L. acidophilus was most effective at mitigating pH decline with both corn and wheat fermentation, in which the greatest acidification occurred (P < 0.05). Exogenous lactobacilli decreased amylolytics, while increasing lactate-utilizers in corn and wheat fermentations (P < 0.0001). In oat fermentations, L. acidophilus and L. reuteri inhibited pH decline and increased lactate-utilizers while decreasing amylolytics (P < 0.0001). For all substrates, L. reuteri additions (regardless of viability) had the lowest number of GPC and the highest number of lactobacilli and lactate-utilizers (P < 0.05). There were no additive effects when lactobacilli were mixed. Exogenous lactobacilli decreased the initial (first 8 h) rate of starch catalysis when wheat was the substrate, but did not decrease total (24 h) starch utilization in any case. These results indicate that exogenous lactobacilli can impact the microbial community and pH of cereal grain fermentations by equine fecal microflora ex vivo. Additionally, dead (autoclaved) exogenous lactobacilli had similar effects as live lactobacilli on fermentation. This latter result indicates that the mechanism by which lactobacilli impact other amylolytic bacteria is not simple resource competition. PMID:28358885

Effect of lactose concentration on batch production of ethanol from cheese whey using Candida pseudotropicalis

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

Ghaly, A.E.; El-Taweel, A.A.

1995-07-01

The effect of lactose concentration on growth of Candida pseudotropicalis and ethanol production from cheese whey under batch conditions was investigated. Four initial lactose concentrations ranging from 50 to 200 g/L (5 to 20% wt/vol) were used. High concentration of lactose had an inhibitory effect on the specific growth rate, lactose utilization rate, and ethanol production rate. The maximum cell concentration was influenced by the initial substrate concentration as well as ethanol concentration. Inhibition of ethanol production was more pronounced at higher initial lactose concentrations. The maximum ethanol yield (96.6% of the theoretical yield) was achieved with the 100 g/Lmore » initial substrate concentration. The results indicated that pH control during alcohol fermentation of cheese whey is not necessary. 41 refs., 12 figs., 1 tab.« less

Method and apparatus for synthesis of arrays of DNA probes

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

Cerrina, Francesco; Sussman, Michael R.; Blattner, Frederick R.

The synthesis of arrays of DNA probes sequences, polypeptides, and the like is carried out using a patterning process on an active surface of a substrate. An image is projected onto the active surface of the substrate utilizing an image former that includes a light source that provides light to a micromirror device comprising an array of electronically addressable micromirrors, each of which can be selectively tilted between one of at least two positions. Projection optics receives the light reflected from the micromirrors along an optical axis and precisely images the micromirrors onto the active surface of the substrate, whichmore » may be used to activate the surface of the substrate. The first level of bases may then be applied to the substrate, followed by development steps, and subsequent exposure of the substrate utilizing a different pattern of micromirrors, with further repeats until the elements of a two dimensional array on the substrate surface have an appropriate base bound thereto. The micromirror array can be controlled in conjunction with a DNA synthesizer supplying appropriate reagents to a flow cell containing the active substrate to control the sequencing of images presented by the micromirror array in coordination of the reagents provided to the substrate.« less

Two-dimensional modeling of water and heat fluxes in green roof substrates

NASA Astrophysics Data System (ADS)

Suarez, F. I.; Sandoval, V. P.

2016-12-01

Due to public concern towards sustainable development, greenhouse gas emissions and energy efficiency, green roofs have become popular in the last years. Green roofs integrate vegetation into infrastructures to reach additional benefits that minimize negative impacts of the urbanization. A properly designed green roof can reduce environmental pollution, noise levels, energetic requirements or surface runoff. The correct performance of green roofs depends on site-specific conditions and on each component of the roof. The substrate and the vegetation layers strongly influence water and heat fluxes on a green roof. The substrate is an artificial media that has an improved performance compared to natural soils as it provides critical resources for vegetation survival: water, nutrients, and a growing media. Hence, it is important to study the effects of substrate properties on green roof performance. The objective of this work is to investigate how the thermal and hydraulic properties affect the behavior of a green roof through numerical modeling. The substrates that were investigated are composed by: crushed bricks and organic soil (S1); peat with perlite (S2); crushed bricks (S3); mineral soil with tree leaves (S4); and a mixture of topsoil and mineral soil (S5). The numerical model utilizes summer-arid meteorological information to evaluate the performance of each substrate. Results show that the area below the water retention curve helps to define the substrate that retains more water. In addition, the non-linearity of the water retention curve can increment the water needed to irrigate the roof. The heat propagation through the roof depends strongly on the hydraulic behavior, meaning that a combination of a substrate with low thermal conductivity and more porosity can reduce the heat fluxes across the roof. Therefore, it can minimize the energy consumed of an air-conditioner system.

Preparation and surface characterization of plasma-treated and biomolecular-micropatterned polymer substrates

NASA Astrophysics Data System (ADS)

Langowski, Bryan Alfred

A micropatterning process creates distinct microscale domains on substrate surfaces that differ from the surfaces' original chemical/physical properties. Numerous micropatterning methods exist, each having relative advantages and disadvantages in terms of cost, ease, reproducibility, and versatility. Polymeric surfaces micropatterned with biomolecules have many applications, but are specifically utilized in tissue engineering as cell scaffolds that attempt to controlled tissue generation in vivo and ex vivo. As the physical and chemical cues presented by micropatterned substrates control resulting cellular behavior, characterization of these cues via surface-sensitive analytical techniques is essential in developing cell scaffolds that mimic complex in vivo physicochemical environments. The initial focus of this thesis is the chemical and physical characterization of plasma-treated, microcontact-printed (muCP) polymeric substrates used to direct nerve cell behavior. Unmodified and oxygen plasma-treated poly(methyl methacrylate) (PMMA) substrates were analyzed by surface sensitive techniques to monitor plasma-induced chemical and physical modifications. Additionally, protein-micropattern homogeneity and size were microscopically evaluated. Lastly, poly(dimethylsiloxane) (PDMS) stamps and contaminated PMMA substrates were characterized by spectroscopic and microscopic methods to identify a contamination source during microcontact printing. The final focus of this thesis is the development of microscale plasma-initiated patterning (muPIP) as a versatile, reproducible micropatterning method. Using muPIP, polymeric substrates were micropatterned with several biologically relevant inks. Polymeric substrates were characterized following muPIP by surface-sensitive techniques to identify the technique's underlying physical and chemical bases. In addition, neural stem cell response to muPIP-generated laminin micropatterns was microscopically and biologically evaluated. Finally, enhanced versatility of muPIP in generating microscale poly-L-lysine gradients was demonstrated.

Protein export through the bacterial flagellar type III export pathway.

PubMed

Minamino, Tohru

2014-08-01

For construction of the bacterial flagellum, which is responsible for bacterial motility, the flagellar type III export apparatus utilizes both ATP and proton motive force across the cytoplasmic membrane and exports flagellar proteins from the cytoplasm to the distal end of the nascent structure. The export apparatus consists of a membrane-embedded export gate made of FlhA, FlhB, FliO, FliP, FliQ, and FliR and a water-soluble ATPase ring complex consisting of FliH, FliI, and FliJ. FlgN, FliS, and FliT act as substrate-specific chaperones that do not only protect their cognate substrates from degradation and aggregation in the cytoplasm but also efficiently transfer the substrates to the export apparatus. The ATPase ring complex facilitates the initial entry of the substrates into the narrow pore of the export gate. The export gate by itself is a proton-protein antiporter that uses the two components of proton motive force, the electric potential difference and the proton concentration difference, for different steps of the export process. A specific interaction of FlhA with FliJ located in the center of the ATPase ring complex allows the export gate to efficiently use proton motive force to drive protein export. The ATPase ring complex couples ATP binding and hydrolysis to its assembly-disassembly cycle for rapid and efficient protein export cycle. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. © 2013 Elsevier B.V. All rights reserved.

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms.

PubMed

Geerts, R; van Ginkel, C G; Plugge, C M

2017-04-01

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine, respectively. Both strains utilized all NAPPs tested as the sole source of carbon, nitrogen and energy for growth. Mineralization of NAPPs was independent of the alkyl chain length and the size of the polyamine moiety. NAPPs degraded in closed bottle tests (CBTs) using both river water and activated sludge. However, ready biodegradability of NAPPs with alkyl chain lengths of 16-18 carbon atoms and polyamine moieties with three and four nitrogen atoms could not be demonstrated. Biodegradation in the CBT was hampered by their limited bioavailability, making assessment of the true ready biodegradability of these highly adsorptive surfactants impossible. All NAPPs are therefore classified as readily biodegradable through read-across. Read-across is justified by the broad substrate specificity of NAPP-degrading microorganisms, their omnipresence and the mineralization of NAPPs.

Crossroads between Bacterial and Mammalian Glycosyltransferases

PubMed Central

Brockhausen, Inka

2014-01-01

Bacterial glycosyltransferases (GT) often synthesize the same glycan linkages as mammalian GT; yet, they usually have very little sequence identity. Nevertheless, enzymatic properties, folding, substrate specificities, and catalytic mechanisms of these enzyme proteins may have significant similarity. Thus, bacterial GT can be utilized for the enzymatic synthesis of both bacterial and mammalian types of complex glycan structures. A comparison is made here between mammalian and bacterial enzymes that synthesize epitopes found in mammalian glycoproteins, and those found in the O antigens of Gram-negative bacteria. These epitopes include Thomsen–Friedenreich (TF or T) antigen, blood group O, A, and B, type 1 and 2 chains, Lewis antigens, sialylated and fucosylated structures, and polysialic acids. Many different approaches can be taken to investigate the substrate binding and catalytic mechanisms of GT, including crystal structure analyses, mutations, comparison of amino acid sequences, NMR, and mass spectrometry. Knowledge of the protein structures and functions helps to design GT for specific glycan synthesis and to develop inhibitors. The goals are to develop new strategies to reduce bacterial virulence and to synthesize vaccines and other biologically active glycan structures. PMID:25368613

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

PubMed

Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

2015-11-17

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Efficient whole cell biocatalyst for formate-based hydrogen production.

PubMed

Kottenhahn, Patrick; Schuchmann, Kai; Müller, Volker

2018-01-01

Molecular hydrogen (H 2 ) is an attractive future energy carrier to replace fossil fuels. Biologically and sustainably produced H 2 could contribute significantly to the future energy mix. However, biological H 2 production methods are faced with multiple barriers including substrate cost, low production rates, and low yields. The C1 compound formate is a promising substrate for biological H 2 production, as it can be produced itself from various sources including electrochemical reduction of CO 2 or from synthesis gas. Many microbes that can produce H 2 from formate have been isolated; however, in most cases H 2 production rates cannot compete with other H 2 production methods. We established a formate-based H 2 production method utilizing the acetogenic bacterium Acetobacterium woodii . This organism can use formate as sole energy and carbon source and possesses a novel enzyme complex, the hydrogen-dependent CO 2 reductase that catalyzes oxidation of formate to H 2 and CO 2 . Cell suspensions reached specific formate-dependent H 2 production rates of 71 mmol g protein -1 h -1 (30.5 mmol g CDW -1 h -1 ) and maximum volumetric H 2 evolution rates of 79 mmol L -1 h -1 . Using growing cells in a two-step closed batch fermentation, specific H 2 production rates reached 66 mmol g CDW -1 h -1 with a volumetric H 2 evolution rate of 7.9 mmol L -1  h -1 . Acetate was the major side product that decreased the H 2 yield. We demonstrate that inhibition of the energy metabolism by addition of a sodium ionophore is suitable to completely abolish acetate formation. Under these conditions, yields up to 1 mol H 2 per mol formate were achieved. The same ionophore can be used in cultures utilizing formate as specific switch from a growing phase to a H 2 production phase. Acetobacterium woodii reached one of the highest formate-dependent specific H 2 productivity rates at ambient temperatures reported so far for an organism without genetic modification and converted the substrate exclusively to H 2 . This makes this organism a very promising candidate for sustainable H 2 production and, because of the reversibility of the A. woodii enzyme, also a candidate for reversible H 2 storage.

Measuring bioenergetics in T cells using a Seahorse Extracellular Flux Analyzer

PubMed Central

van der Windt, Gerritje J.W.; Chang, Chih-Hao; Pearce, Erika L.

2016-01-01

This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XFe96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XFe24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. PMID:27038461

Measuring Bioenergetics in T Cells Using a Seahorse Extracellular Flux Analyzer.

PubMed

van der Windt, Gerritje J W; Chang, Chih-Hao; Pearce, Erika L

2016-04-01

This unit contains several protocols to determine the energy utilization of T cells in real-time using a Seahorse Extracellular Flux Analyzer (http://www.seahorsebio.com). The advantages to using this machine over traditional metabolic assays include the simultaneous measurement of glycolysis and mitochondrial respiration, in real-time, on relatively small numbers of cells, without any radioactivity. The Basic Protocol describes a standard mitochondrial stress test on the XF(e) 96, which yields information about oxidative phosphorylation and glycolysis, two energy-generating pathways. The alternate protocols provide examples of adaptations to the Basic Protocol, including adjustments for the use of the XF(e) 24. A protocol for real-time bioenergetic responses to T cell activation allows for the analysis of immediate metabolic changes after T cell receptor stimulation. Specific substrate utilization can be determined by the use of differential assay media, or the injection of drugs that specifically affect certain metabolic processes. Accurate cell numbers, purity, and viability are critical to obtain reliable results. Copyright © 2016 John Wiley & Sons, Inc.

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

PubMed Central

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

2017-01-01

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

Acetate transport and utilization in the rat brain.

PubMed

Deelchand, Dinesh K; Shestov, Alexander A; Koski, Dee M; Uğurbil, Kâmil; Henry, Pierre-Gilles

2009-05-01

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-13C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR(ace)) increased progressively and reached close to saturation for plasma acetate concentration > 2-3 mM, whereas brain acetate concentration continued to increase. The Michaelis-Menten constant for brain acetate utilization (K(M)(util) = 0.01 +/- 0.14 mM) was much smaller than for acetate transport through the blood-brain barrier (BBB) (K(M)(t) = 4.18 +/- 0.83 mM). The maximum transport capacity of acetate through the BBB (V(max)(t) = 0.96 +/- 0.18 micromol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (V(max)(util) = 0.50 +/- 0.08 micromol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2-3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.

MEMS Based Acoustic Array

NASA Technical Reports Server (NTRS)

Sheplak, Mark (Inventor); Nishida, Toshikaza (Inventor); Humphreys, William M. (Inventor); Arnold, David P. (Inventor)

2006-01-01

Embodiments of the present invention described and shown in the specification aid drawings include a combination responsive to an acoustic wave that can be utilized as a dynamic pressure sensor. In one embodiment of the present invention, the combination has a substrate having a first surface and an opposite second surface, a microphone positioned on the first surface of the substrate and having an input and a first output and a second output, wherein the input receives a biased voltage, and the microphone generates an output signal responsive to the acoustic wave between the first output and the second output. The combination further has an amplifier positioned on the first surface of the substrate and having a first input and a second input and an output, wherein the first input of the amplifier is electrically coupled to the first output of the microphone and the second input of the amplifier is electrically coupled to the second output of the microphone for receiving the output sinual from the microphone. The amplifier is spaced from the microphone with a separation smaller than 0.5 mm.

Nuclear RNA Exosome at 3.1 Å Reveals Substrate Specificities, RNA Paths, and Allosteric Inhibition of Rrp44/Dis3.

PubMed

Zinder, John C; Wasmuth, Elizabeth V; Lima, Christopher D

2016-11-17

The eukaryotic RNA exosome is an essential and conserved 3'-to-5' exoribonuclease complex that degrades or processes nearly every class of cellular RNA. The nuclear RNA exosome includes a 9-subunit non-catalytic core that binds Rrp44 (Dis3) and Rrp6 subunits to modulate their processive and distributive 3'-to-5' exoribonuclease activities, respectively. Here we utilize an engineered RNA with two 3' ends to obtain a crystal structure of an 11-subunit nuclear exosome bound to RNA at 3.1 Å. The structure reveals an extended RNA path to Rrp6 that penetrates into the non-catalytic core; contacts between the non-catalytic core and Rrp44, which inhibit exoribonuclease activity; and features of the Rrp44 exoribonuclease site that support its ability to degrade 3' phosphate RNA substrates. Using reconstituted exosome complexes, we show that 3' phosphate RNA is not a substrate for Rrp6 but is readily degraded by Rrp44 in the nuclear exosome. Copyright © 2016 Elsevier Inc. All rights reserved.

Kinetics of substrate utilization and bacterial growth of crude oil degraded by Pseudomonas aeruginosa.

PubMed

Talaiekhozani, Amirreza; Jafarzadeh, Nematollah; Fulazzaky, Mohamad Ali; Talaie, Mohammad Reza; Beheshti, Masoud

2015-01-01

Pollution associated with crude oil (CO) extraction degrades the quality of waters, threatens drinking water sources and may ham air quality. The systems biology approach aims at learning the kinetics of substrate utilization and bacterial growth for a biological process for which very limited knowledge is available. This study uses the Pseudomonas aeruginosa to degrade CO and determines the kinetic parameters of substrate utilization and bacterial growth modeled from a completely mixed batch reactor. The ability of Pseudomonas aeruginosa can remove 91 % of the total petroleum hydrocarbons and 83 % of the aromatic compounds from oily environment. The value k of 9.31 g of substrate g(-1) of microorganism d(-1) could be far higher than the value k obtained for petrochemical wastewater treatment and that for municipal wastewater treatment. The production of new cells of using CO as the sole carbon and energy source can exceed 2(3) of the existing cells per day. The kinetic parameters are verified to contribute to improving the biological removal of CO from oily environment.

Biochemical characterization and substrate specificity of jojoba fatty acyl-CoA reductase and jojoba wax synthase.

PubMed

Miklaszewska, Magdalena; Banaś, Antoni

2016-08-01

Wax esters are used in industry for production of lubricants, pharmaceuticals and cosmetics. The only natural source of wax esters is jojoba oil. A much wider variety of industrial wax esters-containing oils can be generated through genetic engineering. Biotechnological production of tailor-made wax esters requires, however, a detailed substrate specificity of fatty acyl-CoA reductases (FAR) and wax synthases (WS), the two enzymes involved in wax esters synthesis. In this study we have successfully characterized the substrate specificity of jojoba FAR and jojoba WS. The genes encoding both enzymes were expressed heterologously in Saccharomyces cerevisiae and the activity of tested enzymes was confirmed by in vivo studies and in vitro assays using microsomal preparations from transgenic yeast. Jojoba FAR exhibited the highest in vitro activity toward 18:0-CoA followed by 20:1-CoA and 22:1-CoA. The activity toward other 11 tested acyl-CoAs was low or undetectable as with 18:2-CoA and 18:3-CoA. In assays characterizing jojoba WS combinations of 17 fatty alcohols with 14 acyl-CoAs were tested. The enzyme displayed the highest activity toward 14:0-CoA and 16:0-CoA in combination with C16-C20 alcohols as well as toward C18 acyl-CoAs in combination with C12-C16 alcohols. 20:1-CoA was efficiently utilized in combination with most of the tested alcohols. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

High Carbon Use Efficiency is Not Explained by Production of Storage Compounds

NASA Astrophysics Data System (ADS)

Dijkstra, Paul; van Groenigen, Kees-Jan

2015-04-01

The efficiency with which microbes use substrate to make new microbial biomass (Carbon Use Efficiency or CUE; mol C / mol C) is an important variable in soil and ecosystem C cycling models. Estimates of CUE in soil microbial communities vary widely. It has been hypothesized that high values of CUE are associated with production of storage compounds following a sudden increases in substrate availability during CUE measurements. In that case, these high CUE values would not be representative for balanced microbial growth (i.e. the production of all compounds needed to make new microbial cells). To test this hypothesis, we added position-specific 13C-labeled glucose isotopomers in parallel incubations of a ponderosa pine and piñon-juniper soil. We compared the measured pattern of CO2 release for the six glucose C atoms with patterns of CO2 production expected for balanced growth with a low, medium, or high CUE, and with CO2 production patterns associated with production of storage compounds (glycogen, lipids, or polyhydroxybutyrate). The measured position-specific CO2 production did not match that for production of glycogen, lipids, or polyhydroxybutyrate, but agreed closely with that expected for balanced growth at high CUE and high pentose phosphate pathway activity. We conclude that soil microbial communities utilize glucose substrate for biomass growth with high CUE, and that addition of small amounts of 13C-labeled glucose tracers do not affect CUE or induce storage compounds production. We submit that the measurement of position-specific CO2 production offers a quick and easy way to test biochemically explicit hypotheses concerning microbial growth metabolism.

Cultured representatives of two major phylogroups of human colonic Faecalibacterium prausnitzii can utilize pectin, uronic acids, and host-derived substrates for growth.

PubMed

Lopez-Siles, Mireia; Khan, Tanweer M; Duncan, Sylvia H; Harmsen, Hermie J M; Garcia-Gil, L Jesús; Flint, Harry J

2012-01-01

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution.

Cultured Representatives of Two Major Phylogroups of Human Colonic Faecalibacterium prausnitzii Can Utilize Pectin, Uronic Acids, and Host-Derived Substrates for Growth

PubMed Central

Lopez-Siles, Mireia; Khan, Tanweer M.; Duncan, Sylvia H.; Harmsen, Hermie J. M.; Garcia-Gil, L. Jesús

2012-01-01

Faecalibacterium prausnitzii is one of the most abundant commensal bacteria in the healthy human large intestine, but information on genetic diversity and substrate utilization is limited. Here, we examine the phylogeny, phenotypic characteristics, and influence of gut environmental factors on growth of F. prausnitzii strains isolated from healthy subjects. Phylogenetic analysis based on the 16S rRNA sequences indicated that the cultured strains were representative of F. prausnitzii sequences detected by direct analysis of fecal DNA and separated the available isolates into two phylogroups. Most F. prausnitzii strains tested grew well under anaerobic conditions on apple pectin. Furthermore, F. prausnitzii strains competed successfully in coculture with two other abundant pectin-utilizing species, Bacteroides thetaiotaomicron and Eubacterium eligens, with apple pectin as substrate, suggesting that this species makes a contribution to pectin fermentation in the colon. Many F. prausnitzii isolates were able to utilize uronic acids for growth, an ability previously thought to be confined to Bacteroides spp. among human colonic anaerobes. Most strains grew on N-acetylglucosamine, demonstrating an ability to utilize host-derived substrates. All strains tested were bile sensitive, showing at least 80% growth inhibition in the presence of 0.5 μg/ml bile salts, while inhibition at mildly acidic pH was strain dependent. These attributes help to explain the abundance of F. prausnitzii in the colonic community but also suggest factors in the gut environment that may limit its distribution. PMID:22101049

Planar/dpiX common military avionics AMLCDs: roadmap and production

NASA Astrophysics Data System (ADS)

Wanner, John; Gard, Allen; Roselle, Paul; Lewis, Alan

2000-08-01

This paper reviews the current production approach and status at Planar and dpiX utilizing a common design architecture within a family of cockpit AMLCD displays. The present status of low volume production requirements to support military applications, as well as the unique display formats and performance requirements dictated by the specific cockpit applications has resulted in a manufacturing approach requiring common TFT substrate design flexibility and the use of a common foundation for the assembly of AMLCD displays suitable for a variety of high performance military cockpits.

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

PubMed Central

Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

2012-01-01

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

Atmospheric pressure plasma jet for biomedical applications characterised by passive thermal probe

NASA Astrophysics Data System (ADS)

Mance, Diana; Wiese, Ruben; Kewitz, Thorben; Kersten, Holger

2018-05-01

Atmospheric pressure plasma jets (APPJs) are a promising tool in medicine with extensive possibilities of utilization. For a safe and therapeutically effective application of APPJs, it is necessary to know in detail the physical processes in plasma as well as possible hazards. In this paper, we focus on plasma thermal energy transferred to the substrate, i.e. to a passive thermal probe acting as substrate dummy. Specifically, we examined the dependence of transferred energy on the distance from the plasma source outlet, on the gas flow rate, and on the length of the visible plasma plume. The plasma plume is the plasma carried by the gas flow from the outlet of the source into the ambient air. The results show the distance between the plasma-generating device and the substrate to be the most important determinant of the transferred thermal energy, among the three examined variables. Most importantly for the end-user, the results also show this relation to be non-linear. To describe this relation, we chose a model based on a Boltzmann type of sigmoid function. Based on the results of our modelling and visual inspection of the plasma, we provide sort of a user guide for the adjustment of a suitable energy flux on the (bio) substrate.

Chemical biology-based approaches on fluorescent labeling of proteins in live cells.

PubMed

Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun

2013-05-01

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

A mathematical model for the interactive behavior of sulfate-reducing bacteria and methanogens during anaerobic digestion.

PubMed

Ahammad, S Ziauddin; Gomes, James; Sreekrishnan, T R

2011-09-01

Anaerobic degradation of waste involves different classes of microorganisms, and there are different types of interactions among them for substrates, terminal electron acceptors, and so on. A mathematical model is developed based on the mass balance of different substrates, products, and microbes present in the system to study the interaction between methanogens and sulfate-reducing bacteria (SRB). The performance of major microbial consortia present in the system, such as propionate-utilizing acetogens, butyrate-utilizing acetogens, acetoclastic methanogens, hydrogen-utilizing methanogens, and SRB were considered and analyzed in the model. Different substrates consumed and products formed during the process also were considered in the model. The experimental observations and model predictions showed very good prediction capabilities of the model. Model prediction was validated statistically. It was observed that the model-predicted values matched the experimental data very closely, with an average error of 3.9%.

Microbial Cellulose Utilization: Fundamentals and Biotechnology

PubMed Central

Lynd, Lee R.; Weimer, Paul J.; van Zyl, Willem H.; Pretorius, Isak S.

2002-01-01

Fundamental features of microbial cellulose utilization are examined at successively higher levels of aggregation encompassing the structure and composition of cellulosic biomass, taxonomic diversity, cellulase enzyme systems, molecular biology of cellulase enzymes, physiology of cellulolytic microorganisms, ecological aspects of cellulase-degrading communities, and rate-limiting factors in nature. The methodological basis for studying microbial cellulose utilization is considered relative to quantification of cells and enzymes in the presence of solid substrates as well as apparatus and analysis for cellulose-grown continuous cultures. Quantitative description of cellulose hydrolysis is addressed with respect to adsorption of cellulase enzymes, rates of enzymatic hydrolysis, bioenergetics of microbial cellulose utilization, kinetics of microbial cellulose utilization, and contrasting features compared to soluble substrate kinetics. A biological perspective on processing cellulosic biomass is presented, including features of pretreated substrates and alternative process configurations. Organism development is considered for “consolidated bioprocessing” (CBP), in which the production of cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired products occur in one step. Two organism development strategies for CBP are examined: (i) improve product yield and tolerance in microorganisms able to utilize cellulose, or (ii) express a heterologous system for cellulose hydrolysis and utilization in microorganisms that exhibit high product yield and tolerance. A concluding discussion identifies unresolved issues pertaining to microbial cellulose utilization, suggests approaches by which such issues might be resolved, and contrasts a microbially oriented cellulose hydrolysis paradigm to the more conventional enzymatically oriented paradigm in both fundamental and applied contexts. PMID:12209002

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

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

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

PubMed

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

2014-09-01

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

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

PubMed Central

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

1971-01-01

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

Chemical Vapor Deposition for Ultra-lightweight Thin-film Solar Arrays for Space

NASA Technical Reports Server (NTRS)

Hepp, Aloysius F.; Raffaelle, Ryne P.; Banger, Kulbinder K.; Jin, Michael H.; Lau, Janice E.; Harris, Jerry D.; Cowen, Jonathan E.; Duraj, Stan A.

2002-01-01

The development of thin-film solar cells on flexible, lightweight, space-qualified substrates provides an attractive cost solution to fabricating solar arrays with high specific power, (W/kg). The use of a polycrystalline chalcopyrite absorber layer for thin film solar cells is considered as the next generation photovoltaic devices. A key technical issues outlined in the 2001 U.S. Photovoltaic Roadmap, is the need to develop low cost, high throughput manufacturing for high-efficiency thin film solar cells. At NASA GRC we have focused on the development of new single-source-precursors (SSPs) and their utility to deposit the chalcopyrite semi-conducting layer (CIS) onto flexible substrates for solar cell fabrication. The syntheses and thermal modulation of SSPs via molecular engineering is described. Thin-film fabrication studies demonstrate the SSPs can be used in a spray CVD process, for depositing CIS at reduced temperatures, which display good electrical properties, suitable for PV devices.

Catalyst recognition of cis-1,2-diols enables site-selective functionalization of complex molecules

NASA Astrophysics Data System (ADS)

Sun, Xixi; Lee, Hyelee; Lee, Sunggi; Tan, Kian L.

2013-09-01

Carbohydrates and natural products serve essential roles in nature, and also provide core scaffolds for pharmaceutical agents and vaccines. However, the inherent complexity of these molecules imposes significant synthetic hurdles for their selective functionalization and derivatization. Nature has, in part, addressed these issues by employing enzymes that are able to orient and activate substrates within a chiral pocket, which increases dramatically both the rate and selectivity of organic transformations. In this article we show that similar proximity effects can be utilized in the context of synthetic catalysts to achieve general and predictable site-selective functionalization of complex molecules. Unlike enzymes, our catalysts apply a single reversible covalent bond to recognize and bind to specific functional group displays within substrates. By combining this unique binding selectivity and asymmetric catalysis, we are able to modify the less reactive axial positions within monosaccharides and natural products.

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Pattern Recognition of Adsorbing HP Lattice Proteins

NASA Astrophysics Data System (ADS)

Wilson, Matthew S.; Shi, Guangjie; Wüst, Thomas; Landau, David P.; Schmid, Friederike

2015-03-01

Protein adsorption is relevant in fields ranging from medicine to industry, and the qualitative behavior exhibited by course-grained models could shed insight for further research in such fields. Our study on the selective adsorption of lattice proteins utilizes the Wang-Landau algorithm to simulate the Hydrophobic-Polar (H-P) model with an efficient set of Monte Carlo moves. Each substrate is modeled as a square pattern of 9 lattice sites which attract either H or P monomers, and are located on an otherwise neutral surface. The fully enumerated set of 102 unique surfaces is simulated with each protein sequence. A collection of 27-monomer sequences is used- each of which is non-degenerate and protein-like. Thermodynamic quantities such as the specific heat and free energy are calculated from the density of states, and are used to investigate the adsorption of lattice proteins on patterned substrates. Research supported by NSF.

The influence of calcium supplementation on substrate metabolism during exercise in humans: a randomized controlled trial.

PubMed

Gonzalez, J T; Green, B P; Campbell, M D; Rumbold, P L S; Stevenson, E J

2014-06-01

High calcium intakes enhance fat loss under restricted energy intake. Mechanisms explaining this may involve reduced dietary fat absorption, enhanced lipid utilization and (or) reductions in appetite. This study aimed to assess the impact of 2 weeks of calcium supplementation on substrate utilization during exercise and appetite sensations at rest. Thirteen physically active males completed two 14-d supplemental periods, in a double-blind, randomized crossover design separated by a ⩾4-week washout period. During supplementation, a test-drink was consumed daily containing 400 and 1400 mg of calcium during control (CON) and high-calcium (CAL) periods, respectively. Cycling-based exercise tests were conducted before and after each supplemental period to determine substrate utilization rates and circulating metabolic markers (non-esterified fatty acid, glycerol, glucose and lactate concentrations) across a range of exercise intensities. Visual analog scales were completed in the fasting, rested state to determine subjective appetite sensations. No significant differences between supplements were observed in lipid or carbohydrate utilization rates, nor in circulating metabolic markers (both P>0.05). Maximum rates of lipid utilization were 0.47±0.05 and 0.44±0.05 g/min for CON and CAL, respectively, prior to supplementation and 0.44±0.05 and 0.42±0.05 g/min, respectively, post-supplementation (main effects of time, supplement and time x supplement interaction effect all P>0.05). Furthermore, no significant differences were detected in any subjective appetite sensations (all P>0.05). Two weeks of calcium supplementation does not influence substrate utilization during exercise in physically active males.

Characterization of two-step deglycosylation via oxidation by glycoside oxidoreductase and defining their subfamily

PubMed Central

Kim, Eun-Mi; Seo, Joo-Hyun; Baek, Kiheon; Kim, Byung-Gee

2015-01-01

Herein, we report a two-step deglycosylation mediated by the oxidation of glycoside which is different from traditional glycoside hydrolase (GH) mechanism. Previously, we reported a novel flavin adenine dinucleotide (FAD)-dependent glycoside oxidoreductase (FAD-GO) having deglycosylation activity. Various features of the reaction of FAD-GO such as including mechanism and catalytic residue and substrate specificity were studied. In addition, classification of novel FAD-GO subfamily was attempted. Deglycosylation of glycoside was performed spontaneously via oxidation of 3-OH of glycone moiety by FAD-GO mediated oxidation reaction. His493 residue was identified as a catalytic residue for the oxidation step. Interestingly, this enzyme has broad glycone and aglycon specificities. For the classification of FAD-GO enzyme subfamily, putative FAD-GOs were screened based on the FAD-GO from Rhizobium sp. GIN611 (gi 365822256) using BLAST search. The homologs of R. sp. GIN611 included the putative FAD-GOs from Stenotrophomonas strains, Sphingobacterium strains, Agrobacterium tumefaciens str. C58, and etc. All the cloned FAD-GOs from the three strains catalyzed the deglycosylation via enzymatic oxidation. Based on their substrate specificities, deglycosylation and oxidation activities to various ginsenosides, the FAD-GO subfamily members can be utilized as novel biocatalysts for the production of various aglycones. PMID:26057169

Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

PubMed

Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

2015-08-10

The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

Genomics and Transcriptomics Analyses of the Oil-Accumulating Basidiomycete Yeast Trichosporon oleaginosus: Insights into Substrate Utilization and Alternative Evolutionary Trajectories of Fungal Mating Systems

PubMed Central

Bracharz, Felix; Lorenzen, Jan; Kracht, Octavia N.; Chovatia, Mansi; Daum, Chris; Deshpande, Shweta; Lipzen, Anna; Nolan, Matt; Ohm, Robin A.; Grigoriev, Igor V.; Sun, Sheng; Heitman, Joseph

2015-01-01

ABSTRACT Microbial fermentation of agro-industrial waste holds great potential for reducing the environmental impact associated with the production of lipids for industrial purposes from plant biomass. However, the chemical complexity of many residues currently prevents efficient conversion into lipids, creating a high demand for strains with the ability to utilize all energy-rich components of agricultural residues. Here, we present results of genome and transcriptome analyses of Trichosporon oleaginosus. This oil-accumulating yeast is able to grow on a wide variety of substrates, including pentoses and N-acetylglucosamine, making it an interesting candidate for biotechnological applications. Transcriptomics shows specific changes in gene expression patterns under lipid-accumulating conditions. Furthermore, gene content and expression analyses indicate that T. oleaginosus is well-adapted for the utilization of chitin-rich biomass. We also focused on the T. oleaginosus mating type, because this species is a member of the Tremellomycetes, a group that has been intensively analyzed as a model for the evolution of sexual development, the best-studied member being Cryptococcus neoformans. The structure of the T. oleaginosus mating-type regions differs significantly from that of other Tremellomycetes and reveals a new evolutionary trajectory paradigm. Comparative analysis shows that recruitment of developmental genes to the ancestral tetrapolar mating-type loci occurred independently in the Trichosporon and Cryptococcus lineages, supporting the hypothesis of a trend toward larger mating-type regions in fungi. PMID:26199329

Long-term competition between sulfate reducing and methanogenic bacteria in UASB reactors treating volatile fatty acids.

PubMed

Omil, F; Lens, P; Visser, A; Hulshoff Pol, L W; Lettinga, G

1998-03-20

The competition between acetate utilizing methane-producing bacteria (MB) and sulfate-reducing bacteria (SRB) was studied in mesophilic (30 degrees C) upflow anaerobic sludge bed (UASB) reactors (upward velocity 1 m h-1; pH 8) treating volatile fatty acids and sulfate. The UASB reactors treated a VFA mixture (with an acetate:propionate:butyrate ratio of 5:3:2 on COD basis) or acetate as the sole substrate at different COD:sulfate ratios. The outcome of the competition was evaluated in terms of conversion rates and specific methanogenic and sulfidogenic activities. The COD:sulfate ratio was a key factor in the partitioning of acetate utilization between MB and SRB. In excess of sulfate (COD:sulfate ratio lower than 0.67), SRB became predominant over MB after prolonged reactor operation: 250 and 400 days were required to increase the amount of acetate used by SRB from 50 to 90% in the reactor treating, respectively, the VFA mixture or acetate as the sole substrate. The competition for acetate was further studied by dynamic simulations using a mathematical model based on the Monod kinetic parameters of acetate utilizing SRB and MB. The simulations confirmed the long term nature of the competition between these acetotrophs. A high reactor pH (+/-8), a short solid retention time (<150 days), and the presence of a substantial SRB population in the inoculum may considerably reduce the time required for acetate-utilising SRB to outcompete MB. Copyright 1998 John Wiley & Sons, Inc.

Glucose Oxidase-Mediated Polymerization as a Platform for Dual-Mode Signal Amplification and Biodetection

PubMed Central

Berron, Brad J; Johnson, Leah M; Ba, Xiao; McCall, Joshua D; Alvey, Nicholas J; Anseth, Kristi S; Bowman, Christopher N

2011-01-01

We report the first use of a polymerization-based ELISA substrate solution employing enzymatically mediated radical polymerization as a dual-mode amplification strategy. Enzymes are selectively coupled to surfaces to generate radicals that subsequently lead to polymerization-based amplification (PBA) and biodetection. Sensitivity and amplification of the polymerization-based detection system were optimized in a microwell strip format using a biotinylated microwell surface with a glucose oxidase (GOx)–avidin conjugate. The immobilized GOx is used to initiate polymerization, enabling the detection of the biorecognition event visually or through the use of a plate reader. Assay response is compared to that of an enzymatic substrate utilizing nitroblue tetrazolium in a simplified assay using biotinylated wells. The polymerization substrate exhibits equivalent sensitivity (2 µg/mL of GOx-avidin) and over three times greater signal amplification than this traditional enzymatic substrate since each radical that is enzymatically generated leads to a large number of polymerization events. Enzyme-mediated polymerization proceeds in an ambient atmosphere without the need for external energy sources, which is an improvement upon previous PBA platforms. Substrate formulations are highly sensitive to both glucose and iron concentrations at the lowest enzyme concentrations. Increases in amplification time correspond to higher assay sensitivities with no increase in non-specific signal. Finally, the polymerization substrate generated a signal to noise ratio of 14 at the detection limit (156 ng/mL) in an assay of transforming growth factor-beta. Biotechnol. Bioeng. 2011; 108:1521–1528. © 2011 Wiley Periodicals, Inc. PMID:21337335

PnuC and the utilization of the nicotinamide riboside analog 3-aminopyridine in Haemophilus influenzae.

PubMed

Sauer, Elizabeta; Merdanovic, Melisa; Mortimer, Anne Price; Bringmann, Gerhard; Reidl, Joachim

2004-12-01

The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

ANAEROBIC BIOTRANSFORMATION OF 2,4-DINITROTOLUENE WITH ETHANOL AS PRIMARY SUBSTRATE: MUTUAL EFFECT OF THE SUBSTRATES ON THEIR BIOTRANSFORMATION

EPA Science Inventory

The effect of the initial concentration of 2,4-dinitrotoluene (DNT) on its biotransformation and on the microbial utilization of ethanol was investigated. The culture used in this study was acclimated in a continuous flow laboratory fermentor with 2,4-DNT and ethanol as substrat...

Evaluation and Refinement of a Field-Portable Drinking Water Toxicity Sensor Utilizing Electric Cell-Substrate Impedance Sensing and a Fluidic Biochip

DTIC Science & Technology

2014-01-01

Potential interferences tested were chlorine and chloramine (commonly used for drinking water disinfection ), geosmin and 2-methyl-isoborneol (MIB...Protection Agency maximum residual disinfectant level for chlorine and chloramine is set at 4 mg l1 under the Safe Drinking Water Act and thus would...Evaluation and refinement of a field-portable drinking water toxicity sensor utilizing electric cell–substrate impedance sensing and a fluidic

Bacterial growth and substrate degradation by BTX-oxidizing culture in response to salt stress.

PubMed

Lee, Chi-Yuan; Lin, Ching-Hsing

2006-01-01

Interactions between microbial growth and substrate degradation are important in determining the performance of trickle-bed bioreactors (TBB), especially when salt is added to reduce biomass formation in order to alleviate media clogging. This study was aimed at quantifying salinity effects on bacterial growth and substrate degradation, and at acquiring kinetic information in order to improve the design and operation of TBB. Experiment works began by cultivating a mixed culture in a chemostat reactor receiving artificial influent containing a mixture of benzene, toluene, and xylene (BTX), followed by using the enrichment culture to degrade the individual BTX substrates under a particular salinity, which ranged 0-50 g l(-1) in batch mode. Then, the measured concentrations of biomass and residual substrate versus time were analyzed with the microbial kinetics; moreover, the obtained microbial kinetic constants under various salinities were modeled using noncompetitive inhibition kinetics. For the three substrates the observed bacterial yields appeared to be decreased from 0.51-0.74 to 0.20-0.22 mg mg(-1) and the maximum specific rate of substrate utilization, q, declined from 0.25-0.42 to 0.07-0.11 h(-1), as the salinity increased from 0 to 50 NaCl g l(-1). The NaCl acted as noncompetitive inhibitor, where the modeling inhibitions of the coefficients, K ( T(S)), were 22.7-29.7 g l(-1) for substrate degradation and K ( T(mu)), 13.0-19.0 g l(-1), for biomass formation. The calculated ratios for the bacterial maintenance rate, m (S), to q, further indicated that the percentage energy spent on maintenance increased from 19-24 to 86-91% as salinity level increased from 0 to 50 g l(-1). These results revealed that the bacterial growth was more inhibited than substrate degradation by the BTX oxidizers under the tested salinity levels. The findings from this study demonstrate the potential of applying NaCl salt to control excessive biomass formation in biotrickling filters.

Muscle interleukin-6 and fasting-induced PDH regulation in mouse skeletal muscle.

PubMed

Gudiksen, Anders; Bertholdt, Laerke; Vingborg, Mikkel Birkkjaer; Hansen, Henriette Watson; Ringholm, Stine; Pilegaard, Henriette

2017-03-01

Fasting prompts a metabolic shift in substrate utilization from carbohydrate to predominant fat oxidation in skeletal muscle, and pyruvate dehydrogenase (PDH) is seen as a controlling link between the competitive oxidation of carbohydrate and fat during metabolic challenges like fasting. Interleukin (IL)-6 has been proposed to be released from muscle with concomitant effects on both glucose and fat utilization. The aim was to test the hypothesis that muscle IL-6 has a regulatory impact on fasting-induced suppression of skeletal muscle PDH. Skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and floxed littermate controls (control) were either fed or fasted for 6 or 18 h. Lack of muscle IL-6 elevated the respiratory exchange ratio in the fed and early fasting state, but not with prolonged fasting. Activity of PDH in the active form (PDHa) was higher in fed and fasted IL-6 MKO than in control mice at 18 h, but not at 6 h, whereas lack of muscle IL-6 did not prevent downregulation of PDHa activity in skeletal muscle or changes in plasma and muscle substrate levels in response to 18 h of fasting. Phosphorylation of three of four sites on PDH-E1α increased with 18 h of fasting, but was lower in IL-6 MKO mice than in control. In addition, both PDK4 mRNA and protein increased with 6 and 18 h of fasting in both genotypes, but PDK4 protein was lower in IL-6 MKO than in control. In conclusion, skeletal muscle IL-6 seems to regulate whole body substrate utilization in the fed, but not fasted, state and influence skeletal muscle PDHa activity in a circadian manner. However, skeletal muscle IL-6 is not required for maintaining metabolic flexibility in response to fasting. Copyright © 2017 the American Physiological Society.

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

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

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

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

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE PAGES

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

2017-02-01

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

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

PubMed

Kim, Doyoun; San, Boi Hoa; Moh, Sang Hyun; Park, Hyejin; Kim, Dong Young; Lee, Sangho; Kim, Kyeong Kyu

2010-01-01

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. Copyright 2009 Elsevier Inc. All rights reserved.

A biosensor platform for rapid detection of E. coli in drinking water.

PubMed

Hesari, Nikou; Alum, Absar; Elzein, Mohamad; Abbaszadegan, Morteza

2016-02-01

There remains a need for rapid, specific and sensitive assays for the detection of bacterial indicators for water quality monitoring. In this study, a strategy for rapid detection of Escherichia coli in drinking water has been developed. This strategy is based on the use of the substrate 4-methylumbelliferyl-β-d-glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-d-glucuronidase (GUD) enzyme to yield a fluorogenic 4-methylumbelliferone (4-MU) product that can be quantified and related to the number of E. coli cells present in water samples. In this study, the detection time required for the biosensor response ranged between 20 and 120 min, depending on the number of bacteria in the sample. This approach does not need extensive sample processing with a rapid detection capability. The specificity of the MUG substrate was examined in both, pure cultures of non-target bacterial genera such as Klebsiella, Salmonella, Enterobacter and Bacillus. Non-target substrates that included 4-methylumbelliferyl-β-d-galactopyranoside (MUGal) and l-leucine β-naphthylamide aminopeptidase (LLβ-N) were also investigated to identify nonspecific patterns of enzymatic activities in E. coli. GUD activity was found to be specific for E. coli and no further enzymatic activity was detected by other species. In addition, fluorescence assays were performed for the detection of E. coli to generate standard curves; and the sensitivity of the GUD enzymatic response was measured and repeatedly determined to be less than 10 E. coli cells in a reaction vial. The applicability of the method was tested by performing multiple fluorescence assays under pure and mixed bacterial flora in environmental samples. The results of this study showed that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water. Copyright © 2015 Elsevier Inc. All rights reserved.

Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

ERIC Educational Resources Information Center

Kin, Ng Hong; Ling, Tan Aik

2016-01-01

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

Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria

PubMed Central

Reay, David S.; Nedwell, David B.; Priddle, Julian; Ellis-Evans, J. Cynan

1999-01-01

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. PMID:10347046

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

PubMed

Kong, Muwen; Beckwitt, Emily C; Springall, Luke; Kad, Neil M; Van Houten, Bennett

2017-01-01

Single-molecule approaches to solving biophysical problems are powerful tools that allow static and dynamic real-time observations of specific molecular interactions of interest in the absence of ensemble-averaging effects. Here, we provide detailed protocols for building an experimental system that employs atomic force microscopy and a single-molecule DNA tightrope assay based on oblique angle illumination fluorescence microscopy. Together with approaches for engineering site-specific lesions into DNA substrates, these complementary biophysical techniques are well suited for investigating protein-DNA interactions that involve target-specific DNA-binding proteins, such as those engaged in a variety of DNA repair pathways. In this chapter, we demonstrate the utility of the platform by applying these techniques in the studies of proteins participating in nucleotide excision repair. © 2017 Elsevier Inc. All rights reserved.

Applications of Microbial Cell Sensors

NASA Astrophysics Data System (ADS)

Shimomura-Shimizu, Mifumi; Karube, Isao

Since the first microbial cell sensor was studied by Karube et al. in 1977, many types of microbial cell sensors have been developed as analytical tools. The microbial cell sensor utilizes microbes as a sensing element and a transducer. The characteristics of microbial cell sensors as sensing devices are a complete contrast to those of enzyme sensors or immunosensors, which are highly specific for the substrates of interest, although the specificity of the microbial cell sensor has been improved by genetic modification of the microbe used as the sensing element. Microbial cell sensors have the advantages of tolerance to measuring conditions, a long lifetime, and good cost performance, and have the disadvantage of a long response time. In this review, applications of microbial cell sensors are summarized.

Homogeneous purification and characterization of LePGT1--a membrane-bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon.

PubMed

Ohara, Kazuaki; Mito, Koji; Yazaki, Kazufumi

2013-06-01

Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family. © 2013 The Authors Journal compilation © 2013 FEBS.

High-precision robotic microcontact printing (R-μCP) utilizing a vision guided selectively compliant articulated robotic arm.

PubMed

McNulty, Jason D; Klann, Tyler; Sha, Jin; Salick, Max; Knight, Gavin T; Turng, Lih-Sheng; Ashton, Randolph S

2014-06-07

Increased realization of the spatial heterogeneity found within in vivo tissue microenvironments has prompted the desire to engineer similar complexities into in vitro culture substrates. Microcontact printing (μCP) is a versatile technique for engineering such complexities onto cell culture substrates because it permits microscale control of the relative positioning of molecules and cells over large surface areas. However, challenges associated with precisely aligning and superimposing multiple μCP steps severely limits the extent of substrate modification that can be achieved using this method. Thus, we investigated the feasibility of using a vision guided selectively compliant articulated robotic arm (SCARA) for μCP applications. SCARAs are routinely used to perform high precision, repetitive tasks in manufacturing, and even low-end models are capable of achieving microscale precision. Here, we present customization of a SCARA to execute robotic-μCP (R-μCP) onto gold-coated microscope coverslips. The system not only possesses the ability to align multiple polydimethylsiloxane (PDMS) stamps but also has the capability to do so even after the substrates have been removed, reacted to graft polymer brushes, and replaced back into the system. Plus, non-biased computerized analysis shows that the system performs such sequential patterning with <10 μm precision and accuracy, which is equivalent to the repeatability specifications of the employed SCARA model. R-μCP should facilitate the engineering of complex in vivo-like complexities onto culture substrates and their integration with microfluidic devices.

Parametric studies of metabolic cooperativity in Escherichia coli colonies: Strain and geometric confinement effects

PubMed Central

Cole, John A.; Luthey-Schulten, Zaida

2017-01-01

Characterizing the complex spatial and temporal interactions among cells in a biological system (i.e. bacterial colony, microbiome, tissue, etc.) remains a challenge. Metabolic cooperativity in these systems can arise due to the subtle interplay between microenvironmental conditions and the cells’ regulatory machinery, often involving cascades of intra- and extracellular signalling molecules. In the simplest of cases, as demonstrated in a recent study of the model organism Escherichia coli, metabolic cross-feeding can arise in monoclonal colonies of bacteria driven merely by spatial heterogeneity in the availability of growth substrates; namely, acetate, glucose and oxygen. Another recent study demonstrated that even closely related E. coli strains evolved different glucose utilization and acetate production capabilities, hinting at the possibility of subtle differences in metabolic cooperativity and the resulting growth behavior of these organisms. Taking a first step towards understanding the complex spatio-temporal interactions within microbial populations, we performed a parametric study of E. coli growth on an agar substrate and probed the dependence of colony behavior on: 1) strain-specific metabolic characteristics, and 2) the geometry of the underlying substrate. To do so, we employed a recently developed multiscale technique named 3D dynamic flux balance analysis which couples reaction-diffusion simulations with iterative steady-state metabolic modeling. Key measures examined include colony growth rate and shape (height vs. width), metabolite production/consumption and concentration profiles, and the emergence of metabolic cooperativity and the fractions of cell phenotypes. Five closely related strains of E. coli, which exhibit large variation in glucose consumption and organic acid production potential, were studied. The onset of metabolic cooperativity was found to vary substantially between these five strains by up to 10 hours and the relative fraction of acetate utilizing cells within the colonies varied by a factor of two. Additionally, growth with six different geometries designed to mimic those that might be found in a laboratory, a microfluidic device, and inside a living organism were considered. Geometries were found to have complex, often nonlinear effects on colony growth and cross-feeding with “hard” features resulting in larger effect than “soft” features. These results demonstrate that strain-specific features and spatial constraints imposed by the growth substrate can have significant effects even for microbial populations as simple as isogenic E. coli colonies. PMID:28820904

Parametric studies of metabolic cooperativity in Escherichia coli colonies: Strain and geometric confinement effects.

PubMed

Peterson, Joseph R; Cole, John A; Luthey-Schulten, Zaida

2017-01-01

Characterizing the complex spatial and temporal interactions among cells in a biological system (i.e. bacterial colony, microbiome, tissue, etc.) remains a challenge. Metabolic cooperativity in these systems can arise due to the subtle interplay between microenvironmental conditions and the cells' regulatory machinery, often involving cascades of intra- and extracellular signalling molecules. In the simplest of cases, as demonstrated in a recent study of the model organism Escherichia coli, metabolic cross-feeding can arise in monoclonal colonies of bacteria driven merely by spatial heterogeneity in the availability of growth substrates; namely, acetate, glucose and oxygen. Another recent study demonstrated that even closely related E. coli strains evolved different glucose utilization and acetate production capabilities, hinting at the possibility of subtle differences in metabolic cooperativity and the resulting growth behavior of these organisms. Taking a first step towards understanding the complex spatio-temporal interactions within microbial populations, we performed a parametric study of E. coli growth on an agar substrate and probed the dependence of colony behavior on: 1) strain-specific metabolic characteristics, and 2) the geometry of the underlying substrate. To do so, we employed a recently developed multiscale technique named 3D dynamic flux balance analysis which couples reaction-diffusion simulations with iterative steady-state metabolic modeling. Key measures examined include colony growth rate and shape (height vs. width), metabolite production/consumption and concentration profiles, and the emergence of metabolic cooperativity and the fractions of cell phenotypes. Five closely related strains of E. coli, which exhibit large variation in glucose consumption and organic acid production potential, were studied. The onset of metabolic cooperativity was found to vary substantially between these five strains by up to 10 hours and the relative fraction of acetate utilizing cells within the colonies varied by a factor of two. Additionally, growth with six different geometries designed to mimic those that might be found in a laboratory, a microfluidic device, and inside a living organism were considered. Geometries were found to have complex, often nonlinear effects on colony growth and cross-feeding with "hard" features resulting in larger effect than "soft" features. These results demonstrate that strain-specific features and spatial constraints imposed by the growth substrate can have significant effects even for microbial populations as simple as isogenic E. coli colonies.

Energetics and Application of Heterotrophy in Acetogenic Bacteria.

PubMed

Schuchmann, Kai; Müller, Volker

2016-07-15

Acetogenic bacteria are a diverse group of strictly anaerobic bacteria that utilize the Wood-Ljungdahl pathway for CO2 fixation and energy conservation. These microorganisms play an important part in the global carbon cycle and are a key component of the anaerobic food web. Their most prominent metabolic feature is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates. However, most members also show an outstanding metabolic flexibility for utilizing a vast variety of different substrates. In contrast to autotrophic growth, which is hardly competitive, metabolic flexibility is seen as a key ability of acetogens to compete in ecosystems and might explain the almost-ubiquitous distribution of acetogenic bacteria in anoxic environments. This review covers the latest findings with respect to the heterotrophic metabolism of acetogenic bacteria, including utilization of carbohydrates, lactate, and different alcohols, especially in the model acetogen Acetobacterium woodii Modularity of metabolism, a key concept of pathway design in synthetic biology, together with electron bifurcation, to overcome energetic barriers, appears to be the basis for the amazing substrate spectrum. At the same time, acetogens depend on only a relatively small number of enzymes to expand the substrate spectrum. We will discuss the energetic advantages of coupling CO2 reduction to fermentations that exploit otherwise-inaccessible substrates and the ecological advantages, as well as the biotechnological applications of the heterotrophic metabolism of acetogens. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Energetics and Application of Heterotrophy in Acetogenic Bacteria

PubMed Central

Schuchmann, Kai

2016-01-01

Acetogenic bacteria are a diverse group of strictly anaerobic bacteria that utilize the Wood-Ljungdahl pathway for CO2 fixation and energy conservation. These microorganisms play an important part in the global carbon cycle and are a key component of the anaerobic food web. Their most prominent metabolic feature is autotrophic growth with molecular hydrogen and carbon dioxide as the substrates. However, most members also show an outstanding metabolic flexibility for utilizing a vast variety of different substrates. In contrast to autotrophic growth, which is hardly competitive, metabolic flexibility is seen as a key ability of acetogens to compete in ecosystems and might explain the almost-ubiquitous distribution of acetogenic bacteria in anoxic environments. This review covers the latest findings with respect to the heterotrophic metabolism of acetogenic bacteria, including utilization of carbohydrates, lactate, and different alcohols, especially in the model acetogen Acetobacterium woodii. Modularity of metabolism, a key concept of pathway design in synthetic biology, together with electron bifurcation, to overcome energetic barriers, appears to be the basis for the amazing substrate spectrum. At the same time, acetogens depend on only a relatively small number of enzymes to expand the substrate spectrum. We will discuss the energetic advantages of coupling CO2 reduction to fermentations that exploit otherwise-inaccessible substrates and the ecological advantages, as well as the biotechnological applications of the heterotrophic metabolism of acetogens. PMID:27208103

Fast self-assembly of silver nanoparticle monolayer in hydrophobic environment and its application as SERS substrate

NASA Astrophysics Data System (ADS)

Leiterer, Christian; Zopf, David; Seise, Barbara; Jahn, Franka; Weber, Karina; Popp, Jürgen; Cialla-May, Dana; Fritzsche, Wolfgang

2014-09-01

We present a method which allows the straightforward wet-chemical synthesis of silver nanoparticles (AgNPs), hydrophobic coating assembling into monolayer, and their utilization as substrates for surface-enhanced Raman spectroscopy (SERS). In order to fabricate the SERS-active substrates, AgNPs were synthesized in water by chemical reduction of Ag+, coated with a hydrophobic shell (dodecanethiol), transferred to a non-polar solvent, and finally assembled through precipitation into a SERS-active self-assembled monolayer (SAM). Simple approaches for concentration and purification of the coated AgNPs are shown. The synthesized particles and SAMs were characterized by transmission electron microscopy, optical imaging, and spectroscopic measurements. This manuscript can be used as a do-it-yourself (DIY) tutorial which allows making SAMs from coated AgNPs (<15 nm) in every laboratory within less than 1 h and their utilization as potential low-cost SERS substrates (movie 1-4).

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

PubMed

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

2012-07-01

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

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

PubMed

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

2014-04-01

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

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis.

PubMed

Ang, Ee L; Obbard, Jeffrey P; Zhao, Huimin

2007-02-01

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

Modeling hexavalent chromium removal in a Bacillus sp. fixed-film bioreactor.

PubMed

Nkhalambayausi-Chirwa, Evans M; Wang, Yi-Tin

2004-09-30

A one-dimensional diffusion-reaction model was developed to simulate Cr(VI) reduction in a Bacillus sp. pure culture biofilm reactor with glucose as a sole supplied carbon and energy source. Substrate utilization and Cr(VI) reduction in the biofilm was best represented by a system of (second-order) partial differential equations (PDEs). The PDE system was solved by the (fourth-order) Runge-Kutta method adjusted for mass transport resistance using the (second-order) Crank-Nicholson and Backward Euler finite difference methods. A heuristic procedure (genetic search algorithm) was used to find global optimum values of Cr(VI) reduction and substrate utilization rate kinetic parameters. The fixed-film bioreactor system yielded higher values of the maximum specific Cr(VI) reduction rate coefficient and Cr(VI) reduction capacity (kmc = 0.062 1/h, and Rc = 0.13 mg/mg, respectively) than previously determined in batch reactors (kmc = 0.022 1/h and Rc = 0.012 mg/mg). The model predicted effluent Cr(VI) concentration well with 98.9% confidence (sigmay2 = 2.37 mg2/L2, N = 119) and effluent glucose with 96.4 % confidence (sigmay(w)2 = 5402 mg2/L2, N = 121, w = 100) over a wide range of Cr(VI) loadings (10-498 mg Cr(VI)/L/d). Copyright 2004 Wiley Periodicals, Inc.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Thick-film nickel-metal-hydride battery based on porous ceramic substrates

NASA Astrophysics Data System (ADS)

Do, Jing-Shan; Yu, Sen-Hao; Cheng, Suh-Fen

Nickel-metal-hydride (Ni-MH) batteries are prepared with thick-film and thin-film technologies based on porous ceramic substrates. The porosity and the mean pore diameter of BP ceramic substrates prepared from the argils increases from 19.81% and 0.0432 μm to 29.81% and 0.224 μm, respectively, upon increasing the ethyl cellulose content in the BP argil from 0 to 0.79%. The pore diameter of Al 2O 3 substrates prepared from Al 2O 3 powder is mainly distributed in the range 0.01-0.5 μm. The distribution of the pore diameters of BP ceramic substrates lies in two ranges, namely: 0.04-2 μm and 10-300 μm. Using BP ceramic plates and Al 2O 3 plates as substrates to fabricate thick-film Ni-MH batteries, the optimal electroactive material utilization in the batteries is 77.0 and 71.1%, respectively. On increasing the screen-printing number for preparing the cathode (Ni(OH) 2) from 1 to 3, the discharge capacity of the thick-film battery increases from 0.2917 to 0.7875 mAh, and the utilization in the battery decreases from 71.0 to 53.0%.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

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

Force-controlled inorganic crystallization lithography.

PubMed

Cheng, Chao-Min; LeDuc, Philip R

2006-09-20

Lithography plays a key role in integrated circuits, optics, information technology, biomedical applications, catalysis, and separation technologies. However, inorganic lithography techniques remain of limited utility for applications outside of the typical foci of integrated circuit manufacturing. In this communication, we have developed a novel stamping method that applies pressure on the upper surface of the stamp to regulate the dewetting process of the inorganic buffer and the evaporation rate of the solvent in this buffer between the substrate and the surface of the stamp. We focused on generating inorganic microstructures with specific locations and also on enabling the ability to pattern gradients during the crystallization of the inorganic salts. This approach utilized a combination of lithography with bottom-up growth and assembly of inorganic crystals. This work has potential applications in a variety of fields, including studying inorganic material patterning and small-scale fabrication technology.

Controlled Spalling in (100)-Oriented Germanium by Electroplating

NASA Astrophysics Data System (ADS)

Crouse, Dustin Ray

This work investigates controlled spalling as a method to exfoliate thin films of various thickness from rigid, crystalline germanium (Ge) substrates and to enable substrate reuse for III-V single junction photovoltaic devices. Technological limitations impeding wide-spread adoption of flexible electronics and high-material-cost photovoltaic devices have motivated significant interest in a method to remove devices from their substrates. DC magnetron sputtering has been previously utilized to remove semiconductor devices of various thicknesses from Ge substrates, but this method is expensive and time-consuming. Controlled spalling via high-speed electrodeposition is a fast, inexpensive exfoliation method that utilizes a tensile-stressed metal layer deposited on a (100)-oriented Ge substrate and an external force to mechanically propagate a crack parallel to the surface at a desired depth in the substrate material. Suo and Hutchinson's quantitative models describe critical combinations of film thickness and strain mismatch between a film and substrate at which a stressed bilayer system spontaneously spalls; however, fine control over a wide steady-state spall depth range has been limited by the ability to experimentally tailor strain mismatch caused by residual stress within deposited stressor layers. This work investigates the effect of tuning electroplating current density and electrolyte chemistry on the residual stress in a nickel stressor film and their impact on the achievable spall depth range. Steady-state spall depth is found to increase with increasing stressor layer thickness and decrease with increasing residual stress. By tailoring residual stress through adjusting plating conditions and the electrolyte's phosphorous concentration, wide control over spall depth within Ge substrates from sub-micron to 76microm-thicknesses were achieved. To assess the viability of utilizing controlled spalling for substrate reuse, this dissertation demonstrates the first III-V solar cells (GaInAsP, Eg 1.7 eV) grown directly on a spalled-Ge substrate without any additional surface preparation. Widespread adoption of high-efficiency III-V solar cells has been limited by expensive deposition processes and high material cost of substrates. Substrate reuse offers a promising route towards enabling III-V devices to become cost-competitive for one-sun terrestrial applications. In this study, the quality of spalled Ge surfaces is characterized to assess lattice matching capability between the device layer materials and the substrate. GaAs films grown on spalled Ge substrates by hydride vapor phase epitaxy were single-crystal in nature. III-V solar cells grown on spalled and pristine Ge substrates show nearly equivalent efficiency of 8%, despite the roughness of the spalled-Ge substrate. Principles of fractography were used to deduce that surface roughness originated from non-uniform crack propagation and mixed-mode loading during the spalling process.

Characterization of acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme of human small intestine.

PubMed

Hiramine, Yasushi; Tanabe, Toshizumi

2011-06-01

Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) enzyme plays a significant role in dietary triacylglycerol (TAG) absorption in the small intestine. However, the characteristics of human intestinal DGAT enzyme have not been examined in detail. The aim of our study was to characterize the human intestinal DGAT enzyme by examining acyl-CoA specificity, temperature dependency, and selectivity for 1,2-diacylglycerol (DAG) or 1,3-DAG. We detected DGAT activity of human intestinal microsome and found that the acyl-CoA specificity and temperature dependency of intestinal DGAT coincided with those of recombinant human DGAT1. To elucidate the selectivity of human intestinal DGAT to 1,2-DAG or 1,3-DAG, we conducted acyl-coenzyme A:monoacylglycerol acyltransferase assays using 1- or 2-monoacylglycerol (MAG) as substrates. When 2-MAG was used as acyl acceptor, both 1,2-DAG and TAG were generated; however, when 1-MAG was used, 1,3-DAG was predominantly observed and little TAG was detected. These findings suggest that human small intestinal DGAT, which is mainly encoded by DGAT1, utilizes 1,2-DAG as the substrate to form TAG. This study will contribute to understand the lipid absorption profile in the small intestine.

Why the impact of mechanical stimuli on stem cells remains a challenge.

PubMed

Goetzke, Roman; Sechi, Antonio; De Laporte, Laura; Neuss, Sabine; Wagner, Wolfgang

2018-05-04

Mechanical stimulation affects growth and differentiation of stem cells. This may be used to guide lineage-specific cell fate decisions and therefore opens fascinating opportunities for stem cell biology and regenerative medicine. Several studies demonstrated functional and molecular effects of mechanical stimulation but on first sight these results often appear to be inconsistent. Comparison of such studies is hampered by a multitude of relevant parameters that act in concert. There are notorious differences between species, cell types, and culture conditions. Furthermore, the utilized culture substrates have complex features, such as surface chemistry, elasticity, and topography. Cell culture substrates can vary from simple, flat materials to complex 3D scaffolds. Last but not least, mechanical forces can be applied with different frequency, amplitude, and strength. It is therefore a prerequisite to take all these parameters into consideration when ascribing their specific functional relevance-and to only modulate one parameter at the time if the relevance of this parameter is addressed. Such research questions can only be investigated by interdisciplinary cooperation. In this review, we focus particularly on mesenchymal stem cells and pluripotent stem cells to discuss relevant parameters that contribute to the kaleidoscope of mechanical stimulation of stem cells.

Gene cloning and characterization of arylamine N-acetyltransferase from Bacillus cereus strain 10-L-2.

PubMed

Takenaka, Shinji; Cheng, Minyi; Mulyono; Koshiya, Atsushi; Murakami, Shuichiro; Aoki, Kenji

2009-01-01

Bacillus cereus strain 10-L-2 synthesizes two arylamine N-acetyltransferases (Nat-a and Nat-b) with broad substrate specificities toward aniline and its derivatives. In southern blot analysis using probes encoding the NH2-terminus of Nat-b and a conserved region of N-acetyltransferases, digested total DNA of strain 10-L-2 showed one positive band. We cloned and sequenced the gene encoding Nat-b. The NH2-terminal amino acid sequence predicted from the open reading frame (768 base pairs) corresponded to that of purified Nat-b. The cloned Nat-b gene was expressed in Escherichia coli. The expressed enzyme (BcNAT) from the recombinant strain was partially purified and characterized. Nat-b from strain 10-L-2 and BcNAT from the recombinant strain were slightly different from each others in substrate specificity and thermo-stability. We examined the biotransformations of 2-aminophenols and phenylenediamines by the whole cells of the recombinant strain. The cells converted these compounds into their corresponding acetanilides. Only one amino group of phenylenediamines was acetylated. The cells utilized 4-nitroacetanilide as an acetyl donor instead of acetyl-CoA. 4-Aminoacetanilide was produced and 4-nitroaniline was released almost stoichiometrically.

N-mustard analogs of S-adenosyl-L-methionine as biochemical probes of protein arginine methylation.

PubMed

Hymbaugh Bergman, Sarah J; Comstock, Lindsay R

2015-08-01

Nucleosomes, the fundamental building blocks of eukaryotic chromatin, undergo post-synthetic modifications and play a major role in the regulation of transcriptional processes. Combinations of these modifications, including methylation, regulate chromatin structure, determining its different functional states and playing a central role in differentiation. The biological significance of cellular methylation, particularly on chromatin, is widely recognized, yet we know little about the mechanisms that link biological methylation events. To characterize and fully understand protein methylation, we describe here novel N-mustard analogs of S-adenosyl-l-methionine (SAM) as biochemical tools to better understand protein arginine methylation events using protein arginine methyltransferase 1 (PRMT1). Specifically, azide- and alkyne-functionalized N-mustard analogs serve as cofactor mimics of SAM and are enzymatically transferred to a model peptide substrate in a PRMT1-dependent fashion. Once incorporated, the resulting alkynes and azides can be modified through chemoselective ligations, including click chemistry and the Staudinger ligation. These results readily demonstrate the feasibility of utilizing N-mustard analogs as biochemical tools to site-specifically label substrates of PRMT1 and serve as an alternative approach to study protein methylation events. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

Carbon source utilization patterns of Bacillus simplex ecotypes do not reflect their adaptation to ecologically divergent slopes in 'Evolution Canyon', Israel.

PubMed

Sikorski, Johannes; Pukall, Rüdiger; Stackebrandt, Erko

2008-10-01

The 'Evolution Canyons' I and II in Israel are model habitats to study adaptation and speciation of bacteria in the environment. These canyons represent similar ecological replicates, separated by 40 km, with a strongly sun-exposed and hot 'African' south-facing slope (SFS) vs. a cooler and mesic-lush 'European' north-facing slope (NFS). Previously, among 131 Bacillus simplex isolates, distinct genetic lineages (ecotypes), each specific for either SFS or NFS, were identified, suggesting a temperature-driven slope-specific adaptation. Here, we asked whether the ecological heterogeneity of SFS vs. NFS also affected carbon utilization abilities, as determined using the Biolog assay. Contrary to expectation, a correlation between substrate utilization patterns and the ecological origin of strains was not found. Rather, the patterns split according to the two major phylogenetic lineages each of which contain SFS and NFS ecotypes. We conclude that traits related to the general energy metabolism, as far as assessed here, are neither shaped by the major abiotic features of 'Evolution Canyon', namely solar radiation, temperature, and drought, nor by the soil characteristics. We further conclude that some traits diverge rather neutrally from each other, whereas other, more environmentally related traits are shaped by natural selection and show evolutionary convergence.

PPARδ activation in human myotubes increases mitochondrial fatty acid oxidative capacity and reduces glucose utilization by a switch in substrate preference.

PubMed

Feng, Yuan Z; Nikolić, Nataša; Bakke, Siril S; Boekschoten, Mark V; Kersten, Sander; Kase, Eili T; Rustan, Arild C; Thoresen, G Hege

2014-02-01

The role of peroxisome proliferator-activated receptor δ (PPARδ) activation on global gene expression and mitochondrial fuel utilization were investigated in human myotubes. Only 21 genes were up-regulated and 3 genes were down-regulated after activation by the PPARδ agonist GW501516. Pathway analysis showed up-regulated mitochondrial fatty acid oxidation, TCA cycle and cholesterol biosynthesis. GW501516 increased oleic acid oxidation and mitochondrial oxidative capacity by 2-fold. Glucose uptake and oxidation were reduced, but total substrate oxidation was not affected, indicating a fuel switch from glucose to fatty acid. Cholesterol biosynthesis was increased, but lipid biosynthesis and mitochondrial content were not affected. This study confirmed that the principal effect of PPARδ activation was to increase mitochondrial fatty acid oxidative capacity. Our results further suggest that PPARδ activation reduced glucose utilization through a switch in mitochondrial substrate preference by up-regulating pyruvate dehydrogenase kinase isozyme 4 and genes involved in lipid metabolism and fatty acid oxidation.

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Methods of making monolayers

DOEpatents

Alford, Kentin L [Pasco, WA; Simmons, Kevin L [Kennewick, WA; Samuels, William D [Richland, WA; Zemanian, Thomas S [Richland, WA; Liu, Jun [Albuquerque, NM; Shin, Yongsoon [Richland, WA; Fryxell, Glen E [Kennewick, WA

2009-12-08

The invention pertains to methods of forming monolayers on various surfaces. The surfaces can be selected from a wide array of materials, including, for example, aluminum dioxide, silicon dioxide, carbon and SiC. The substrates can be planar or porous. The monolayer is formed under enhanced pressure conditions. The monolayer contains functionalized molecules, and accordingly functionalizes a surface of the substrate. The properties of the functionalized substrate can enhance the substrate's applicability for numerous purposes including, for example, utilization in extracting contaminants, or incorporation into a polymeric matrix.

Methods of making monolayers

DOEpatents

Alford, Kentin L [Pasco, WA; Simmons, Kevin L [Kennewick, WA; Samuels, William D [Richland, WA; Zemanian, Thomas S [Richland, WA; Liu, Jun [Albuquerque, NM; Shin, Yongsoon [Richland, WA; Fryxell, Glen E [Kennewick, WA

2009-09-15

The invention pertains to methods of forming monolayers on various surfaces. The surfaces can be selected from a wide array of materials, including, for example, aluminum dioxide, silicon dioxide, carbon and SiC. The substrates can be planar or porous. The monolayer is formed under enhanced pressure conditions. The monolayer contains functionalized molecules, and accordingly functionalizes a surface of the substrate. The properties of the functionalized substrate can enhance the substrate's applicability for numerous purposes including, for example, utilization in extracting contaminants, or incorporation into a polymeric matrix.

Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: mushroom production, lignocellulolytic enzymes activity and substrate utilization.

PubMed

Isikhuemhen, Omoanghe S; Mikiashvili, Nona A; Kelkar, Vinaya

2009-06-01

The degradation and utilization of solid waste (SW) from anaerobic digestion of poultry litter by Agrocybe aegerita was evaluated through mushroom production, loss of organic matter (LOM), lignocellulolytic enzymes activity, lignocellulose degradation and mushroom nutrients content. Among the substrate combinations (SCs) tested, substrates composed of 10-20% SW, 70-80% wheat straw and 10% millet was found to produce the highest mushroom yield (770.5 and 642.9 g per 1.5 kg of substrate). LOM in all SCs tested varied between 8.8 and 48.2%. A. aegerita appears to degrade macromolecule components (0.6-21.8% lignin, 33.1-55.2% cellulose and 14-53.9% hemicellulose) during cultivation on the different SCs. Among the seven extracellular enzymes monitored, laccase, peroxidase and CMCase activities were higher before fruiting; while xylanase showed higher activities after fruiting. A source of carbohydrates (e.g., millet) in the substrate is needed in order to obtain yield and biological efficiency comparable to other commercially cultivated exotic mushrooms.

Capturing the response of Clostridium acetobutylicum to chemical stressors using a regulated genome-scale metabolic model

DOE PAGES

Dash, Satyakam; Mueller, Thomas J.; Venkataramanan, Keerthi P.; ...

2014-10-14

Clostridia are anaerobic Gram-positive Firmicutes containing broad and flexible systems for substrate utilization, which have been used successfully to produce a range of industrial compounds. Clostridium acetobutylicum has been used to produce butanol on an industrial scale through acetone-butanol-ethanol (ABE) fermentation. A genome-scale metabolic (GSM) model is a powerful tool for understanding the metabolic capacities of an organism and developing metabolic engineering strategies for strain development. The integration of stress related specific transcriptomics information with the GSM model provides opportunities for elucidating the focal points of regulation.

A novel assay of DGAT activity based on high temperature GC/MS of triacylglycerol.

PubMed

Greer, Michael S; Zhou, Ting; Weselake, Randall J

2014-08-01

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-(14)C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.

Optimization of Hyalella azteca IQ Toxicity Test{trademark} for prediction of 28-day sediment toxicity tests

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

Novotny, A.N.; Ezzard, C.L.; Douglas, W.S.

1995-12-31

The IQ Toxicity Test, which is a rapid screening toxicity test consisting of the observation of in-vivo inhibition of an enzymatic process using a fluorescent substrate, has proven successful for the determination of 24 and 48-hour EC50`s of D. magna, C. dubia, D. pulex and M. bahia. The application of this concept to utilize the freshwater amphipod Hyalella azteca may be an excellent way in which to reduce the standard 28-day chronic sediment toxicity test to possibly one hour`s time. This study incorporates an additive experimental design to explore the effects of and interactions between five specific variables: size ofmore » the amphipod, exposure time to the toxicant, concentration of substrate, exposure time to the substrate, and length of time starved prior to testing. The results of the IQ toxicity test were compared to those of a 28-day chronic sediment toxicity test. Preliminary data indicate that there is an optimal combination of these variables which results in a concise, reproducible toxicity test for use with Hyalella azteca, and would potentially be applicable to other freshwater amphipods in the future.« less

High-resolution proton NMR studies of intracellular metabolites in yeast using 13C decoupling

NASA Astrophysics Data System (ADS)

Sillerud, Laurel O.; Alger, Jeffry R.; Shulman, Robert G.

The resolution and specificity of 1H NMR in studies of yeast cellular metabolism were increased by feeding a 13C-labeled substrate and observing 1H difference spectra in the presence and absence of 13C decoupling fields. [2- 13C]Acetate was utilized as a respiratory substrate in an aerobic suspension of Saccharomyces cerevisiae. The broad cellular background proton resonances are removed by the technique, leaving only signals from the protons of the substrate, or its metabolites, that are coupled to 13C. Spectra of the yeast suspension after acetate feeding show the disappearance of label from the acetate pool and the subsequent appearance of 13C in glutamate C 3 and C 4 and in aspartate C 3. These results are in accord with the known fluxes of metabolites. Selective single-frequency 13C decoupling was used to provide assignments for the difference signals. The limitations on single-frequency decoupling coming from finite decoupling fields are investigated. The technique shows a potential for application in a wide variety of systems where the resolution of the 13C spectrum may be combined with the sensitivity for proton detection to observe metabolites that have been previously unobservable.

Substrate specificity of sheep liver sorbitol dehydrogenase.

PubMed Central

Lindstad, R I; Köll, P; McKinley-McKee, J S

1998-01-01

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546

Process for producing nickel electrode having lightweight substrate

NASA Technical Reports Server (NTRS)

Lim, Hong S. (Inventor)

1996-01-01

A nickel electrode having a lightweight porous nickel substrate is subjected to a formation cycle involving heavy overcharging and under-discharging in a KOH electrolyte having a concentration of 26% to 31%, resulting in electrodes displaying high active material utilization.

Fixation of CO2 and CO on a diverse range of carbohydrates using anaerobic, non-photosynthetic mixotrophy.

PubMed

Maru, Biniam T; Munasinghe, Pradeep C; Gilary, Hadar; Jones, Shawn W; Tracy, Bryan P

2018-04-01

Biological CO2 fixation is an important technology that can assist in combating climate change. Here, we show an approach called anaerobic, non-photosynthetic mixotrophy can result in net CO2 fixation when using a reduced feedstock. This approach uses microbes called acetogens that are capable of concurrent utilization of both organic and inorganic substrates. In this study, we investigated the substrate utilization of 17 different acetogens, both mesophilic and thermophilic, on a variety of different carbohydrates and gases. Compared to most model acetogen strains, several non-model mesophilic strains displayed greater substrate flexibility, including the ability to utilize disaccharides, glycerol and an oligosaccharide, and growth rates. Three of these non-model strains (Blautia producta, Clostridium scatologenes and Thermoanaerobacter kivui) were chosen for further characterization, under a variety of conditions including H2- or syngas-fed sugar fermentations and a CO2-fed glycerol fermentation. In all cases, CO2 was fixed and carbon yields approached 100%. Finally, the model acetogen C. ljungdahlii was engineered to utilize glucose, a non-preferred sugar, while maintaining mixotrophic behavior. This work demonstrates the flexibility and robustness of anaerobic, non-photosynthetic mixotrophy as a technology to help reduce CO2 emissions.

Analysis of Histone Deacetylase-Dependent Effects on Cell Migration Using the Stripe Assay.

PubMed

Mertsch, Sonja; Thanos, Solon

2017-01-01

For normal embryonic development/morphogenesis, cell migration and homing are well-orchestrated and important events requiring specific cellular mechanisms. In diseases such as cancer deregulated cell migration represents a major problem. Therefore, numerous efforts are under way to understand the molecular mechanisms of tumor cell migration and to generate more efficient tumor therapies. Cell migration assays are one of the most commonly used functional assays. The wound-healing assay or the Boyden chamber assay are variations of these assays. Nearly all of them are two-dimensional assays and the cells can only migrate on one substrate at a time. This is in contrast to the in vivo situation where the cells are faced simultaneously with different surfaces and interact with different cell types. To approach this in vivo situation we used a modified version of the stripe assay designed by Bonhoeffer and colleagues to examine mechanisms of axonal guidance. The design of this assay allows cells to decide between two different substrates offered at the same time. Utilizing alternating neuronal substrates for migration analyses we can partially mimic the complex in vivo situation for brain tumor cells. Here we describe the detailed protocol to perform a modified version of the stripe assay in order to observe substrate-dependent migration effects in vitro, to analyze the effect of Rho-dependent kinases (ROCKS), of histone deacetylases (HDACs) and of other molecules on glioma cells.

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

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.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Scintillator assembly for alpha radiation detection and an associated method of making

DOEpatents

Lauf, R.J.; McElhaney, S.A.; Bates, J.B.

1994-07-26

A scintillator assembly for use in conjunction with a photomultiplier or the like in the detection of alpha radiation utilizes a substrate or transparent yttrium aluminum garnet and a relatively thin film of cerium-doped yttrium aluminum garnet coated upon the substrate. The film material is applied to the substrate in a sputtering process, and the applied film and substrate are annealed to effect crystallization of the film upon the substrate. The resultant assembly provides relatively high energy resolution during use in a detection instrument and is sufficiently rugged for use in field environments. 4 figs.

Scintillator assembly for alpha radiation detection and an associated method of making

DOEpatents

Lauf, Robert J.; McElhaney, Stephanie A.; Bates, John B.

1994-01-01

A scintillator assembly for use in conjunction with a photomultiplier or the like in the detection of alpha radiation utilizes a substrate or transparent yttrium aluminum garnet and a relatively thin film of cerium-doped yttrium aluminum garnet coated upon the substrate. The film material is applied to the substrate in a sputtering process, and the applied film and substrate are annealed to effect crystallization of the film upon the substrate. The resultant assembly provides relatively high energy resolution during use in a detection instrument and is sufficiently rugged for use in field environments.

Silicon on insulator self-aligned transistors

DOEpatents

McCarthy, Anthony M.

2003-11-18

A method for fabricating thin-film single-crystal silicon-on-insulator (SOI) self-aligned transistors. Standard processing of silicon substrates is used to fabricate the transistors. Physical spaces, between the source and gate, and the drain and gate, introduced by etching the polysilicon gate material, are used to provide connecting implants (bridges) which allow the transistor to perform normally. After completion of the silicon substrate processing, the silicon wafer is bonded to an insulator (glass) substrate, and the silicon substrate is removed leaving the transistors on the insulator (glass) substrate. Transistors fabricated by this method may be utilized, for example, in flat panel displays, etc.

Characterizing Protease Specificity: How Many Substrates Do We Need?

PubMed Central

Schauperl, Michael; Fuchs, Julian E.; Waldner, Birgit J.; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

Molecular biomimetics: utilizing nature's molecular ways in practical engineering.

PubMed

Tamerler, Candan; Sarikaya, Mehmet

2007-05-01

In nature, proteins are the machinery that accomplish many functions through their specific recognition and interactions in biological systems from single-celled to multicellular organisms. Biomolecule-material interaction is accomplished via molecular specificity, leading to the formation of controlled structures and functions at all scales of dimensional hierarchy. Through evolution, molecular recognition and, consequently, functions developed through successive cycles of mutation and selection. Using biology as a guide, we can now understand, engineer and control peptide-material interactions and exploit these to tailor novel materials and systems for practical applications. We adapted combinatorial biology protocols to display peptide libraries, either on the cell surface or on phages, to select short peptides specific to a variety of practical materials systems. Following the selection step, we determined the kinetics and stability of peptide binding experimentally to understand the bound peptide structure via modeling and its assembly via atomic force microscopy. The peptides were further engineered to have multiple repeats or their amino acid sequences varied to tailor their function. Both nanoparticles and flat inorganic substrates containing multimaterials patterned at the nano- and microscales were used for self-directed immobilization of molecular constructs. The molecular biomimetic approach opens up new avenues for the design and utilization of multifunctional molecular systems with wide ranging applications, from tissue engineering, drug delivery and biosensors, to nanotechnology and bioremediation. Here we give examples of protein-mediated functional materials in biology, peptide selection and engineering with affinity to inorganics, demonstrate potential utilizations in materials science, engineering and medicine, and describe future prospects.

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

PubMed Central

2013-01-01

Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

Silicon Nanowire Growth at Chosen Positions and Orientations

NASA Technical Reports Server (NTRS)

Getty, Stephanie A.

2009-01-01

It is now possible to grow silicon nanowires at chosen positions and orientations by a method that involves a combination of standard microfabrication processes. Because their positions and orientations can be chosen with unprecedented precision, the nanowires can be utilized as integral parts of individually electronically addressable devices in dense arrays. Nanowires made from silicon and perhaps other semiconductors hold substantial promise for integration into highly miniaturized sensors, field-effect transistors, optoelectronic devices, and other electronic devices. Like bulk semiconductors, inorganic semiconducting nanowires are characterized by electronic energy bandgaps that render them suitable as means of modulating or controlling electronic signals through electrostatic gating, in response to incident light, or in response to molecules of interest close to their surfaces. There is now potential for fabricating arrays of uniform, individually electronically addressable nanowires tailored to specific applications. The method involves formation of metal catalytic particles at the desired positions on a substrate, followed by heating the substrate in the presence of silane gas. The figure illustrates an example in which a substrate includes a silicon dioxide surface layer that has been etched into an array of pillars and the catalytic (in this case, gold) particles have been placed on the right-facing sides of the pillars. The catalytic thermal decomposition of the silane to silicon and hydrogen causes silicon columns (the desired nanowires) to grow outward from the originally catalyzed spots on the substrate, carrying the catalytic particles at their tips. Thus, the position and orientation of each silicon nanowire is determined by the position of its originally catalyzed spot on the substrate surface, and the orientation of the nanowire is perpendicular to the substrate surface at the originally catalyzed spot.

CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions.

PubMed

Galetin, Aleksandra; Ito, Kiyomi; Hallifax, David; Houston, J Brian

2005-07-01

The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homo- or heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more "pragmatic" substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. Ki estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings.

Programmable RNA recognition and cleavage by CRISPR/Cas9.

PubMed

O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

2014-12-11

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

Programmable RNA recognition and cleavage by CRISPR/Cas9

PubMed Central

O’Connell, Mitchell R.; Oakes, Benjamin L.; Sternberg, Samuel H.; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A.

2014-01-01

The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA:DNA complementarity to identify target sites for sequence-specific doublestranded DNA (dsDNA) cleavage1-5. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, the protospacer adjacent motif (PAM), next to and on the strand opposite the 20-nucleotide target site in dsDNA4-7. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in many cell types and organisms8, but it has been thought to be incapable of targeting RNA5. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalyzed DNA cleavage7. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous mRNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable and tagless transcript recognition. PMID:25274302

The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

PubMed Central

King, Steven C

2004-01-01

Background In establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters. Results Using the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for [14C]GABA (4-aminobutyrate) versus [3H]NA (nipecotic acid). Conclusions The TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Δ(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (ΔGb) that translocation catalysts rely upon to decrease activation energy (). TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity). PMID:15548327

Crystallographic analysis and structure-guided engineering of NADPH-dependent Ralstonia sp. alcohol dehydrogenase toward NADH cosubstrate specificity.

PubMed

Lerchner, Alexandra; Jarasch, Alexander; Meining, Winfried; Schiefner, André; Skerra, Arne

2013-11-01

The NADP⁺-dependent alcohol dehydrogenase from Ralstonia sp. (RasADH) belongs to the protein superfamily of short-chain dehydrogenases/reductases (SDRs). As an enzyme that accepts different types of substrates--including bulky-bulky as well as small-bulky secondary alcohols or ketones--with high stereoselectivity, it offers potential as a biocatalyst for industrial biotechnology. To understand substrate and cosubstrate specificities of RasADH we determined the crystal structure of the apo-enzyme as well as its NADP⁺-bound state with resolutions down to 2.8 Å. RasADH displays a homotetrameric quaternary structure that can be described as a dimer of homodimers while in each subunit a seven-stranded parallel β-sheet, flanked by three α-helices on each side, forms a Rossmann fold-type dinucleotide binding domain. Docking of the well-known substrate (S)-1-phenylethanol clearly revealed the structural determinants of stereospecificity. To favor practical RasADH application in the context of established cofactor recycling systems, for example, those involving an NADH-dependent amino acid dehydrogenase, we attempted to rationally change its cosubstrate specificity from NADP⁺ to NAD⁺ utilizing the structural information that NADP⁺ specificity is largely governed by the residues Asn15, Gly37, Arg38, and Arg39. Furthermore, an extensive sequence alignment with homologous dehydrogenases that have different cosubstrate specificities revealed a modified general SDR motif ASNG (instead of NNAG) at positions 86-89 of RasADH. Consequently, we constructed mutant enzymes with one (G37D), four (N15G/G37D/R38V/R39S), and six (N15G/G37D/R38V/R39S/A86N/S88A) amino acid exchanges. RasADH (N15G/G37D/R38V/R39S) was better able to accept NAD⁺ while showing much reduced catalytic efficiency with NADP⁺, leading to a change in NADH/NADPH specificity by a factor of ∼3.6 million. © 2013 Wiley Periodicals, Inc.

SapF-mediated heme-iron utilization enhances persistence and coordinates biofilm architecture of Haemophilus

PubMed Central

Vogel, Andrew R.; Szelestey, Blake R.; Raffel, Forrest K.; Sharpe, Samantha W.; Gearinger, Rachel L.; Justice, Sheryl S.; Mason, Kevin M.

2012-01-01

Non-typeable Haemophilus influenzae (NTHI) is a common commensal bacterium that resides in the human upper respiratory tract of healthy individuals. NTHI is also a known causative agent of multiple diseases including sinusitis, otitis media, as well as exacerbates disease severity of patients with cystic fibrosis and chronic obstructive pulmonary disease. We have previously shown that the Sap transporter mediates resistance to host antimicrobial peptides (AMPs) and import of the iron-containing compound heme. Here, we analyzed the contribution of the Sap structural ATPase protein, SapF, in these essential functions. In contrast to SapD, SapF was dispensable for NTHI survival when exposed to AMPs in vitro. SapF was responsible for heme utilization and recovery of depleted internal heme-iron stores. Further, a loss of SapF resulted in morphological plasticity and enhanced community development and biofilm architecture, suggesting the potential role of heme-iron availability in coordinating the complexity of NTHI biofilm architecture. SapF was required for colonization of the nasopharynx and acute infection of the middle ear, as SapF deficiency correlated with a statistically significant decrease in NTHI persistence in vivo. These data suggest that SapF is required for proper heme utilization which directly impacts NTHI survival. Thus, these studies further support a role for the Sap complex in the transport of multiple substrates and further defines substrate specificity for the two ATPase subunits. Given the multiple essential functions provided by the Sap transporter, this complex could prove to be an effective therapeutic target for the treatment of NTHI diseases. PMID:22919633

Magnetic tunnel junctions utilizing diamond-like carbon tunnel barriers

NASA Astrophysics Data System (ADS)

Cadieu, F. J.; Chen, Li; Li, Biao

2002-05-01

We have devised a method whereby thin particulate-free diamond-like carbon films can be made with good adhesion onto even room-temperature substrates. The method employs a filtered ionized carbon beam created by the vacuum impact of a high-energy, approximately 1 J per pulse, 248 nm excimer laser onto a carbon target. The resultant deposition beam can be steered and deflected by magnetic and electric fields to paint a specific substrate area. An important aspect of this deposition method is that the resultant films are particulate free and formed only as the result of atomic species impact. The vast majority of magnetic tunnel junctions utilizing thin metallic magnetic films have employed a thin oxidized layer of aluminum to form the tunnel barrier. This has presented reproducibility problems because the indicated optimal barrier thickness is only approximately 13 Å thick. Magnetic tunnel junctions utilizing Co and permalloy films made by evaporation and sputtering have been fabricated with an intervening diamond-like carbon tunnel barrier. The diamond-like carbon thickness profile has been tapered so that seven junctions with different barrier thickness can be formed at once. Magnetoresistive (MR) measurements made between successive permalloy strip ends include contributions from two junctions and from the permalloy and Co strips that act as current leads to the junctions. Magnetic tunnel junctions with thicker carbon barriers exhibit MR effects that are dominated by that of the permalloy strips. Since these tunnel barriers are formed without the need for oxygen, complete tunnel junctions can be formed with all high-vacuum processing.

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10.

PubMed Central

Janssen, D B; Scheper, A; Dijkhuizen, L; Witholt, B

1985-01-01

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms. Images PMID:3994371

Viral Activation of Cellular Metabolism

PubMed Central

Sanchez, Erica L.; Lagunoff, Michael

2015-01-01

To ensure optimal environments for their replication and spread, viruses have evolved to alter many host cell pathways. In the last decade, metabolomic studies have shown that eukaryotic viruses induce large-scale alterations in host cellular metabolism. Most viruses examined to date induce aerobic glycolysis also known as the Warburg effect. Many viruses tested also induce fatty acid synthesis as well as glutaminolysis. These modifications of carbon source utilization by infected cells can increase available energy for virus replication and virion production, provide specific cellular substrates for virus particles and create viral replication niches while increasing infected cell survival. Each virus species also likely requires unique metabolic changes for successful spread and recent research has identified additional virus-specific metabolic changes induced by many virus species. A better understanding of the metabolic alterations required for each virus may lead to novel therapeutic approaches through targeted inhibition of specific cellular metabolic pathways. PMID:25812764

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.

Alteration in substrate specificity of horse liver alcohol dehydrogenase by an acyclic nicotinamide analog of NAD(+).

PubMed

Malver, Olaf; Sebastian, Mina J; Oppenheimer, Norman J

2014-11-01

A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. Copyright © 2014 Elsevier B.V. All rights reserved.

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis.

PubMed

Hardin, Shane C; Tang, Guo-Qing; Scholz, Anke; Holtgraewe, Daniela; Winter, Heike; Huber, Steven C

2003-09-01

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.

Exploring Nitrilase Sequence Space for Enantioselective Catalysis†

PubMed Central

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

2004-01-01

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

Structure-Specificity Relationships of an Intracellular Xylanase from Geobacillus stearothermophilus

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

Solomon,V.; Teplitsky, A.; Shulami, S.

2007-01-01

Geobacillus stearothermophilus T-6 is a thermophilic Gram-positive bacterium that produces two selective family 10 xylanases which both take part in the complete degradation and utilization of the xylan polymer. The two xylanases exhibit significantly different substrate specificities. While the extracellular xylanase (XT6; MW 43.8 kDa) hydrolyzes the long and branched native xylan polymer, the intracellular xylanase (IXT6; MW 38.6 kDa) preferentially hydrolyzes only short xylo-oligosaccharides. In this study, the detailed three-dimensional structure of IXT6 is reported, as determined by X-ray crystallography. It was initially solved by molecular replacement and then refined at 1.45 {angstrom} resolution to a final R factormore » of 15.0% and an R{sub free} of 19.0%. As expected, the structure forms the classical ({alpha}/{beta}){sub 8} fold, in which the two catalytic residues (Glu134 and Glu241) are located on the inner surface of the central cavity. The structure of IXT6 was compared with the highly homologous extracellular xylanase XT6, revealing a number of structural differences between the active sites of the two enzymes. In particular, structural differences derived from the unique subdomain in the carboxy-terminal region of XT6, which is completely absent in IXT6. These structural modifications may account for the significant differences in the substrate specificities of these otherwise very similar enzymes.« less

Efficient utilization of crude glycerol as fermentation substrate in the synthesis of poly(3-hydroxybutyrate) biopolymers

USDA-ARS?s Scientific Manuscript database

One refined and 2 crude glycerol samples were utilized to produce poly(3-hydroxybutyrate) (PHB) by Pseudomonas oleovorans NRRL B-14682. Fermentation conditions were determined to efficiently utilize glycerol while maintaining PHB yields. A batch culture protocol including 1% glycerol and an aerati...

Metabolic remodeling of substrate utilization during heart failure progression.

PubMed

Chen, Liang; Song, Jiangping; Hu, Shengshou

2018-05-23

Heart failure (HF) is a clinical syndrome caused by a decline in cardiac systolic or diastolic function, which leaves the heart unable to pump enough blood to meet the normal physiological requirements of the human body. It is a serious disease burden worldwide affecting nearly 23 million patients. The concept that heart failure is "an engine out of fuel" has been generally accepted and metabolic remodeling has been recognized as an important aspect of this condition; it is characterized by defects in energy production and changes in metabolic pathways involved in the regulation of essential cellular functions such as the process of substrate utilization, the tricarboxylic acid cycle, oxidative phosphorylation, and high-energy phosphate metabolism. Advances in second-generation sequencing, proteomics, and metabolomics have made it possible to perform comprehensive tests on genes and metabolites that are crucial in the process of HF, thereby providing a clearer and comprehensive understanding of metabolic remodeling during HF. In recent years, new metabolic changes such as ketone bodies and branched-chain amino acids were demonstrated as alternative substrates in end-stage HF. This systematic review focuses on changes in metabolic substrate utilization during the progression of HF and the underlying regulatory mechanisms. Accordingly, the conventional concepts of metabolic remodeling characteristics are reviewed, and the latest developments, particularly multi-omics studies, are compiled.

Method of forming crystalline silicon devices on glass

DOEpatents

McCarthy, Anthony M.

1995-01-01

A method for fabricating single-crystal silicon microelectronic components on a silicon substrate and transferring same to a glass substrate. This is achieved by utilizing conventional silicon processing techniques for fabricating components of electronic circuits and devices on bulk silicon, wherein a bulk silicon surface is prepared with epitaxial layers prior to the conventional processing. The silicon substrate is bonded to a glass substrate and the bulk silicon is removed leaving the components intact on the glass substrate surface. Subsequent standard processing completes the device and circuit manufacturing. This invention is useful in applications requiring a transparent or insulating substrate, particularly for display manufacturing. Other applications include sensors, actuators, optoelectronics, radiation hard electronics, and high temperature electronics.

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

Hooper, R. J.; Adams, D. P.; Hirschfeld, D.

The rapid release of energy from reactive multilayer foils can create extreme local temperature gradients near substrate materials. To fully exploit the potential of these materials, a better understanding of the interaction between the substrate or filler material and the foil is needed. In particular, this work investigates how variations in local properties within the substrate (i.e. differences between properties in constituent phases) can affect heat transport into the substrate. Furthermore, this can affect the microstructural evolution observed within the substrate, which may affect the final joint properties. The effect of the initial substrate microstructure on microstructural evolution within themore » heat-affected zone is evaluated experimentally in two Sn-Zn alloys and numerical techniques are utilized to inform the analysis.« less

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

DOE PAGES

Bansal, Sunil; Durrett, Timothy P.

2016-11-08

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

PubMed Central

Bansal, Sunil; Durrett, Timothy P.

2016-01-01

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants. PMID:27688773

Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

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

Bansal, Sunil; Durrett, Timothy P.

Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. Inmore » vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. As a result, this improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants.« less

Utility of Adenosine Monophosphate Detection System for Monitoring the Activities of Diverse Enzyme Reactions.

PubMed

Mondal, Subhanjan; Hsiao, Kevin; Goueli, Said A

Adenosine monophosphate (AMP) is a key cellular metabolite regulating energy homeostasis and signal transduction. AMP is also a product of various enzymatic reactions, many of which are dysregulated during disease conditions. Thus, monitoring the activities of these enzymes is a primary goal for developing modulators for these enzymes. In this study, we demonstrate the versatility of an enzyme-coupled assay that quantifies the amount of AMP produced by any enzymatic reaction regardless of its substrates. We successfully implemented it to enzyme reactions that use adenosine triphosphate (ATP) as a substrate (aminoacyl tRNA synthetase and DNA ligase) by an elaborate strategy of removing residual ATP and converting AMP produced into ATP; so it can be detected using luciferase/luciferin and generating light. We also tested this assay to measure the activities of AMP-generating enzymes that do not require ATP as substrate, including phosphodiesterases (cyclic adenosine monophosphate) and Escherichia coli DNA ligases (nicotinamide adenine dinucleotide [NAD + ]). In a further elaboration of the AMP-Glo platform, we coupled it to E. coli DNA ligase, enabling measurement of NAD + and enzymes that use NAD + like monoadenosine and polyadenosine diphosphate-ribosyltransferases. Sulfotransferases use 3'-phosphoadenosine-5'-phosphosulfate as the universal sulfo-group donor and phosphoadenosine-5'-phosphate (PAP) is the universal product. PAP can be quantified by converting PAP to AMP by a Golgi-resident PAP-specific phosphatase, IMPAD1. By coupling IMPAD1 to the AMP-Glo system, we can measure the activities of sulfotransferases. Thus, by utilizing the combinations of biochemical enzymatic conversion of various cellular metabolites to AMP, we were able to demonstrate the versatility of the AMP-Glo assay.

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Investigation of the strain-sensitive superconducting transition of BaFe1.8Co0.2As2 thin films utilizing piezoelectric substrates

NASA Astrophysics Data System (ADS)

Trommler, S.; Hänisch, J.; Iida, K.; Kurth, F.; Schultz, L.; Holzapfel, B.; Hühne, R.

2014-05-01

The preparation of biaxially textured BaFe1.8Co0.2As2 thin films has been optimized on MgO single crystals and transfered to piezoelectric (001) Pb(Mg1/3Nb2/3)0.72Ti0.28O3 substrates. By utilizing the inverse piezoelectric effect the lattice parameter of these substrates can be controlled applying an electric field, leading to a induction of biaxial strain into the superconducting layer. High electric fields were used to achieve a total strain of up to 0.05% at low temperatures. A sharpening of the resistive transition and a shift of about 0.6 K to higher temperatures was found at a compressive strain of 0.035%.

Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization.

PubMed

Whitaker, William B; Sandoval, Nicholas R; Bennett, Robert K; Fast, Alan G; Papoutsakis, Eleftherios T

2015-06-01

Synthetic methylotrophy is the development of non-native methylotrophs that can utilize methane and methanol as sole carbon and energy sources or as co-substrates with carbohydrates to produce metabolites as biofuels and chemicals. The availability of methane (from natural gas) and its oxidation product, methanol, has been increasing, while prices have been decreasing, thus rendering them as attractive fermentation substrates. As they are more reduced than most carbohydrates, methane and methanol, as co-substrates, can enhance the yields of biologically produced metabolites. Here we discuss synthetic biology and metabolic engineering strategies based on the native biology of aerobic methylotrophs for developing synthetic strains grown on methanol, with Escherichia coli as the prototype. Copyright © 2015. Published by Elsevier Ltd.

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

PubMed Central

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

2017-01-01

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

Oil palm waste and synthetic zeolite: an alternative soil-less growth substrate for lettuce production as a waste management practice.

PubMed

Jayasinghe, Guttila Y; Tokashiki, Yoshihiro; Kitou, Makato; Kinjo, Kazutoshi

2008-12-01

A study was conducted to assess the characteristics and the prospective utilization of oil palm waste (OP) and synthetic zeolite (SZ) developed by coal fly ash, as an alternative substrate to peat and commercial perlite for lettuce (Lactuca sativa L.) production. The SZ, OP, sphagnum peat (PE), perlite (PL) and two different SZ-OP mixtures (v/v) at the ratio of 1 : 3 and 1 : 10 were utilized as the substrates under this study. The substrates formulated by mixing SZ with OP at the ratio of 1 : 3 and 1 : 10 showed improved substrate physical and chemical properties such as air space, bulk density, particle density, water-holding capacity, pH and electrical conductivity (EC), which were in the ideal substrate range when compared with PL. Furthermore, the water-holding capacity of the substrate having a 1 : 10 mixing ratio of SZ with OP was higher than that of the PL by 28.23%, whereas the bulk density was lower than that of PL by 35%. A greenhouse experiment was carried out to assess the influence of the substrates on the growth and development of lettuce. The shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and number of leaves per plant of the lettuce grown in the 1 : 10 mixing ratio of SZ and OP were the highest, which showed increased values compared with that of PL by 11.56, 9.77, 3.48, 17.35 and 16.53%, respectively. The shoot fresh weight, shoot dry weight, root fresh weight, root dry weight and number of leaves per plant of the lettuce grown in the 1 : 10 mixing ratio of SZ and OP showed increased percentages compared with that of PE by 12.12, 11.37, 3.74, 23.66 and 17.50%, respectively. In addition, the growth and yield parameters of lettuce grown in the 1 : 3 mixing ratio and the OP did not show any significant difference with PL and PE but differed from the 1 : 10 mixing ratio. The results of the study suggest that the SZ-OP-based substrates and OP can be successfully utilized as alternatives to the commercial perlite and to substitute the conventional peat substrate for lettuce cultivation. In addition, this can be proposed as an alternative waste management practice.

MODELING SUBSTRATE TRANSPORT INTO BIOFILMS: ROLE OF MULTIPLE IONS AND PH EFFECTS

EPA Science Inventory

Steady-state substrate utilization in biofilms has traditionally been modeled by coupling Fickian diffusion with Monod reaction kinetics. An inherent assumption in most of the previous models was that the pH remains constant within the biofilm. Experiments have shown differences ...

Promising Hard Carbon Coatings on Cu Substrates: Corrosion and Tribological Performance with Theoretical Aspect

NASA Astrophysics Data System (ADS)

Kumar, A. Madhan; Babu, R. Suresh; Obot, I. B.; Adesina, Akeem Yusuf; Ibrahim, Ahmed; de Barros, A. L. F.

2018-05-01

Protecting the surface of metals and alloys against corrosion and wear is of abundant importance owing to their widespread applications. In the present work, we report the improved anticorrosion and tribo-mechanical performance of copper (Cu) by a hard carbon (HC) coating synthesized in different pyrolysis temperature. Structural and surface characterization with roughness measurements was systematically investigated using various techniques. Effect of pyrolysis temperature on the corrosion behavior of coated Cu substrates in 0.6 M NaCl solution was evaluated via electrochemical impedance spectroscopy, potentiodynamic polarization. Pin-on-disk wear test of coated Cu substrate showed the influence of the pyrolysis temperature on the wear resistance performance of the HC coatings. According to the obtained results, it could be concluded that the HC coatings synthesized at 1100 °C revealed an enhanced comprehensive performance, revealing their possible utilization as a protective coating for Cu substrates in chloride environment. Monte Carlo simulations have been utilized to elucidate the interaction between the Cu surface and HC coatings.

Promising Hard Carbon Coatings on Cu Substrates: Corrosion and Tribological Performance with Theoretical Aspect

NASA Astrophysics Data System (ADS)

Kumar, A. Madhan; Babu, R. Suresh; Obot, I. B.; Adesina, Akeem Yusuf; Ibrahim, Ahmed; de Barros, A. L. F.

2018-01-01

Protecting the surface of metals and alloys against corrosion and wear is of abundant importance owing to their widespread applications. In the present work, we report the improved anticorrosion and tribo-mechanical performance of copper (Cu) by a hard carbon (HC) coating synthesized in different pyrolysis temperature. Structural and surface characterization with roughness measurements was systematically investigated using various techniques. Effect of pyrolysis temperature on the corrosion behavior of coated Cu substrates in 0.6 M NaCl solution was evaluated via electrochemical impedance spectroscopy, potentiodynamic polarization. Pin-on-disk wear test of coated Cu substrate showed the influence of the pyrolysis temperature on the wear resistance performance of the HC coatings. According to the obtained results, it could be concluded that the HC coatings synthesized at 1100 °C revealed an enhanced comprehensive performance, revealing their possible utilization as a protective coating for Cu substrates in chloride environment. Monte Carlo simulations have been utilized to elucidate the interaction between the Cu surface and HC coatings.

Asp- and Glu-specific Novel Dipeptidyl Peptidase 11 of Porphyromonas gingivalis Ensures Utilization of Proteinaceous Energy Sources*

PubMed Central

Ohara-Nemoto, Yuko; Shimoyama, Yu; Kimura, Shigenobu; Kon, Asako; Haraga, Hiroshi; Ono, Toshio; Nemoto, Takayuki K.

2011-01-01

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp22. A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the kcat/Km value was higher for Asp than Glu. Those activities were lost by substitution of Ser652 in P. endodontalis and Ser655 in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg670 is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg670 interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods. PMID:21896480

Asp- and Glu-specific novel dipeptidyl peptidase 11 of Porphyromonas gingivalis ensures utilization of proteinaceous energy sources.

PubMed

Ohara-Nemoto, Yuko; Shimoyama, Yu; Kimura, Shigenobu; Kon, Asako; Haraga, Hiroshi; Ono, Toshio; Nemoto, Takayuki K

2011-11-04

Porphyromonas gingivalis and Porphyromonas endodontalis, asaccharolytic black-pigmented anaerobes, are predominant pathogens of human chronic and periapical periodontitis, respectively. They incorporate di- and tripeptides from the environment as carbon and energy sources. In the present study we cloned a novel dipeptidyl peptidase (DPP) gene of P. endodontalis ATCC 35406, designated as DPP11. The DPP11 gene encoded 717 amino acids with a molecular mass of 81,090 Da and was present as a 75-kDa form with an N terminus of Asp(22). A homology search revealed the presence of a P. gingivalis orthologue, PGN0607, that has been categorized as an isoform of authentic DPP7. P. gingivalis DPP11 was exclusively cell-associated as a truncated 60-kDa form, and the gene ablation retarded cell growth. DPP11 specifically removed dipeptides from oligopeptides with the penultimate N-terminal Asp and Glu and has a P2-position preference to hydrophobic residues. Optimum pH was 7.0, and the k(cat)/K(m) value was higher for Asp than Glu. Those activities were lost by substitution of Ser(652) in P. endodontalis and Ser(655) in P. gingivalis DPP11 to Ala, and they were consistently decreased with increasing NaCl concentration. Arg(670) is a unique amino acid completely conserved in all DPP11 members distributed in the genera Porphyromonas, Bacteroides, and Parabacteroides, whereas this residue is converted to Gly in all authentic DPP7 members. Substitution analysis suggested that Arg(670) interacts with an acidic residue of the substrate. Considered to preferentially utilize acidic amino acids, DPP11 ensures efficient degradation of oligopeptide substrates in these Gram-negative anaerobic rods.

Reciprocal Prioritization to Dietary Glycans by Gut Bacteria in a Competitive Environment Promotes Stable Coexistence.

PubMed

Tuncil, Yunus E; Xiao, Yao; Porter, Nathan T; Reuhs, Bradley L; Martens, Eric C; Hamaker, Bruce R

2017-10-10

When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron , we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs) were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome. IMPORTANCE The microorganisms that reside in the human colon fulfill their energy requirements mainly from diet- and host-derived complex carbohydrates. Members of this ecosystem possess poorly understood strategies to prioritize and compete for these nutrients. Based on direct carbohydrate measurements and corresponding transcriptional analyses, our findings showed that individual bacterial species exhibit different preferences for the same set of glycans and that this prioritization is maintained in a competitive environment, which may promote stable coexistence. Such understanding of gut bacterial glycan utilization will be essential to eliciting predictable changes in the gut microbiota to improve health through the diet. Copyright © 2017 Tuncil et al.

Design and fabrication of 6.1-.ANG. family semiconductor devices using semi-insulating A1Sb substrate

DOEpatents

Sherohman, John W [Livermore, CA; Coombs, III, Arthur W.; Yee, Jick Hong [Livermore, CA; Wu, Kuang Jen J [Cupertino, CA

2007-05-29

For the first time, an aluminum antimonide (AlSb) single crystal substrate is utilized to lattice-match to overlying semiconductor layers. The AlSb substrate establishes a new design and fabrication approach to construct high-speed, low-power electronic devices while establishing inter-device isolation. Such lattice matching between the substrate and overlying semiconductor layers minimizes the formation of defects, such as threaded dislocations, which can decrease the production yield and operational life-time of 6.1-.ANG. family heterostructure devices.

Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

PubMed Central

Diallinas, George

2014-01-01

Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439

The automated array assembly task of the low-cost silicon solar array project, phase 2

NASA Technical Reports Server (NTRS)

Coleman, M. G.; Pryor, R. A.; Sparks, T. G.; Legge, R.; Saltzman, D. L.

1980-01-01

Several specific processing steps as part of a total process sequence for manufacturing silicon solar cells were studied. Ion implantation was identified as the preferred process step for impurity doping. Unanalyzed beam ion implantation was shown to have major cost advantages over analyzed beam implantation. Further, high quality cells were fabricated using a high current unanalyzed beam. Mechanically masked plasma patterning of silicon nitride was shown to be capable of forming fine lines on silicon surfaces with spacings between mask and substrate as great as 250 micrometers. Extensive work was performed on advances in plated metallization. The need for the thick electroless palladium layer was eliminated. Further, copper was successfully utilized as a conductor layer utilizing nickel as a barrier to copper diffusion into the silicon. Plasma etching of silicon for texturing and saw damage removal was shown technically feasible but not cost effective compared to wet chemical etching techniques.

Single molecule characterization of DNA binding and strand displacement reactions on lithographic DNA origami microarrays.

PubMed

Scheible, Max B; Pardatscher, Günther; Kuzyk, Anton; Simmel, Friedrich C

2014-03-12

The combination of molecular self-assembly based on the DNA origami technique with lithographic patterning enables the creation of hierarchically ordered nanosystems, in which single molecules are positioned at precise locations on multiple length scales. Based on a hybrid assembly protocol utilizing DNA self-assembly and electron-beam lithography on transparent glass substrates, we here demonstrate a DNA origami microarray, which is compatible with the requirements of single molecule fluorescence and super-resolution microscopy. The spatial arrangement allows for a simple and reliable identification of single molecule events and facilitates automated read-out and data analysis. As a specific application, we utilize the microarray to characterize the performance of DNA strand displacement reactions localized on the DNA origami structures. We find considerable variability within the array, which results both from structural variations and stochastic reaction dynamics prevalent at the single molecule level.

Optimization of free ammonia concentration for nitrite accumulation in shortcut biological nitrogen removal process.

PubMed

Chung, Jinwook; Shim, Hojae; Park, Seong-Jun; Kim, Seung-Jin; Bae, Wookeun

2006-03-01

A shortcut biological nitrogen removal (SBNR) utilizes the concept of a direct conversion of ammonium to nitrite and then to nitrogen gas. A successful SBNR requires accumulation of nitrite in the system and inhibition of the activity of nitrite oxidizers. A high concentration of free ammonia (FA) inhibits nitrite oxidizers, but unfortunately decreases the ammonium removal rate as well. Therefore, the optimal range of FA concentration is necessary not only to stabilize nitrite accumulation but also to achieve maximum ammonium removal. In order to derive such optimal FA concentrations, the specific substrate utilization rates of ammonium and nitrite oxidizers were measured. The optimal FA concentration range appeared to be 5-10 mg/L for the adapted sludge. The simulated results from the modified inhibition model expressed by FA and ammonium/nitrite concentrations were shown very similar to the experimental results.

Structural and functional analysis of betaine aldehyde dehydrogenase from Staphylococcus aureus.

PubMed

Halavaty, Andrei S; Rich, Rebecca L; Chen, Chao; Joo, Jeong Chan; Minasov, George; Dubrovska, Ievgeniia; Winsor, James R; Myszka, David G; Duban, Mark; Shuvalova, Ludmilla; Yakunin, Alexander F; Anderson, Wayne F

2015-05-01

When exposed to high osmolarity, methicillin-resistant Staphylococcus aureus (MRSA) restores its growth and establishes a new steady state by accumulating the osmoprotectant metabolite betaine. Effective osmoregulation has also been implicated in the acquirement of a profound antibiotic resistance by MRSA. Betaine can be obtained from the bacterial habitat or produced intracellularly from choline via the toxic betaine aldehyde (BA) employing the choline dehydrogenase and betaine aldehyde dehydrogenase (BADH) enzymes. Here, it is shown that the putative betaine aldehyde dehydrogenase SACOL2628 from the early MRSA isolate COL (SaBADH) utilizes betaine aldehyde as the primary substrate and nicotinamide adenine dinucleotide (NAD(+)) as the cofactor. Surface plasmon resonance experiments revealed that the affinity of NAD(+), NADH and BA for SaBADH is affected by temperature, pH and buffer composition. Five crystal structures of the wild type and three structures of the Gly234Ser mutant of SaBADH in the apo and holo forms provide details of the molecular mechanisms of activity and substrate specificity/inhibition of this enzyme.

Advanced Solar Panel Designs

NASA Technical Reports Server (NTRS)

Ralph, E. L.; Linder, E. B.

1995-01-01

Solar panel designs that utilize new high-efficiency solar cells and lightweight rigid panel technologies are described. The resulting designs increase the specific power (W/kg) achievable in the near-term and are well suited to meet the demands of higher performance small satellites (smallsats). Advanced solar panel designs have been developed and demonstrated on two NASA SBIR contracts at Applied Solar. The first used 19% efficient, large area (5.5 cm x 6.5 cm) GaAs/Ge solar cells with a lightweight rigid graphite epoxy isogrid substrate configuration. A 1,445 sq cm coupon was fabricated and tested to demonstrate 60 W/kg with a high potential of achieving 80 W/kg. The second panel design used new 22% efficiency, dual-junction GaInP2/GaAs/Ge solar cells combined with a lightweight aluminum core/graphite fiber mesh facesheet substrate. A 1,445 sq cm coupon was fabricated and tested to demonstrate 105 W/kg with the potential of achieving 115 W/kg.

Chemoselective Nitro Group Reduction and Reductive Dechlorination Initiate Degradation of 2-Chloro-5-Nitrophenol by Ralstonia eutropha JMP134

PubMed Central

Schenzle, Andreas; Lenke, Hiltrud; Spain, Jim C.; Knackmuss, Hans-Joachim

1999-01-01

Ralstonia eutropha JMP134 utilizes 2-chloro-5-nitrophenol as a sole source of nitrogen, carbon, and energy. The initial steps for degradation of 2-chloro-5-nitrophenol are analogous to those of 3-nitrophenol degradation in R. eutropha JMP134. 2-Chloro-5-nitrophenol is initially reduced to 2-chloro-5-hydroxylaminophenol, which is subject to an enzymatic Bamberger rearrangement yielding 2-amino-5-chlorohydroquinone. The chlorine of 2-amino-5-chlorohydroquinone is removed by a reductive mechanism, and aminohydroquinone is formed. 2-Chloro-5-nitrophenol and 3-nitrophenol induce the expression of 3-nitrophenol nitroreductase, of 3-hydroxylaminophenol mutase, and of the dechlorinating activity. 3-Nitrophenol nitroreductase catalyzes chemoselective reduction of aromatic nitro groups to hydroxylamino groups in the presence of NADPH. 3-Nitrophenol nitroreductase is active with a variety of mono-, di-, and trinitroaromatic compounds, demonstrating a relaxed substrate specificity of the enzyme. Nitrosobenzene serves as a substrate for the enzyme and is converted faster than nitrobenzene. PMID:10347008

Neural substrates of trait ruminations in depression

PubMed Central

Mandell, Darcy; Siegle, Greg; Shutt, Luann; Feldmiller, Josh; Thase, Michael E.

2014-01-01

Rumination in depression is a risk factor for longer, more intense, and harder-to-treat depressions. But there appear to be multiple types of depressive rumination – whether they all share these vulnerability mechanisms, and thus would benefit from the same types of clinical attention is unclear. In the current study, we examined neural correlates of empirically-derived dimensions of trait rumination in 35 depressed participants. These individuals and 29 never-depressed controls completed 17 self-report measures of rumination and an alternating emotion-processing/executive-control task during functional magnetic resonance imaging (fMRI) assessment. We examined associations of regions of interest—the amygdala and other cortical regions subserving a potential role in deficient cognitive control and elaborative emotion-processing—with trait rumination. Rumination of all types was generally associated with increased sustained amygdala reactivity. When controlling for amygdala reactivity, distinct activity patterns in hippocampus were also associated with specific dimensions of rumination. We discuss the possibly utility of targeting more basic biological substrates of emotional reactivity in depressed patients who frequently ruminate. PMID:24661157

A comparison of enhancement techniques for footwear impressions on dark and patterned fabrics.

PubMed

Farrugia, Kevin J; Bandey, Helen; Dawson, Lorna; Daéid, Niamh Nic

2013-11-01

The use of chemical enhancement techniques on porous substrates, such as fabrics, poses several challenges predominantly due to the occurrence of background staining and diffusion as well as visualization difficulties. A range of readily available chemical and lighting techniques were utilized to enhance footwear impressions made in blood, soil, and urine on dark and patterned fabrics. Footwear impressions were all prepared at a set force using a specifically built footwear rig. In most cases, results demonstrated that fluorescent chemical techniques were required for visualization as nonfluorescent techniques provided little or no contrast with the background. Occasionally, this contrast was improved by oblique lighting. Successful results were obtained for the enhancement of footwear impressions in blood; however, the enhancement of footwear impressions in urine and soil on dark and patterned fabrics was much more limited. The results demonstrate that visualization and fluorescent enhancement on porous substrates such as fabrics is possible. © 2013 American Academy of Forensic Sciences.

ARTD1 (PARP1) activation and NAD+ in DNA repair and cell death

PubMed Central

Fouquerel, Elise; Sobol, Robert W.

2014-01-01

Nicotinamide adenine dinucleotide, NAD+, is a small metabolite coenzyme that is essential for the progress of crucial cellular pathways including glycolysis, the tricarboxylic acid cycle (TCA) and mitochondrial respiration. These processes consume and produce both oxidative and reduced forms of NAD (NAD+ and NADH). NAD+ is also important for ADP(ribosyl)ation reactions mediated by the ADP-ribosyltransferase enzymes (ARTDs) or deacetylation reactions catalysed by the sirtuins (SIRTs) which use NAD+ as a substrate. In this review, we highlight the significance of NAD+ catabolism in DNA repair and cell death through its utilization by ARTDs and SIRTs. We summarize the current findings on the involvement of ARTD1 activity in DNA repair and most specifically its involvement in the trigger of cell death mediated by energy depletion. By sharing the same substrate, the activities of ARTDs and SIRTs are tightly linked and dependent on each other and are thereby involved in the same cellular processes that play an important role in cancer biology, inflammatory diseases and ischemia/reperfusion. PMID:25283336

MICROBIAL METABOLISM OF AROMATIC COMPOUNDS I.

PubMed Central

Tabak, Henry H.; Chambers, Cecil W.; Kabler, Paul W.

1964-01-01

Tabak, Henry H. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), Cecil W. Chambers, and Paul W. Kabler. Microbial metabolism of aromatic carbon compounds. I. Decomposition of phenolic compounds and aromatic hydrocarbons by phenol-adapted bacteria. J. Bacteriol. 87:910–919. 1964.—Bacteria from soil and related environments were selected or adapted to metabolize phenol, hydroxy phenols, nitrophenols, chlorophenols, methylphenols, alkylphenols, and arylphenols when cultured in mineral salts media with the specific substrate as the sole source of carbon. A phenol-adapted culture (substrate-induced enzyme synthesis proven) was challenged in respirometric tests with 104 related compounds; probable significant oxidative activity occurred with 65. Dihydric phenols were generally oxidized; trihydric phenols were not. Cresols and dimethylphenols were oxidized; adding a chloro group increased resistance. Benzoic and hydroxybenzoic acids were oxidized; sulfonated, methoxylated, nitro, and chlorobenzoic acids were not; m-toluic acid was utilized but not the o- and p-isomers. Benzaldehyde and p-hydroxybenzaldehyde were oxidized. In general, nitro- and chloro-substituted compounds and the benzenes were difficult to oxidize. PMID:14137630

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE PAGES

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

2016-03-31

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

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

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

Microwave GaAs Integrated Circuits On Quartz Substrates

NASA Technical Reports Server (NTRS)

Siegel, Peter H.; Mehdi, Imran; Wilson, Barbara

1994-01-01

Integrated circuits for use in detecting electromagnetic radiation at millimeter and submillimeter wavelengths constructed by bonding GaAs-based integrated circuits onto quartz-substrate-based stripline circuits. Approach offers combined advantages of high-speed semiconductor active devices made only on epitaxially deposited GaAs substrates with low-dielectric-loss, mechanically rugged quartz substrates. Other potential applications include integration of antenna elements with active devices, using carrier substrates other than quartz to meet particular requirements using lifted-off GaAs layer in membrane configuration with quartz substrate supporting edges only, and using lift-off technique to fabricate ultrathin discrete devices diced separately and inserted into predefined larger circuits. In different device concept, quartz substrate utilized as transparent support for GaAs devices excited from back side by optical radiation.

Kinetic mechanism of Toxoplasma gondii adenosine kinase and the highly efficient utilization of adenosine

PubMed Central

Naguib, Fardos N. M.; Rais, Reem H.; Al Safarjalani, Omar N.; el Kouni, Mahmoud H.

2015-01-01

Toxoplasma gondii has an extraordinarily ability to utilize adenosine (Ado) as the primary source of all necessary purines in this parasite which lacks de novo purine biosynthesis. The activity of T. gondii adenosine kinase (TgAK, EC 2.7.1.20) is responsible for this efficient salvage of Ado in T. gondii. To fully understand this remarkable efficiency of TgAK in the utilization of Ado, complete kinetic parameters of this enzyme are necessary. Initial velocity and product inhibition studies of TgAK demonstrated that the basic mechanism of this enzyme is a hybrid random bi-uni ping-pong uni-bi. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation and a binding pattern indicating that binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KAdo = 0.002 ± 0.0002 mM, KATP = 0.05 ± 0.008 mM, and Vmax = 920 ± 35 μmol/min/mg protein. Ado exhibited substrate inhibition suggesting the presence of more than one binding site for Ado on the enzyme. ATP relieved substrate inhibition by Ado. Thus, Ado also binds to the ATP binding site. AMP was competitive with ATP, inferring that AMP binds to the same site as ATP. AMP, ADP and ATP were non-competitive with Ado, therefore, none of these nucleotides binds to the Ado binding site. Combining ATP with ADP was additive. Therefore, the binding of either ATP or ADP does not interfere with the binding of the other. It is concluded that for every ATP consumed, TgAK generates three new AMPs. These findings along with the fact that a wide range of nucleoside 5′-mono, di, and triphosphates could substitute for ATP as phosphate donors in this reaction may explain the efficient and central role played by TgAK in the utilization of Ado as the major source from which all other purines can be synthesized in T. gondii. PMID:26112826

Effect of selective and nonselective beta-blockers on resting energy production rate and total body substrate utilization in chronic heart failure.

PubMed

Podbregar, Matej; Voga, Gorazd

2002-12-01

In chronic heart failure (CHF) beta-blockers reduce myocardial oxygen consumption and improve myocardial efficiency by shifting myocardial substrate utilization from increased free fatty acid oxidation to increased glucose oxidation. The effect of selective and nonselective beta-blockers on total body resting energy production rate (EPR) and substrate utilization is not known. Twenty-six noncachectic patients with moderately severe heart failure (New York Heart Association class II or III, left ventricular ejection fraction < 0.40) were treated with carvedilol (37.5 +/- 13.5 mg/12 h) or bisoprolol (5.4 +/- 3.0 mg/d) for 6 months. Indirect calorimetry was performed before and after 6 months of treatment. Resting EPR was decreased in carvedilol (5.021 +/- 0.803 to 4.552 +/- 0.615 kJ/min, P <.001) and bisoprolol group (5.230 +/- 0.828 to 4.978 +/- 0.640 kJ/min, P <.05; nonsignificant difference between groups). Lipid oxidation rate decreased in carvedilol and remained unchanged in bisoprolol group (2.4 +/- 1.4 to 1.5 +/- 0.9 mg m(2)/kg min versus 2.7 +/- 1.1 to 2.5 +/- 1.1 mg m(2)/kg min, P <.05). Glucose oxidation rate was increased only in carvedilol (2.6 +/- 1.4 to 4.4 +/- 1.6 mg m(2)/kg min, P <.05), but did not change in bisoprolol group. Both selective and nonselective beta-blockers reduce total body resting EPR in noncachectic CHF patients. Carvedilol compared to bisoprolol shifts total body substrate utilization from lipid to glucose oxidation.

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

PubMed Central

Lee, Rachel S.; House, Colin M.; Cristiano, Briony E.; Hannan, Ross D.; Pearson, Richard B.; Hannan, Katherine M.

2011-01-01

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ. PMID:21869924

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

PubMed Central

Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

2017-01-01

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

Performance of microbial fuel cell double chamber using mozzarella cheese whey substrate

NASA Astrophysics Data System (ADS)

Darmawan, M. D.; Hawa, L. C.; Argo, B. D.

2018-03-01

Nowadays the availability of electric energy is decreasing, hence there is a need for innovation of electric energy producer alternative; one of them is microbial fuel cell (MFC). MFC is a bioelectrochemical system generated by bacterial metabolism that utilizes organic substrate. One of the substrates that can be used is whey, a waste generated from cheese production. Therefore, this study aimed to determine the power of potential current and voltage generated from the use of whey cheese as a substrate for bacterial metabolism. In this research, double chamber system was used in microbial fuel cell reactor by using cheese whey as substrate at anode and potassium permanganate as cathode and utilizing membrane nafion 212 as membrane of proton exchange. The variable of experiment was bacteria type. The types of bacteria used in this study were Lactobacillus bulgaricus, Streptococcus thermophillus and Lactobacillus casei. While the operating time used was 100 hours. The highest current produced was 74.6 μA and the highest voltage was 529.3 mV produced by Lactobacillus bulgaricus bacteria. In this study, it was also found that the death phase of the three bacteria was at 70-80 hours.

Cellulosic ethanol production via consolidated bioprocessing by a novel thermophilic anaerobic bacterium isolated from a Himalayan hot spring.

PubMed

Singh, Nisha; Mathur, Anshu S; Tuli, Deepak K; Gupta, Ravi P; Barrow, Colin J; Puri, Munish

2017-01-01

Cellulose-degrading thermophilic anaerobic bacterium as a suitable host for consolidated bioprocessing (CBP) has been proposed as an economically suited platform for the production of second-generation biofuels. To recognize the overall objective of CBP, fermentation using co-culture of different cellulolytic and sugar-fermenting thermophilic anaerobic bacteria has been widely studied as an approach to achieving improved ethanol production. We assessed monoculture and co-culture fermentation of novel thermophilic anaerobic bacterium for ethanol production from real substrates under controlled conditions. In this study, Clostridium sp. DBT-IOC-C19, a cellulose-degrading thermophilic anaerobic bacterium, was isolated from the cellulolytic enrichment cultures obtained from a Himalayan hot spring. Strain DBT-IOC-C19 exhibited a broad substrate spectrum and presented single-step conversion of various cellulosic and hemicellulosic substrates to ethanol, acetate, and lactate with ethanol being the major fermentation product. Additionally, the effect of varying cellulose concentrations on the fermentation performance of the strain was studied, indicating a maximum cellulose utilization ability of 10 g L -1 cellulose. Avicel degradation kinetics of the strain DBT-IOC-C19 displayed 94.6% degradation at 5 g L -1 and 82.74% degradation at 10 g L -1 avicel concentration within 96 h of fermentation. In a comparative study with Clostridium thermocellum DSM 1313, the ethanol and total product concentrations were higher by the newly isolated strain on pretreated rice straw at an equivalent substrate loading. Three different co-culture combinations were used on various substrates that presented two-fold yield improvement than the monoculture during batch fermentation. This study demonstrated the direct fermentation ability of the novel thermophilic anaerobic bacteria on various cellulosic and hemicellulosic substrates into ethanol without the aid of any exogenous enzymes, representing CBP-based fermentation approach. Here, the broad substrate utilization spectrum of isolated cellulolytic thermophilic anaerobic bacterium was shown to be of potential utility. We demonstrated that the co-culture strategy involving novel strains is efficient in improving ethanol production from real substrate.

[Myocardial substrate support before and after mitral commissurotomy].

PubMed

Ovchinnikov, I V; Guliamov, D S; Andres, Iu P; Amanov, A A; Khodzhaev, M M

1981-07-01

The utilization by the myocardium of 8 substrates of the carboxydrate-lipid metabolism was assessed in 44 patients with the 3d and 4th stages of mitral stenosis before and after mitral commissurotomy by the method of the arterio-venous difference. The character of the myocardium supply with substrates with different energetic value was established to depend and to be a sufficiently objective criterium of prognosis of cardiac insufficiency in the early postoperative period.

Glucose Transporters in Cardiac Metabolism and Hypertrophy

PubMed Central

Shao, Dan; Tian, Rong

2016-01-01

The heart is adapted to utilize all classes of substrates to meet the high-energy demand, and it tightly regulates its substrate utilization in response to environmental changes. Although fatty acids are known as the predominant fuel for the adult heart at resting stage, the heart switches its substrate preference toward glucose during stress conditions such as ischemia and pathological hypertrophy. Notably, increasing evidence suggests that the loss of metabolic flexibility associated with increased reliance on glucose utilization contribute to the development of cardiac dysfunction. The changes in glucose metabolism in hypertrophied hearts include altered glucose transport and increased glycolysis. Despite the role of glucose as an energy source, changes in other nonenergy producing pathways related to glucose metabolism, such as hexosamine biosynthetic pathway and pentose phosphate pathway, are also observed in the diseased hearts. This article summarizes the current knowledge regarding the regulation of glucose transporter expression and translocation in the heart during physiological and pathological conditions. It also discusses the signaling mechanisms governing glucose uptake in cardiomyocytes, as well as the changes of cardiac glucose metabolism under disease conditions. PMID:26756635

Metabolism of Nitrogen Oxides in Ammonia-Oxidizing Bacteria

NASA Astrophysics Data System (ADS)

Kozlowski, J.; Stein, L. Y.

2014-12-01

Ammonia-oxidizing bacteria (AOB) are key microorganisms in the transformation of nitrogen intermediates in most all environments. Until recently there was very little work done to elucidate the physiology of ammonia-oxidizing bacteria cultivated from variable trophic state environments. With a greater variety of ammonia-oxidizers now in pure culture the importance of comparative physiological and genomic analysis is crucial. Nearly all known physiology of ammonia-oxidizing bacteria lies within the Nitrosomonas genus with Nitrosomonas europaea strain ATCC 19718 as the model. To more broadly characterize and understand the nature of obligate ammonia chemolithotrophy and the contribution of AOB to production of nitrogen oxides, Nitrosomonas spp. and Nitrosospira spp. isolated from variable trophic states and with sequenced genomes, were utilized. Instantaneous ammonia- and hydroxylamine-oxidation kinetics as a function of oxygen and substrate concentration were measured using an oxygen micro-sensor. The pathway intermediates nitric oxide and nitrous oxide were measured in real time using substrate-specific micro-sensors to elucidate whether production of these molecules is stoichiometric with rates of substrate oxidation. Genomic inventory was compared among the strains to identify specific pathways and modules to explain physiological differences in kinetic rates and production of N-oxide intermediates as a condition of their adaptation to different ammonium concentrations. This work provides knowledge of how nitrogen metabolism is differentially controlled in AOB that are adapted to different concentrations of ammonium. Overall, this work will provide further insight into the control of ammonia oxidizing chemolithotrophy across representatives of the Nitrosomonas and Nitrosospira genus, which can then be applied to examine additional genome-sequenced AOB isolates.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.

PubMed

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-07-29

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology.

The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass

PubMed Central

Maki, Miranda; Leung, Kam Tin; Qin, Wensheng

2009-01-01

Lignocellulosic biomass is a renewable and abundant resource with great potential for bioconversion to value-added bioproducts. However, the biorefining process remains economically unfeasible due to a lack of biocatalysts that can overcome costly hurdles such as cooling from high temperature, pumping of oxygen/stirring, and, neutralization from acidic or basic pH. The extreme environmental resistance of bacteria permits screening and isolation of novel cellulases to help overcome these challenges. Rapid, efficient cellulase screening techniques, using cellulase assays and metagenomic libraries, are a must. Rare cellulases with activities on soluble and crystalline cellulose have been isolated from strains of Paenibacillus and Bacillus and shown to have high thermostability and/or activity over a wide pH spectrum. While novel cellulases from strains like Cellulomonas flavigena and Terendinibacter turnerae, produce multifunctional cellulases with broader substrate utilization. These enzymes offer a framework for enhancement of cellulases including: specific activity, thermalstability, or end-product inhibition. In addition, anaerobic bacteria like the clostridia offer potential due to species capable of producing compound multienzyme complexes called cellulosomes. Cellulosomes provide synergy and close proximity of enzymes to substrate, increasing activity towards crystalline cellulose. This has lead to the construction of designer cellulosomes enhanced for specific substrate activity. Furthermore, cellulosome-producing Clostridium thermocellum and its ability to ferment sugars to ethanol; its amenability to co-culture and, recent advances in genetic engineering, offer a promising future in biofuels. The exploitation of bacteria in the search for improved enzymes or strategies provides a means to upgrade feasibility for lignocellulosic biomass conversion, ultimately providing means to a 'greener' technology. PMID:19680472

Assessing methanotrophy and carbon fixation for biofuel production by Methanosarcina acetivorans

DOE PAGES

Nazem-Bokaee, Hadi; Gopalakrishnan, Saratram; Ferry, James G.; ...

2016-01-17

Methanosarcina acetivorans is a model archaeon with renewed interest due to its unique reversible methane production pathways. However, the mechanism and relevant pathways implicated in (co)utilizing novel carbon substrates in this organism are still not fully understood. This paper provides a comprehensive inventory of thermodynamically feasible routes for anaerobic methane oxidation, co-reactant utilization, and maximum carbon yields of major biofuel candidates by M. acetivorans. Here, an updated genome-scale metabolic model of M. acetivorans is introduced (iMAC868 containing 868 genes, 845 reactions, and 718 metabolites) by integrating information from two previously reconstructed metabolic models (i.e., iVS941 and iMB745), modifying 17 reactions,more » adding 24 new reactions, and revising 64 gene-proteinreaction associations based on newly available information. The new model establishes improved predictions of growth yields on native substrates and is capable of correctly predicting the knockout outcomes for 27 out of 28 gene deletion mutants. By tracing a bifurcated electron flow mechanism, the iMAC868 model predicts thermodynamically feasible (co)utilization pathway of methane and bicarbonate using various terminal electron acceptors through the reversal of the aceticlastic pathway. In conclusion, this effort paves the way in informing the search for thermodynamically feasible ways of (co)utilizing novel carbon substrates in the domain Archaea.« less

Effects of space flight, clinorotation, and centrifugation on the substrate utilization efficiency of E. coli

NASA Technical Reports Server (NTRS)

Brown, Robert B.; Klaus, D.; Todd, P.

2002-01-01

Cultures of Escherichia coli grown in space reached a 25% higher average final cell population than those in comparably matched ground controls (p<0.05). However, both groups consumed the same quantity of glucose, which suggests that space flight not only stimulated bacterial growth as has been previously reported, but also resulted in a 25% more efficient utilization of the available nutrients. Supporting experiments performed in "simulated weightlessness" under clinorotation produced similar trends of increased growth and efficiency, but to a lesser extent in absolute values. These experiments resulted in increases of 12% and 9% in average final cell population (p<0.05), while the efficiency of substrate utilization improved by 6% and 9% relative to static controls (p=0.12 and p<0.05, respectively). In contrast, hypergravity, produced by centrifugation, predictably resulted in the opposite effect--a decrease of 33% to 40% in final cell numbers with corresponding 29% to 40% lower net growth efficiencies (p<0.01). Collectively, these findings support the hypothesis that the increased bacterial growth observed in weightlessness is a result of reduced extracellular mass transport that occurs in the absence of sedimentation and buoyancy-driven convection, which consequently also improves substrate utilization efficiency in suspended cultures.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Biodegradation kinetics of thin-stillage treatment by Aspergillus awamori and characterization of recovered chitosan.

PubMed

Ray, S Ghosh; Ghangrekar, M M

2016-02-01

An attempt has been made to provide solution for distillery wastewater using fungal pretreatment followed by an anaerobic process to achieve higher organic matter removal, which is a challenge at present with currently adopted technologies. Submerged growth kinetics of distillery wastewater supernatant by Aspergillus awamori was also evaluated. The proposed kinetic models using a logistic equation for fungal growth and the Leudeking-Piret equation for product formation were validated experimentally, and substrate consumption equation was derived using estimated kinetic coefficients. Up to 59.6 % chemical oxygen demand (COD) and 70 % total organic carbon (TOC) removals were observed in 96 h of fungal incubation. Maximum specific growth rate of fungi, coefficient of biomass yield on substrate and growth-associated product formation coefficient were estimated to be 0.07 ± 0.01 h(-1), 0.614 kg biomass/kg utilized COD and 0.215 kg CO2/kg utilized TOC, respectively. The chitosan recovery of 0.072-0.078 kg/kg of dry mycelium was obtained using dilute sulphuric acid extraction, showing high purity and characteristic chitosan properties according to FTIR and XRD analyses. After anaerobic treatment of the fungal pretreated effluent with COD concentration of 7.920 ± 0.120 kg COD/m(3) (organic loading rate of 3.28 kg COD/m(3) day), overall COD reduction of 91.07 % was achieved from distillery wastewater.

Arylsulfotransferase from Clostridium innocuum-A new enzyme catalyst for sulfation of phenol-containing compounds.

PubMed

Mozhaev, Vadim V; Khmelnitsky, Yuri L; Sanchez-Riera, Fernando; Maurina-Brunker, Julie; Rosson, Reinhardt A; Grund, Alan D

2002-06-05

Arylsulfotransferase (AST, EC 2.8.2.22), an enzyme capable of sulfating a wide range of phenol-containing compounds was purified from a Clostridium innocuum isolate (strain 554). The enzyme has a molecular weight of 320 kDa and is composed of four subunits. Unlike many mammalian and plant arylsulfotransferases, AST from Clostridium utilizes arylsulfates, including p-nitrophenyl sulfate, as sulfate donors, and is not reactive with 3-phosphoadenosine-5'-phosphosulfate (PAPS). The enzyme possesses broad substrate specificity and is active with a variety of phenols, quinones and flavonoids, but does not utilize primary and secondary alcohols and sugars as substrates. Arylsulfotransferase tolerates the presence of 10 vol% of polar cosolvents (dimethyl formamide, acetonitrile, methanol), but loses significant activity at higher solvent concentrations of 30-40 vol%. The enzyme retains high arylsulfotransferase activity in biphasic systems composed of water and nonpolar solvents, such as cyclohexane, toluene and chloroform, while in biphasic systems with more polar solvents (ethyl acetate, 2-pentanone, methyl tert-butyl ether, and butyl acetate) the enzyme activity is completely lost. High yields of AST-catalyzed sulfation were achieved in reactions with several phenols and tyrosine-containing peptides. Overall, AST studied in this work is a promising biocatalyst in organic synthesis to afford efficient sulfation of phenolic compounds under mild reaction conditions. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 567-575, 2002.

Method of forming crystalline silicon devices on glass

DOEpatents

McCarthy, A.M.

1995-03-21

A method is disclosed for fabricating single-crystal silicon microelectronic components on a silicon substrate and transferring same to a glass substrate. This is achieved by utilizing conventional silicon processing techniques for fabricating components of electronic circuits and devices on bulk silicon, wherein a bulk silicon surface is prepared with epitaxial layers prior to the conventional processing. The silicon substrate is bonded to a glass substrate and the bulk silicon is removed leaving the components intact on the glass substrate surface. Subsequent standard processing completes the device and circuit manufacturing. This invention is useful in applications requiring a transparent or insulating substrate, particularly for display manufacturing. Other applications include sensors, actuators, optoelectronics, radiation hard electronics, and high temperature electronics. 7 figures.

Epoxy bond and stop etch fabrication method

DOEpatents

Simmons, Jerry A.; Weckwerth, Mark V.; Baca, Wes E.

2000-01-01

A class of epoxy bond and stop etch (EBASE) microelectronic fabrication techniques is disclosed. The essence of such techniques is to grow circuit components on top of a stop etch layer grown on a first substrate. The first substrate and a host substrate are then bonded together so that the circuit components are attached to the host substrate by the bonding agent. The first substrate is then removed, e.g., by a chemical or physical etching process to which the stop etch layer is resistant. EBASE fabrication methods allow access to regions of a device structure which are usually blocked by the presence of a substrate, and are of particular utility in the fabrication of ultrafast electronic and optoelectronic devices and circuits.

Influence of mare uterine tubal fluids on the metabolism of stallion sperm.

PubMed

Engle, C E; Foley, C W; Witherspoon, D M; Scarth, R D; Goetsch, D D

1975-08-01

Three experiments were conducted on the metabolism of stallion sperm. In experiment 1, whole and washed sperm were incubated under aerobic and anaerobic enviroments and analyzed before and after controlled incubation for motility, pH, lactic acid, glucose, fructose, and O2 comsumption. In experiment 2, whole and washed sperm were incubated aerobically and anaerobically with and without uterine tubal fluids. Experiment 3 was the same as experiment 2, except added substrates of glucose and lactic acid were studied. The same examinations were made in experiments 2 and 3 as for experiment 1. Motility decreased significantly during incubation for all treatments, with the greatest decrease occurring for whole semen where only trace amounts of substrate (fructose) were present. Exogenous glucose plus uterine tubal fluid maintained sperm motility better than did added lactate. However, sperm respiration rates were highest when exogenous lactate was the only substrate in the incubation medium. The mean pH values for gel-free stallion semen at the start of controlled aerobic and anaerobic incubation were 7.08 and 7.34. Lactic acid accummulation for 1 hour increased from 0.05 mg to 0.09 mg/10(9) sperm when uterine tubal fluid was added to the incubation medium. Washed spermatozoa incubated in 0.03 M glucose plus uterine tubal fluid utilized less glucose than did sperm incubated in the glucose medium. These results, along with the increased oxygen utilization (ZO2) values produced by adding uterine tubal fluid to the incubation mediums, might indicate utilization of a uterine tubal substrate. Added uterine tubal fluid resulted in increased ZO2 values (expressed in mul of O2 utilized by 10(8) sperm in 1 hour at 37 C) for whole semen from 10.45 to 12.63. Washed spermatozoa also respired at a significantly greater rate than whole sperm. Respiration rates were greater for sperm incubated with 0.01 M lactic acid than for any other substrate or experiment.

Comparison of metabolic substrates in alligators and several birds of prey.

PubMed

Sweazea, Karen L; McMurtry, John P; Elsey, Ruth M; Redig, Patrick; Braun, Eldon J

2014-08-01

On average, avian blood glucose concentrations are 1.5-2 times those of mammals of similar mass and high concentrations of insulin are required to lower blood glucose. Whereas considerable data exist for granivorous species, few data are available for plasma metabolic substrate and glucoregulatory hormone concentrations for carnivorous birds and alligators. Birds and mammals with carnivorous diets have higher metabolic rates than animals consuming diets with less protein whereas alligators have low metabolic rates. Therefore, the present study was designed to compare substrate and glucoregulatory hormone concentrations in several birds of prey and a phylogenetically close relative of birds, the alligator. The hypothesis was that the combination of carnivorous diets and high metabolic rates favored the evolution of greater protein and fatty acid utilization leading to insulin resistance and high plasma glucose concentrations in carnivorous birds. In contrast, it was hypothesized that alligators would have low substrate utilization attributable to a low metabolic rate. Fasting plasma substrate and glucoregulatory hormone concentrations were compared for bald eagles (Haliaeetus leucocephalus), great horned owls (Bubo virginianus), red-tailed hawks (Buteo jamaicensis), and American alligators (Alligator mississippiensis). Avian species had high circulating β-hydroxybutyrate (10-21 mg/dl) compared to alligators (2.81 ± 0.16 mg/dl). In mammals high concentrations of this byproduct of fatty acid utilization are correlated with insulin resistance. Fasting glucose and insulin concentrations were positively correlated in eagles whereas no relationship was found between these variables for owls, hawks or alligators. Additionally, β-hydroxybutyrate concentrations were low in alligators. Similar to carnivorous mammals, ingestion of a high protein diet may have favored the utilization of fatty acids and protein for energy thereby promoting the development of insulin resistance and gluconeogenesis-induced high plasma glucose concentrations during periods of fasting in birds of prey. Copyright © 2014 Elsevier GmbH. All rights reserved.

GaAs Monolithic Microwave Subsystem Technology Base

DTIC Science & Technology

1980-01-01

To provide a captive source of reliable, high-quality GaAs substrates, a new crystal growth and substrate preparation facility which utilizes a high...Symp. GaAs and Related Compounds, Inst. Phys. Conf. Ser. 24, 6. 20. Wood, Woodcock and Harris (1978) GaAs and Related Compounds, Inst. Phys. Conf

Chemical Sensing Systems that Utilize Soft Electronics on Thin Elastomeric Substrates with Open Cellular Designs

PubMed Central

Lee, Yoon Kyeung; Jang, Kyung-In; Ma, Yinji; Koh, Ahyeon; Chen, Hang; Jung, Han Na; Kim, Yerim; Kwak, Jean Won; Wang, Liang; Xue, Yeguang; Yang, Yiyuan; Tian, Wenlong; Jiang, Yu; Zhang, Yihui; Feng, Xue; Huang, Yonggang

2017-01-01

A collection of materials and device architectures are introduced for thin, stretchable arrays of ion sensors that mount on open cellular substrates to facilitate solution exchange for use in biointegrated electronics. The results include integration strategies and studies of fundamental characteristics in chemical sensing and mechanical response. The latter involves experimental measurements and theoretical simulations that establish important considerations in the design of low modulus, stretchable properties in cellular substrates, and in the realization of advanced capabilities in spatiotemporal mapping of chemicals' gradients. As the chemical composition of extracellular fluids contains valuable information related to biological function, the concepts introduced here have potential utility across a range of skin- and internal-organ-integrated electronics where soft mechanics, fluidic permeability, and advanced chemical sensing capabilities are key requirements. PMID:28989338

Application of continuous substrate feeding to the ABE fermentation: Relief of product inhibition using extraction, perstraction, stripping, and pervaporation

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

Qureshi, N.; Maddox, I.S.; Friedl, A.

1992-09-01

The technique of continuous substrate feeding has been applied to the batch fermentation process using freely suspended cells, for ABE (acetone-butanol-ethanol) production. To avoid the product inhibition which normally restricts ABE production to less than 20 g/L and sugar utilization to 60 g/L, a product removal technique has been integrated into the fermentation process. The techniques investigated were liquid-liquid extraction, perstraction, gas-stripping, and pervaporation. By using a substrate of whey permeate, the reactor productivity has been improved over that observed in a traditional batch fermentation, while equivalent lactose utilization and ABE production values of 180 g and 69 g, respectively,more » have been achieved in a 1-L culture volume. 17 refs., 14 figs., 5 tabs.« less

Retinal Processing: Polarization Vision in Teleost Fishes

DTIC Science & Technology

2005-07-26

conspecific visual communication network utilizing polarized light signals in the coral reef environment. These observations have provoked interest in the... visual communication net- reflection off non-metallic substrates produces predom- work utilizing polarized light signals in the coral reef inantly

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

An Outer Membrane Protein Involved in the Uptake of Glucose Is Essential for Cytophaga hutchinsonii Cellulose Utilization

PubMed Central

Zhou, Hong; Wang, Xia; Yang, Tengteng; Zhang, Weixin; Chen, Guanjun

2016-01-01

Cytophaga hutchinsonii specializes in cellulose digestion by employing a collection of novel cell-associated proteins. Here, we identified a novel gene locus, CHU_1276, that is essential for C. hutchinsonii cellulose utilization. Disruption of CHU_1276 in C. hutchinsonii resulted in complete deficiency in cellulose degradation, as well as compromised assimilation of cellobiose or glucose at a low concentration. Further analysis showed that CHU_1276 was an outer membrane protein that could be induced by cellulose and low concentrations of glucose. Transcriptional profiling revealed that CHU_1276 exerted a profound effect on the genome-wide response to both glucose and Avicel and that the mutant lacking CHU_1276 displayed expression profiles very different from those of the wild-type strain under different culture conditions. Specifically, comparison of their transcriptional responses to cellulose led to the identification of a gene set potentially regulated by CHU_1276. These results suggest that CHU_1276 plays an essential role in cellulose utilization, probably by coordinating the extracellular hydrolysis of cellulose substrate with the intracellular uptake of the hydrolysis product in C. hutchinsonii. PMID:26773084

Cloning and characterization of a Candida albicans maltase gene involved in sucrose utilization.

PubMed Central

Geber, A; Williamson, P R; Rex, J H; Sweeney, E C; Bennett, J E

1992-01-01

In order to isolate the structural gene involved in sucrose utilization, we screened a sucrose-induced Candida albicans cDNA library for clones expressing alpha-glucosidase activity. The C. albicans maltase structural gene (CAMAL2) was isolated. No other clones expressing alpha-glucosidase activity. were detected. A genomic CAMAL2 clone was obtained by screening a size-selected genomic library with the cDNA clone. DNA sequence analysis reveals that CAMAL2 encodes a 570-amino-acid protein which shares 50% identity with the maltase structural gene (MAL62) of Saccharomyces carlsbergensis. The substrate specificity of the recombinant protein purified from Escherichia coli identifies the enzyme as a maltase. Northern (RNA) analysis reveals that transcription of CAMAL2 is induced by maltose and sucrose and repressed by glucose. These results suggest that assimilation of sucrose in C. albicans relies on an inducible maltase enzyme. The family of genes controlling sucrose utilization in C. albicans shares similarities with the MAL gene family of Saccharomyces cerevisiae and provides a model system for studying gene regulation in this pathogenic yeast. Images PMID:1400249

Pedestal substrate for coated optics

DOEpatents

Hale, Layton C.; Malsbury, Terry N.; Patterson, Steven R.

2001-01-01

A pedestal optical substrate that simultaneously provides high substrate dynamic stiffness, provides low surface figure sensitivity to mechanical mounting hardware inputs, and constrains surface figure changes caused by optical coatings to be primarily spherical in nature. The pedestal optical substrate includes a disk-like optic or substrate section having a top surface that is coated, a disk-like base section that provides location at which the substrate can be mounted, and a connecting cylindrical section between the base and optics or substrate sections. The connecting cylindrical section may be attached via three spaced legs or members. However, the pedestal optical substrate can be manufactured from a solid piece of material to form a monolith, thus avoiding joints between the sections, or the disk-like base can be formed separately and connected to the connecting section. By way of example, the pedestal optical substrate may be utilized in the fabrication of optics for an extreme ultraviolet (EUV) lithography imaging system, or in any optical system requiring coated optics and substrates with reduced sensitivity to mechanical mounts.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate-intensity exercise in human skeletal muscle.

PubMed

Vigelsø, A; Gram, M; Dybboe, R; Kuhlman, A B; Prats, C; Greenhaff, P L; Constantin-Teodosiu, D; Birk, J B; Wojtaszewski, J F P; Dela, F; Helge, J W

2016-04-15

This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

The effect of age and unilateral leg immobilization for 2 weeks on substrate utilization during moderate‐intensity exercise in human skeletal muscle

PubMed Central

Gram, M.; Dybboe, R.; Kuhlman, A. B.; Prats, C.; Greenhaff, P. L.; Constantin‐Teodosiu, D.; Birk, J. B.; Wojtaszewski, J. F. P.; Dela, F.; Helge, J. W.

2016-01-01

Key points This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate‐intensity exercise.Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2 weeks on leg substrate utilization in young and older men during two‐legged dynamic knee‐extensor moderate‐intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise.Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise.Using a combined whole‐leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms. Abstract Age and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2 weeks of unilateral leg immobilization on substrate utilization across the legs during moderate‐intensity exercise in young (n = 17; 23 ± 1 years old) and older men (n = 15; 68 ± 1 years old), while the contralateral leg served as the control. After immobilization, the participants performed two‐legged isolated knee‐extensor exercise at 20 ± 1 W (∼50% maximal work capacity) for 45 min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl‐CoA carboxylase 2 and AMP‐activated protein kinase (AMPK)γ3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane‐associated fatty acid binding protein and AMPKγ3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole‐leg and molecular differences in fatty acid mobilization could explain the age‐ and immobilization‐induced IMTG accumulation. PMID:26801521

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

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

Utilisation of mucin glycans by the human gut symbiont Ruminococcus gnavus is strain-dependent.

PubMed

Crost, Emmanuelle H; Tailford, Louise E; Le Gall, Gwenaelle; Fons, Michel; Henrissat, Bernard; Juge, Nathalie

2013-01-01

Commensal bacteria often have an especially rich source of glycan-degrading enzymes which allow them to utilize undigested carbohydrates from the food or the host. The species Ruminococcus gnavus is present in the digestive tract of ≥90% of humans and has been implicated in gut-related diseases such as inflammatory bowel diseases (IBD). Here we analysed the ability of two R. gnavus human strains, E1 and ATCC 29149, to utilize host glycans. We showed that although both strains could assimilate mucin monosaccharides, only R. gnavus ATCC 29149 was able to grow on mucin as a sole carbon source. Comparative genomic analysis of the two R. gnavus strains highlighted potential clusters and glycoside hydrolases (GHs) responsible for the breakdown and utilization of mucin-derived glycans. Transcriptomic and functional activity assays confirmed the importance of specific GH33 sialidase, and GH29 and GH95 fucosidases in the mucin utilisation pathway. Notably, we uncovered a novel pathway by which R. gnavus ATCC 29149 utilises sialic acid from sialylated substrates. Our results also demonstrated the ability of R. gnavus ATCC 29149 to produce propanol and propionate as the end products of metabolism when grown on mucin and fucosylated glycans. These new findings provide molecular insights into the strain-specificity of R. gnavus adaptation to the gut environment advancing our understanding of the role of gut commensals in health and disease.

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

Gene transfer device utilizing micron-spiked electrodes produced by the self-organization phenomenon of Fe-alloy.

PubMed

Miyano, Naoki; Inoue, Yuuki; Teramura, Yuji; Fujii, Keisuke; Tsumori, Fujio; Iwata, Hiroo; Kotera, Hidetoshi

2008-07-01

In the diffusional phase transformation of two-phase alloys, the new phase precipitates form the matrix phase at specific temperatures, followed by the formation of a mixed microstructure comprising the precipitate and the matrix. It has been found that by specific chemical-etching treatment, the precipitate in Fe-25Cr-6Ni alloy projects substantially and clusters at the surface. The configuration of the precipitate has an extremely high aspect ratio: it is several microns in width and several tens of microns in length (known as micron-spiked). This study targets the development of a gene transfer device with a micro-spike produced based on the self-organization phenomenon of the Fe-25Cr-6Ni alloy. With this spike-projected device, we tried to efficiently transfer plasmid DNA into adherent cells by electric pulse-triggered gene transfer using a plasmid-loaded electrode (electroporation-based reverse transfection). The spiked structure was applied to a substrate of the device to allow efficient gene transfer into adherent cells, although the general substrate was flat and had a smooth surface. The results suggest that this unique spike-projected device has potential applications in gene transfer devices for the analysis of the human genome in the post-genome period.

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

PubMed Central

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

2014-01-01

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

Molecular basis of P450 OleTJE: an investigation of substrate binding mechanism and major pathways

NASA Astrophysics Data System (ADS)

Du, Juan; Liu, Lin; Guo, Li Zhong; Yao, Xiao Jun; Yang, Jian Ming

2017-05-01

Cytochrome P450 OleTJE has attracted much attention for its ability to catalyze the decarboxylation of long chain fatty acids to generate alkenes, which are not only biofuel molecule, but also can be used broadly for making lubricants, polymers and detergents. In this study, the molecular basis of the binding mechanism of P450 OleTJE for arachidic acid, myristic acid, and caprylic acid was investigated by utilizing conventional molecular dynamics simulation and binding free energy calculations. Moreover, random acceleration molecular dynamics (RAMD) simulations were performed to uncover the most probable access/egress channels for different fatty acids. The predicted binding free energy shows an order of arachidic acid < myristic acid < caprylic acid. Key residues interacting with three substrates and residues specifically binding to one of them were identified. The RAMD results suggest the most likely channel for arachidic acid, myristic acid, and caprylic acid are 2e/2b, 2a and 2f/2a, respectively. It is suggested that the reaction is easier to carry out in myristic acid bound system than those in arachidic acid and caprylic acid bound system based on the distance of Hβ atom of substrate relative to P450 OleTJE Compound I states. This study provided novel insight to understand the substrate preference mechanism of P450 OleTJE and valuable information for rational enzyme design for short chain fatty acid decarboxylation.

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.

PubMed

Stogios, Peter J; Shakya, Tushar; Evdokimova, Elena; Savchenko, Alexei; Wright, Gerard D

2011-01-21

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.

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

Stogios, Peter J.; Shakya, Tushar; Evdokimova, Elena

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity,more » indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.« less

The Effect of Substrate Microstructure on the Heat-Affected Zone Size in Sn-Zn Alloys Due to Adjoining Ni-Al Reactive Multilayer Foil Reaction

DOE PAGES

Hooper, R. J.; Adams, D. P.; Hirschfeld, D.; ...

2015-08-05

The rapid release of energy from reactive multilayer foils can create extreme local temperature gradients near substrate materials. To fully exploit the potential of these materials, a better understanding of the interaction between the substrate or filler material and the foil is needed. In particular, this work investigates how variations in local properties within the substrate (i.e. differences between properties in constituent phases) can affect heat transport into the substrate. Furthermore, this can affect the microstructural evolution observed within the substrate, which may affect the final joint properties. The effect of the initial substrate microstructure on microstructural evolution within themore » heat-affected zone is evaluated experimentally in two Sn-Zn alloys and numerical techniques are utilized to inform the analysis.« less

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.

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

PubMed Central

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

Building Integrated Photovoltaic Module-Based on Aluminum Substrate With Forced Water Cooling.

PubMed

Pang, Wei; Zhang, Yongzhe; Cui, Yanan; Yu, Hongwen; Liu, Yu; Yan, Hui

2018-04-01

The increase of operating temperature on a photovoltaic (PV) cell degrades its electrical efficiency. This paper is organized to describe our latest design of an aluminum substrate-based photovoltaic/thermal (PV/T) system. The electrical efficiency of the proposed PV/T can be increased by ∼ 20% in comparison with a conventional glass substrate-based PV. The work will benefit hybrid utilization of solar energy in development of building integrated photovoltaic systems.

Reusable tamper-indicating security seal

DOEpatents

Ryan, Michael J.

1983-01-01

The invention teaches means for detecting unauthorized tampering or substitutions of a device, and has particular utility when applied on a "seal" device used to secure a location or thing. The seal has a transparent body wall, and a first indicia, viz., a label identification is formed on the inside surface of this wall. Second and third indicia are formed on the outside surface of the transparent wall, and each of these indicia is transparent to allow the parallax angled viewing of the first indicia through these indicia. The second indicia is in the form of a broadly uniform pattern, viz, many small spaced dots; while the third indicia is in the form of easily memorized objects, such as human faces, made on a substrate by means of halftone printing. The substrate is lapped over the outside surface of the transparent wall. A thin cocoon of a transparent material, generally of the same material as the substrate such as plastic, is formed over the seal body and specifically over the transparent wall and the second and third indicia formed thereon. This cocoon is seamless and has walls of nonuniform thickness. Both the genuineness of the seal and whether anyone has attempted to compromise the seal can thus be visually determined upon inspection.

Production of methoxylated flavonoids in yeast using ring A hydroxylases and flavonoid O-methyltransferases from sweet basil.

PubMed

Berim, Anna; Gang, David R

2018-07-01

Numerous methoxylated flavonoids exhibit pronounced bioactivities. Their biotechnological production and diversification are therefore of interest to pharmaceutical and nutraceutical industries. We used a set of enzymes from sweet basil (Ocimum basilicum) to construct five strains of Saccharomyces cerevisiae producing 8- and/or 6-substituted, methoxylated flavones from their natural precursor apigenin. After identifying several growth parameters affecting the overall yields and flux, we applied optimized conditions and explored the ability of the generated strains to utilize alternative substrates. The yeast cells produced substantial amounts of 6-hydroxylated, methylated derivatives of naringenin and luteolin while the corresponding derivatives of flavonol kaempferol were only detected in trace amounts. Analysis of the intermediates and by-products of the different bioconversions suggested that the substrate specificity of both the hydroxylases and the flavonoid O-methyltransferases is imposing barriers on yields obtained with alternative substrates and highlighted steps that appear to represent bottlenecks en route to increasing the strains' efficiencies. Additionally, analysis of flavonoid localization during fermentation revealed unequal distribution with strong intracellular accumulation of a number of methylated flavonoids and extracellular enrichment of several pathway intermediates. This work establishes a platform for the production of complex methoxylated flavonoids and discusses strategies for its improvement.

Ionic Driven Embedment of Hyaluronic Acid Coated Liposomes in Polyelectrolyte Multilayer Films for Local Therapeutic Delivery

NASA Astrophysics Data System (ADS)

Hayward, Stephen L.; Francis, David M.; Sis, Matthew J.; Kidambi, Srivatsan

2015-10-01

The ability to control the spatial distribution and temporal release of a therapeutic remains a central challenge for biomedical research. Here, we report the development and optimization of a novel substrate mediated therapeutic delivery system comprising of hyaluronic acid covalently functionalized liposomes (HALNPs) embedded into polyelectrolyte multilayer (PEM) platform via ionic stabilization. The PEM platform was constructed from sequential deposition of Poly-L-Lysine (PLL) and Poly(Sodium styrene sulfonate) (SPS) “(PLL/SPS)4.5” followed by adsorption of anionic HALNPs. An adsorption affinity assay and saturation curve illustrated the preferential HALNP deposition density for precise therapeutic loading. (PLL/SPS)2.5 capping layer on top of the deposited HALNP monolayer further facilitated complete nanoparticle immobilization, cell adhesion, and provided nanoparticle confinement for controlled linear release profiles of the nanocarrier and encapsulated cargo. To our knowledge, this is the first study to demonstrate the successful embedment of a translatable lipid based nanocarrier into a substrate that allows for temporal and spatial release of both hydrophobic and hydrophilic drugs. Specifically, we have utilized our platform to deliver chemotherapeutic drug Doxorubicin from PEM confined HALNPs. Overall, we believe the development of our HALNP embedded PEM system is significant and will catalyze the usage of substrate mediated delivery platforms in biomedical applications.

Kinetic study on anaerobic oxidation of methane coupled to denitrification.

PubMed

Yu, Hou; Kashima, Hiroyuki; Regan, John M; Hussain, Abid; Elbeshbishy, Elsayed; Lee, Hyung-Sool

2017-09-01

Monod kinetic parameters provide information required for kinetic analysis of anaerobic oxidation of methane coupled to denitrification (AOM-D). This information is critical for engineering AOM-D processes in wastewater treatment facilities. We first experimentally determined Monod kinetic parameters for an AOM-D enriched culture and obtained the following values: maximum specific growth rate (μ max ) 0.121/d, maximum substrate-utilization rate (q max ) 28.8mmol CH 4 /g cells-d, half maximum-rate substrate concentration (K s ) 83μΜ CH 4 , growth yield (Y) 4.76gcells/mol CH 4 , decay coefficient (b) 0.031/d, and threshold substrate concentration (S min ) 28.8μM CH 4 . Clone library analysis of 16S rRNA and mcrA gene fragments suggested that AOM-D reactions might have occurred via the syntrophic interaction between denitrifying bacteria (e.g., Ignavibacterium, Acidovorax, and Pseudomonas spp.) and hydrogenotrophic methanogens (Methanobacterium spp.), supporting reverse methanogenesis-dependent AOM-D in our culture. High μ max and q max , and low K s for the AOM-D enrichment imply that AOM-D could play a significant role in mitigating atmospheric methane efflux. In addition, these high kinetic features suggest that engineered AOM-D systems may provide a sustainable alternative to nitrogen removal in wastewater treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Process for forming epitaxial perovskite thin film layers using halide precursors

DOEpatents

Clem, Paul G.; Rodriguez, Mark A.; Voigt, James A.; Ashley, Carol S.

2001-01-01

A process for forming an epitaxial perovskite-phase thin film on a substrate. This thin film can act as a buffer layer between a Ni substrate and a YBa.sub.2 Cu.sub.3 O.sub.7-x superconductor layer. The process utilizes alkali or alkaline metal acetates dissolved in halogenated organic acid along with titanium isopropoxide to dip or spin-coat the substrate which is then heated to about 700.degree. C. in an inert gas atmosphere to form the epitaxial film on the substrate. The YBCO superconductor can then be deposited on the layer formed by this invention.

High efficiency epitaxial GaAs/GaAs and GaAs/Ge solar cell technology using OM/CVD

NASA Technical Reports Server (NTRS)

Wang, K. L.; Yeh, Y. C. M.; Stirn, R. J.; Swerdling, S.

1980-01-01

A technology for fabricating high efficiency, thin film GaAs solar cells on substrates appropriate for space and/or terrestrial applications was developed. The approach adopted utilizes organometallic chemical vapor deposition (OM-CVD) to form a GaAs layer epitaxially on a suitably prepared Ge epi-interlayer deposited on a substrate, especially a light weight silicon substrate which can lead to a 300 watt per kilogram array technology for space. The proposed cell structure is described. The GaAs epilayer growth on single crystal GaAs and Ge wafer substrates were investigated.

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

PubMed

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

2011-01-01

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

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

PubMed Central

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

2011-01-01

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

THE LOCALIZATION OF CHOLINESTERASE ACTIVITY IN RAT CARDIAC MUSCLE BY ELECTRON MICROSCOPY

PubMed Central

Karnovsky, Morris J.

1964-01-01

A method has been developed for localizing sites of cholinesterase activity in rat cardiac muscle by electron microscopy. The method utilizes thiocholine esters as substrates, and is believed to be dependent on the reduction of ferricyanide to ferrocyanide by thiocholine released by enzymatic activity. The ferrocyanide thus formed is captured by copper to form fine, electron-opaque deposits of copper ferrocyanide, which sharply delineate sites of enzymatic activity at the ultrastructural level. Cholinesterase activity in formalin-fixed heart muscle was localized: (a) in longitudinal elements of the sarcoplasmic reticulum, but not in the T, or transverse, elements; and (b) in the A band, with virtually no activity noted in the M band, or in the H zone. The I band was also negative. No activity was detected in the sarcolemma, or in invaginations of the sarcolemma at the level of the Z band. The perinuclear element of the sarcoplasmic (endoplasmic) reticulum was frequently strongly positive. Activity at all sites was completely abolished by omitting the substrates, or by inhibition with eserine 10-4 M and diisopropylfluorophosphate 10-5 M. Eserine 10-5 M completely inhibited reaction in the sarcoplasmic reticulum, and virtually abolished that in the A band. These observations, together with the use of the relatively specific substrates and suitable controls to eliminate non-enzymatic staining, indicate that cholinesterase activity was being demonstrated. The activity in rat heart against different substrates was that of non-specific cholinesterases, in accordance with biochemical data. The activity in the A band was considered to be probably due to myosincholinesterase. It is proposed that the localization of cholinesterases in myocardium at the ultrastructural level should be taken into account in considering the possible functions of these myocardial enzymes, and it is hoped that knowledge of their localization will open up new avenues of approach in considering their physiological role in myocardium, which at present is not definitely known. PMID:14222810

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

PubMed

Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

2015-04-15

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress.

PubMed

Chandrashekarappa, Dakshayini G; McCartney, Rhonda R; O'Donnell, Allyson F; Schmidt, Martin C

2016-12-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. Copyright © 2016 Elsevier Inc. All rights reserved.

The β subunit of yeast AMP-activated protein kinase directs substrate specificity in response to alkaline stress

PubMed Central

Chandrashekarappa, Dakshayini G.; McCartney, Rhonda R.; O’Donnell, Allyson F.; Schmidt, Martin C.

2016-01-01

Saccharomyces cerevisiae express three isoforms of Snf1 kinase that differ by which β subunit is present, Gal83, Sip1 or Sip2. Here we investigate the abundance, activation, localization and signaling specificity of the three Snf1 isoforms. The relative abundance of these isoforms was assessed by quantitative immunoblotting using two different protein extraction methods and by fluorescence microscopy. The Gal83 containing isoform is the most abundant in all assays while the abundance of the Sip1 and Sip2 isoforms is typically underestimated especially in glass-bead extractions. Earlier studies to assess Snf1 isoform function utilized gene deletions as a means to inactivate specific isoforms. Here we use point mutations in Gal83 and Sip2 and a 17 amino acid C-terminal truncation of Sip1 to inactivate specific isoforms without affecting their abundance or association with the other subunits. The effect of low glucose and alkaline stresses was examined for two Snf1 phosphorylation substrates, the Mig1 and Mig2 proteins. Any of the three isoforms was capable of phosphorylating Mig1 in response to glucose stress. In contrast, the Gal83 isoform of Snf 1 was both necessary and sufficient for the phosphorylation of the Mig2 protein in response to alkaline stress. Alkaline stress led to the activation of all three isoforms yet only the Gal83 isoform translocates to the nucleus and phosphorylates Mig2. Deletion of the SAK1 gene blocked nuclear translocation of Gal83 and signaling to Mig2. These data strongly support the idea that Snf1 signaling specificity is mediated by localization of the different Snf1 isoforms. PMID:27592031

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular and tumor delivery of enzymes.

PubMed

Kim, Kyoung-Ran; Hwang, Dohyeon; Kim, Juhyeon; Lee, Chang-Yong; Lee, Wonseok; Yoon, Dae Sung; Shin, Dongyun; Min, Sun-Joon; Kwon, Ick Chan; Chung, Hak Suk; Ahn, Dae-Ro

2018-06-28

Despite the extremely high substrate specificity and catalytically amplified activity of enzymes, the lack of efficient cellular internalization limits their application as therapeutics. To overcome this limitation and to harness enzymes as practical biologics for targeting intracellular functions, we developed the streptavidin-mirror DNA tetrahedron hybrid as a platform for intracellular delivery of various enzymes. The hybrid consists of streptavidin, which provides a stoichiometrically controlled loading site for the enzyme cargo and an L-DNA (mirror DNA) tetrahedron, which provides the intracellular delivery potential. Due to the cell-penetrating ability of the mirror DNA tetrahedron of this hybrid, enzymes loaded on streptavidin can be efficiently delivered into the cells, intracellularly expressing their activity. In addition, we demonstrate tumor delivery of enzymes in an animal model by utilizing the potential of the hybrid to accumulate in tumors. Strikingly, the hybrid is able to transfer the apoptotic enzyme specifically into tumor cells, leading to strong suppression of tumor growth without causing significant damage to other tissues. These results suggest that the hybrid may allow anti-proliferative enzymes and proteins to be utilized as anticancer drugs. Copyright © 2018 Elsevier B.V. All rights reserved.

Methods for fabricating thin film III-V compound solar cell

DOEpatents

Pan, Noren; Hillier, Glen; Vu, Duy Phach; Tatavarti, Rao; Youtsey, Christopher; McCallum, David; Martin, Genevieve

2011-08-09

The present invention utilizes epitaxial lift-off in which a sacrificial layer is included in the epitaxial growth between the substrate and a thin film III-V compound solar cell. To provide support for the thin film III-V compound solar cell in absence of the substrate, a backing layer is applied to a surface of the thin film III-V compound solar cell before it is separated from the substrate. To separate the thin film III-V compound solar cell from the substrate, the sacrificial layer is removed as part of the epitaxial lift-off. Once the substrate is separated from the thin film III-V compound solar cell, the substrate may then be reused in the formation of another thin film III-V compound solar cell.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

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

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

Effect of telmisartan on selected adipokines, insulin sensitivity, and substrate utilization during insulin-stimulated conditions in patients with metabolic syndrome and impaired fasting glucose.

PubMed

Wohl, Petr; Krusinová, Eva; Hill, Martin; Kratochvílová, Simona; Zídková, Katerina; Kopecký, Jan; Neskudla, Tomás; Pravenec, Michal; Klementová, Marta; Vrbíková, Jana; Wohl, Pavel; Mlejnek, Petr; Pelikánová, Terezie

2010-10-01

Telmisartan improves glucose and lipid metabolism in rodents. This study evaluated the effect of telmisartan on insulin sensitivity, substrate utilization, selected plasma adipokines and their expressions in subcutaneous adipose tissue (SAT) in metabolic syndrome. Twelve patients with impaired fasting glucose completed the double-blind, randomized, crossover trial. Patients received telmisartan (160 mg/day) or placebo for 3 weeks and vice versa with a 2-week washout period. At the end of each period, a hyperinsulinemic euglycemic clamp (HEC) combined with indirect calorimetry was performed. During HEC (0, 30, and 120 min), plasma levels of adipokines were measured and a needle biopsy (0 and 30 min) of SAT was performed. Fasting plasma glucose was lower after telmisartan compared with placebo (P<0.05). There were no differences in insulin sensitivity and substrate utilization. We found no differences in basal plasma adiponectin, resistin and tumour necrosis factor α (TNFα), but an increase was found in basal leptin, after telmisartan treatment. Insulin-stimulated plasma adiponectin (P<0.05), leptin and resistin (P<0.001) were increased, whereas TNFα was decreased (P<0.05) after telmisartan treatment. Expression of resistin, but not adiponectin, TNFα and leptin was increased after telmisartan treatment. Despite the decrease in fasting plasma glucose, telmisartan does not improve insulin sensitivity and substrate utilization. Telmisartan increases plasma leptin as well as insulin-stimulated plasma adiponectin, leptin and resistin, and decreases plasma TNFα during HEC. Changes in plasma adipokines cannot be explained by their expressions in SAT. The changes in plasma adipokines might be involved in the metabolic effects of telmisartan in metabolic syndrome.

Amino acid and glucose uptake by rat brown adipose tissue. Effect of cold-exposure and acclimation.

PubMed Central

López-Soriano, F J; Fernández-López, J A; Mampel, T; Villarroya, F; Iglesias, R; Alemany, M

1988-01-01

The net uptake/release of glucose, lactate and amino acids from the bloodstream by the interscapular brown adipose tissue of control, cold-exposed and cold-acclimated rats was estimated by measurement of arteriovenous differences in their concentrations. In the control animals amino acids contributed little to the overall energetic needs of the tissue; glucose uptake was more than compensated by lactate efflux. Cold-exposure resulted in an enhancement of amino acid utilization and of glucose uptake, with high lactate efflux. There was a net glycine and proline efflux that partly compensated the positive nitrogen balance of the tissue; amino acids accounted for about one-third of the energy supplied by glucose to the tissue. Cold-acclimation resulted in a very high increase in glucose uptake, with a parallel decrease in lactate efflux and amino acid consumption. Branched-chain amino acids, however, were more actively utilized. This was related with a much higher alanine efflux, in addition to that of glycine and proline. It is suggested that most of the glucose used during cold-exposure is returned to the bloodstream as lactate under conditions of active lipid utilization, amino acids contributing their skeletons largely in anaplerotic pathways. On the other hand, cold-acclimation resulted in an important enhancement of glucose utilization, with lowered amino acid oxidation. Amino acids are thus used as metabolic substrates by the brown adipose tissue of rats under conditions of relatively scarce substrate availability, but mainly as anaplerotic substrates, in parallel to glucose. Cold-acclimation results in a shift of the main substrates used in thermogenesis from lipid to glucose, with a much lower need for amino acids. PMID:3421924

Turnover-Dependent Inactivation of the Nitrogenase MoFe-Protein at High pH

PubMed Central

2013-01-01

Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725–13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis. PMID:24392967

Turnover-dependent inactivation of the nitrogenase MoFe-protein at high pH.

PubMed

Yang, Kun-Yun; Haynes, Chad A; Spatzal, Thomas; Rees, Douglas C; Howard, James B

2014-01-21

Proton uptake accompanies the reduction of all known substrates by nitrogenase. As a consequence, a higher pH should limit the availability of protons as a substrate essential for turnover, thereby increasing the proportion of more highly reduced forms of the enzyme for further study. The utility of the high-pH approach would appear to be problematic in view of the observation reported by Pham and Burgess [(1993) Biochemistry 32, 13725-13731] that the MoFe-protein undergoes irreversible protein denaturation above pH 8.65. In contrast, we found by both enzyme activity and crystallographic analyses that the MoFe-protein is stable when incubated at pH 9.5. We did observe, however, that at higher pHs and under turnover conditions, the MoFe-protein is slowly inactivated. While a normal, albeit low, level of substrate reduction occurs under these conditions, the MoFe-protein undergoes a complex transformation; initially, the enzyme is reversibly inhibited for substrate reduction at pH 9.5, yet in a second, slower process, the MoFe-protein becomes irreversibly inactivated as measured by substrate reduction activity at the optimal pH of 7.8. The final inactivated MoFe-protein has an increased hydrodynamic radius compared to that of the native MoFe-protein, yet it has a full complement of iron and molybdenum. Significantly, the modified MoFe-protein retains the ability to specifically interact with its nitrogenase partner, the Fe-protein, as judged by the support of ATP hydrolysis and by formation of a tight complex with the Fe-protein in the presence of ATP and aluminum fluoride. The turnover-dependent inactivation coupled to conformational change suggests a mechanism-based transformation that may provide a new probe of nitrogenase catalysis.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference.

PubMed

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path.

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference

PubMed Central

Jiang, Xuan; Luo, Xi; Zhou, Ning-Yi

2015-01-01

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path. PMID:26544851

c-Myc alters substrate utilization and O-GlcNAc protein posttranslational modifications without altering cardiac function during early aortic constriction

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

Ledee, Dolena; Smith, Lincoln; Bruce, Margaret

Pressure overload cardiac hypertrophy alters substrate metabolism. Prior work showed that myocardial inactivation of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we hypothesize that Myc regulates substrate preferences for the citric acid cycle during pressure overload hypertrophy from transverse aortic constriction (TAC) and that these metabolic changes impact cardiac function and growth. To test this hypothesis, we subjected mice with cardiac specific, inducible Myc inactivation (MycKO-TAC) and non-transgenic littermates (Cont-TAC) to transverse aortic constriction (TAC; n=7/group). A separate group underwent sham surgery (Sham, n=5). After two weeks, function was measured in isolated workingmore » hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. Compared to Sham, Cont-TAC had increased free fatty acid fractional contribution with a concurrent decrease in unlabeled (predominately glucose) contribution. The changes in free fatty acid and unlabeled fractional contributions were abrogated by Myc inactivation during TAC (MycKO-TAC). Additionally, protein posttranslational modification by O-GlcNAc was significantly greater in Cont-TAC versus both Sham and MycKO-TAC. Lastly, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy.« less

c-Myc alters substrate utilization and O-GlcNAc protein posttranslational modifications without altering cardiac function during early aortic constriction

DOE PAGES

Ledee, Dolena; Smith, Lincoln; Bruce, Margaret; ...

2015-08-12

Pressure overload cardiac hypertrophy alters substrate metabolism. Prior work showed that myocardial inactivation of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we hypothesize that Myc regulates substrate preferences for the citric acid cycle during pressure overload hypertrophy from transverse aortic constriction (TAC) and that these metabolic changes impact cardiac function and growth. To test this hypothesis, we subjected mice with cardiac specific, inducible Myc inactivation (MycKO-TAC) and non-transgenic littermates (Cont-TAC) to transverse aortic constriction (TAC; n=7/group). A separate group underwent sham surgery (Sham, n=5). After two weeks, function was measured in isolated workingmore » hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. Compared to Sham, Cont-TAC had increased free fatty acid fractional contribution with a concurrent decrease in unlabeled (predominately glucose) contribution. The changes in free fatty acid and unlabeled fractional contributions were abrogated by Myc inactivation during TAC (MycKO-TAC). Additionally, protein posttranslational modification by O-GlcNAc was significantly greater in Cont-TAC versus both Sham and MycKO-TAC. Lastly, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy.« less

A Novel Mechanism for Desulfation of Mucin: Identification and Cloning of a Mucin-Desulfating Glycosidase (Sulfoglycosidase) from Prevotella Strain RS2

PubMed Central

Rho, Jung-hyun; Wright, Damian P.; Christie, David L.; Clinch, Keith; Furneaux, Richard H.; Roberton, Anthony M.

2005-01-01

A novel enzyme which may be important in mucin degradation has been discovered in the mucin-utilizing anaerobe Prevotella strain RS2. This enzyme cleaves terminal 2-acetamido-2-deoxy-β-d-glucopyranoside 6-sulfate (6-SO3-GlcNAc) residues from sulfomucin and from the model substrate 4-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside 6-sodium sulfate. The existence of this mucin-desulfating glycosidase (sulfoglycosidase) suggests an alternative mechanism by which this bacterium may desulfate sulfomucins, by glycosidic removal of a sulfated sugar from mucin oligosaccharide chains. Previously, mucin desulfation was thought to take place by the action of a specific desulfating enzyme, which then allowed glycosidases to remove desulfated sugar. Sulfate removal from sulfomucins is thought to be a rate-limiting step in mucin degradation by bacteria in the regions of the digestive tract with a significant bacterial flora. The sulfoglycosidase was induced by growth of the Prevotella strain on mucin and was purified 284-fold from periplasmic extracts. Tryptic digestion and sequencing of peptides from the 100-kDa protein enabled the sulfoglycosidase gene to be cloned and sequenced. Active recombinant enzyme was made in an Escherichia coli expression system. The sulfoglycosidase shows sequence similarity to hexosaminidases. The only other enzyme that has been shown to remove 6-SO3-GlcNAc from glycoside substrates is the human lysosomal enzyme β-N-acetylhexosaminidase A, point mutations in which cause the inheritable, lysosomal storage disorder Tay-Sachs disease. The human enzyme removes GlcNAc from glycoside substrates also, in contrast to the Prevotella enzyme, which acts on a nonsulfated substrate at a rate that is only 1% of the rate observed with a sulfated substrate. PMID:15716424

c-Myc Alters Substrate Utilization and O-GlcNAc Protein Posttranslational Modifications without Altering Cardiac Function during Early Aortic Constriction

PubMed Central

Ledee, Dolena; Smith, Lincoln; Bruce, Margaret; Kajimoto, Masaki; Isern, Nancy; Portman, Michael A.; Olson, Aaron K.

2015-01-01

Hypertrophic stimuli cause transcription of the proto-oncogene c-Myc (Myc). Prior work showed that myocardial knockout of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we assessed the interplay between Myc, substrate oxidation and cardiac function during early pressure overload hypertrophy. Mice with cardiac specific, inducible Myc knockout (MycKO-TAC) and non-transgenic littermates (Cont-TAC) were subjected to transverse aortic constriction (TAC; n = 7/group). Additional groups underwent sham surgery (Cont-Sham and MycKO-Sham, n = 5 per group). After two weeks, function was measured in isolated working hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. In sham hearts, Myc knockout did not affect cardiac function or substrate preferences for the citric acid cycle. However, Myc knockout altered fractional contributions during TAC. The unlabeled fractional contribution increased in MycKO-TAC versus Cont-TAC, whereas ketone and free fatty acid fractional contributions decreased. Additionally, protein posttranslational modifications by O-GlcNAc were significantly greater in Cont-TAC versus both Cont-Sham and MycKO-TAC. In conclusion, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy, which may regulate Myc-induced metabolic changes. PMID:26266538

Performance-Enhancing Methods for Au Film over Nanosphere Surface-Enhanced Raman Scattering Substrate and Melamine Detection Application

PubMed Central

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm−1 and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect. PMID:24886913

Performance-enhancing methods for Au film over nanosphere surface-enhanced Raman scattering substrate and melamine detection application.

PubMed

Wang, Jun Feng; Wu, Xue Zhong; Xiao, Rui; Dong, Pei Tao; Wang, Chao Guang

2014-01-01

A new high-performance surface-enhanced Raman scattering (SERS) substrate with extremely high SERS activity was produced. This SERS substrate combines the advantages of Au film over nanosphere (AuFON) substrate and Ag nanoparticles (AgNPs). A three order enhancement of SERS was observed when Rhodamine 6G (R6G) was used as a probe molecule to compare the SERS effects of the new substrate and commonly used AuFON substrate. These new SERS substrates can detect R6G down to 1 nM. The new substrate was also utilized to detect melamine, and the limit of detection (LOD) is 1 ppb. A linear relationship was also observed between the SERS intensity at Raman peak 682 cm(-1) and the logarithm of melamine concentrations ranging from 10 ppm to 1 ppb. This ultrasensitive SERS substrate is a promising tool for detecting trace chemical molecules because of its simple and effective fabrication procedure, high sensitivity and high reproducibility of the SERS effect.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Soft lithography using perfluorinated polyether molds and PRINT technology for fabrication of 3-D arrays on glass substrates

NASA Astrophysics Data System (ADS)

Wiles, Kenton B.; Wiles, Natasha S.; Herlihy, Kevin P.; Maynor, Benjamin W.; Rolland, Jason P.; DeSimone, Joseph M.

2006-03-01

The fabrication of nanometer size structures and complex devices for microelectronics is of increasing importance so as to meet the challenges of large-scale commercial applications. Soft lithography typically employs elastomeric polydimethylsiloxane (PDMS) molds to replicate micro- and nanoscale features. However, the difficulties of PDMS for nanoscale fabrication include inherent incompatibility with organic liquids and the production of a residual scum or flash layer that link features where the nano-structures meet the substrate. An emerging technologically advanced technique known as Pattern Replication in Non-wetting Templates (PRINT) avoids both of these dilemmas by utilizing photocurable perfluorinated polyether (PFPE) rather than PDMS as the elastomeric molding material. PFPE is a liquid at room temperature that exhibits low modulus and high gas permeability when cured. The highly fluorinated PFPE material allows for resistance to swelling by organic liquids and very low surface energies, thereby preventing flash layer formation and ease of separation of PFPE molds from the substrates. These enhanced characteristics enable easy removal of the stamp from the molded material, thereby minimizing damage to the nanoscale features. Herein we describe that PRINT can be operated in two different modes depending on whether the objects to be molded are to be removed and harvested (i.e. to make shape specific organic particles) or whether scum free objects are desired which are adhered onto the substrate (i.e. for scum free pattern generation using imprint lithography). The former can be achieved using a non-reactive, low surface energy substrate (PRINT: Particle Replication in Non-wetting Templates) and the latter can be achieved using a reactive, low surface energy substrate (PRINT: Pattern Replication in Non-wetting Templates). We show that the PRINT technology can been used to fabricate nano-particle arrays covalently bound to a glass substrate with no scum layer. The nanometer size arrays were fabricated using a PFPE mold and a self-assembled monolayer (SAM) fluorinated glass substrate that was also functionalized with free-radically reactive SAM methacrylate moieties. The molded polymeric materials were covalently bound to the glass substrate through thermal curing with the methacrylate groups to permit three dimensional array fabrication. The low surface energies of the PFPE mold and fluorinated glass substrate allowed for no flash layer formation, permitting well resolved structures.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

Maximizing power generation from dark fermentation effluents in microbial fuel cell by selective enrichment of exoelectrogens and optimization of anodic operational parameters.

PubMed

Varanasi, Jhansi L; Sinha, Pallavi; Das, Debabrata

2017-05-01

To selectively enrich an electrogenic mixed consortium capable of utilizing dark fermentative effluents as substrates in microbial fuel cells and to further enhance the power outputs by optimization of influential anodic operational parameters. A maximum power density of 1.4 W/m 3 was obtained by an enriched mixed electrogenic consortium in microbial fuel cells using acetate as substrate. This was further increased to 5.43 W/m 3 by optimization of influential anodic parameters. By utilizing dark fermentative effluents as substrates, the maximum power densities ranged from 5.2 to 6.2 W/m 3 with an average COD removal efficiency of 75% and a columbic efficiency of 10.6%. A simple strategy is provided for selective enrichment of electrogenic bacteria that can be used in microbial fuel cells for generating power from various dark fermentative effluents.

RNA-Catalyzed RNA Ligation on an External RNA Template

NASA Technical Reports Server (NTRS)

McGinness, Kathleen E.; Joyce, Gerald F.

2002-01-01

Variants of the hc ligase ribozyme, which catalyzes ligation of the 3' end of an RNA substrate to the 5' end of the ribozyme, were utilized to evolve a ribozyme that catalyzes ligation reactions on an external RNA template. The evolved ribozyme catalyzes the joining of an oligonucleotide 3'-hydroxyl to the 5'-triphosphate of an RNA hairpin molecule. The ribozyme can also utilize various substrate sequences, demonstrating a largely sequence-independent mechanism for substrate recognition. The ribozyme also carries out the ligation of two oligonucleotides that are bound at adjacent positions on a complementary template. Finally, it catalyzes addition of mononucleoside '5-triphosphates onto the '3 end of an oligonucleotide primer in a template-dependent manner. The development of ribozymes that catalyze polymerase-type reactions contributes to the notion that an RNA world could have existed during the early history of life on Earth.

Utilization of plasmas for graphene synthesis

NASA Astrophysics Data System (ADS)

Shashurin, Alexey; Keidar, Michael

2013-10-01

Graphene is a one-atom-thick planar sheet of carbon atoms that are densely packed in a honeycomb crystal lattice. Grapheen has tremendous range of potential applications ranging from high-speed transistors to electrochemical energy storage devices and biochemical sensors. Methods of graphene synthesis include mechanical exfoliation, epitaxial growth on SiC, CVD and colloidal suspensions. In this work the utilization of plasmas in synthesis process is considered. Types of carbonaceous structures produced by the anodic arc and regions of their synthesis were studied. Ultimate role of substrate temperature and transformations occurring with various carbonaceous structures generated in plasma discharge were considered. Formation of graphene film on copper substrate was detected at temperatures around the copper melting point. The film was consisted of several layers graphene flakes having typical sizes of about 200 nm. Time required for crystallization of graphene on externally heated substrates was determined. This work was supported by National Science Foundation (NSF Grant No. CBET-1249213).

Bacteria and Fungi Respond Differently to Multifactorial Climate Change in a Temperate Heathland, Traced with 13C-Glycine and FACE CO2

PubMed Central

Andresen, Louise C.; Dungait, Jennifer A. J.; Bol, Roland; Selsted, Merete B.; Ambus, Per; Michelsen, Anders

2014-01-01

It is vital to understand responses of soil microorganisms to predicted climate changes, as these directly control soil carbon (C) dynamics. The rate of turnover of soil organic carbon is mediated by soil microorganisms whose activity may be affected by climate change. After one year of multifactorial climate change treatments, at an undisturbed temperate heathland, soil microbial community dynamics were investigated by injection of a very small concentration (5.12 µg C g−1 soil) of 13C-labeled glycine (13C2, 99 atom %) to soils in situ. Plots were treated with elevated temperature (+1°C, T), summer drought (D) and elevated atmospheric carbon dioxide (510 ppm [CO2]), as well as combined treatments (TD, TCO2, DCO2 and TDCO2). The 13C enrichment of respired CO2 and of phospholipid fatty acids (PLFAs) was determined after 24 h. 13C-glycine incorporation into the biomarker PLFAs for specific microbial groups (Gram positive bacteria, Gram negative bacteria, actinobacteria and fungi) was quantified using gas chromatography-combustion-stable isotope ratio mass spectrometry (GC-C-IRMS). Gram positive bacteria opportunistically utilized the freshly added glycine substrate, i.e. incorporated 13C in all treatments, whereas fungi had minor or no glycine derived 13C-enrichment, hence slowly reacting to a new substrate. The effects of elevated CO2 did suggest increased direct incorporation of glycine in microbial biomass, in particular in G+ bacteria, in an ecosystem subjected to elevated CO2. Warming decreased the concentration of PLFAs in general. The FACE CO2 was 13C-depleted (δ13C = 12.2‰) compared to ambient (δ13C = ∼−8‰), and this enabled observation of the integrated longer term responses of soil microorganisms to the FACE over one year. All together, the bacterial (and not fungal) utilization of glycine indicates substrate preference and resource partitioning in the microbial community, and therefore suggests a diversified response pattern to future changes in substrate availability and climatic factors. PMID:24454793

Thin film photovoltaic device with multilayer substrate

DOEpatents

Catalano, Anthony W.; Bhushan, Manjul

1984-01-01

A thin film photovoltaic device which utilizes at least one compound semiconductor layer chosen from Groups IIB and VA of the Periodic Table is formed on a multilayer substrate The substrate includes a lowermost support layer on which all of the other layers of the device are formed. Additionally, an uppermost carbide or silicon layer is adjacent to the semiconductor layer. Below the carbide or silicon layer is a metal layer of high conductivity and expansion coefficient equal to or slightly greater than that of the semiconductor layer.

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

PubMed

Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak

2018-01-01

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Continuous ethanol production from cheese whey fermentation by Candida pseudotropicalis

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

Ghaly, A.E.; El-Taweel, A.A.

1997-12-01

Three pilot-scale continuous mix reactors of 5-L volume each were used to study the effects of retention time (18--42 hours) and initial substrate concentration (50--150 g/L) on the cell yield, lactose consumption, and maximum ethanol concentration during continuous fermentation of cheese whey using the yeast Candida pseudotropicalis. A microaeration rate of 480 mL/min and a nutrient supplement (yeast extract) concentration of 0.1% vol/vol were used. The results indicated that the dissolved oxygen concentration, temperature, cell concentration, lactose utilization rate, and ethanol concentration were affected by hydraulic retention time and initial substrate concentration. The highest cell concentration of 5.46 g/L andmore » the highest ethanol concentration of 57.96 g/L (with a maximum ethanol yield of 99.6% from the theoretical yield) were achieved at the 42-hour hydraulic retention time and the 150 g/L initial substrate concentration, whereas the highest cell yield was observed at the 50 g/L initial substrate concentration and the 36-hour hydraulic retention time. Lactose utilizations of 98, 91, and 83% were obtained with 50, 100, and 150 g/L initial substrate concentrations at the 42-hour hydraulic retention time. A pH control system was found unnecessary.« less

Effect of various pretreatment methods on anaerobic mixed microflora to enhance biohydrogen production utilizing dairy wastewater as substrate.

PubMed

Venkata Mohan, S; Lalit Babu, V; Sarma, P N

2008-01-01

Influence of different pretreatment methods applied on anaerobic mixed inoculum was evaluated for selectively enriching the hydrogen (H(2)) producing mixed culture using dairy wastewater as substrate. The experimental data showed the feasibility of molecular biohydrogen generation utilizing dairy wastewater as primary carbon source through metabolic participation. However, the efficiency of H(2) evolution and substrate removal efficiency were found to be dependent on the type of pretreatment procedure adopted on the parent inoculum. Among the studied pretreatment methods, chemical pretreatment (2-bromoethane sulphonic acid sodium salt (0.2 g/l); 24 h) procedure enabled higher H(2) yield along with concurrent substrate removal efficiency. On the contrary, heat-shock pretreatment (100 degrees C; 1 h) procedure resulted in relatively low H(2) yield. Compared to control experiments all the adopted pretreatment methods documented higher H(2) generation efficiency. In the case of combination experiments, integration of pH (pH 3; adjusted with ortho-phosphoric acid; 24 h) and chemical pretreatment evidenced higher H(2) production. Data envelopment analysis (DEA), a frontier analysis technique model was successfully applied to enumerate the relative efficiency of different pretreatment methods studied by considered pretreatment procedures as input and cumulative H(2) production rate and substrate degradation rate as corresponding two outputs.

Biochemical analysis with microfluidic systems.

PubMed

Bilitewski, Ursula; Genrich, Meike; Kadow, Sabine; Mersal, Gaber

2003-10-01

Microfluidic systems are capillary networks of varying complexity fabricated originally in silicon, but nowadays in glass and polymeric substrates. Flow of liquid is mainly controlled by use of electroosmotic effects, i.e. application of electric fields, in addition to pressurized flow, i.e. application of pressure or vacuum. Because electroosmotic flow rates depend on the charge densities on the walls of capillaries, they are influenced by substrate material, fabrication processes, surface pretreatment procedures, and buffer additives. Microfluidic systems combine the properties of capillary electrophoretic systems and flow-through analytical systems, and thus biochemical analytical assays have been developed utilizing and integrating both aspects. Proteins, peptides, and nucleic acids can be separated because of their different electrophoretic mobility; detection is achieved with fluorescence detectors. For protein analysis, in particular, interfaces between microfluidic chips and mass spectrometers were developed. Further levels of integration of required sample-treatment steps were achieved by integration of protein digestion by immobilized trypsin and amplification of nucleic acids by the polymerase chain reaction. Kinetic constants of enzyme reactions were determined by adjusting different degrees of dilution of enzyme substrates or inhibitors within a single chip utilizing mainly the properties of controlled dosing and mixing liquids within a chip. For analysis of kinase reactions, however, a combination of a reaction step (enzyme with substrate and inhibitor) and a separation step (enzyme substrate and reaction product) was required. Microfluidic chips also enable separation of analytes from sample matrix constituents, which can interfere with quantitative determination, if they have different electrophoretic mobilities. In addition to analysis of nucleic acids and enzymes, immunoassays are the third group of analytical assays performed in microfluidic chips. They utilize either affinity capillary electrophoresis as a homogeneous assay format, or immobilized antigens or antibodies in heterogeneous assays with serial supply of reagents and washing solutions.

Energy substrate utilization with and without exogenous carbohydrate intake in boys and men exercising in the heat.

PubMed

Leites, Gabriela T; Cunha, Giovani S; Chu, Lisa; Meyer, Flavia; Timmons, Brian W

2016-11-01

Little is known about energy yield during exercise in the heat in boys compared with men. To investigate substrate utilization with and without exogenous carbohydrate (CHO exo ) intake, seven boys [11.2 ± 0.2 (SE) yr] and nine men (24.0 ± 1.1 yr) cycled (4 × 20-min bouts) at a fixed metabolic heat production (Ḣ p ) per unit body mass (6 W/kg) in a climate chamber (38°C and 50% relative humidity), on two occasions. Participants consumed a 13 C-enriched 8% CHO beverage (CARB) or placebo beverage (CONT) in a double-blinded, counterbalanced manner. Substrate utilization was calculated for the last 60 min of exercise. CHO exo oxidation rate (2.0 ± 0.3 vs. 2.5 ± 0.2 mg·kg fat-free mass -1 ·min -1 , P = 0.02) and CHO exo oxidation efficiency (12.8 ± 0.6 vs. 16.0 ± 0.9%, P = 0.01) were lower in boys compared with men exercising in the heat. Total carbohydrate (CHO total ), endogenous CHO (CHO endo ), and total fat (Fat total ) remained stable in boys and men (P > 0.05) during CARB, whereas CHO total oxidation rate decreased (P < 0.001) and Fat total oxidation rate increased over time similarly in boys and men during CONT (P < 0.001). The relative contribution of CHO exo to total energy yield increased over time in both groups (P < 0.001). In conclusion, endogenous substrate metabolism and the relative contribution of fuels to total energy yield were not different between groups. The ingestion of a CHO beverage during exercise in the heat may be as beneficial for boys as men to spare endogenous substrate. Copyright © 2016 the American Physiological Society.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses.

PubMed

Sabra, Wael; Bommareddy, Rajesh Reddy; Maheshwari, Garima; Papanikolaou, Seraphim; Zeng, An-Ping

2017-05-08

Unlike the well-studied backer yeast where catabolite repression represents a burden for mixed substrate fermentation, Yarrowia lipolytica, an oleaginous yeast, is recognized for its potential to produce single cell oils and citric acid from different feedstocks. These versatilities of Y. lipolytica with regards to substrate utilization make it an attractive host for biorefinery application. However, to develop a commercial process for the production of citric acid by Y. lipolytica, it is necessary to better understand the primary metabolism and its regulation, especially for growth on mixed substrate. Controlling the dissolved oxygen concentration (pO 2 ) in Y. lipolytica cultures enhanced citric acid production significantly in cultures grown on glucose in mono- or dual substrate fermentations, whereas with glycerol as mono-substrate no significant effect of pO 2 was found on citrate production. Growth on mixed substrate with glucose and glycerol revealed a relative preference of glycerol utilization by Y. lipolytica. Under optimized conditions with pO 2 control, the citric acid titer on glucose in mono- or in dual substrate cultures was 55 and 50 g/L (with productivity of 0.6 g/L*h in both cultures), respectively, compared to a maximum of 18 g/L (0.2 g/L*h) with glycerol in monosubstrate culture. Additionally, in dual substrate fermentation, glycerol limitation was found to trigger citrate consumption despite the presence of enough glucose in pO 2 -limited culture. The metabolic behavior of this yeast on different substrates was investigated at transcriptomic and 13 C-based fluxomics levels. Upregulation of most of the genes of the pentose phosphate pathway was found in cultures with highest citrate production with glucose in mono- or in dual substrate fermentation with pO 2 control. The activation of the glyoxylate cycle in the oxygen limited cultures and the imbalance caused by glycerol limitation might be the reason for the re-consumption of citrate in dual substrate fermentations. This study provides interesting targets for metabolic engineering of this industrial yeast.

Mapping Microbial Carbon Substrate Utilization Across Permafrost Thaw

NASA Astrophysics Data System (ADS)

Anderson, D.; Rich, V. I.; Hodgkins, S. B.; Tfaily, M.; Chanton, J.

2014-12-01

Permafrost thaw is likely to create a substantial positive feedback to climate warming, as previously frozen carbon becomes bioavailable and is released to the atmosphere. Microbes mediate this release, while also consuming "new" carbon from plant inputs and middle-aged soil carbon pools in the seasonally-thawed active layer overlying permafrost. This carbon consumption releases carbon dioxide (CO2) and methane (CH4), both potent greenhouse gases. To investigate microbial carbon cycling in this changing habitat, we examined how microbial communities' carbon substrate degradation changes along a natural permafrost thaw gradient in Stordalen Mire (68.35°N, 19.05°E), northern Sweden. At this location, intermediate thaw creates Sphagnum moss-dominated bogs, while complete thaw results in Eriophorum sedge-dominated fens. The progression of thaw results in increasing organic matter lability (Hodgkins et al, 2014), shifting microbial community composition (Mondav & Woodcroft et al 2014), and changing carbon gas emissions (McCalley et al, in review). However, the inter-relationship of the first two in producing the third remains unclear. We analyzed microbial carbon substrate utilization in the intermediate-thaw and full-thaw sites by two incubation-based methods. We used Biolog EcoPlates, which contain 31 ecologically relevant carbon substrates and a colorimetric marker of their consumption, and into which we added a soil liquid suspension. In addition, we performed mason-jar incubations of peat with carbon substrate amendments and measured CH4 and CO2 emissions. Preliminary Biolog Ecoplate incubations showed that intermediate-thaw features responded faster and more strongly overall to a wide range of substrates relative to the full-thaw features. Preliminary mason jar incubations showed that acetate amendment elicited the greatest response increase in CH4 production and the second greatest increase in CO2 production relative to the controls, in samples from both habitats. In addition, the lowest CH4 and CO2 production was seen in amendments of sphagnum acid. It is important to understand the carbon substrate utilization occurring at these initial and advanced thaw features to speculate the degree to which various carbon inputs are being metabolized to produce the observed gas emissions.

Simultaneous Heterotrophic Nitrification and Aerobic Denitrification by Chryseobacterium sp. R31 Isolated from Abattoir Wastewater

PubMed Central

Kundu, Pradyut; Pramanik, Arnab; Dasgupta, Arpita; Mukherjee, Somnath; Mukherjee, Joydeep

2014-01-01

A heterotrophic carbon utilizing microbe (R31) capable of simultaneous nitrification and denitrification (SND) was isolated from wastewater of an Indian slaughterhouse. From an initial COD value of 583.0 mg/L, 95.54% was removed whilst, from a starting NH4 +-N concentration of 55.7 mg/L, 95.87% was removed after 48 h contact. The concentrations of the intermediates hydroxylamine, nitrite, and nitrate were low, thus ensuring nitrogen removal. Aerobic denitrification occurring during ammonium removal by R31 was confirmed by utilization of both nitrate and nitrite as nitrogen substrates. Glucose and succinate were superior while acetate and citrate were poor substrates for nitrogen removal. Molecular phylogenetic identification, supported by chemotaxonomic and physiological properties, assigned R31 as a close relative of Chryseobacterium haifense. The NH4 +-N utilization rate and growth of strain R31 were found to be higher at C/N = 10 in comparison to those achieved with C/N ratios of 5 and 20. Monod kinetic coefficients, half saturation concentration (K s), maximum rate of substrate utilization (k), yield coefficient, (Y) and endogenous decay coefficient (K d) indicated potential application of R31 in large-scale SND process. This is the first report on concomitant carbon oxidation, nitrification, and denitrification in the genus Chryseobacterium and the associated kinetic coefficients. PMID:24991552

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

HARM processing techniques for MEMS and MOEMS devices using bonded SOI substrates and DRIE

NASA Astrophysics Data System (ADS)

Gormley, Colin; Boyle, Anne; Srigengan, Viji; Blackstone, Scott C.

2000-08-01

Silicon-on-Insulator (SOI) MEMS devices (1) are rapidly gaining popularity in realizing numerous solutions for MEMS, especially in the optical and inertia application fields. BCO recently developed a DRIE trench etch, utilizing the Bosch process, and refill process for high voltage dielectric isolation integrated circuits on thick SOI substrates. In this paper we present our most recently developed DRIE processes for MEMS and MOEMS devices. These advanced etch techniques are initially described and their integration with silicon bonding demonstrated. This has enabled process flows that are currently being utilized to develop optical router and filter products for fiber optics telecommunications and high precision accelerometers.

Methods and energy storage devices utilizing electrolytes having surface-smoothing additives

DOEpatents

Xu, Wu; Zhang, Jiguang; Graff, Gordon L; Chen, Xilin; Ding, Fei

2015-11-12

Electrodeposition and energy storage devices utilizing an electrolyte having a surface-smoothing additive can result in self-healing, instead of self-amplification, of initial protuberant tips that give rise to roughness and/or dendrite formation on the substrate and anode surface. For electrodeposition of a first metal (M1) on a substrate or anode from one or more cations of M1 in an electrolyte solution, the electrolyte solution is characterized by a surface-smoothing additive containing cations of a second metal (M2), wherein cations of M2 have an effective electrochemical reduction potential in the solution lower than that of the cations of M1.

Analytical determination of critical crack size in solar cells

NASA Technical Reports Server (NTRS)

Chen, C. P.

1988-01-01

Although solar cells usually have chips and cracks, no material specifications concerning the allowable crack size on solar cells are available for quality assurance and engineering design usage. Any material specifications that the cell manufacturers use were developed for cosmetic reasons that have no technical basis. Therefore, the Applied Solar Energy Corporation (ASEC) has sponsored a continuing program for the fracture mechanics evaluation of GaAs. Fracture mechanics concepts were utilized to develop an analytical model that can predict the critical crack size of solar cells. This model indicates that the edge cracks of a solar cell are more critical than its surface cracks. In addition, the model suggests that the material specifications on the allowable crack size used for Si solar cells should not be applied to GaAs solar cells. The analytical model was applied to Si and GaAs solar cells, but it would also be applicable to the semiconductor wafers of other materials, such as a GaAs thin film on a Ge substrate, using appropriate input data.

Chemical surface deposition of ultra-thin semiconductors

DOEpatents

McCandless, Brian E.; Shafarman, William N.

2003-03-25

A chemical surface deposition process for forming an ultra-thin semiconducting film of Group IIB-VIA compounds onto a substrate. This process eliminates particulates formed by homogeneous reactions in bath, dramatically increases the utilization of Group IIB species, and results in the formation of a dense, adherent film for thin film solar cells. The process involves applying a pre-mixed liquid coating composition containing Group IIB and Group VIA ionic species onto a preheated substrate. Heat from the substrate causes a heterogeneous reaction between the Group IIB and VIA ionic species of the liquid coating composition, thus forming a solid reaction product film on the substrate surface.

Flexible substrate-based devices for point-of-care diagnostics

PubMed Central

Wang, ShuQi; Chinnasamy, Thiruppathiraja; Lifson, Mark; Inci, Fatih; Demirci, Utkan

2016-01-01

Point-of-care (POC) diagnostics play an important role in delivering healthcare, particularly for clinical management and disease surveillance in both developed and developing countries. Currently, the majority of POC diagnostics utilize paper substrates owing to their affordability, disposability, and mass production capability. Recently, flexible polymer substrates have been investigated due to their enhanced physicochemical properties, potential to be integrated into wearable devices with wireless communications for personalized health monitoring, and ability to be customized for POC diagnostics. Here, we focus on the latest advances in developing flexible substrate-based diagnostic devices, including paper and polymers, and their clinical applications at the POC. PMID:27344425

Flexible Substrate-Based Devices for Point-of-Care Diagnostics.

PubMed

Wang, ShuQi; Chinnasamy, Thiruppathiraja; Lifson, Mark A; Inci, Fatih; Demirci, Utkan

2016-11-01

Point-of-care (POC) diagnostics play an important role in delivering healthcare, particularly for clinical management and disease surveillance in both developed and developing countries. Currently, the majority of POC diagnostics utilize paper substrates owing to affordability, disposability, and mass production capability. Recently, flexible polymer substrates have been investigated due to their enhanced physicochemical properties, potential to be integrated into wearable devices with wireless communications for personalized health monitoring, and ability to be customized for POC diagnostics. Here, we focus on the latest advances in developing flexible substrate-based diagnostic devices, including paper and polymers, and their clinical applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

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.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

PubMed

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

Soft lithographic functionalization and patterning oxide-free silicon and germanium.

PubMed

Bowers, Carleen M; Toone, Eric J; Clark, Robert L; Shestopalov, Alexander A

2011-12-16

The development of hybrid electronic devices relies in large part on the integration of (bio)organic materials and inorganic semiconductors through a stable interface that permits efficient electron transport and protects underlying substrates from oxidative degradation. Group IV semiconductors can be effectively protected with highly-ordered self-assembled monolayers (SAMs) composed of simple alkyl chains that act as impervious barriers to both organic and aqueous solutions. Simple alkyl SAMs, however, are inert and not amenable to traditional patterning techniques. The motivation for immobilizing organic molecular systems on semiconductors is to impart new functionality to the surface that can provide optical, electronic, and mechanical function, as well as chemical and biological activity. Microcontact printing (μCP) is a soft-lithographic technique for patterning SAMs on myriad surfaces. Despite its simplicity and versatility, the approach has been largely limited to noble metal surfaces and has not been well developed for pattern transfer to technologically important substrates such as oxide-free silicon and germanium. Furthermore, because this technique relies on the ink diffusion to transfer pattern from the elastomer to substrate, the resolution of such traditional printing is essentially limited to near 1 μm. In contrast to traditional printing, inkless μCP patterning relies on a specific reaction between a surface-immobilized substrate and a stamp-bound catalyst. Because the technique does not rely on diffusive SAM formation, it significantly expands the diversity of patternable surfaces. In addition, the inkless technique obviates the feature size limitations imposed by molecular diffusion, facilitating replication of very small (<200 nm) features. However, up till now, inkless μCP has been mainly used for patterning relatively disordered molecular systems, which do not protect underlying surfaces from degradation. Here, we report a simple, reliable high-throughput method for patterning passivated silicon and germanium with reactive organic monolayers and demonstrate selective functionalization of the patterned substrates with both small molecules and proteins. The technique utilizes a preformed NHS-reactive bilayered system on oxide-free silicon and germanium. The NHS moiety is hydrolyzed in a pattern-specific manner with a sulfonic acid-modified acrylate stamp to produce chemically distinct patterns of NHS-activated and free carboxylic acids. A significant limitation to the resolution of many μCP techniques is the use of PDMS material which lacks the mechanical rigidity necessary for high fidelity transfer. To alleviate this limitation we utilized a polyurethane acrylate polymer, a relatively rigid material that can be easily functionalized with different organic moieties. Our patterning approach completely protects both silicon and germanium from chemical oxidation, provides precise control over the shape and size of the patterned features, and gives ready access to chemically discriminated patterns that can be further functionalized with both organic and biological molecules. The approach is general and applicable to other technologically-relevant surfaces.

GxySBA ABC Transporter of Agrobacterium tumefaciens and Its Role in Sugar Utilization and vir Gene Expression

PubMed Central

Zhao, Jinlei

2014-01-01

Monosaccharides available in the extracellular milieu of Agrobacterium tumefaciens can be transported into the cytoplasm, or via the periplasmic sugar binding protein, ChvE, play a critical role in controlling virulence gene expression. The ChvE-MmsAB ABC transporter is involved in the utilization of a wide range of monosaccharide substrates but redundant transporters are likely given the ability of a chvE-mmsAB deletion strain to grow, albeit more slowly, in the presence of particular monosaccharides. In this study, a putative ABC transporter encoded by the gxySBA operon is identified and shown to be involved in the utilization of glucose, xylose, fucose, and arabinose, which are also substrates for the ChvE-MmsAB ABC transporter. Significantly, GxySBA is also shown to be the first characterized glucosamine ABC transporter. The divergently transcribed gene gxyR encodes a repressor of the gxySBA operon, the function of which can be relieved by a subset of the transported sugars, including glucose, xylose, and glucosamine, and this substrate-induced expression can be repressed by glycerol. Furthermore, deletion of the transporter can increase the sensitivity of the virulence gene expression system to certain sugars that regulate it. Collectively, the results reveal a remarkably diverse set of substrates for the GxySBA transporter and its contribution to the repression of sugar sensitivity by the virulence-controlling system, thereby facilitating the capacity of the bacterium to distinguish between the soil and plant environments. PMID:24957625

Molecular Basis of Signaling Specificity of Insulin and IGF Receptors: Neglected Corners and Recent Advances

PubMed Central

Siddle, Kenneth

2011-01-01

Insulin and insulin-like growth factor (IGF) receptors utilize common phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways to mediate a broad spectrum of “metabolic” and “mitogenic” responses. Specificity of insulin and IGF action in vivo must in part reflect expression of receptors and responsive pathways in different tissues but it is widely assumed that it is also determined by the ligand binding and signaling mechanisms of the receptors. This review focuses on receptor-proximal events in insulin/IGF signaling and examines their contribution to specificity of downstream responses. Insulin and IGF receptors may differ subtly in the efficiency with which they recruit their major substrates (IRS-1 and IRS-2 and Shc) and this could influence effectiveness of signaling to “metabolic” and “mitogenic” responses. Other substrates (Grb2-associated binder, downstream of kinases, SH2Bs, Crk), scaffolds (RACK1, β-arrestins, cytohesins), and pathways (non-receptor tyrosine kinases, phosphoinositide kinases, reactive oxygen species) have been less widely studied. Some of these components appear to be specifically involved in “metabolic” or “mitogenic” signaling but it has not been shown that this reflects receptor-preferential interaction. Very few receptor-specific interactions have been characterized, and their roles in signaling are unclear. Signaling specificity might also be imparted by differences in intracellular trafficking or feedback regulation of receptors, but few studies have directly addressed this possibility. Although published data are not wholly conclusive, no evidence has yet emerged for signaling mechanisms that are specifically engaged by insulin receptors but not IGF receptors or vice versa, and there is only limited evidence for differential activation of signaling mechanisms that are common to both receptors. Cellular context, rather than intrinsic receptor activity, therefore appears to be the major determinant of whether responses to insulin and IGFs are perceived as “metabolic” or “mitogenic.” PMID:22649417

Lightweight Electrode For Nickel/Hydrogen Cell

NASA Technical Reports Server (NTRS)

Britton, Doris L.

1994-01-01

Improved substrate for nickel electrode increases specific energy of nickel/hydrogen cell. Consists of 50 percent by weight nickel fiber, 35 percent nickel powder, and 15 percent cobalt powder. Porosity and thickness of nickel electrodes affect specific energy, initial performance, and cycle life of cell. Substrate easily manufactured with much larger porosities than those of heavy-sintered state-of-art nickel substrate.

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

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

Degradation of 1,3-Dichloropropene by Pseudomonas cichorii 170

PubMed Central

Poelarends, Gerrit J.; Wilkens, Marga; Larkin, Michael J.; van Elsas, Jan Dirk; Janssen, Dick B.

1998-01-01

The gram-negative bacterium Pseudomonas cichorii 170, isolated from soil that was repeatedly treated with the nematocide 1,3-dichloropropene, could utilize low concentrations of 1,3-dichloropropene as a sole carbon and energy source. Strain 170 was also able to grow on 3-chloroallyl alcohol, 3-chloroacrylic acid, and several 1-halo-n-alkanes. This organism produced at least three different dehalogenases: a hydrolytic haloalkane dehalogenase specific for haloalkanes and two 3-chloroacrylic acid dehalogenases, one specific for cis-3-chloroacrylic acid and the other specific for trans-3-chloroacrylic acid. The haloalkane dehalogenase and the trans-3-chloroacrylic acid dehalogenase were expressed constitutively, whereas the cis-3-chloroacrylic acid dehalogenase was inducible. The presence of these enzymes indicates that 1,3-dichloropropene is hydrolyzed to 3-chloroallyl alcohol, which is oxidized in two steps to 3-chloroacrylic acid. The latter compound is then dehalogenated, probably forming malonic acid semialdehyde. The haloalkane dehalogenase gene, which is involved in the conversion of 1,3-dichloropropene to 3-chloroallyl alcohol, was cloned and sequenced, and this gene turned out to be identical to the previously studied dhaA gene of the gram-positive bacterium Rhodococcus rhodochrous NCIMB13064. Mutants resistant to the suicide substrate 1,2-dibromoethane lacked haloalkane dehalogenase activity and therefore could not utilize haloalkanes for growth. PCR analysis showed that these mutants had lost at least part of the dhaA gene. PMID:9687453

Insight on specificity of uracil permeases of the NAT/NCS2 family from analysis of the transporter encoded in the pyrimidine utilization operon of Escherichia coli.

PubMed

Botou, Maria; Lazou, Panayiota; Papakostas, Konstantinos; Lambrinidis, George; Evangelidis, Thomas; Mikros, Emmanuel; Frillingos, Stathis

2018-04-01

The uracil permease UraA of Escherichia coli is a structurally known prototype for the ubiquitous Nucleobase-Ascorbate Transporter (NAT) or Nucleobase-Cation Symporter-2 (NCS2) family and represents a well-defined subgroup of bacterial homologs that remain functionally unstudied. Here, we analyze four of these homologs, including RutG of E. coli which shares 35% identity with UraA and is encoded in the catabolic rut (pyrimidine utilization) operon. Using amplified expression in E. coli K-12, we show that RutG is a high-affinity permease for uracil, thymine and, at low efficiency, xanthine and recognizes also 5-fluorouracil and oxypurinol. In contrast, UraA and the homologs from Acinetobacter calcoaceticus and Aeromonas veronii are permeases specific for uracil and 5-fluorouracil. Molecular docking indicates that thymine is hindered from binding to UraA by a highly conserved Phe residue which is absent in RutG. Site-directed replacement of this Phe with Ala in the three uracil-specific homologs allows high-affinity recognition and/or transport of thymine, emulating the RutG profile. Furthermore, all RutG orthologs from enterobacteria retain an Ala at this position, implying that they can use both uracil and thymine and, possibly, xanthine as substrates and provide the bacterial cell with a range of catabolizable nucleobases. © 2018 John Wiley & Sons Ltd.

Reprogramming the Chemodiversity of Terpenoid Cyclization by Remolding the Active Site Contour of epi-Isozizaene Synthase

PubMed Central

2015-01-01

The class I terpenoid cyclase epi-isozizaene synthase (EIZS) utilizes the universal achiral isoprenoid substrate, farnesyl diphosphate, to generate epi-isozizaene as the predominant sesquiterpene cyclization product and at least five minor sesquiterpene products, making EIZS an ideal platform for the exploration of fidelity and promiscuity in a terpenoid cyclization reaction. The hydrophobic active site contour of EIZS serves as a template that enforces a single substrate conformation, and chaperones subsequently formed carbocation intermediates through a well-defined mechanistic sequence. Here, we have used the crystal structure of EIZS as a guide to systematically remold the hydrophobic active site contour in a library of 26 site-specific mutants. Remolded cyclization templates reprogram the reaction cascade not only by reproportioning products generated by the wild-type enzyme but also by generating completely new products of diverse structure. Specifically, we have tripled the overall number of characterized products generated by EIZS. Moreover, we have converted EIZS into six different sesquiterpene synthases: F96A EIZS is an (E)-β-farnesene synthase, F96W EIZS is a zizaene synthase, F95H EIZS is a β-curcumene synthase, F95M EIZS is a β-acoradiene synthase, F198L EIZS is a β-cedrene synthase, and F96V EIZS and W203F EIZS are (Z)-γ-bisabolene synthases. Active site aromatic residues appear to be hot spots for reprogramming the cyclization cascade by manipulating the stability and conformation of critical carbocation intermediates. A majority of mutant enzymes exhibit only relatively modest 2–100-fold losses of catalytic activity, suggesting that residues responsible for triggering substrate ionization readily tolerate mutations deeper in the active site cavity. PMID:24517311

Discovery of new enzymes and metabolic pathways by using structure and genome context.

PubMed

Zhao, Suwen; Kumar, Ritesh; Sakai, Ayano; Vetting, Matthew W; Wood, B McKay; Brown, Shoshana; Bonanno, Jeffery B; Hillerich, Brandan S; Seidel, Ronald D; Babbitt, Patricia C; Almo, Steven C; Sweedler, Jonathan V; Gerlt, John A; Cronan, John E; Jacobson, Matthew P

2013-10-31

Assigning valid functions to proteins identified in genome projects is challenging: overprediction and database annotation errors are the principal concerns. We and others are developing computation-guided strategies for functional discovery with 'metabolite docking' to experimentally derived or homology-based three-dimensional structures. Bacterial metabolic pathways often are encoded by 'genome neighbourhoods' (gene clusters and/or operons), which can provide important clues for functional assignment. We recently demonstrated the synergy of docking and pathway context by 'predicting' the intermediates in the glycolytic pathway in Escherichia coli. Metabolite docking to multiple binding proteins and enzymes in the same pathway increases the reliability of in silico predictions of substrate specificities because the pathway intermediates are structurally similar. Here we report that structure-guided approaches for predicting the substrate specificities of several enzymes encoded by a bacterial gene cluster allowed the correct prediction of the in vitro activity of a structurally characterized enzyme of unknown function (PDB 2PMQ), 2-epimerization of trans-4-hydroxy-L-proline betaine (tHyp-B) and cis-4-hydroxy-D-proline betaine (cHyp-B), and also the correct identification of the catabolic pathway in which Hyp-B 2-epimerase participates. The substrate-liganded pose predicted by virtual library screening (docking) was confirmed experimentally. The enzymatic activities in the predicted pathway were confirmed by in vitro assays and genetic analyses; the intermediates were identified by metabolomics; and repression of the genes encoding the pathway by high salt concentrations was established by transcriptomics, confirming the osmolyte role of tHyp-B. This study establishes the utility of structure-guided functional predictions to enable the discovery of new metabolic pathways.

Radiation tolerant back biased CMOS VLSI

NASA Technical Reports Server (NTRS)

Maki, Gary K. (Inventor); Gambles, Jody W. (Inventor); Hass, Kenneth J. (Inventor)

2003-01-01

A CMOS circuit formed in a semiconductor substrate having improved immunity to total ionizing dose radiation, improved immunity to radiation induced latch up, and improved immunity to a single event upset. The architecture of the present invention can be utilized with the n-well, p-well, or dual-well processes. For example, a preferred embodiment of the present invention is described relative to a p-well process wherein the p-well is formed in an n-type substrate. A network of NMOS transistors is formed in the p-well, and a network of PMOS transistors is formed in the n-type substrate. A contact is electrically coupled to the p-well region and is coupled to first means for independently controlling the voltage in the p-well region. Another contact is electrically coupled to the n-type substrate and is coupled to second means for independently controlling the voltage in the n-type substrate. By controlling the p-well voltage, the effective threshold voltages of the n-channel transistors both drawn and parasitic can be dynamically tuned. Likewise, by controlling the n-type substrate, the effective threshold voltages of the p-channel transistors both drawn and parasitic can also be dynamically tuned. Preferably, by optimizing the threshold voltages of the n-channel and p-channel transistors, the total ionizing dose radiation effect will be neutralized and lower supply voltages can be utilized for the circuit which would result in the circuit requiring less power.

Two-Dimensional Micropatterns of Self-Assembled Poly(N-isopropylacrylamide) Microgels for Patterned Adhesion and Temperature-Responsive Detachment of Fibroblasts

PubMed Central

Tsai, Hsin-Yi; Vats, Kanika; Yates, Matthew Z.; Benoit, Danielle S. W.

2013-01-01

Thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) microgels were patterned on polystyrene substrates via dip coating, creating cytocompatible substrates that provided spatial control over cell adhesion. This simple dip coating method, which exploits variable substrate withdrawal speeds form particle suspension formed stripes of densely-packed PNIPAM microgels, while spacings between the stripes contained sparsely-distributed PNIPAM microgels. The assembly of three different PNIPAM microgel patterns, namely patterns composed of 50 μm stripes/50 μm spacings, 50 μm stripes/100 μm spacings, and 100 μm stripes/100 μm spacings was verified using high-resolution optical micrographs and ImageJ analysis. PNIPAM microgels existed as monolayers within stripes and spacings, as revealed by atomic force microscopy (AFM). Upon cell seeding on PNIPAM micropatterned substrates, NIH3T3 fibroblast cells preferentially adhered within spacings to form cell patterns. Three days after cell seeding, cells proliferated to form confluent cell layers. The thermoresponsiveness of the underlying PNIPAM microgels was then utilized to recover fibroblast cell sheets from substrates simply by lowering the temperature, without disrupting the underlying PNIPAM microgel patterns. Harvested cell sheets similar to these have been used for multiple tissue engineering applications. Also, this simple, low cost, template-free dip coating technique can be utilized to micropattern multifunctional PNIPAM microgels, generating complex stimuli-responsive substrates to study cell-material interactions and allow drug delivery to cells in a spatially and temporally-controlled manners. PMID:23968193

Caspase selective reagents for diagnosing apoptotic mechanisms.

PubMed

Poreba, Marcin; Groborz, Katarzyna; Navarro, Mario; Snipas, Scott J; Drag, Marcin; Salvesen, Guy S

2018-05-10

Apical caspases initiate and effector caspases execute apoptosis. Reagents that can distinguish between caspases, particularly apical caspases-8, 9, and 10 are scarce and generally nonspecific. Based upon a previously described large-scale screen of peptide-based caspase substrates termed HyCoSuL, we sought to develop reagents to distinguish between apical caspases in order to reveal their function in apoptotic cell death paradigms. To this end, we selected tetrapeptide-based sequences that deliver optimal substrate selectivity and converted them to inhibitors equipped with a detectable tag (activity-based probes-ABPs). We demonstrate a strong relationship between substrate kinetics and ABP kinetics. To evaluate the utility of selective substrates and ABPs, we examined distinct apoptosis pathways in Jurkat T lymphocyte and MDA-MB-231 breast cancer lines triggered to undergo cell death via extrinsic or intrinsic apoptosis. We report the first highly selective substrate appropriate for quantitation of caspase-8 activity during apoptosis. Converting substrates to ABPs promoted loss-of-activity and selectivity, thus we could not define a single ABP capable of detecting individual apical caspases in complex mixtures. To overcome this, we developed a panel strategy utilizing several caspase-selective ABPs to interrogate apoptosis, revealing the first chemistry-based approach to uncover the participation of caspase-8, but not caspase-9 or -10 in TRAIL-induced extrinsic apoptosis. We propose that using select panels of ABPs can provide information regarding caspase-8 apoptotic signaling more faithfully than can single, generally nonspecific reagents.

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Linking protein motion to enzyme catalysis.

PubMed

Singh, Priyanka; Abeysinghe, Thelma; Kohen, Amnon

2015-01-13

Enzyme motions on a broad range of time scales can play an important role in various intra- and intermolecular events, including substrate binding, catalysis of the chemical conversion, and product release. The relationship between protein motions and catalytic activity is of contemporary interest in enzymology. To understand the factors influencing the rates of enzyme-catalyzed reactions, the dynamics of the protein-solvent-ligand complex must be considered. The current review presents two case studies of enzymes-dihydrofolate reductase (DHFR) and thymidylate synthase (TSase)-and discusses the role of protein motions in their catalyzed reactions. Specifically, we will discuss the utility of kinetic isotope effects (KIEs) and their temperature dependence as tools in probing such phenomena.

Beginnings of microbiology and biochemistry: the contribution of yeast research.

PubMed

Barnett, James A

2003-03-01

With improvements in microscopes early in the nineteenth century, yeasts were seen to be living organisms, although some famous scientists ridiculed the idea and their influence held back the development of microbiology. In the 1850s and 1860s, yeasts were established as microbes and responsible for alcoholic fermentation, and this led to the study of the rôle of bacteria in lactic and other fermentations, as well as bacterial pathogenicity. At this time, there were difficulties in distinguishing between the activities of microbes and of extracellular enzymes. Between 1884 and 1894, Emil Fischer's study of sugar utilization by yeasts generated an understanding of enzymic specificity and the nature of enzyme-substrate complexes.

Compostability and biodegradation study of PLA-wheat straw and PLA-soy straw based green composites in simulated composting bioreactor.

PubMed

Pradhan, Ranjan; Misra, Manjusri; Erickson, Larry; Mohanty, Amar

2010-11-01

A laboratory scale simulated composting facility (as per ASTM D 5338) was designed and utilized to determine and evaluate the extent of degradation of polylactic acid (PLA), untreated wheat and soy straw and injection moulded composites of PLA-wheat straw (70:30) and PLA-soy straw (70:30). The outcomes of the study revealed the suitability of the test protocol, validity of the test system and defined the compostability of the composites of PLA with unmodified natural substrate. The study would help to design composites using modified soy straw and wheat straw as reinforcement/filler to satisfy ASTM D 6400 specifications. Copyright 2010 Elsevier Ltd. All rights reserved.

Dynamic analysis of CO₂ labeling and cell respiration using membrane-inlet mass spectrometry.

PubMed

Yang, Tae Hoon

2014-01-01

Here, we introduce a mass spectrometry-based analytical method and relevant technical details for dynamic cell respiration and CO2 labeling analysis. Such measurements can be utilized as additional information and constraints for model-based (13)C metabolic flux analysis. Dissolved dynamics of oxygen consumption and CO2 mass isotopomer evolution from (13)C-labeled tracer substrates through different cellular processes can be precisely measured on-line using a miniaturized reactor system equipped with a membrane-inlet mass spectrometer. The corresponding specific rates of physiologically relevant gases and CO2 mass isotopomers can be quantified within a short-term range based on the liquid-phase dynamics of dissolved fermentation gases.

Breath carbon stable isotope ratios identify changes in energy balance and substrate utilization in humans.

PubMed

Whigham, L D; Butz, D E; Johnson, L K; Schoeller, D A; Abbott, D H; Porter, W P; Cook, M E

2014-09-01

Rapid detection of shifts in substrate utilization and energy balance would provide a compelling biofeedback tool for individuals attempting weight loss. As a proof of concept, we tested whether the natural abundance of exhaled carbon stable isotope ratios (breath δ(13)C) reflects shifts between negative and positive energy balance. Volunteers (n=5) consumed a 40% energy-restricted diet for 6 days followed by 50% excess on day 7. Breath was sampled immediately before and 1 h and 2 h after breakfast, lunch and dinner. Exhaled breath δ(13)C values were measured by cavity ring-down spectroscopy. Using repeated measures analysis of variance (ANOVA) followed by Dunnett's contrasts, pre-breakfast breath values on days 2-6 were compared with day 1, and postprandial day 7 time points were compared with pre-breakfast day 7. Energy restriction diminished pre-breakfast breath δ(13)C by day 3 (P<0.05). On day 7, increased energy intake was first detected immediately before dinner (-23.8±0.6 vs -21.9±0.7‰, P=0.002 (means±s.d.)), and breath δ(13)C remained elevated at least 2 h post dinner. In conclusion, when shifting between negative and positive energy balance, breath δ(13)C showed anticipated isotopic changes. Although additional research is needed to determine specificity and repeatability, this method may provide a biomarker for marked increases in caloric intake.

Trapping and Collection of Lymphocytes Using Microspot Array Chip and Magnetic Beads

NASA Astrophysics Data System (ADS)

Hashioka, Shingi; Obata, Tsutomu; Tokimitsu, Yoshiharu; Fujiki, Satoshi; Nakazato, Hiroyoshi; Muraguchi, Atsushi; Kishi, Hiroyuki; Tanino, Katsumi

2006-04-01

A microspot array chip, which has microspots of a magnetic thin film patterned on a glass substrate, was fabricated for trapping individual cells and for measuring their cellular response. The chip was easily fabricated by conventional semiconductor fabrication techniques on a mass production level as a disposable medical device. When a solution of lymphocyte-bound-magnetic beads was poured into the magnetized chip, each lymphocyte was trapped on each microspot of the magnetic thin film. The trapped cells were easily recovered from the chip using a micromanipulator. The micro-spot array chip can be utilized for arraying live cells and for measuring the response of each cell. The chip will be useful for preparing on array of different kinds of cells and for analyzing cellular response at the single cell level. The chip will be particularly useful for detecting antigen-specific B-lymphocytes and antigen-specific antibody complementary deoxyribonucleic acid (cDNA).

Specificity of hammerhead ribozyme cleavage.

PubMed Central

Hertel, K J; Herschlag, D; Uhlenbeck, O C

1996-01-01

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

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

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

PubMed Central

Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

2012-01-01

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

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.

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

PubMed Central

Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

2008-01-01

SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

PubMed Central

Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

2014-01-01

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

A multiwell format assay for heparanase.

PubMed

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.