Sample records for broader substrate specificity
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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.
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Sayer, Christopher; Isupov, Michail N; Westlake, Aaron; Littlechild, Jennifer A
2013-04-01
The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron
2013-04-01
The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes. The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes showmore » activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.« less
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Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.
2013-01-01
The crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases. PMID:23519665
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Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri
2018-02-01
Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
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Reichhardt, Charles; Olson Reichhardt, Cynthia Jane
2016-12-20
Here, we review the depinning and nonequilibrium phases of collectively interacting particle systems driven over random or periodic substrates. This type of system is relevant to vortices in type-II superconductors, sliding charge density waves, electron crystals, colloids, stripe and pattern forming systems, and skyrmions, and could also have connections to jamming, glassy behaviors, and active matter. These systems are also ideal for exploring the broader issues of characterizing transient and steady state nonequilibrium flow phases as well as nonequilibrium phase transitions between distinct dynamical phases, analogous to phase transitions between different equilibrium states. We discuss the differences between elastic andmore » plastic depinning on random substrates and the different types of nonequilibrium phases which are associated with specific features in the velocity-force curves, fluctuation spectra, scaling relations, and local or global particle ordering. We describe how these quantities can change depending on the dimension, anisotropy, disorder strength, and the presence of hysteresis. Within the moving phase we discuss how there can be a transition from a liquid-like state to dynamically ordered moving crystal, smectic, or nematic states. Systems with periodic or quasiperiodic substrates can have multiple nonequilibrium second or first order transitions in the moving state between chaotic and coherent phases, and can exhibit hysteresis. We also discuss systems with competing repulsive and attractive interactions, which undergo dynamical transitions into stripes and other complex morphologies when driven over random substrates. Throughout this work we highlight open issues and future directions such as absorbing phase transitions, nonequilibrium work relations, inertia, the role of non-dissipative dynamics such as Magnus effects, and how these results could be extended to the broader issues of plasticity in crystals, amorphous solids, and jamming phenomena.« less
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NASA Astrophysics Data System (ADS)
Reichhardt, C.; Olson Reichhardt, C. J.
2017-02-01
We review the depinning and nonequilibrium phases of collectively interacting particle systems driven over random or periodic substrates. This type of system is relevant to vortices in type-II superconductors, sliding charge density waves, electron crystals, colloids, stripe and pattern forming systems, and skyrmions, and could also have connections to jamming, glassy behaviors, and active matter. These systems are also ideal for exploring the broader issues of characterizing transient and steady state nonequilibrium flow phases as well as nonequilibrium phase transitions between distinct dynamical phases, analogous to phase transitions between different equilibrium states. We discuss the differences between elastic and plastic depinning on random substrates and the different types of nonequilibrium phases which are associated with specific features in the velocity-force curves, fluctuation spectra, scaling relations, and local or global particle ordering. We describe how these quantities can change depending on the dimension, anisotropy, disorder strength, and the presence of hysteresis. Within the moving phase we discuss how there can be a transition from a liquid-like state to dynamically ordered moving crystal, smectic, or nematic states. Systems with periodic or quasiperiodic substrates can have multiple nonequilibrium second or first order transitions in the moving state between chaotic and coherent phases, and can exhibit hysteresis. We also discuss systems with competing repulsive and attractive interactions, which undergo dynamical transitions into stripes and other complex morphologies when driven over random substrates. Throughout this work we highlight open issues and future directions such as absorbing phase transitions, nonequilibrium work relations, inertia, the role of non-dissipative dynamics such as Magnus effects, and how these results could be extended to the broader issues of plasticity in crystals, amorphous solids, and jamming phenomena.
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Albuquerque, Maria G E; Carvalho, Gilda; Kragelund, Caroline; Silva, Ana F; Barreto Crespo, Maria T; Reis, Maria A M; Nielsen, Per H
2013-01-01
The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor. PMID:22810062
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Structural and Biochemical Characterization of Cinnamoyl-CoA Reductases1
Walker, Alexander M.
2017-01-01
Cinnamoyl-coenzyme A reductase (CCR) catalyzes the reduction of hydroxycinnamoyl-coenzyme A (CoA) esters using NADPH to produce hydroxycinnamyl aldehyde precursors in lignin synthesis. The catalytic mechanism and substrate specificity of cinnamoyl-CoA reductases from sorghum (Sorghum bicolor), a strategic plant for bioenergy production, were deduced from crystal structures, site-directed mutagenesis, and kinetic and thermodynamic analyses. Although SbCCR1 displayed higher affinity for caffeoyl-CoA or p-coumaroyl-CoA than for feruloyl-CoA, the enzyme showed significantly higher activity for the latter substrate. Through molecular docking and comparisons between the crystal structures of the Vitis vinifera dihydroflavonol reductase and SbCCR1, residues threonine-154 and tyrosine-310 were pinpointed as being involved in binding CoA-conjugated phenylpropanoids. Threonine-154 of SbCCR1 and other CCRs likely confers strong substrate specificity for feruloyl-CoA over other cinnamoyl-CoA thioesters, and the T154Y mutation in SbCCR1 led to broader substrate specificity and faster turnover. Through data mining using our structural and biochemical information, four additional putative CCR genes were discovered from sorghum genomic data. One of these, SbCCR2, displayed greater activity toward p-coumaroyl-CoA than did SbCCR1, which could imply a role in the synthesis of defense-related lignin. Taken together, these findings provide knowledge about critical residues and substrate preference among CCRs and provide, to our knowledge, the first three-dimensional structure information for a CCR from a monocot species. PMID:27956488
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Substrate specificity of platypus venom L-to-D-peptide isomerase.
Bansal, Paramjit S; Torres, Allan M; Crossett, Ben; Wong, Karen K Y; Koh, Jennifer M S; Geraghty, Dominic P; Vandenberg, Jamie I; Kuchel, Philip W
2008-04-04
The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.
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Jak, Amy J.; Bangen, Katherine J.; Wierenga, Christina E.; Delano-Wood, Lisa; Corey-Bloom, Jody; Bondi, Mark W.
2010-01-01
The original conceptualization of mild cognitive impairment (MCI) was primarily as an amnestic disorder representing an intermediate stage between normal aging and Alzheimer’s dementia (AD). More recently, broader conceptualizations of MCI have emerged that also encompass cognitive domains other than memory. These characterizations delineate clinical subtypes that commonly include amnestic and non-amnestic forms, and that involve single and multiple cognitive domains. With the advent of these broader classifications, more specific information is emerging regarding the neuropsychological presentation of individuals with MCI, risk for dementia associated with different subtypes of MCI, and neuropathologic substrates connected to the clinical subtypes. This review provides an overview of this burgeoning literature specific to clinical subtypes of MCI. Focus is primarily on neuropsychological and structural neuroimaging findings specific to clinical subtypes of MCI as well as the issue of daily functioning. Although investigations of non-amnestic subtypes using advanced neuroimaging techniques and clinical trials are quite limited, we briefly review these topics in MCI because these data provide a framework for future investigations specifically examining additional clinical subtypes of MCI. Finally, the review comments on select methodological issues involved in studying this heterogeneous population, and future directions to continue to improve our understanding of MCI and its clinical subtypes are offered. PMID:19501714
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A single molecule perspective on the functional diversity of in vitro evolved β-glucuronidase.
Liebherr, Raphaela B; Renner, Max; Gorris, Hans H
2014-04-23
The mechanisms that drive the evolution of new enzyme activity have been investigated by comparing the kinetics of wild-type and in vitro evolved β-glucuronidase (GUS) at the single molecule level. Several hundred single GUS molecules were separated in large arrays of 62,500 ultrasmall reaction chambers etched into the surface of a fused silica slide to observe their individual substrate turnover rates in parallel by fluorescence microscopy. Individual GUS molecules feature long-lived but divergent activity states, and their mean activity is consistent with classic Michaelis-Menten kinetics. The large number of single molecule substrate turnover rates is representative of the activity distribution within an entire enzyme population. Partially evolved GUS displays a much broader activity distribution among individual enzyme molecules than wild-type GUS. The broader activity distribution indicates a functional division of work between individual molecules in a population of partially evolved enzymes that-as so-called generalists-are characterized by their promiscuous activity with many different substrates.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Borole, A P
The recovery and conversion of heavy oils is limited due to the high viscosity of these crudes and their high heteroatom content. Conventional technology relies on thermochemical hydrogenation and hydrodesulfurization to address these problems and is energy intensive due to the high operating temperature and pressure. This project was initiated to explore biological catalysts for adding hydrogen to the heavy oil molecules. Biological enzymes are efficient at hydrogen splitting at very mild conditions such as room temperature and pressure, however, they are very specific in terms of the substrates they hydrogenate. The goal of the project was to investigate howmore » the specificity of these enzymes can be altered to develop catalysts for oil upgrading. Three approaches were used. First was to perform chemical modification of the enzyme surface to improve binding of other non-natural substrates. Second approach was to expose the deeply buried catalytic active site of the enzyme by removal of protein scaffolding to enable better interaction with other substrates. The third approach was based on molecular biology to develop genetically engineered systems for enabling targeted structural changes in the enzyme. The first approach was found to be limited in success due to the non-specificity of the chemical modification and inability to target the region near the active site or the site of substrate binding. The second approach produced a smaller catalyst capable of catalyzing hydrogen splitting, however, further experimentation is needed to address reproducibility and stability issues. The third approach which targeted cloning of hydrogenase in alternate hosts demonstrated progress, although further work is necessary to complete the cloning process. The complex nature of the hydrogenase enzyme structure-function relationship and role of various ligands in the protein require significant more research to better understand the enzyme and to enable success in strategies in developing catalysts with broader specificity as that required for crude upgrading.« less
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YTPdb: a wiki database of yeast membrane transporters.
Brohée, Sylvain; Barriot, Roland; Moreau, Yves; André, Bruno
2010-10-01
Membrane transporters constitute one of the largest functional categories of proteins in all organisms. In the yeast Saccharomyces cerevisiae, this represents about 300 proteins ( approximately 5% of the proteome). We here present the Yeast Transport Protein database (YTPdb), a user-friendly collaborative resource dedicated to the precise classification and annotation of yeast transporters. YTPdb exploits an evolution of the MediaWiki web engine used for popular collaborative databases like Wikipedia, allowing every registered user to edit the data in a user-friendly manner. Proteins in YTPdb are classified on the basis of functional criteria such as subcellular location or their substrate compounds. These classifications are hierarchical, allowing queries to be performed at various levels, from highly specific (e.g. ammonium as a substrate or the vacuole as a location) to broader (e.g. cation as a substrate or inner membranes as location). Other resources accessible for each transporter via YTPdb include post-translational modifications, K(m) values, a permanently updated bibliography, and a hierarchical classification into families. The YTPdb concept can be extrapolated to other organisms and could even be applied for other functional categories of proteins. YTPdb is accessible at http://homes.esat.kuleuven.be/ytpdb/. Copyright © 2010 Elsevier B.V. All rights reserved.
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Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi
2012-08-01
The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.
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Low dielectric polyimide aerogels as substrates for lightweight patch antennas.
Meador, Mary Ann B; Wright, Sarah; Sandberg, Anna; Nguyen, Baochau N; Van Keuls, Frederick W; Mueller, Carl H; Rodríguez-Solís, Rafael; Miranda, Félix A
2012-11-01
The dielectric properties and loss tangents of low-density polyimide aerogels have been characterized at various frequencies. Relative dielectric constants as low as 1.16 were measured for polyimide aerogels made from 2,2'-dimethylbenzidine (DMBZ) and biphenyl 3,3',4,4'-tetracarbozylic dianhydride (BPDA) cross-linked with 1,3,5-triaminophenoxybenzene (TAB). This formulation was used as the substrate to fabricate and test prototype microstrip patch antennas and benchmark against state of practice commercial antenna substrates. The polyimide aerogel antennas exhibited broader bandwidth, higher gain, and lower mass than the antennas made using commercial substrates. These are very encouraging results, which support the potential advantages of the polyimide aerogel-based antennas for aerospace applications.
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Fabrication of broadband antireflection coating at terahertz frequency using a hot emboss method
NASA Astrophysics Data System (ADS)
Li, YunZhou; Cai, Bin; Zhu, YiMing
2014-11-01
We fabricated a terahertz anti-reflective structure on a polystylene by using a hot-embossing method. Polystylene was spin-coated onto a silicon substrate and then transformed by using a metallic mould comprising a bunch of Chinese acupuncture needles. The transformation layer yielded gradient refractive index profiles on the substrate which can reduce the surface reflection effectively. The samples were evaluated by a terahertz time-domain spectroscope. Compared with a bare silicon substrate, we observed an increase of ~30% in the transmittance. We also observed broader bandwidth properties compared with a single-layer antireflective structure. The process imposes no substrate limiting; i.e., it has great potential to be applied onto various THz devices.
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Dual ion beam assisted deposition of biaxially textured template layers
Groves, James R.; Arendt, Paul N.; Hammond, Robert H.
2005-05-31
The present invention is directed towards a process and apparatus for epitaxial deposition of a material, e.g., a layer of MgO, onto a substrate such as a flexible metal substrate, using dual ion beams for the ion beam assisted deposition whereby thick layers can be deposited without degradation of the desired properties by the material. The ability to deposit thicker layers without loss of properties provides a significantly broader deposition window for the process.
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Roy, Hervé; Ibba, Michael
2009-01-01
Aminoacylphosphatidylglycerol synthases (aaPGSs) are multiple peptide resistance factors that transfer amino acids from aminoacyl-tRNAs to phosphatidylglycerol (PG) in the cytoplasmic membrane. Aminoacylation of PG is used by bacteria to decrease the net negative charge of the cell envelope, diminishing affinity for charged molecules and allowing for adaptation to environmental changes. Lys-PGS, which transfers lysine to PG, is essential for the virulence of certain pathogens, providing resistance to both host cationic antimicrobial peptides and therapeutic antibiotics. Ala-PGS was also recently described, but little is known about the possible activities of other members of the highly diverse aaPGS family of proteins. Systematic deletion of the predicted membrane-inserted domains of several aaPGSs revealed that the carboxyl-terminal hydrophilic domain alone is sufficient for aminoacylphosphatidylglycerol transferase catalytic activity. In contrast to previously characterized aaPGSs, the Enterococcus faecium enzyme used an expanded repertoire of amino acids to modify PG with Ala, Arg, or Lys. Reexamination of previously characterized aaPGSs also revealed broader than anticipated substrate specificity, for example Bacillus subtilis Lys-PGS was shown to also catalyze Ala-PG synthesis. The relaxed substrate specificities of these aaPGSs allows for more elaborate remodeling of membrane lipids than previously thought, potentially providing bacteria that harbor these enzymes resistance to a broad spectrum of antibiotics and environmental stresses. PMID:19734140
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Code of Federal Regulations, 2012 CFR
2012-04-01
... the issues addressed in the broader review. The broader review should identify and evaluate those... consideration. The broader review should also establish the policy, standard or process to be followed in the site specific review. The Finding of No Significant Impact (FONSI) with respect to the broader...
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Code of Federal Regulations, 2011 CFR
2011-04-01
... the issues addressed in the broader review. The broader review should identify and evaluate those... consideration. The broader review should also establish the policy, standard or process to be followed in the site specific review. The Finding of No Significant Impact (FONSI) with respect to the broader...
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Code of Federal Regulations, 2014 CFR
2014-04-01
... the issues addressed in the broader review. The broader review should identify and evaluate those... consideration. The broader review should also establish the policy, standard or process to be followed in the site specific review. The Finding of No Significant Impact (FONSI) with respect to the broader...
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Code of Federal Regulations, 2013 CFR
2013-04-01
... the issues addressed in the broader review. The broader review should identify and evaluate those... consideration. The broader review should also establish the policy, standard or process to be followed in the site specific review. The Finding of No Significant Impact (FONSI) with respect to the broader...
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Code of Federal Regulations, 2010 CFR
2010-04-01
... the issues addressed in the broader review. The broader review should identify and evaluate those... consideration. The broader review should also establish the policy, standard or process to be followed in the site specific review. The Finding of No Significant Impact (FONSI) with respect to the broader...
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Álvaro-Benito, Miguel; Sainz-Polo, M. Angela; González-Pérez, David; González, Beatriz; Plou, Francisco J.; Fernández-Lobato, María; Sanz-Aparicio, Julia
2012-01-01
Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2–1)/β(2–6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides. PMID:22511773
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Effect of the substrate on the insulator-metal transition of vanadium dioxide films
NASA Astrophysics Data System (ADS)
Kovács, György J.; Bürger, Danilo; Skorupa, Ilona; Reuther, Helfried; Heller, René; Schmidt, Heidemarie
2011-03-01
Single-phase vanadium dioxide films grown on (0001) sapphire and (001) silicon substrates show a very different insulator-metal electronic transition. A detailed description of the growth mechanisms and the substrate-film interaction is given, and the characteristics of the electronic transition are described by the morphology and grain boundary structure. (Tri-)epitaxy-stabilized columnar growth of VO2 takes place on the sapphire substrate, whereas on silicon the expected Zone II growth is identified. We have found that in the case of the Si substrate the reasons for the broader hysteresis and the lower switching amplitude are the formation of an amorphous insulating VOx (x > 2.6) phase coexisting with VO2 and the high vanadium vacancy concentration of the VO2. These phenomena are the result of the excess oxygen during the growth and the interaction between the silicon substrate and the growing film.
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Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.; ...
2017-04-09
The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.
The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putativemore » monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less
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Musumeci, Matías A; Lozada, Mariana; Rial, Daniela V; Mac Cormack, Walter P; Jansson, Janet K; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M
2017-04-09
The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.
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Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M.
2017-01-01
The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer–Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments. PMID:28397770
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Flexible organic light emitting diodes fabricated on biocompatible silk fibroin substrate
NASA Astrophysics Data System (ADS)
Liu, Yuqiang; Xie, Yuemin; Liu, Yuan; Song, Tao; Zhang, Ke-Qin; Liao, Liangsheng; Sun, Baoquan
2015-10-01
Flexible and biodegradable electronics are currently under extensive investigation for biocompatible and environmentally-friendly applications. Synthetic plastic foils are widely used as substrates for flexible electronics. But typical plastic substrates such as polyethylene naphthalate (PEN) could not be degraded in a natural bio-environment. A great demand still exists for a next-generation biocompatible and biodegradable substrate for future application. For example, electronic devices can be potentially integrated into the human body. In this work, we demonstrate that the biocompatible and biodegradable natural silk fibroin (SF) films embedded with silver nanowires (AgNWs) mesh could be employed as conductive transparent substrates to fabricate flexible organic light emitting diodes (OLEDs). Compared with commercial PEN substrates coated with indium tin oxide, the AgNWs/SF composite substrates exhibit a similar sheet resistance of 12 Ω sq-1, a lower surface roughness, as well as a broader light transmission range. Flexible OLEDs based on AgNWs/SF substrates achieve a current efficiency of 19 cd A-1, demonstrating the potential of the flexible AgNWs/SF films as conductive and transparent substrates for next-generation biodegradable devices.
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The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass.
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.
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The prospects of cellulase-producing bacteria for the bioconversion of lignocellulosic biomass
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
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Varga, Elisabeth; Malachova, Alexandra; Nguyen, Nhung Thi; Lorenz, Cindy; Haltrich, Dietmar; Berthiller, Franz; Adam, Gerhard
2015-01-01
Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A Km of 5.4 mM and a Vmax of 16 μmol min−1 mg−1 were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples. PMID:25979885
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Blaukopf, Markus; Yuen, Macaire M.S.; Withers, Stephen G.; Mattsson, Jim; Russell, John H.; Bohlmann, Jörg
2015-01-01
Western redcedar (WRC; Thuja plicata) produces high amounts of oxygenated thujone monoterpenoids associated with resistance against herbivore feeding, particularly ungulate browsing. Thujones and other monoterpenoids accumulate in glandular structures in the foliage of WRC. Thujones are produced from (+)-sabinene by sabinol and sabinone. Using metabolite analysis, enzyme assays with WRC tissue extracts, cloning, and functional characterization of cytochrome P450 monooxygenases, we established that trans-sabin-3-ol but not cis-sabin-3-ol is the intermediate in thujone biosynthesis in WRC. Based on transcriptome analysis, full-length complementary DNA cloning, and characterization of expressed P450 proteins, we identified CYP750B1 and CYP76AA25 as the enzymes that catalyze the hydroxylation of (+)-sabinene to trans-sabin-3-ol. Gene-specific transcript analysis in contrasting WRC genotypes producing high and low amounts of monoterpenoids, including a glandless low-terpenoid clone, as well as assays for substrate specificity supported a biological role of CYP750B1 in α- and β-thujone biosynthesis. This P450 belongs to the apparently gymnosperm-specific CYP750 family and is, to our knowledge, the first member of this family to be functionally characterized. In contrast, CYP76AA25 has a broader substrate spectrum, also converting the sesquiterpene farnesene and the herbicide isoproturon, and its transcript profiles are not well correlated with thujone accumulation. PMID:25829465
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Modeling the evolution of drug resistance in malaria.
Hecht, David; Fogel, Gary B
2012-12-01
Plasmodium falciparum, the causal agent of malaria, continues to evolve resistance to frontline therapeutics such as chloroquine and sulfadoxine-pyrimethamine. Here we study the amino acid replacements in dihydrofolate reductase (DHFR) that confer resistance to pyrimethamine while still binding the natural DHFR substrate, 7,8-dihydrofolate, and cofactor, NADPH. The chain of amino acid replacements that has led to resistance can be inferred in a computer, leading to a broader understanding of the coevolution between the drug and target. This in silico approach suggests that only a small set of specific active site replacements in the proper order could have led to the resistant strains in the wild today. A similar approach can be used on any target of interest to anticipate likely pathways of future resistance for more effective drug development.
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Gorlin, Yelena; Jaramillo, Thomas F.
2014-01-01
The selection of an appropriate substrate is an important initial step for many studies of electrochemically active materials. In order to help researchers with the substrate selection process, we employ a consistent experimental methodology to evaluate the electrochemical reactivity and stability of seven potential substrate materials for electrocatalyst and photoelectrode evaluation. Using cyclic voltammetry with a progressively increased scan range, we characterize three transparent conducting oxides (indium tin oxide, fluorine-doped tin oxide, and aluminum-doped zinc oxide) and four opaque conductors (gold, stainless steel 304, glassy carbon, and highly oriented pyrolytic graphite) in three different electrolytes (sulfuric acid, sodium acetate, and sodium hydroxide). We determine the inert potential window for each substrate/electrolyte combination and make recommendations about which materials may be most suitable for application under different experimental conditions. Furthermore, the testing methodology provides a framework for other researchers to evaluate and report the baseline activity of other substrates of interest to the broader community. PMID:25357131
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Benck, Jesse D.; Pinaud, Blaise A.; Gorlin, Yelena; ...
2014-10-30
The selection of an appropriate substrate is an important initial step for many studies of electrochemically active materials. In order to help researchers with the substrate selection process, we employ a consistent experimental methodology to evaluate the electrochemical reactivity and stability of seven potential substrate materials for electrocatalyst and photoelectrode evaluation. Using cyclic voltammetry with a progressively increased scan range, we characterize three transparent conducting oxides (indium tin oxide, fluorine-doped tin oxide, and aluminum-doped zinc oxide) and four opaque conductors (gold, stainless steel 304, glassy carbon, and highly oriented pyrolytic graphite) in three different electrolytes (sulfuric acid, sodium acetate, andmore » sodium hydroxide). Here, we determine the inert potential window for each substrate/electrolyte combination and make recommendations about which materials may be most suitable for application under different experimental conditions. Furthermore, the testing methodology provides a framework for other researchers to evaluate and report the baseline activity of other substrates of interest to the broader community.« less
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Ray, Thomas A; Roy, Suva; Kozlowski, Christopher; Wang, Jingjing; Cafaro, Jon; Hulbert, Samuel W; Wright, Christopher V; Field, Greg D
2018-01-01
A common strategy by which developing neurons locate their synaptic partners is through projections to circuit-specific neuropil sublayers. Once established, sublayers serve as a substrate for selective synapse formation, but how sublayers arise during neurodevelopment remains unknown. Here, we identify the earliest events that initiate formation of the direction-selective circuit in the inner plexiform layer of mouse retina. We demonstrate that radially migrating newborn starburst amacrine cells establish homotypic contacts on arrival at the inner retina. These contacts, mediated by the cell-surface protein MEGF10, trigger neuropil innervation resulting in generation of two sublayers comprising starburst-cell dendrites. This dendritic scaffold then recruits projections from circuit partners. Abolishing MEGF10-mediated contacts profoundly delays and ultimately disrupts sublayer formation, leading to broader direction tuning and weaker direction-selectivity in retinal ganglion cells. Our findings reveal a mechanism by which differentiating neurons transition from migratory to mature morphology, and highlight this mechanism’s importance in forming circuit-specific sublayers. PMID:29611808
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In silico ligand binding studies of cyanogenic β-glucosidase, dhurrinase-2 from Sorghum bicolor.
Mahajan, Chavi; Patel, Krunal; Khan, Bashir M; Rawal, Shuban S
2015-07-01
Dhurrinase, a cyanogenic β-glucosidase from Sorghum bicolor is the key enzyme responsible for the hydrolysis of dhurrin to produce toxic hydrogen cyanide, as a part of plant defence mechanism. Dhurrinase 1 (SbDhr1) and dhurrinase 2 (SbDhr2), two isozymes have been isolated and characterized from S. bicolor. However, there is no information in the literature about the three dimensional (3D) structure of SbDhr2 and molecular interactions involved between the protein and ligand. In this study, the three dimensional structure of SbDhr2 was built based on homology modeling by using the X-ray crystallographic structure of its close homologue SbDhr1 as the template. The generated 3D model was energy minimized and the quality was validated by Ramachndran plot, various bioinformatic tools and their relevant parameters. Stability, folding-unfolding and flexibility of the modeled SbDhr2 was evaluated on the basis of RMSD, radius of gyration (Rg) and RMSF values respectively, obtained through molecular dynamic (MD) simulation. Further, molecular docking was performed with its natural substrate dhurrin, one substrate analogue, three un-natural substrates, and one inhibitor. Analysis of molecular interactions in the SbDhr2-ligand complexes revealed the key amino acid residues responsible to stabilize the ligands within the binding pocket through non-bonded interactions and some of them were found to be conserved (Glu239, Tyr381, Trp426, Glu454, Trp511). Reasonably broader substrate specificity of SbDhr2 was explained through the wider entrance passage observed in comparison to SbDhr1.
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Temperature dependent surface and spectral modifications of nano V2O5 films
NASA Astrophysics Data System (ADS)
Manthrammel, M. Aslam; Fatehmulla, A.; Al-Dhafiri, A. M.; Alshammari, A. S.; Khan, Aslam
2017-03-01
Nanocrystalline V2O5 films have been deposited on glass substrates at 300°C substrate temperature using thermal evaporation technique and were subjected to thermal annealing at different temperatures 350, 400, and 550°C. X-ray diffraction (XRD) spectra exhibit sharper and broader characteristic peaks respectively indicating the rearrangement of nanocrystallite phases with annealing temperatures. Other phases of vanadium oxides started emerging with the rise in annealing temperature and the sample converted completely to VO2 (B) phase at 550°C annealing. FESEM images showed an increase in crystallite size with 350 and 400°C annealing temperatures followed by a decrease in crystallite size for the sample annealed at 550°C. Transmission spectra showed an initial redshift of the fundamental band edge with 350 and 400°C while a blue shift for the sample annealed at 550°C, which was in agreement with XRD and SEM results. The films exhibited smart window properties as well as nanorod growth at specific annealing temperatures. Apart from showing the PL and defect related peaks, PL studies also supported the observations made in the transmission spectra.
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Engineering and Applications of fungal laccases for organic synthesis
Kunamneni, Adinarayana; Camarero, Susana; García-Burgos, Carlos; Plou, Francisco J; Ballesteros, Antonio; Alcalde, Miguel
2008-01-01
Laccases are multi-copper containing oxidases (EC 1.10.3.2), widely distributed in fungi, higher plants and bacteria. Laccase catalyses the oxidation of phenols, polyphenols and anilines by one-electron abstraction, with the concomitant reduction of oxygen to water in a four-electron transfer process. In the presence of small redox mediators, laccase offers a broader repertory of oxidations including non-phenolic substrates. Hence, fungal laccases are considered as ideal green catalysts of great biotechnological impact due to their few requirements (they only require air, and they produce water as the only by-product) and their broad substrate specificity, including direct bioelectrocatalysis. Thus, laccases and/or laccase-mediator systems find potential applications in bioremediation, paper pulp bleaching, finishing of textiles, bio-fuel cells and more. Significantly, laccases can be used in organic synthesis, as they can perform exquisite transformations ranging from the oxidation of functional groups to the heteromolecular coupling for production of new antibiotics derivatives, or the catalysis of key steps in the synthesis of complex natural products. In this review, the application of fungal laccases and their engineering by rational design and directed evolution for organic synthesis purposes are discussed. PMID:19019256
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Strain Effects in Epitaxial VO2 Thin Films on Columnar Buffer-Layer TiO2/Al2O3 Virtual Substrates.
Breckenfeld, Eric; Kim, Heungsoo; Burgess, Katherine; Charipar, Nicholas; Cheng, Shu-Fan; Stroud, Rhonda; Piqué, Alberto
2017-01-18
Epitaxial VO 2 /TiO 2 thin film heterostructures were grown on (100) (m-cut) Al 2 O 3 substrates via pulsed laser deposition. We have demonstrated the ability to reduce the semiconductor-metal transition (SMT) temperature of VO 2 to ∼44 °C while retaining a 4 order of magnitude SMT using the TiO 2 buffer layer. A combination of electrical transport and X-ray diffraction reciprocal space mapping studies help examine the specific strain states of VO 2 /TiO 2 /Al 2 O 3 heterostructures as a function of TiO 2 film growth temperatures. Atomic force microscopy and transmission electron microscopy analyses show that the columnar microstructure present in TiO 2 buffer films is responsible for the partially strained VO 2 film behavior and subsequently favorable transport characteristics with a lower SMT temperature. Such findings are of crucial importance for both the technological implementation of the VO 2 system, where reduction of its SMT temperature is widely sought, as well as the broader complex oxide community, where greater understanding of the evolution of microstructure, strain, and functional properties is a high priority.
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Cytochrome P450 monooxygenases: perspectives for synthetic application.
Urlacher, Vlada B; Eiben, Sabine
2006-07-01
Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.
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Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus
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
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Biology and Beyond: Domain Specificity in a Broader Developmental Context
ERIC Educational Resources Information Center
Keil, Frank C.
2007-01-01
The assumption of domain specificity has been invaluable to the study of the emergence of biological thought in young children. Yet, domains of thought must be understood within a broader context that explains how those domains relate to the surrounding cultures, to different kinds of cognitive constraints, to framing effects, to abilities to…
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Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R
2016-11-21
Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.
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Unexpected expansion of tRNA substrate recognition by the yeast m1G9 methyltransferase Trm10.
Swinehart, William E; Henderson, Jeremy C; Jackman, Jane E
2013-08-01
N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m1G9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m1G9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m1G9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m1G9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wild-type yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.
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Defining efficient enzyme-cofactor pairs for bioorthogonal profiling of protein methylation
DOE Office of Scientific and Technical Information (OSTI.GOV)
Islam, Kabirul; Chen, Yuling; Wu, Hong
2013-11-18
Protein methyltransferase (PMT)-mediated posttranslational modification of histone and nonhistone substrates modulates stability, localization, and interacting partners of target proteins in diverse cellular contexts. These events play critical roles in normal biological processes and are frequently deregulated in human diseases. In the course of identifying substrates of individual PMTs, bioorthogonal profiling of protein methylation (BPPM) has demonstrated its merits. In this approach, specific PMTs are engineered to process S-adenosyl-L-methionine (SAM) analogs as cofactor surrogates and label their substrates with distinct chemical modifications for target elucidation. Despite the proof-of-concept advancement of BPPM, few efforts have been made to explore its generality. Withmore » two cancer-relevant PMTs, EuHMT1 (GLP1/KMT1D) and EuHMT2 (G9a/KMT1C), as models, we defined the key structural features of engineered PMTs and matched SAM analogs that can render the orthogonal enzyme–cofactor pairs for efficient catalysis. Here we have demonstrated that the presence of sulfonium-β-sp 2 carbon and flexible, medium-sized sulfonium-δ-substituents are crucial for SAM analogs as BPPM reagents. The bulky cofactors can be accommodated by tailoring the conserved Y1211/Y1154 residues and nearby hydrophobic cavities of EuHMT1/2. Profiling proteome-wide substrates with BPPM allowed identification of >500 targets of EuHMT1/2 with representative targets validated using native EuHMT1/2 and SAM. This finding indicates that EuHMT1/2 may regulate many cellular events previously unrecognized to be modulated by methylation. The present work, therefore, paves the way to a broader application of the BPPM technology to profile methylomes of diverse PMTs and elucidate their downstream functions.« less
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Dynamic kirigami structures for integrated solar tracking.
Lamoureux, Aaron; Lee, Kyusang; Shlian, Matthew; Forrest, Stephen R; Shtein, Max
2015-09-08
Optical tracking is often combined with conventional flat panel solar cells to maximize electrical power generation over the course of a day. However, conventional trackers are complex and often require costly and cumbersome structural components to support system weight. Here we use kirigami (the art of paper cutting) to realize novel solar cells where tracking is integral to the structure at the substrate level. Specifically, an elegant cut pattern is made in thin-film gallium arsenide solar cells, which are then stretched to produce an array of tilted surface elements which can be controlled to within ±1°. We analyze the combined optical and mechanical properties of the tracking system, and demonstrate a mechanically robust system with optical tracking efficiencies matching conventional trackers. This design suggests a pathway towards enabling new applications for solar tracking, as well as inspiring a broader range of optoelectronic and mechanical devices.
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Dynamic kirigami structures for integrated solar tracking
Lamoureux, Aaron; Lee, Kyusang; Shlian, Matthew; Forrest, Stephen R.; Shtein, Max
2015-01-01
Optical tracking is often combined with conventional flat panel solar cells to maximize electrical power generation over the course of a day. However, conventional trackers are complex and often require costly and cumbersome structural components to support system weight. Here we use kirigami (the art of paper cutting) to realize novel solar cells where tracking is integral to the structure at the substrate level. Specifically, an elegant cut pattern is made in thin-film gallium arsenide solar cells, which are then stretched to produce an array of tilted surface elements which can be controlled to within ±1°. We analyze the combined optical and mechanical properties of the tracking system, and demonstrate a mechanically robust system with optical tracking efficiencies matching conventional trackers. This design suggests a pathway towards enabling new applications for solar tracking, as well as inspiring a broader range of optoelectronic and mechanical devices. PMID:26348820
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Cognitive Control Network Contributions to Memory-Guided Visual Attention
Rosen, Maya L.; Stern, Chantal E.; Michalka, Samantha W.; Devaney, Kathryn J.; Somers, David C.
2016-01-01
Visual attentional capacity is severely limited, but humans excel in familiar visual contexts, in part because long-term memories guide efficient deployment of attention. To investigate the neural substrates that support memory-guided visual attention, we performed a set of functional MRI experiments that contrast long-term, memory-guided visuospatial attention with stimulus-guided visuospatial attention in a change detection task. Whereas the dorsal attention network was activated for both forms of attention, the cognitive control network (CCN) was preferentially activated during memory-guided attention. Three posterior nodes in the CCN, posterior precuneus, posterior callosal sulcus/mid-cingulate, and lateral intraparietal sulcus exhibited the greatest specificity for memory-guided attention. These 3 regions exhibit functional connectivity at rest, and we propose that they form a subnetwork within the broader CCN. Based on the task activation patterns, we conclude that the nodes of this subnetwork are preferentially recruited for long-term memory guidance of visuospatial attention. PMID:25750253
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Napora-Wijata, Kamila; Strohmeier, Gernot A.; Sonavane, Manoj N.; Avi, Manuela; Robins, Karen; Winkler, Margit
2013-01-01
Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. PMID:24970175
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Napora-Wijata, Kamila; Strohmeier, Gernot A; Sonavane, Manoj N; Avi, Manuela; Robins, Karen; Winkler, Margit
2013-08-12
Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases.
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Peel-and-Stick: Fabricating Thin Film Solar Cell on Universal Substrates
Lee, Chi Hwan; Kim, Dong Rip; Cho, In Sun; William, Nemeth; Wang, Qi; Zheng, Xiaolin
2012-01-01
Fabrication of thin-film solar cells (TFSCs) on substrates other than Si and glass has been challenging because these nonconventional substrates are not suitable for the current TFSC fabrication processes due to poor surface flatness and low tolerance to high temperature and chemical processing. Here, we report a new peel-and-stick process that circumvents these fabrication challenges by peeling off the fully fabricated TFSCs from the original Si wafer and attaching TFSCs to virtually any substrates regardless of materials, flatness and rigidness. With the peel-and-stick process, we integrated hydrogenated amorphous silicon (a-Si:H) TFSCs on paper, plastics, cell phone and building windows while maintaining the original 7.5% efficiency. The new peel-and-stick process enables further reduction of the cost and weight for TFSCs and endows TFSCs with flexibility and attachability for broader application areas. We believe that the peel-and-stick process can be applied to thin film electronics as well. PMID:23277871
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Buttet, Géraldine F.; Holliger, Christof
2013-01-01
Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed. PMID:23995945
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Peel-and-Stick: Fabricating Thin Film Solar Cell on Universal Substrates
NASA Astrophysics Data System (ADS)
Lee, Chi Hwan; Kim, Dong Rip; Cho, In Sun; William, Nemeth; Wang, Qi; Zheng, Xiaolin
2012-12-01
Fabrication of thin-film solar cells (TFSCs) on substrates other than Si and glass has been challenging because these nonconventional substrates are not suitable for the current TFSC fabrication processes due to poor surface flatness and low tolerance to high temperature and chemical processing. Here, we report a new peel-and-stick process that circumvents these fabrication challenges by peeling off the fully fabricated TFSCs from the original Si wafer and attaching TFSCs to virtually any substrates regardless of materials, flatness and rigidness. With the peel-and-stick process, we integrated hydrogenated amorphous silicon (a-Si:H) TFSCs on paper, plastics, cell phone and building windows while maintaining the original 7.5% efficiency. The new peel-and-stick process enables further reduction of the cost and weight for TFSCs and endows TFSCs with flexibility and attachability for broader application areas. We believe that the peel-and-stick process can be applied to thin film electronics as well.
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Sequestration of CDH1 by MAD2L2 prevents premature APC/C activation prior to anaphase onset
Listovsky, Tamar
2013-01-01
The switch from activation of the anaphase-promoting complex/cyclosome (APC/C) by CDC20 to CDH1 during anaphase is crucial for accurate mitosis. APC/CCDC20 ubiquitinates a limited set of substrates for subsequent degradation, including Cyclin B1 and Securin, whereas APC/CCDH1 has a broader specificity. This switch depends on dephosphorylation of CDH1 and the APC/C, and on the degradation of CDC20. Here we show, in human cells, that the APC/C inhibitor MAD2L2 also contributes to ensuring the sequential activation of the APC/C by CDC20 and CDH1. In prometaphase, MAD2L2 sequestered free CDH1 away from the APC/C. At the onset of anaphase, MAD2L2 was rapidly degraded by APC/CCDC20, releasing CDH1 to activate the dephosphorylated APC/C. Loss of MAD2L2 led to premature association of CDH1 with the APC/C, early destruction of APC/CCDH1 substrates, and accelerated mitosis with frequent mitotic aberrations. Thus, MAD2L2 helps to ensure a robustly bistable switch between APC/CCDC20 and APC/CCDH1 during the metaphase-to-anaphase transition, thereby contributing to mitotic fidelity. PMID:24100295
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Studies related to the development of the Viking 1975 labeled release experiment
NASA Technical Reports Server (NTRS)
Devincenzi, D. L.; Deal, P. H.
1976-01-01
The labeled release life detection experiment on the Viking 1975 Mars mission is based on the concept that microorganisms will metabolize radioactive organic substrates in a nutrient medium and release radioactive carbon dioxide. Several experiments, using laboratory equipment, were carried out to evaluate various aspects of the concept. Results indicate: (1) label is released by sterilization-treated soil, (2) substantial quantities of label are retained in solution under basic conditions, (3) the substrate used, as well as position of label in the molecule, affect release of label, (4) label release is depressed by radiolytic decomposition of substrates, and (5) About 100,000 organisms are required to produce a detectable response. These results, suggest additional areas for testing, add to the data base for interpretation of flight results, and have significance for broader application of this technique for assessing microbial activity.
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Zuend, Stephan J; Jacobsen, Eric N
2007-12-26
The mechanism of the enantioselective cyanosilylation of ketones catalyzed by tertiary amino-thiourea derivatives was investigated using a combination of experimental and theoretical methods. The kinetic analysis is consistent with a cooperative mechanism in which both the thiourea and the tertiary amine of the catalyst are involved productively in the rate-limiting cyanide addition step. Density functional theory calculations were used to distinguish between mechanisms involving thiourea activation of ketone or of cyanide in the enantioselectivity-determining step. The strong correlation obtained between experimental and calculated ee's for a range of substrates and catalysts provides support for the most favorable calculated transition structures involving amine-bound HCN adding to thiourea-bound ketone. The calculations suggest that enantioselectivity arises from direct interactions between the ketone substrate and the amino-acid derived portion of the catalyst. On the basis of this insight, more enantioselective catalysts with broader substrate scope were prepared and evaluated experimentally.
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Orthogonal Luciferase-Luciferin Pairs for Bioluminescence Imaging.
Jones, Krysten A; Porterfield, William B; Rathbun, Colin M; McCutcheon, David C; Paley, Miranda A; Prescher, Jennifer A
2017-02-15
Bioluminescence imaging with luciferase-luciferin pairs is widely used in biomedical research. Several luciferases have been identified in nature, and many have been adapted for tracking cells in whole animals. Unfortunately, the optimal luciferases for imaging in vivo utilize the same substrate and therefore cannot easily differentiate multiple cell types in a single subject. To develop a broader set of distinguishable probes, we crafted custom luciferins that can be selectively processed by engineered luciferases. Libraries of mutant enzymes were iteratively screened with sterically modified luciferins, and orthogonal enzyme-substrate "hits" were identified. These tools produced light when complementary enzyme-substrate partners interacted both in vitro and in cultured cell models. Based on their selectivity, these designer pairs will bolster multicomponent imaging and enable the direct interrogation of cell networks not currently possible with existing tools. Our screening platform is also general and will expedite the identification of more unique luciferases and luciferins, further expanding the bioluminescence toolkit.
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Lateral epitaxy of atomically sharp WSe 2/WS 2 heterojunctions on silicon dioxide substrates
DOE Office of Scientific and Technical Information (OSTI.GOV)
Chen, Jianyi; Zhou, Wu; Tang, Wei
Here, in recent years, 2-D transition-metal dichalcogenides (TMDCs) have received great interests because of the broader possibilities offered by their tunable band gaps, as opposed to gapless graphene which precludes application in digital electronics. TMDCs exhibit an indirect-to-direct band gap transition at the single atomic sheet state as well as optically accessible spin degree of freedom in valleytronics.
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Lateral epitaxy of atomically sharp WSe 2/WS 2 heterojunctions on silicon dioxide substrates
Chen, Jianyi; Zhou, Wu; Tang, Wei; ...
2016-09-30
Here, in recent years, 2-D transition-metal dichalcogenides (TMDCs) have received great interests because of the broader possibilities offered by their tunable band gaps, as opposed to gapless graphene which precludes application in digital electronics. TMDCs exhibit an indirect-to-direct band gap transition at the single atomic sheet state as well as optically accessible spin degree of freedom in valleytronics.
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Kosović, Marin; Balarin, Maja; Ivanda, Mile; Đerek, Vedran; Marciuš, Marijan; Ristić, Mira; Gamulin, Ozren
2015-12-01
Microporous and macro-mesoporous silicon templates for surface-enhanced Raman scattering (SERS) substrates were produced by anodization of low doped p-type silicon wafers. By immersion plating in AgNO3, the templates were covered with silver metallic film consisting of different silver nanostructures. Scanning electron microscopy (SEM) micrographs of these SERS substrates showed diverse morphology with significant difference in an average size and size distribution of silver nanoparticles. Ultraviolet-visible-near-infrared (UV-Vis-NIR) reflection spectroscopy showed plasmonic absorption at 398 and 469 nm, which is in accordance with the SEM findings. The activity of the SERS substrates was tested using rhodamine 6G (R6G) dye molecules and 514.5 nm laser excitation. Contrary to the microporous silicon template, the SERS substrate prepared from macro-mesoporous silicon template showed significantly broader size distribution of irregular silver nanoparticles as well as localized surface plasmon resonance closer to excitation laser wavelength. Such silver morphology has high SERS sensitivity that enables ultralow concentration detection of R6G dye molecules up to 10(-15) M. To our knowledge, this is the lowest concentration detected of R6G dye molecules on porous silicon-based SERS substrates, which might even indicate possible single molecule detection.
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Code of Federal Regulations, 2011 CFR
2011-07-01
... review (§ 1508.28). Whenever a broad environmental impact statement has been prepared (such as a program... statement or environmental assessment need only summarize the issues discussed in the broader statement and incorporate discussions from the broader statement by reference and shall concentrate on the issues specific...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... review (§ 1508.28). Whenever a broad environmental impact statement has been prepared (such as a program... statement or environmental assessment need only summarize the issues discussed in the broader statement and incorporate discussions from the broader statement by reference and shall concentrate on the issues specific...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... review (§ 1508.28). Whenever a broad environmental impact statement has been prepared (such as a program... statement or environmental assessment need only summarize the issues discussed in the broader statement and incorporate discussions from the broader statement by reference and shall concentrate on the issues specific...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... review (§ 1508.28). Whenever a broad environmental impact statement has been prepared (such as a program... statement or environmental assessment need only summarize the issues discussed in the broader statement and incorporate discussions from the broader statement by reference and shall concentrate on the issues specific...
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Code of Federal Regulations, 2014 CFR
2014-07-01
... review (§ 1508.28). Whenever a broad environmental impact statement has been prepared (such as a program... statement or environmental assessment need only summarize the issues discussed in the broader statement and incorporate discussions from the broader statement by reference and shall concentrate on the issues specific...
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Improved Muscarinic Antagonists as Anticholinesterase Antidotes
1990-02-01
could be ad-ninistered more readily in suspected cases of poisoning -6ithout fear of overdose . Second, the side effects would be of a shorter duration...acetylcholinesterase inactivation and hydrolyzes a broader range of substrates.3 ,12 4 Serum cholinesterase hydrolyzes choline esters containing a variety of...a lower hydrolytic rate. The enzyme is more sensitive to the structure of the choline moiety. Addition of an a- or fý methyl group to choline or
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Federico, Lorenzo; Yang, Liping; Brandon, Jason; Panchatcharam, Manikandan; Ren, Hongmei; Mueller, Paul; Sunkara, Manjula; Escalante-Alcalde, Diana; Morris, Andrew J; Smyth, Susan S
2018-01-01
Dephosphorylation of phosphatidic acid (PA) is the penultimate step in triglyceride synthesis. Adipocytes express soluble intracellular PA-specific phosphatases (Lipins) and broader specificity membrane-associated lipid phosphate phosphatases (LPPs) that can also dephosphorylate PA. Inactivation of lipin1 causes lipodystrophy in mice due to defective developmental adipogenesis. Triglyceride synthesis is diminished but not ablated by inactivation of lipin1 in differentiated adipocytes implicating other PA phosphatases in this process. To investigate the possible role of LPPs in adipocyte lipid metabolism and signaling we made mice with adipocyte-targeted inactivation of LPP3 encoded by the Plpp3(Ppap2b) gene. Adipocyte LPP3 deficiency resulted in blunted ceramide and sphingomyelin accumulation during diet-induced adipose tissue expansion, accumulation of the LPP3 substrate sphingosine 1- phosphate, and reduced expression of serine palmitoyl transferase. However, adiposity was unaffected by LPP3 deficiency on standard, high fat diet or Western diets, although Western diet-fed mice with adipocyte LPP3 deficiency exhibited improved glucose tolerance. Our results demonstrate functional compartmentalization of lipid phosphatase activity in adipocytes and identify an unexpected role for LPP3 in the regulation of diet-dependent sphingolipid synthesis that may impact on insulin signaling.
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Sucksmith, E; Roth, I; Hoekstra, R A
2011-12-01
Diagnosis, intervention and support for people with autism can be assisted by research into the aetiology of the condition. Twin and family studies indicate that autism spectrum conditions are highly heritable; genetic relatives of people with autism often show milder expression of traits characteristic for autism, referred to as the Broader Autism Phenotype (BAP). In the past decade, advances in the biological and behavioural sciences have facilitated a more thorough examination of the BAP from multiple levels of analysis. Here, the candidate phenotypic traits delineating the BAP are summarised, including key findings from neuroimaging studies examining the neural substrates of the BAP. We conclude by reviewing the value of further research into the BAP, with an emphasis on deriving heritable endophenotypes which will reliably index autism susceptibility and offer neurodevelopmental mechanisms that bridge the gap between genes and a clinical autism diagnosis.
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Bheda, Poonam; Swatkoski, Stephen; Fiedler, Katherine L; Boeke, Jef D; Cotter, Robert J; Wolberger, Cynthia
2012-04-17
Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.
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Synthesis of α,β-unsaturated aldehydes as potential substrates for bacterial luciferases.
Brodl, Eveline; Ivkovic, Jakov; Tabib, Chaitanya R; Breinbauer, Rolf; Macheroux, Peter
2017-02-15
Bacterial luciferase catalyzes the monooxygenation of long-chain aldehydes such as tetradecanal to the corresponding acid accompanied by light emission with a maximum at 490nm. In this study even numbered aldehydes with eight, ten, twelve and fourteen carbon atoms were compared with analogs having a double bond at the α,β-position. These α,β-unsaturated aldehydes were synthesized in three steps and were examined as potential substrates in vitro. The luciferase of Photobacterium leiognathi was found to convert these analogs and showed a reduced but significant bioluminescence activity compared to tetradecanal. This study showed the trend that aldehydes, both saturated and unsaturated, with longer chain lengths had higher activity in terms of bioluminescence than shorter chain lengths. The maximal light intensity of (E)-tetradec-2-enal was approximately half with luciferase of P. leiognathi, compared to tetradecanal. Luciferases of Vibrio harveyi and Aliivibrio fisheri accepted these newly synthesized substrates but light emission dropped drastically compared to saturated aldehydes. The onset and the decay rate of bioluminescence were much slower, when using unsaturated substrates, indicating a kinetic effect. As a result the duration of the light emission is doubled. These results suggest that the substrate scope of bacterial luciferases is broader than previously reported. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.
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NASA Astrophysics Data System (ADS)
Fluegel, B.; Rice, A. D.; Mascarenhas, A.
2018-05-01
Resonant electronic Raman (ER) scattering is used to compare the below-gap excitations in molecular-beam epitaxially grown GaAs and in undoped semi-insulating GaAs substrates. The measurement geometry was designed to eliminate common measurement artifacts caused by the high optical transmission below the fundamental absorption edge. In epitaxial GaAs, ER is a clear Raman signal from the two-electron transitions of donors, eliminating an ambiguity encountered in previous results. In semi-insulating GaAs, ER occurs in a much broader dispersive band well below the bound exciton energies. The difference in the two materials may be due to the occupation of the substrate acceptor states in the presence of the midgap state EL2.
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NASA Astrophysics Data System (ADS)
Stapel, Janina G.; Schwamborn, Georg; Schirrmeister, Lutz; Horsfield, Brian; Mangelsdorf, Kai
2018-04-01
In this study the organic matter (OM) in several permafrost cores from Bol'shoy Lyakhovsky Island in NE Siberia was investigated. In the context of the observed global warming the aim was to evaluate the potential of freeze-locked OM from different depositional ages to act as a substrate provider for microbial production of greenhouse gases from thawing permafrost. To assess this potential, the concentrations of free and bound acetate, which form an appropriate substrate for methanogenesis, were determined. The largest free-acetate (in pore water) and bound-acetate (organic-matrix-linked) substrate pools were present in interstadial marine isotope stage (MIS) 3 and stadial MIS 4 Yedoma permafrost deposits. In contrast, deposits from the last interglacial MIS 5e (Eemian) contained only a small pool of substrates. The Holocene (MIS 1) deposits revealed a significant bound-acetate pool, representing a future substrate potential upon release during OM degradation. Additionally, pyrolysis experiments on the OM allocated an increased aliphatic character to the MIS 3 and 4 Late Pleistocene deposits, which might indicate less decomposed and presumably more easily degradable OM. Biomarkers for past microbial communities, including those for methanogenic archaea, also showed the highest abundance during MIS 3 and 4, which indicated OM-stimulated microbial degradation and presumably greenhouse gas production during time of deposition. On a broader perspective, Arctic warming will increase and deepen permafrost thaw and favor substrate availability from older freeze-locked permafrost deposits. Thus, the Yedoma deposits especially showed a high potential for providing substrates relevant for microbial greenhouse gas production.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Lee, Brady D.; Apel, William A.; Sheridan, Peter P.
Background Metabolism of carbon bound in wheat arabinoxylan (WAX) polysaccharides by bacteria requires a number of glycoside hydrolases active toward different bonds between sugars and other molecules. Alicyclobacillus acidocaldarius is a Gram-positive thermoacidophilic bacterium capable of growth on a variety of mono-, di-, oligo-, and polysaccharides. Nineteen proposed glycoside hydrolases have been annotated in the A. acidocaldarius Type Strain ATCC27009/DSM 446 genome. Results Molecular analysis using high-density oligonucleotide microarrays was performed on A. acidocaldarius strain ATCC27009 when growing on WAX. When a culture growing exponentially at the expense of arabinoxylan saccharides was challenged with glucose or xylose, most glycoside hydrolasesmore » were down-regulated. Interestingly, regulation was more intense when xylose was added to the culture than when glucose was added, a clear departure from classical carbon catabolite repression demonstrated by many Gram-positive bacteria. In silico analyses of the regulated glycoside hydrolases, along with the results from the microarray analyses, yielded a potential mechanism for arabinoxylan metabolism by A. acidocaldarius. Glycoside hydrolases expressed by this strain may have broad substrate specificity, and initial hydrolysis is catalyzed by an extracellular xylanase, while subsequent steps are likely performed inside the growing cell. Conclusions Glycoside hydrolases, for the most part, appear to be found in clusters, throughout the A. acidocaldarius genome. Not all of the glycoside hydrolase genes found at loci within these clusters were regulated during the experiment, indicating that a specific subset of the 19 glycoside hydrolase genes found in A. acidocaldarius were used during metabolism of WAX. While specific functions of the glycoside hydrolases was not tested as part of the research discussed, many of the glycoside hydrolases found in the A. acidocaldarius Type Strain appear to have a broader substrate range than represented by the glycoside hydrolase family in which the enzymes were categorized.« less
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Molecular Genetics of Mycobacteriophages
HATFULL, GRAHAM F.
2014-01-01
Mycobacteriophages have provided numerous essential tools for mycobacterial genetics, including delivery systems for transposons, reporter genes, and allelic exchange substrates, and components for plasmid vectors and mutagenesis. Their genetically diverse genomes also reveal insights into the broader nature of the phage population and the evolutionary mechanisms that give rise to it. The substantial advances in our understanding of the biology of mycobacteriophages including a large collection of completely sequenced genomes indicates a rich potential for further contributions in tuberculosis genetics and beyond. PMID:25328854
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Fluegel, B.; Rice, A. D.; Mascarenhas, A.
Resonant electronic Raman (ER) scattering is used to compare the below-gap excitations in molecular-beam epitaxially grown GaAs and in undoped semi-insulating GaAs substrates. The measurement geometry was designed to eliminate common measurement artifacts caused by the high optical transmission below the fundamental absorption edge. In epitaxial GaAs, ER is a clear Raman signal from the two-electron transitions of donors, eliminating an ambiguity encountered in previous results. In semi-insulating GaAs, ER occurs in a much broader dispersive band well below the bound exciton energies. Furthermore, the difference in the two materials may be due to the occupation of the substrate acceptormore » states in the presence of the midgap state EL2.« less
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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
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New Chromogenic Agar Medium for the Identification of Candida spp.
Cooke, Venitia M.; Miles, R. J.; Price, R. G.; Midgley, G.; Khamri, W.; Richardson, A. C.
2002-01-01
A new chromogenic agar medium (Candida diagnostic agar [CDA]) for differentiation of Candida spp. is described. This medium is based on Sabouraud dextrose agar (Oxoid CM41) and contains (per liter) 40.0 g of glucose, 10.0 g of mycological peptone, and 15.0 g of agar along with a novel chromogenic glucosaminidase substrate, ammonium 4-{2-[4-(2-acetamido-2-deoxy-β-d-glucopyranosyloxy)-3-methoxyphenyl]-vinyl}-1-(propan-3-yl-oate)-quinolium bromide (0.32 g liter−1). The glucosaminidase substrate in CDA was hydrolyzed by Candida albicans and Candida dubliniensis, yielding white colonies with deep-red spots on a yellow transparent background after 24 to 48 h of incubation at 37°C. Colonies of Candida tropicalis and Candida kefyr were uniformly pink, and colonies of other Candida spp., including Candida glabrata and Candida parapsilosis, were white. CDA was evaluated by using 115 test strains of Candida spp. and other clinically important yeasts and was compared with two commercially available chromogenic agars (Candida ID agar [bioMerieux] and CHROMagar Candida [CHROMagar Company Ltd.]). On all three agars, colonies of C. albicans were not distinguished from colonies of C. dubliniensis. However, for the group containing C. albicans plus C. dubliniensis, both the sensitivity and the specificity of detection when CDA was used were 100%, compared with values of 97.6 and 100%, respectively, with CHROMagar Candida and 100 and 96.8%, respectively, with Candida ID agar. In addition, for the group containing C. tropicalis plus C. kefyr, the sensitivity and specificity of detection when CDA was used were also 100%, compared with 72.7 and 98.1%, respectively, with CHROMagar Candida. Candida ID agar did not differentiate C. tropicalis and C. kefyr strains but did differentiate members of a broader group (C. tropicalis, C. kefyr, Candida lusitaniae plus Candida guilliermondii); the sensitivity and specificity of detection for members of this group were 94.7 and 93.8%, respectively. In addition to the increased sensitivity and/or specificity of Candida detection when CDA was used, differentiation of colony types on CDA (red spotted, pink, or no color) was unambiguous and did not require precise assessment of colony color. PMID:12089051
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Logemann, Elke; Tavernaro, Annette; Schulz, Wolfgang; Somssich, Imre E.; Hahlbrock, Klaus
2000-01-01
The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells. PMID:10677554
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Economic Stimulus: Issues and Policies
2009-04-03
have sought to contain damages spilling over from housing and financial markets to the broader economy, including monetary policy, which is the...compensation in June and November of 2008. Over the past few months, the government has also intervened in specific financial markets , including financial ...into the financial markets has been passed to avoid the spread of financial instability into the broader market but there are disadvantages
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Economic Stimulus: Issues and Policies
2009-01-23
have sought to contain damages spilling over from housing and financial markets to the broader economy, including monetary policy, which is the...compensation in June and November of 2008. Over the past few months, the government has also intervened in specific financial markets , including financial ...intervention into the financial markets has been passed to avoid the spread of financial instability into the broader market but there are disadvantages
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Expanding the product profile of a microbial alkane biosynthetic pathway.
Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B
2013-01-18
Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.
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Cognitive Control Network Contributions to Memory-Guided Visual Attention.
Rosen, Maya L; Stern, Chantal E; Michalka, Samantha W; Devaney, Kathryn J; Somers, David C
2016-05-01
Visual attentional capacity is severely limited, but humans excel in familiar visual contexts, in part because long-term memories guide efficient deployment of attention. To investigate the neural substrates that support memory-guided visual attention, we performed a set of functional MRI experiments that contrast long-term, memory-guided visuospatial attention with stimulus-guided visuospatial attention in a change detection task. Whereas the dorsal attention network was activated for both forms of attention, the cognitive control network(CCN) was preferentially activated during memory-guided attention. Three posterior nodes in the CCN, posterior precuneus, posterior callosal sulcus/mid-cingulate, and lateral intraparietal sulcus exhibited the greatest specificity for memory-guided attention. These 3 regions exhibit functional connectivity at rest, and we propose that they form a subnetwork within the broader CCN. Based on the task activation patterns, we conclude that the nodes of this subnetwork are preferentially recruited for long-term memory guidance of visuospatial attention. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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Rocker, Andrea; Peschke, Madeleine; Kittilä, Tiia; Sakson, Roman; Brieke, Clara; Meinhart, Anton
2018-04-27
Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.
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Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate
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
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NASA Technical Reports Server (NTRS)
Lopez, J. C.; Ryan, S.; Blankenship, R. E.
1996-01-01
The sequence of the Chloroflexus aurantiacus open reading frame thought to be the C. aurantiacus homolog of the Rhodobacter capsulatus bchG gene is reported. The BchG gene product catalyzes esterification of bacteriochlorophyllide a by geranylgeraniol-PPi during bacteriochlorophyll a biosynthesis. Homologs from Arabidopsis thaliana, Synechocystis sp. strain PCC6803, and C. aurantiacus were identified in database searches. Profile analysis identified three related polyprenyltransferase enzymes which attach an aliphatic alcohol PPi to an aromatic substrate. This suggests a broader relationship between chlorophyll synthases and other polyprenyltransferases.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Flemish, Joseph; Soer, Wouter
2015-11-30
Patterned sapphire substrate (PSS) technology has proven to be an effective approach to improve efficacy and reduce cost of light-emitting diodes (LEDs). The volume emission from the transparent substrate leads to high package efficiency, while the simple and robust architecture of PSS-based LEDs enables low cost. PSS substrates have gained wide use in mid-power LEDs over the past years. In this project, Lumileds has developed and industrialized PSS and epitaxy technology for high- power flip-chip LEDs to bring these benefits to a broader range of applications and accelerate the adoption of energy-efficient solid-state lighting (SSL). PSS geometries were designed formore » highly efficient light extraction in a flip-chip architecture and high-volume manufacturability, and corresponding sapphire patterning and epitaxy manufacturing processes were integrally developed. Concurrently, device and package architectures were developed to take advantage of the PSS flip-chip die in different types of products that meet application needs. The developed PSS and epitaxy technology has been fully implemented in manufacturing at Lumileds’ San Jose, CA location, and incorporated in illumination-grade LED products that have been successfully introduced to the market, including LUXEON Q and LUXEON FlipChip White.« less
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Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II*♦
Domsic, John F.; Avvaru, Balendu Sankara; Kim, Chae Un; Gruner, Sol M.; Agbandje-McKenna, Mavis; Silverman, David N.; McKenna, Robert
2008-01-01
The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO2-pressurized, cryo-cooled crystals to capture the first step of CO2 hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO2, for both the holo and the apo (without zinc) enzyme to 1.1Å resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO2 and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675–3679). These structures provide insight into the long hypothesized binding of CO2 in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO2. This method may also have a much broader implication for the study of other enzymes for which CO2 is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins. PMID:18768466
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Nanostructured Electrocatalysts for All-Vanadium Redox Flow Batteries.
Park, Minjoon; Ryu, Jaechan; Cho, Jaephil
2015-10-01
Vanadium redox reactions have been considered as a key factor affecting the energy efficiency of the all-vanadium redox flow batteries (VRFBs). This redox reaction determines the reaction kinetics of whole cells. However, poor kinetic reversibility and catalytic activity towards the V(2+)/V(3+) and VO(2+)/VO2(+) redox couples on the commonly used carbon substrate limit broader applications of VRFBs. Consequently, modified carbon substrates have been extensively investigated to improve vanadium redox reactions. In this Focus Review, recent progress on metal- and carbon-based nanomaterials as an electrocatalyst for VRFBs is discussed in detail, without the intention to provide a comprehensive review on the whole components of the system. Instead, the focus is mainly placed on the redox chemistry of vanadium ions at a surface of various metals, different dimensional carbons, nitrogen-doped carbon nanostructures, and metal-carbon composites. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Intramolecular bonds resolved on a semiconductor surface
NASA Astrophysics Data System (ADS)
Sweetman, Adam; Jarvis, Samuel P.; Rahe, Philipp; Champness, Neil R.; Kantorovich, Lev; Moriarty, Philip
2014-10-01
Noncontact atomic force microscopy (NC-AFM) is now routinely capable of obtaining submolecular resolution, readily resolving the carbon backbone structure of planar organic molecules adsorbed on metal substrates. Here we show that the same resolution may also be obtained for molecules adsorbed on a reactive semiconducting substrate. Surprisingly, this resolution is routinely obtained without the need for deliberate tip functionalization. Intriguingly, we observe two chemically distinct apex types capable of submolecular imaging. We characterize our tip apices by "inverse imaging" of the silicon adatoms of the Si (111)-7×7 surface and support our findings with detailed density functional theory (DFT) calculations. We also show that intramolecular resolution on individual molecules may be readily obtained at 78 K, rather than solely at 5 K as previously demonstrated. Our results suggest a wide range of tips may be capable of producing intramolecular contrast for molecules adsorbed on semiconductor surfaces, leading to a much broader applicability for submolecular imaging protocols.
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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
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Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity
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
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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.
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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…
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Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.
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.
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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.
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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.
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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
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Weilenmann, Hans-Ueli; Engeli, Barbara; Bucheli-Witschel, Margarete; Egli, Thomas
2004-10-01
A Gram-negative, ethylenediaminetetraacetic acid (EDTA)-degrading bacterium (deposited at the German Culture Collection as strain DSM 9103) utilising EDTA as the only source of carbon, energy and nitrogen was isolated from a mixed EDTA-degrading population that was originally enriched in a column system from a mixture of activated sludge and soil. Chemotaxonomic analysis of quinones, polar lipids and fatty acids allowed allocation of the isolate to the alpha-subclass of Proteobacteria. 16S rDNA sequencing and phylogenetic analysis revealed highest similarity to the Mesorhizobium genus followed by the Aminobacter genus. However, the EDTA-degrading strain apparently forms a new branch within the Phyllobacteriaceae/Mesorhizobia family. Growth of the strain was rather slow not only on EDTA (micro(max) = 0.05 h(-1)) but also on other substrates. Classical substrate utilisation testing in batch culture suggested a quite restricted carbon source spectrum with only lactate, glutamate, and complexing agents chemically related to EDTA (nitrilotriacetate, iminodiacetate and ethylenediaminedisuccinate) supporting growth. However, when EDTA-limited continuous cultures of strain DSM 9103 were pulsed with fumarate, succinate, glucose or acetate, these substrates were assimilated immediately. Apparently, the strain can use a broader spectrum than indicated by traditional substrate testing techniques. The EDTA species CaEDTA and MgEDTA served as growth substrates of the strain because in the mineral medium employed EDTA was predicted to be mainly present in the form of these two complexes. The bacterium was not able to degrade Fe3+-complexed EDTA.
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Substrate specificity of the ubiquitin and Ubl proteases
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
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Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease
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
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Cleavage Entropy as Quantitative Measure of Protease Specificity
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
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Evidence for an Inducible Nucleotide-Dependent Acetone Carboxylase in Rhodococcus rhodochrous B276
Clark, Daniel D.; Ensign, Scott A.
1999-01-01
The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grown R. rhodochrous readily metabolized acetone. In contrast, R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria. PMID:10217764
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The Pharmacogenetic Footprint of ACE Inhibition: A Population-Based Metabolomics Study.
Altmaier, Elisabeth; Menni, Cristina; Heier, Margit; Meisinger, Christa; Thorand, Barbara; Quell, Jan; Kobl, Michael; Römisch-Margl, Werner; Valdes, Ana M; Mangino, Massimo; Waldenberger, Melanie; Strauch, Konstantin; Illig, Thomas; Adamski, Jerzy; Spector, Tim; Gieger, Christian; Suhre, Karsten; Kastenmüller, Gabi
2016-01-01
Angiotensin-I-converting enzyme (ACE) inhibitors are an important class of antihypertensives whose action on the human organism is still not fully understood. Although it is known that ACE especially cleaves COOH-terminal dipeptides from active polypeptides, the whole range of substrates and products is still unknown. When analyzing the action of ACE inhibitors, effects of genetic variation on metabolism need to be considered since genetic variance in the ACE gene locus was found to be associated with ACE-concentration in blood as well as with changes in the metabolic profiles of a general population. To investigate the interactions between genetic variance at the ACE-locus and the influence of ACE-therapy on the metabolic status we analyzed 517 metabolites in 1,361 participants from the KORA F4 study. We replicated our results in 1,964 individuals from TwinsUK. We observed differences in the concentration of five dipeptides and three ratios of di- and oligopeptides between ACE inhibitor users and non-users that were genotype dependent. Such changes in the concentration affected major homozygotes, and to a lesser extent heterozygotes, while minor homozygotes showed no or only small changes in the metabolite status. Two of these resulting dipeptides, namely aspartylphenylalanine and phenylalanylserine, showed significant associations with blood pressure which qualifies them-and perhaps also the other dipeptides-as readouts of ACE-activity. Since so far ACE activity measurement is substrate specific due to the usage of only one oligopeptide, taking several dipeptides as potential products of ACE into account may provide a broader picture of the ACE activity.
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Substrate specificity of sheep liver sorbitol dehydrogenase.
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
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Peng, Wenjie; Pranskevich, Jennifer; Nycholat, Corwin; Gilbert, Michel; Wakarchuk, Warren; Paulson, James C; Razi, Nahid
2012-01-01
Poly-N-acetyllactosamine extensions on N- and O-linked glycans are increasingly recognized as biologically important structural features, but access to these structures has not been widely available. Here, we report a detailed substrate specificity and catalytic efficiency of the bacterial β3-N-acetylglucosaminyltransferase (β3GlcNAcT) from Helicobacter pylori that can be adapted to the synthesis of a rich diversity of glycans with poly-LacNAc extensions. This glycosyltransferase has surprisingly broad acceptor specificity toward type-1, -2, -3 and -4 galactoside motifs on both linear and branched glycans, found commonly on N-linked, O-linked and I-antigen glycans. This finding enables the production of complex ligands for glycan-binding studies. Although the enzyme shows preferential activity for type 2 (Galβ1-4GlcNAc) acceptors, it is capable of transferring N-acetylglucosamine (GlcNAc) in β1-3 linkage to type-1 (Galβ1-3GlcNAc) or type-3/4 (Galβ1-3GalNAcα/β) sequences. Thus, by alternating the use of the H. pylori β3GlcNAcT with galactosyltransferases that make the β1-4 or β1-3 linkages, various N-linked, O-linked and I-antigen acceptors could be elongated with type-2 and type-1 LacNAc repeats. Finally, one-pot incubation of di-LacNAc biantennary N-glycopeptide with the β3GlcNAcT and GalT-1 in the presence of uridine diphosphate (UDP)-GlcNAc and UDP-Gal, yielded products with 15 additional LacNAc units on the precursor, which was seen as a series of sequential ion peaks representing alternative additions of GlcNAc and Gal residues, on matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Overall, our data demonstrate a broader substrate specificity for the H. pylori β3GlcNAcT than previously recognized and demonstrate its ability as a potent resource for preparative chemo-enzymatic synthesis of complex glycans. PMID:22786570
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Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.
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.
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E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.
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.
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Glycan microarray screening assay for glycosyltransferase specificities.
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.
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The Evolving Evaluation Process for NSF Broader Impacts
NASA Astrophysics Data System (ADS)
Straub, J. A.; Lawrence, J. E.
2016-12-01
The National Science Foundation (NSF) supports basic research in all non-medical fields of fundamental science that benefit society. To pursue this goal, NSF uses two merit review criteria: intellectual merit and broader impacts. As defined by NSF, intellectual merit "encompasses the potential to advance knowledge," while broader impacts "encompasses the potential to benefit society and contribute to the achievement of specific, desired societal outcomes." Articulating compelling broader impacts is increasingly critical as limited available funding means that both sets of criteria will impact the final proposal outcome. Although societal relevance has been valued by NSF since the foundation was established, recent events have placed increased emphasis on its importance: the America COMPETES Act encouraged increased efforts across agencies in educating the future STEM workforce (2007); NSF prioritized broader STEM participation (2008); the Obama administration issued a memo on transparency and open government (2009); and the National Science Board revised the NSF merit review criteria to emphasize that the same five elements should be considered for both merit review criteria (2012). Principal Investigators, reviewers (including panelists), and Program Officers are being asked to justify how the broader impacts contribute significantly to the project. As broader impacts become increasingly emphasized in the merit review process, it is important to understand not only how Principal Investigators are responding, but how reviewers are evaluating this aspect of proposals. To examine how reviewers are responding to this change in NSF's evaluation policy, an assessment of broader impacts in the Division of Earth Sciences is being conducted. The data were analyzed to see how reviewers have shifted their feedback in the last ten years. Data so far suggest that policy changes to the Grant Proposal Guide in 2012 have caused a notable shift to reviewers being more evaluative of broader impacts, rather than descriptive, in their review of proposals in the core research programs.
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Characterizing Protease Specificity: How Many Substrates Do We Need?
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
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Combined sequence and structure analysis of the fungal laccase family.
Kumar, S V Suresh; Phale, Prashant S; Durani, S; Wangikar, Pramod P
2003-08-20
Plant and fungal laccases belong to the family of multi-copper oxidases and show much broader substrate specificity than other members of the family. Laccases have consequently been of interest for potential industrial applications. We have analyzed the essential sequence features of fungal laccases based on multiple sequence alignments of more than 100 laccases. This has resulted in identification of a set of four ungapped sequence regions, L1-L4, as the overall signature sequences that can be used to identify the laccases, distinguishing them within the broader class of multi-copper oxidases. The 12 amino acid residues in the enzymes serving as the copper ligands are housed within these four identified conserved regions, of which L2 and L4 conform to the earlier reported copper signature sequences of multi-copper oxidases while L1 and L3 are distinctive to the laccases. The mapping of regions L1-L4 on to the three-dimensional structure of the Coprinus cinerius laccase indicates that many of the non-copper-ligating residues of the conserved regions could be critical in maintaining a specific, more or less C-2 symmetric, protein conformational motif characterizing the active site apparatus of the enzymes. The observed intraprotein homologies between L1 and L3 and between L2 and L4 at both the structure and the sequence levels suggest that the quasi C-2 symmetric active site conformational motif may have arisen from a structural duplication event that neither the sequence homology analysis nor the structure homology analysis alone would have unraveled. Although the sequence and structure homology is not detectable in the rest of the protein, the relative orientation of region L1 with L2 is similar to that of L3 with L4. The structure duplication of first-shell and second-shell residues has become cryptic because the intraprotein sequence homology noticeable for a given laccase becomes significant only after comparing the conservation pattern in several fungal laccases. The identified motifs, L1-L4, can be useful in searching the newly sequenced genomes for putative laccase enzymes. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 386-394, 2003.
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NASA Astrophysics Data System (ADS)
McKenna, Ann F.; Hynes, Morgan M.; Johnson, Amy M.; Carberry, Adam R.
2016-07-01
Product archaeology as an educational approach asks engineering students to consider and explore the broader societal and global impacts of a product's manufacturing, distribution, use, and disposal on people, economics, and the environment. This study examined the impact of product archaeology in a project-based engineering design course on student attitudes and perceptions about engineering and abilities to extend and refine knowledge about broader contexts. Two design scenarios were created: one related to dental hygiene and one related to vaccination delivery. Design scenarios were used to (1) assess knowledge of broader contexts, and (2) test variability of student responses across different contextual situations. Results from pre- to post-surveying revealed improved student perceptions of knowledge of broader contexts. Significant differences were observed between the two design scenarios. The findings support the assumption that different design scenarios elicit consideration of different contexts and design scenarios can be constructed to target specific contextual considerations.
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Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility.
Shlevkov, Evgeny; Kramer, Tal; Schapansky, Jason; LaVoie, Matthew J; Schwarz, Thomas L
2016-10-11
The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy.
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Miro phosphorylation sites regulate Parkin recruitment and mitochondrial motility
Shlevkov, Evgeny; Kramer, Tal; Schapansky, Jason; LaVoie, Matthew J.; Schwarz, Thomas L.
2016-01-01
The PTEN-induced putative kinase 1 (PINK1)/Parkin pathway can tag damaged mitochondria and trigger their degradation by mitophagy. Before the onset of mitophagy, the pathway blocks mitochondrial motility by causing Miro degradation. PINK1 activates Parkin by phosphorylating both Parkin and ubiquitin. PINK1, however, has other mitochondrial substrates, including Miro (also called RhoT1 and -2), although the significance of those substrates is less clear. We show that mimicking PINK1 phosphorylation of Miro on S156 promoted the interaction of Parkin with Miro, stimulated Miro ubiquitination and degradation, recruited Parkin to the mitochondria, and via Parkin arrested axonal transport of mitochondria. Although Miro S156E promoted Parkin recruitment it was insufficient to trigger mitophagy in the absence of broader PINK1 action. In contrast, mimicking phosphorylation of Miro on T298/T299 inhibited PINK1-induced Miro ubiquitination, Parkin recruitment, and Parkin-dependent mitochondrial arrest. The effects of the T298E/T299E phosphomimetic were dominant over S156E substitution. We propose that the status of Miro phosphorylation influences the decision to undergo Parkin-dependent mitochondrial arrest, which, in the context of PINK1 action on other substrates, can restrict mitochondrial dynamics before mitophagy. PMID:27679849
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Metabolic regulation in solventogenic clostridia: regulators, mechanisms and engineering.
Yang, Yunpeng; Nie, Xiaoqun; Jiang, Yuqian; Yang, Chen; Gu, Yang; Jiang, Weihong
2018-02-22
Solventogenic clostridia, a group of important industrial microorganisms, have exceptional substrate and product diversity, capable of producing a series of two-carbon and even long-chain chemicals and fuels by using various substrates, including sugars, cellulose and hemicellulose, and C1 gases. For the sake of in-depth understanding and engineering these anaerobic microorganisms for broader applications, studies on metabolic regulation of solventogenic clostridia had been extensively carried out during the past ten years, based on the rapid development of various genetic tools. To date, a number of regulators that are essential for cell physiological and metabolic processes have been identified in clostridia, and the relevant mechanisms have also been dissected, providing a wealth of valuable information for metabolic engineering. Here, we reviewed the latest research progresses on the metabolic regulation for chemical production and substrate utilization in solventogenic clostridia, by focusing on three typical Clostridium species, the saccharolytic C. acetobutylicum and C. beijerinckii, as well as the gas-fermenting C. ljungdahlii. On this basis, future directions in the study and remodeling of clostridial regulation systems, were proposed for effective modification of these industrially important anaerobes. Copyright © 2018 Elsevier Inc. All rights reserved.
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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.
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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.
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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
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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
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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.
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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
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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
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Novel nano-OLED based probes for very high resolution optical microscopy
NASA Astrophysics Data System (ADS)
Zhao, Yiying
Near-field scanning optical microscopy (NSOM) has been applied in the study of nanomaterials, microelectronics, photonics, plasmonics, cells, and molecules. However, conventional NSOM relies on optically pumped probes, suffering low optical transmission, heating of the tip, and poor reproducibility of probe fabrication, increasing the cost, impeding usability, reducing practical imaging resolution, and limiting NSOM's utility. In this thesis, I demonstrate a novel probe based on a nanoscale, electrically pumped organic light-emitting device (OLED) formed on the tip of a low-cost, commercially available atomic force microscopy (AFM) probe. I describe the structure, fabrication, and principles of this novel probe's operation, and discuss its potential to overcome the limitations of conventional NSOM probes. The broader significance of this work in the field of organic optoelectronics is also discussed. Briefly, OLEDs consist of organic thin films sandwiched between two electrodes. Under bias, electrons and holes are injected into the organic layers, leading to radiative recombination. Depositing a small molecular OLED in vacuum onto a pyramid-tipped AFM probe results in a laminar structure that is highly curved at the tip. Simple electrical modeling predicts concentration of electric field and localized electron injection into the organic layers at the tip, improving the local charge balance in an otherwise electron-starved OLED. Utilizing an "inverted" OLED structure (i.e. cathode on the "bottom"), light emission is localized to sub-200 nm sized, green light emitting regions on probe vertices; light output power in the range of 0.1-0.5 nanowatts was observed, comparable to that of typical fiber based NSOM probes but with greater power efficiency. Massive arrays of similar sub-micron OLEDs were also fabricated by depositing onto textured silicon substrates, demonstrating the superior scalability of the probe fabrication process (e.g. relative to pulled glass fibers). The investigation of the effect of non-planar substrate geometry on charge injection, transport and recombination provides broader insights into OLEDs made on rough substrates, general understanding of OLED operation (e.g. filamentary charge conduction) and degradation, and potentially helps to improve technologically important "inverted" OLED structures.
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Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.
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.
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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
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Biocatalytic route to C-3'-azido/-hydroxy-C-4'-spiro-oxetanoribonucleosides.
Kumar, Manish; Sharma, Vivek K; Kumar, Rajesh; Prasad, Ashok K
2015-11-19
The lipase, Novozyme(®)-435, exclusively deacetylates the 5-O-acetyl over 4-C-acetyloxymethyl group of almost identical reactivity in 5-O-acetyl-4-C-acetyloxymethyl-3-azido-3-deoxy-1,2-O-isopropylidene-α-D-ribofuranose that led to the development of first and efficient synthesis of 3'-azido-/3'-amino-C-4'-spiro-oxetanoribonucleosides T, U, C and A in 20-24% overall yields. The X-ray study on the compound obtained by tosylation of lipase-mediated monodeacetylated product unambiguously confirmed the point of diastereoselective monodeacetylation on diacetoxy-azido-ribofuranose derivative. The capability of Novozyme(®)-435 for selective deacylation of 5-O-acetyl group in 5-O-acetyl-4-C-acetyloxymethyl-3-O-benzyl-1,2-O-isopropylidene-α-D-ribofuranose recently discovered by us has been successfully used for the synthesis of C-4'-spiro-oxetanoribonucleosides A and C in good yields. These results clearly indicate that the broader substrate specificity and highly selective capability of Novozyme(®)-435 for carrying out acetylation/deacetylation reactions can be utilized for the development of environment friendly selective methodologies in organic synthesis. Copyright © 2015 Elsevier Ltd. All rights reserved.
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The role of novel chitin-like polysaccharides in Alzheimer disease.
Castellani, Rudy J; Perry, George; Smith, Mark A
2007-12-01
While controversy over the role of carbohydrates in amyloidosis has existed since the initial recognition of amyloid, current understanding of the role of polysaccharides in the pathogenesis of amyloid deposition of Alzheimer disease and other amyloidoses is limited to studies of glyco-conjugates such as heparin sulfate proteoglycan. We hypothesized that polysaccharides may play a broader role in light of 1) the impaired glucose utilization in Alzheimer disease; 2) the demonstration of amylose in the Alzheimer disease brain; 3) the role of amyloid in Alzheimer disease pathogenesis. Specifically, as with glucose polymers (amyloid), we wanted to explore whether glucosamine polymers such as chitin were being synthesized and deposited as a result of impaired glucose utilization and aberrant hexosamine pathway activation. To this end, using calcofluor histochemistry, we recently demonstrated that amyloid plaques and blood vessels affected by amyloid angiopathy in subjects with sporadic and familial Alzheimer disease elicit chitin-type characteristics. Since chitin is a highly insoluble molecule and a substrate for glycan-protein interactions, chitin-like polysaccharides within the Alzheimer disease brain could provide a scaffolding for amyloid-beta deposition. As such, glucosamine may facilitate the process of amyloidosis, and /or provide neuroprotection in the Alzheimer disease brain.
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Colom, Roberto; Solomon, Jeffrey; Krueger, Frank; Forbes, Chad; Grafman, Jordan
2012-01-01
Although cognitive neuroscience has made remarkable progress in understanding the involvement of the prefrontal cortex in executive control, the broader functional networks that support high-level cognition and give rise to general intelligence remain to be well characterized. Here, we investigated the neural substrates of the general factor of intelligence (g) and executive function in 182 patients with focal brain damage using voxel-based lesion–symptom mapping. The Wechsler Adult Intelligence Scale and Delis–Kaplan Executive Function System were used to derive measures of g and executive function, respectively. Impaired performance on these measures was associated with damage to a distributed network of left lateralized brain areas, including regions of frontal and parietal cortex and white matter association tracts, which bind these areas into a coordinated system. The observed findings support an integrative framework for understanding the architecture of general intelligence and executive function, supporting their reliance upon a shared fronto-parietal network for the integration and control of cognitive representations and making specific recommendations for the application of the Wechsler Adult Intelligence Scale and Delis–Kaplan Executive Function System to the study of high-level cognition in health and disease. PMID:22396393
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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
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less
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Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...
2015-02-18
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less
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Antihistamines in drivers, aircrew and occupations of risk.
Jáuregui, I; Ferrer, M; Montoro, J; Dávila, I; Bartra, J; del Cuvillo, A; Mullol, J; Sastre, J; Valero, A
2013-01-01
The most commonly occurring allergic diseases can involve a daytime drowsiness associated with the condition itself. The antihistamines used in their treatment can also have central effects and affect certain occupations concerned with risk, road safety and maritime and air navigation. Cognitive tests, experimental studies and epidemiological data recommend avoiding 1st generation antihistamines for people who must drive regularly and/or professions concerned with safety. Although there are no comparative studies on real driving between 1st and 2nd generation antihistamines, in this type of patients there should be a preference for prescribing those with least possible central effect, especially those which are a good substrate for transmembrane transporter pumps such as P-glycoprotein and therefore have a low capacity for crossing the hematoencephalic barrier, thus allowing a broader window for therapy. In this sense, bilastine is a good P-glycoprotein substrate and shows good tolerance at CNS level, in both psychometric trials and real driving test protocols, even at double the dose recommended in the technical file.
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Substrate-specific regulation of ubiquitination by the anaphase-promoting complex
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
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Exploring the specific features of interfacial enzymology based on lipase studies.
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.
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Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.
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.
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Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*
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
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Substrate Sorting by a Supercharged Nanoreactor
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
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Allosteric regulation of rhomboid intramembrane proteolysis.
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.
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Allosteric regulation of rhomboid intramembrane proteolysis
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
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Evolutionary dynamics of enzymes.
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.
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Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display
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
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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.
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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
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NASA Astrophysics Data System (ADS)
Berdnikov, Y.; Zhiglinsky, A. A.; Rylkova, M. V.; Dubrovskii, V. G.
2017-11-01
We present a model for kinetic broadening effects on the length distributions of Au-catalyzed III-V nanowires obtained in the growth regime with adatom diffusion from the substrate and the nanowire sidewalls to the top. We observe three different regimes for the length distribution evolution with time. For short growth times, the length distribution is sub-Poissonian, converting to broader than Poissonian with increasing the mean length above a certain threshold value. After the diffusion flux from the nanowire sidewalls has stabilized, the length distribution variance increases linearly with the mean length, as in the Poissonian process.
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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.
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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.
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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.
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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.
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Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*
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
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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
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Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang
2017-01-01
Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface. PMID:28087594
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Srivastava, Ruchi; Dervillez, Xavier; Khan, Arif A.; Chentoufi, Aziz A.; Chilukuri, Sravya; Shukr, Nora; Fazli, Yasmin; Ong, Nicolas N.; Afifi, Rasha E.; Osorio, Nelson; Geertsema, Roger; Nesburn, Anthony B.
2016-01-01
ABSTRACT Persistent pathogens, such as herpes simplex virus 1 (HSV-1), have evolved a variety of immune evasion strategies to avoid being detected and destroyed by the host's immune system. A dynamic cross talk appears to occur between the HSV-1 latency-associated transcript (LAT), the only viral gene that is abundantly transcribed during latency, and the CD8+ T cells that reside in HSV-1 latently infected human and rabbit trigeminal ganglia (TG). The reactivation phenotype of TG that are latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT+ TG) is significantly higher than TG latently infected with LAT-null mutant (i.e., LAT− TG). Whether LAT promotes virus reactivation by selectively shaping a unique repertoire of HSV-specific CD8+ T cells in LAT+ TG is unknown. In the present study, we assessed the frequency, function, and exhaustion status of TG-resident CD8+ T cells specific to 40 epitopes derived from HSV-1 gB, gD, VP11/12, and VP13/14 proteins, in human leukocyte antigen (HLA-A*0201) transgenic rabbits infected ocularly with LAT+ versus LAT– virus. Compared to CD8+ T cells from LAT– TG, CD8+ T cells from LAT+ TG (i) recognized a broader selection of nonoverlapping HSV-1 epitopes, (ii) expressed higher levels of PD-1, TIM-3, and CTLA-4 markers of exhaustion, and (iii) produced less tumor necrosis factor alpha, gamma interferon, and granzyme B. These results suggest a novel immune evasion mechanism by which the HSV-1 LAT may contribute to the shaping of a broader repertoire of exhausted HSV-specific CD8+ T cells in latently infected TG, thus allowing for increased viral reactivation. IMPORTANCE A significantly larger repertoire of dysfunctional (exhausted) HSV-specific CD8+ T cells were found in the TG of HLA transgenic rabbits latently infected with wild-type HSV-1 or with LAT-rescued mutant (i.e., LAT+ TG) than in a more restricted repertoire of functional HSV-specific CD8+ T cells in the TG of HLA transgenic rabbits latently infected with LAT-null mutant (i.e., LAT– TG). These findings suggest that the HSV-1 LAT locus interferes with the host cellular immune response by shaping a broader repertoire of exhausted HSV-specific CD8+ T cells within the latency/reactivation TG site. PMID:26842468
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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.
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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.
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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.
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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.
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Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.
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.
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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
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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.
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Length distributions of nanowires: Effects of surface diffusion versus nucleation delay
NASA Astrophysics Data System (ADS)
Dubrovskii, Vladimir G.
2017-04-01
It is often thought that the ensembles of semiconductor nanowires are uniform in length due to the initial organization of the growth seeds such as lithographically defined droplets or holes in the substrate. However, several recent works have already demonstrated that most nanowire length distributions are broader than Poissonian. Herein, we consider theoretically the length distributions of non-interacting nanowires that grow by the material collection from the entire length of their sidewalls and with a delay of nucleation of the very first nanowire monolayer. The obtained analytic length distribution is controlled by two parameters that describe the strength of surface diffusion and the nanowire nucleation rate. We show how the distribution changes from the symmetrical Polya shape without the nucleation delay to a much broader and asymmetrical one for longer delays. In the continuum limit (for tall enough nanowires), the length distribution is given by a power law times an incomplete gamma-function. We discuss interesting scaling properties of this solution and give a recipe for analyzing and tailoring the experimental length histograms of nanowires which should work for a wide range of material systems and growth conditions.
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Riparian forest restoration: Conflicting goals, trade-offs, and measures of success
Heather L. Bateman; David M. Merritt; J. Bradley Johnson
2012-01-01
Restoration projects can have varying goals, depending on the specific focus, rationale, and aims for restoration. When restoration projects use project-specific goals to define activities and gauge success without considering broader ecological context, determination of project implications and success can be confounding. We used case studies from the Middle Rio...
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Writing Essentials | Center for Cancer Research
To effectively communicate research results, the manuscript should be carefully structured to tell a compelling story. As a rule, the introduction should bring the reader from a broad understanding of the topic to the specific question being addressed. In contrast, the discussion should transition the reader from the specific results to their broader implications.
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Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.
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.
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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.
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A Scientist's Guide to Achieving Broader Impacts through K-12 STEM Collaboration.
Komoroske, Lisa M; Hameed, Sarah O; Szoboszlai, Amber I; Newsom, Amanda J; Williams, Susan L
2015-03-01
The National Science Foundation and other funding agencies are increasingly requiring broader impacts in grant applications to encourage US scientists to contribute to science education and society. Concurrently, national science education standards are using more inquiry-based learning (IBL) to increase students' capacity for abstract, conceptual thinking applicable to real-world problems. Scientists are particularly well suited to engage in broader impacts via science inquiry outreach, because scientific research is inherently an inquiry-based process. We provide a practical guide to help scientists overcome obstacles that inhibit their engagement in K-12 IBL outreach and to attain the accrued benefits. Strategies to overcome these challenges include scaling outreach projects to the time available, building collaborations in which scientists' research overlaps with curriculum, employing backward planning to target specific learning objectives, encouraging scientists to share their passion, as well as their expertise with students, and transforming institutional incentives to support scientists engaging in educational outreach.
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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.
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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.
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Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.
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.
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Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.
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.
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Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos
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
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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.
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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.
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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.
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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
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Integrating clinical communication with clinical reasoning and the broader medical curriculum.
Cary, Julie; Kurtz, Suzanne
2013-09-01
The objectives of this paper are to discuss the results of a workshop conducted at EACH 2012. Specifically, we will (1) examine the link between communication, clinical reasoning, and medical problem solving, (2) explore strategies for (a) integrating clinical reasoning, medical problem solving, and content from the broader curriculum into clinical communication teaching and (b) integrating communication into the broader curriculum, and (3) discuss benefits gained from such integration. Salient features from the workshop were recorded and will be presented here, as well as a case example to illustrate important connections between clinical communication and clinical reasoning. Potential links between clinical communication, clinical reasoning, and medical problem solving as well as strategies to integrate clinical communication teaching and the broader curricula in human and veterinary medicine are enumerated. Participants expressed enthusiasm and keen interest in integration of clinical communication teaching and clinical reasoning during this workshop, came to the idea of the interdependence of these skills easily, and embraced the rationale immediately. Valuing the importance of communication as clinical skill and embracing the interdependence between communication and thought processes related to clinical reasoning and medical problem solving will be beneficial in teaching programs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.
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Specificity of hammerhead ribozyme cleavage.
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
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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
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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
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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
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Dissecting substrate specificities of the mitochondrial AFG3L2 protease.
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.
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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
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Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays
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
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A multiwell format assay for heparanase.
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.
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NASA Astrophysics Data System (ADS)
Sutton, M.; Marchetti, A.
2016-02-01
Broader impacts have become a vital component of scientific research projects. A variety of outreach avenues are available to assist scientists in reaching larger audiences, however, the translation of cutting-edge scientific content and concepts can be challenging. Collaborating with educators is a viable option to assist researchers in fulfilling NSF's broader impact requirements. A broader impacts model based on collaborations between a teacher and 28 researchers from 14 institutions will demonstrate successful science outreach and engagement through interactions between teachers, researchers, students, and general audiences. Communication styles (i.e., blogs, social media) and outreach data incorporated by researchers and the teacher will be shared to illustrate the magnitude of the broader impacts achieved with this partnership. Inquiry-based investigations and activities developed to translate the science into the classroom will also be demonstrated, including the use of real scientific data collected during the research cruise. "Finding Microbe Needles in a Haystack of Oceans" provides an understanding of how remote sensing technology is used to locate specific ocean environments (e.g. High Nutrient Low Chlorophyll - HNLC) that support diverse microbial food webs. A board game ("Diatom Adventures©") designed to explore the physiology of microbial organisms and microscopic food webs will also be demonstrated. The tentative nature of science requires a constant vigil to stay abreast of the latest hypotheses and discoveries. Researcher/Teacher collaborations allow each professional to focus on his/her strengths while meeting broader impact requirements. These partnerships encourage lifelong learning as educators observe and work with scientists first-hand and then follow appropriate scope, sequence, and pedagogy to assist various audiences in understanding the innovative technologies being used to explore new scientific frontiers.
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Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl
2015-07-01
Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.
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Studying the Interface between Nanomaterials and Biomolecules
NASA Astrophysics Data System (ADS)
Torelli, Marco Diego
As engineered nanomaterials become ubiquitous among society, their inevitable entrance into the environment invites questions as to potential implications. As the field of nanotechnology progresses, responsible development of nanomaterials requires a broad availability of useful tools. To this aim, this work seeks to improve analytical abilities to address fundamental molecular interactions of nanomaterials with biological systems that can be expanded broadly, divided into the following: (1) A model applicable to X-ray photoelectron spectroscopy was developed and validated to correct the over-estimated signal for core:shell nanomaterials that can occur at small particle sizes approaching the electron attenuation length of the material being investigated. (2) To understand the role of underlying substrate in particle interactions, diamond and gold functionalized with a protein resisting molecule (hexaethylene glycol) were compared to test the ability of each to resist adsorption of charged proteins. It was demonstrated that the underlying substrate can have an effect on the ability of to properly resist proteins, with charged proteins adsorbing to gold, believed to be due to the ability of gold to form an image dipole. (3) To advance the use of nanodiamond in biological settings, methods to create robust chemical linkages at single digit sizes were developed. Alkene based oligo(ethylene glycol) molecules were successfully photochemically grafted to fully disaggregated detonation nanodiamond. Because the scalability of such methods currently limits such functionalization broadly, polyelectrolytic wrapping of nanodiamond was developed as a useful and scalable method to produce diamond nanoparticles with varying amine based functionalities. (4) Phage display was adapted as a method to determine chemical functionalities that interact with anatase titanium dioxide below 20 nm. In contrast to finding specific, individual inorganic binding sequences, we lowered the selection stringency to allow for a broader number of peptides to be samples. While no statistical size dependent differences were observed in the amino acid chemistries that interact with anatase TiO2, chemical functionalities and motifs that appear to be important for interaction with nano-anatase were identified. Specifically, positively charged and aromatic motifs working in concert were found to be important.
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Straus, Marco R.; Whittaker, Gary R.
2017-01-01
Cleavage activation of the hemagglutinin (HA) protein by host proteases is a crucial step in the infection process of influenza A viruses (IAV). However, IAV exists in eighteen different HA subtypes in nature and their cleavage sites vary considerably. There is uncertainty regarding which specific proteases activate a given HA in the human respiratory tract. Understanding the relationship between different HA subtypes and human-specific proteases will be valuable in assessing the pandemic potential of circulating viruses. Here we utilized fluorogenic peptides mimicking the HA cleavage motif of representative IAV strains causing disease in humans or of zoonotic/pandemic potential and tested them with a range of proteases known to be present in the human respiratory tract. Our results show that peptides from the H1, H2 and H3 subtypes are cleaved efficiently by a wide range of proteases including trypsin, matriptase, human airway tryptase (HAT), kallikrein-related peptidases 5 (KLK5) and 12 (KLK12) and plasmin. Regarding IAVs currently of concern for human adaptation, cleavage site peptides from H10 viruses showed very limited cleavage by respiratory tract proteases. Peptide mimics from H6 viruses showed broader cleavage by respiratory tract proteases, while H5, H7 and H9 subtypes showed variable cleavage; particularly matriptase appeared to be a key protease capable of activating IAVs. We also tested HA substrate specificity of Factor Xa, a protease required for HA cleavage in chicken embryos and relevant for influenza virus production in eggs. Overall our data provide novel tool allowing the assessment of human adaptation of IAV HA subtypes. PMID:28358853
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Visual cues for woodpeckers: light reflectance of decayed wood varies by decay fungus
O'Daniels, Sean T.; Kesler, Dylan C.; Mihail, Jeanne D.; Webb, Elisabeth B.; Werner, Scott J.
2018-01-01
The appearance of wood substrates is likely relevant to bird species with life histories that require regular interactions with wood for food and shelter. Woodpeckers detect decayed wood for cavity placement or foraging, and some species may be capable of detecting trees decayed by specific fungi; however, a mechanism allowing for such specificity remains unidentified. We hypothesized that decay fungi associated with woodpecker cavity sites alter the substrate reflectance in a species-specific manner that is visually discriminable by woodpeckers. We grew 10 species of wood decay fungi from pure cultures on sterile wood substrates of 3 tree species. We then measured the relative reflectance spectra of decayed and control wood wafers and compared them using the receptor noise-limited (RNL) color discrimination model. The RNL model has been used in studies of feather coloration, egg shells, flowers, and fruit to model how the colors of objects appear to birds. Our analyses indicated 6 of 10 decayed substrate/control comparisons were above the threshold of discrimination (i.e., indicating differences discriminable by avian viewers), and 12 of 13 decayed substrate comparisons were also above threshold for a hypothetical woodpecker. We conclude that woodpeckers should be capable of visually detecting decayed wood on trees where bark is absent, and they should also be able to detect visually species-specific differences in wood substrates decayed by fungi used in this study. Our results provide evidence for a visual mechanism by which woodpeckers could identify and select substrates decayed by specific fungi, which has implications for understanding ecologically important woodpecker–fungus interactions.
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Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.
Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R
2009-09-15
Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.
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Cole, Susan P. C.
2014-01-01
The multidrug resistance protein 1 (MRP1) encoded by ABCC1 was originally discovered as a cause of multidrug resistance in tumor cells. However, it is now clear that MRP1 serves a broader role than simply mediating the ATP-dependent efflux of drugs from cells. The antioxidant GSH and the pro-inflammatory cysteinyl leukotriene C4 have been identified as key physiological organic anions effluxed by MRP1, and an ever growing body of evidence indicates that additional lipid-derived mediators are also substrates of this transporter. As such, MRP1 is a multitasking transporter that likely influences the etiology and progression of a host of human diseases. PMID:25281745
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Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.
Campbell, T B; Cech, T R
1995-01-01
Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519
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Tokai, Shota; Bito, Tomohiro; Shimizu, Katsuhiko; Arima, Jiro
2017-05-27
Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating. Copyright © 2017 Elsevier Inc. All rights reserved.
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Nano-cones for broadband light coupling to high index substrates
NASA Astrophysics Data System (ADS)
Buencuerpo, J.; Torné, L.; Álvaro, R.; Llorens, J. M.; Dotor, M. L.; Ripalda, J. M.
2016-12-01
The moth-eye structure has been proposed several times as an antireflective coating to replace the standard optical thin films. Here, we experimentally demonstrate the feasibility of a dielectric moth-eye structure as an antireflective coating for high-index substrates, like GaAs. The fabricated photonic crystal has Si3N4 cones in a square lattice, sitting on top of a TiO2 index matching layer. This structure attains 1.4% of reflectance power losses in the operation spectral range of GaAs solar cells (440-870 nm), a 12.5% relative reduction of reflection power losses in comparison with a standard bilayer. The work presented here considers a fabrication process based on laser interference lithography and dry etching, which are compatible with solar cell devices. The experimental results are consistent with scattering matrix simulations of the fabricated structures. In a broader spectral range (400-1800 nm), the simulation estimates that the nanostructure also significantly outperforms the standard bilayer coating (3.1% vs. 4.5% reflection losses), a result of interest for multijunction tandem solar cells.
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Moring, J. Bruce; Braun, Christopher L.; Pearson, Daniel K.
2014-01-01
There were no statistically significant differences between the stream velocities associated with seine hauls of the Rio Grande silvery minnow and Tamaulipas shiner. Stream velocities associated with the seine hauls that included Rio Grande silvery minnow indicate that this species is predominantly found in low-velocity mesohabitats. Velocities associated with seine hauls that included the Tamaulipas shiner represented a much broader overall range of velocities than those associated with Rio Grande silvery minnow collections. No statistically significant differences were found between the depths for seine hauls that included Rio Grande silvery minnow or Tamaulipas shiner. The Rio Grande silvery minnow was more commonly collected in seine hauls from mesohabitats dominated by cobble substrates and less frequently collected in mesohabitats with substrates dominated by fine-sized silt and clay particles, gravels, and sands, in that order. In contrast, the Tamaulipas shiner was broadly distributed among mesohabitats characterized as having gravel, cobble, and silt and clay.
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Obstructive Sleep Apnea Treatment and Atrial Fibrillation: A Need for Definitive Evidence.
Nalliah, Chrishan J; Sanders, Prashanthan; Kalman, Jonathan M
2016-08-01
Prevalence rates of atrial fibrillation (AF) and obstructive sleep apnea (OSA) are rising on a global scale. Epidemiological data have consistently demonstrated an independent association between the 2 conditions. Investigators pose that pathophysiologic features of OSA enable progression of the AF substrate; these features include abnormalities of gas exchange, autonomic remodeling, atrial stretch, and inflammation. Furthermore, many of the mechanistic perturbations that impact the AF substrate in OSA can be substantially attenuated by effective treatment with continuous positive airway pressure (CPAP). Clear associations of OSA treatment and improved AF control have been observed across multiple clinical contexts. However, the precision and generalizability of these findings are unclear in view of the data's observational nature. Although risk factor management has emerged as a critical component of AF treatment, effective control of many AF risk factors can be challenging in the longer term. In view of the efficacy and sustainability of CPAP therapy, OSA raises its profile as a prime candidate for intervention. However, translation of this strategy to the broader framework for AF management requires robust data from randomized controlled trials. © 2016 Wiley Periodicals, Inc.
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Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin
2014-01-01
The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277
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Novel Syntrophic Populations Dominate an Ammonia-Tolerant Methanogenic Microbiome.
Frank, J A; Arntzen, M Ø; Sun, L; Hagen, L H; McHardy, A C; Horn, S J; Eijsink, V G H; Schnürer, A; Pope, P B
2016-01-01
Biogas reactors operating with protein-rich substrates have high methane potential and industrial value; however, they are highly susceptible to process failure because of the accumulation of ammonia. High ammonia levels cause a decline in acetate-utilizing methanogens and instead promote the conversion of acetate via a two-step mechanism involving syntrophic acetate oxidation (SAO) to H 2 and CO 2 , followed by hydrogenotrophic methanogenesis. Despite the key role of syntrophic acetate-oxidizing bacteria (SAOB), only a few culturable representatives have been characterized. Here we show that the microbiome of a commercial, ammonia-tolerant biogas reactor harbors a deeply branched, uncultured phylotype (unFirm_1) accounting for approximately 5% of the 16S rRNA gene inventory and sharing 88% 16S rRNA gene identity with its closest characterized relative. Reconstructed genome and quantitative metaproteomic analyses imply unFirm_1's metabolic dominance and SAO capabilities, whereby the key enzymes required for acetate oxidation are among the most highly detected in the reactor microbiome. While culturable SAOB were identified in genomic analyses of the reactor, their limited proteomic representation suggests that unFirm_1 plays an important role in channeling acetate toward methane. Notably, unFirm_1-like populations were found in other high-ammonia biogas installations, conjecturing a broader importance for this novel clade of SAOB in anaerobic fermentations. IMPORTANCE The microbial production of methane or "biogas" is an attractive renewable energy technology that can recycle organic waste into biofuel. Biogas reactors operating with protein-rich substrates such as household municipal or agricultural wastes have significant industrial and societal value; however, they are highly unstable and frequently collapse due to the accumulation of ammonia. We report the discovery of a novel uncultured phylotype (unFirm_1) that is highly detectable in metaproteomic data generated from an ammonia-tolerant commercial reactor. Importantly, unFirm_1 is proposed to perform a key metabolic step in biogas microbiomes, whereby it syntrophically oxidizes acetate to hydrogen and carbon dioxide, which methanogens then covert to methane. Only very few culturable syntrophic acetate-oxidizing bacteria have been described, and all were detected at low in situ levels compared to unFirm_1. Broader comparisons produced the hypothesis that unFirm_1 is a key mediator toward the successful long-term stable operation of biogas production using protein-rich substrates.
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T'jollyn, Huybrecht; Snoeys, Jan; Van Bocxlaer, Jan; De Bock, Lies; Annaert, Pieter; Van Peer, Achiel; Allegaert, Karel; Mannens, Geert; Vermeulen, An; Boussery, Koen
2017-06-01
Although the measurement of cytochrome P450 (CYP) contributions in metabolism assays is straightforward, determination of actual in vivo contributions might be challenging. How representative are in vitro for in vivo CYP contributions? This article proposes an improved strategy for the determination of in vivo CYP enzyme-specific metabolic contributions, based on in vitro data, using an in vitro-in vivo extrapolation (IVIVE) approach. Approaches are exemplified using tramadol as model compound, and CYP2D6 and CYP3A4 as involved enzymes. Metabolism data for tramadol and for the probe substrates midazolam (CYP3A4) and dextromethorphan (CYP2D6) were gathered in human liver microsomes (HLM) and recombinant human enzyme systems (rhCYP). From these probe substrates, an activity-adjustment factor (AAF) was calculated per CYP enzyme, for the determination of correct hepatic clearance contributions. As a reference, tramadol CYP contributions were scaled-back from in vivo data (retrograde approach) and were compared with the ones derived in vitro. In this view, the AAF is an enzyme-specific factor, calculated from reference probe activity measurements in vitro and in vivo, that allows appropriate scaling of a test drug's in vitro activity to the 'healthy volunteer' population level. Calculation of an AAF, thus accounts for any 'experimental' or 'batch-specific' activity difference between in vitro HLM and in vivo derived activity. In this specific HLM batch, for CYP3A4 and CYP2D6, an AAF of 0.91 and 1.97 was calculated, respectively. This implies that, in this batch, the in vitro CYP3A4 activity is 1.10-fold higher and the CYP2D6 activity 1.97-fold lower, compared to in vivo derived CYP activities. This study shows that, in cases where the HLM pool does not represent the typical mean population CYP activities, AAF correction of in vitro metabolism data, optimizes CYP contributions in the prediction of hepatic clearance. Therefore, in vitro parameters for any test compound, obtained in a particular batch, should be corrected with the AAF for the respective enzymes. In the current study, especially the CYP2D6 contribution was found, to better reflect the average in vivo situation. It is recommended that this novel approach is further evaluated using a broader range of compounds.
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Slider thickness promotes lubricity: from 2D islands to 3D clusters
NASA Astrophysics Data System (ADS)
Guerra, Roberto; Tosatti, Erio; Vanossi, Andrea
2016-05-01
The sliding of three-dimensional clusters and two-dimensional islands adsorbed on crystal surfaces represents an important test case to understand friction. Even for the same material, monoatomic islands and thick clusters will not as a rule exhibit the same friction, but specific differences have not been explored. Through realistic molecular dynamics simulations of the static friction of gold on graphite, an experimentally relevant system, we uncover as a function of gold thickness a progressive drop of static friction from monolayer islands, that are easily pinned, towards clusters, that slide more readily. The main ingredient contributing to this thickness-induced lubricity appears to be the increased effective rigidity of the atomic contact, acting to reduce the cluster interdigitation with the substrate. A second element which plays a role is the lateral contact size, which can accommodate the solitons typical of the incommensurate interface only above a critical contact diameter, which is larger for monolayer islands than for thick clusters. The two effects concur to make clusters more lubric than islands, and large sizes more lubric than smaller ones. These conclusions are expected to be of broader applicability in diverse nanotribological systems, where the role played by static, and dynamic, friction is generally quite important.
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Slider thickness promotes lubricity: from 2D islands to 3D clusters.
Guerra, Roberto; Tosatti, Erio; Vanossi, Andrea
2016-06-07
The sliding of three-dimensional clusters and two-dimensional islands adsorbed on crystal surfaces represents an important test case to understand friction. Even for the same material, monoatomic islands and thick clusters will not as a rule exhibit the same friction, but specific differences have not been explored. Through realistic molecular dynamics simulations of the static friction of gold on graphite, an experimentally relevant system, we uncover as a function of gold thickness a progressive drop of static friction from monolayer islands, that are easily pinned, towards clusters, that slide more readily. The main ingredient contributing to this thickness-induced lubricity appears to be the increased effective rigidity of the atomic contact, acting to reduce the cluster interdigitation with the substrate. A second element which plays a role is the lateral contact size, which can accommodate the solitons typical of the incommensurate interface only above a critical contact diameter, which is larger for monolayer islands than for thick clusters. The two effects concur to make clusters more lubric than islands, and large sizes more lubric than smaller ones. These conclusions are expected to be of broader applicability in diverse nanotribological systems, where the role played by static, and dynamic, friction is generally quite important.
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Two Distinct Scene-Processing Networks Connecting Vision and Memory.
Baldassano, Christopher; Esteva, Andre; Fei-Fei, Li; Beck, Diane M
2016-01-01
A number of regions in the human brain are known to be involved in processing natural scenes, but the field has lacked a unifying framework for understanding how these different regions are organized and interact. We provide evidence from functional connectivity and meta-analyses for a new organizational principle, in which scene processing relies upon two distinct networks that split the classically defined parahippocampal place area (PPA). The first network of strongly connected regions consists of the occipital place area/transverse occipital sulcus and posterior PPA, which contain retinotopic maps and are not strongly coupled to the hippocampus at rest. The second network consists of the caudal inferior parietal lobule, retrosplenial complex, and anterior PPA, which connect to the hippocampus (especially anterior hippocampus), and are implicated in both visual and nonvisual tasks, including episodic memory and navigation. We propose that these two distinct networks capture the primary functional division among scene-processing regions, between those that process visual features from the current view of a scene and those that connect information from a current scene view with a much broader temporal and spatial context. This new framework for understanding the neural substrates of scene-processing bridges results from many lines of research, and makes specific functional predictions.
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Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa
2013-01-01
Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.
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Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa
2013-01-01
Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121
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A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.
Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan
2017-12-22
Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6 L mol -1 s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J
2018-02-28
The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.
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[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].
Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian
2014-03-01
In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.
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Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin
2014-04-01
Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.
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Assessment of indexing trends with specific and general terms for herbal medicine.
Bartol, Tomaz
2012-12-01
Concepts for medicinal plants are represented by a variety of associated general terms with specific indexing patterns in databases, which may not consistently reflect growth of records. The objectives of this study are to assess the development in databases by identifying general terms that describe herbal medicine with optimal retrieval recall and to identify possible special trends in co-occurrence of specific and general concepts. Different search strategies are tested in cab abstracts, medline and web of science. Specific terms (Origanum and Salvia) are employed. Relevant general terms (e.g. 'Plants, Medicinal', Phytotherapy, Herbal drugs) are identified, along with indexing trends and co-occurrences. Growth trends, in specific (narrower) terms, are similar among databases. General terms, however, exhibit dissimilar trends, sometimes almost opposing one another. Co-occurrence of specific and general terms is changing over time. General terms may not denote definite development of trends as the use of terms differs amongst databases, making it difficult to correctly assess possible numbers of relevant records. Perceived increase can, sometimes, be attributed to an increased occurrence of a more general term alongside the specific one. Thesaurus-controlled databases may yield more hits, because of 'up-posted' (broader) terms. Use of broader terms is helpful as it enhances retrieval of relevant documents. © 2012 The authors. Health Information and Libraries Journal © 2012 Health Libraries Group.
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Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...
2018-03-23
Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Poudel, Suresh; Giannone, Richard J.; Basen, Mirko
Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less
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Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L
2018-01-01
Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.
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Template-Free Mesoporous Electrochromic Films on Flexible Substrates from Tungsten Oxide Nanorods
Heo, Sungyeon; Kim, Jongwook; Ong, Gary K.; ...
2017-08-08
Low-temperature processed mesoporous nanocrystal thin films are platforms for fabricating functional composite thin films on flexible substrates. Using a random arrangement of anisotropic nanocrystals can be a facile solution to generate pores without templates. However, the tendency for anisotropic particles to spontaneously assemble into a compact structure must be overcome. Here in this paper, we present a method to achieve random networking of nanorods during solution phase deposition by switching their ligand-stabilized colloidal nature into a charge-stabilized nature by a ligand-stripping chemistry. Ligand-stripped tungsten suboxide (WO 2.72) nanorods result in uniform mesoporous thin films owing to repulsive electrostatic forces preventingmore » nanorods from densely packing. Porosity and pore size distribution of thin films are controlled by changing the aspect ratio of the nanorods. This template-free mesoporous structure, achieved without annealing, provides a framework for introducing guest components, therefore enabling our fabrication of inorganic nanocomposite electrochromic films on flexible substrates. Following infilling of niobium polyoxometalate clusters into pores and successive chemical condensation, a WO x–NbO x composite film is produced that selectively controls visible and near-infrared light transmittance without any annealing required. The composite shows rapid switching kinetics and can be stably cycled between optical states over 2000 times. This simple strategy of using anisotropic nanocrystals gives insight into mesoporous thin film fabrication with broader applications for flexible devices.« less
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Template-Free Mesoporous Electrochromic Films on Flexible Substrates from Tungsten Oxide Nanorods
DOE Office of Scientific and Technical Information (OSTI.GOV)
Heo, Sungyeon; Kim, Jongwook; Ong, Gary K.
Low-temperature processed mesoporous nanocrystal thin films are platforms for fabricating functional composite thin films on flexible substrates. Using a random arrangement of anisotropic nanocrystals can be a facile solution to generate pores without templates. However, the tendency for anisotropic particles to spontaneously assemble into a compact structure must be overcome. Here in this paper, we present a method to achieve random networking of nanorods during solution phase deposition by switching their ligand-stabilized colloidal nature into a charge-stabilized nature by a ligand-stripping chemistry. Ligand-stripped tungsten suboxide (WO 2.72) nanorods result in uniform mesoporous thin films owing to repulsive electrostatic forces preventingmore » nanorods from densely packing. Porosity and pore size distribution of thin films are controlled by changing the aspect ratio of the nanorods. This template-free mesoporous structure, achieved without annealing, provides a framework for introducing guest components, therefore enabling our fabrication of inorganic nanocomposite electrochromic films on flexible substrates. Following infilling of niobium polyoxometalate clusters into pores and successive chemical condensation, a WO x–NbO x composite film is produced that selectively controls visible and near-infrared light transmittance without any annealing required. The composite shows rapid switching kinetics and can be stably cycled between optical states over 2000 times. This simple strategy of using anisotropic nanocrystals gives insight into mesoporous thin film fabrication with broader applications for flexible devices.« less
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Jenab, Ehsan; Temelli, Feral; Curtis, Jonathan M
2013-12-01
The processing parameters in enzymatic reactions using CO2-expanded (CX) lipids have strong effects on the physical properties of liquid phase, degree of interesterification, and physicochemical properties of the final reaction products. CX-canola oil and fully hydrogenated canola oil (FHCO) were interesterified using Lipozyme TL IM in a high pressure stirred batch reactor. The effects of immobilised enzyme load, pressure, substrate ratio and reaction time on the formation of mixed triacylglycerols (TG) from trisaturated and triunsaturated TG were investigated. The optimal immobilised enzyme load, pressure, substrate ratio and time for the degree of interesterification to reach the highest equilibrium state were 6% (w/v) of initial substrates, 10 MPa, blend with 30% (w/w) of FHCO and 2h, respectively. The physicochemical properties of the initial blend and interesterified products with different FHCO ratios obtained at optimal reaction conditions were determined in terms of TG composition, thermal behaviour and solid fat content (SFC). The amounts of saturated and triunsaturated TG decreased while the amounts of mixed TG increased as a result of interesterification. Thus, the interesterified product had a lower melting point, and broader melting and plasticity ranges compared to the initial blends. These findings are important for better understanding of CX-lipid reactions and for optimal formulation of base-stocks of margarine and confectionary fats to meet industry demands. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni
2014-01-01
Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354
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Quest for business intelligence in health care.
Van De Graaff, Joe; Cameron, Austin
2013-02-01
In an era of reform, providers are examining more forward-thinking business intelligence strategies, according to a recent study. Enterprise business intelligence tool sets offer a breadth of design and functionality that often are capable of serving the enterprise. One limitation of broader tool sets is that they may lack needed application-specific functionality or prebuilt healthcare content for a specific department.
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Host specificity and the probability of discovering species of helminth parasites.
Poulin, R; Mouillot, D
2005-06-01
Different animal species have different probabilities of being discovered and described by scientists, and these probabilities are determined to a large extent by the biological characteristics of these species. For instance, species with broader geographical ranges are more likely to be encountered by collectors than species with restricted distributions; indeed, the size of the geographical range is often the best predictor of a species' date of description. For parasitic organisms, host specificity may be similarly linked to the probability of a species being found. Here, using data on 170 helminth species parasitic in freshwater fishes, we show that host specificity is associated with the year in which the helminths were described. Helminths that exploit more host species, and to a lesser degree those that exploit a broader taxonomic range of host species, tend to be discovered earlier than the more host-specific helminths. This pattern was observed across all helminth species, as well as within the different helminth taxa (trematodes, cestodes, nematodes and acanthocephalans). Our results demonstrate that the parasite species known at any given point in time are not a random subset of existing species, but rather a biased subset with respect to the parasites' biological properties.
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Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate
ERIC Educational Resources Information Center
Linenberger, Kimberly J.; Bretz, Stacey Lowery
2015-01-01
Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…
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Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram
2009-12-11
Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.
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High Throughput Transcriptomics @ USEPA (Toxicology ...
The ideal chemical testing approach will provide complete coverage of all relevant toxicological responses. It should be sensitive and specific It should identify the mechanism/mode-of-action (with dose-dependence). It should identify responses relevant to the species of interest. Responses should ideally be translated into tissue-, organ-, and organism-level effects. It must be economical and scalable. Using a High Throughput Transcriptomics platform within US EPA provides broader coverage of biological activity space and toxicological MOAs and helps fill the toxicological data gap. Slide presentation at the 2016 ToxForum on using High Throughput Transcriptomics at US EPA for broader coverage biological activity space and toxicological MOAs.
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Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi
2004-10-15
Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.
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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.
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Ultra-low roughness magneto-rheological finishing for EUV mask substrates
NASA Astrophysics Data System (ADS)
Dumas, Paul; Jenkins, Richard; McFee, Chuck; Kadaksham, Arun J.; Balachandran, Dave K.; Teki, Ranganath
2013-09-01
EUV mask substrates, made of titania-doped fused silica, ideally require sub-Angstrom surface roughness, sub-30 nm flatness, and no bumps/pits larger than 1 nm in height/depth. To achieve the above specifications, substrates must undergo iterative global and local polishing processes. Magnetorheological finishing (MRF) is a local polishing technique which can accurately and deterministically correct substrate figure, but typically results in a higher surface roughness than the current requirements for EUV substrates. We describe a new super-fine MRF® polishing fluid whichis able to meet both flatness and roughness specifications for EUV mask blanks. This eases the burden on the subsequent global polishing process by decreasing the polishing time, and hence the defectivity and extent of figure distortion.
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Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong
2017-06-13
Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.
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Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.
Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe
2016-06-08
Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.
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Insights into the Specificity of Lysine Acetyltransferases
Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...
2014-11-07
Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less
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Liu, Yanping; Yu, Faquan
2011-04-08
Magnetic iron oxide nanoparticles (MION) were recently found to act as a peroxidase with intrinsic advantages over natural counterparts. Their limited affinity toward catalysis substrates, however, dramatically reduces their utility. In this paper, some effective groups were screened out and conjugated on MION as substrate-specific modifications for improving MION's affinity to substrates and hence utility. Nanoparticles of four different superficial structures were synthesized and characterized by TEM, size, zeta potential and SQUID, and assayed for peroxidase activity. Glucose detection was selected as an application model system to evaluate the bonus thereof. Catalysis was found to follow Michaelis-Menten kinetics. Sulfhydryl groups incorporated on MION (SH-MION) notably improve the affinity toward a substrate (hydrogen peroxide) and so do amino groups (NH₂-MION) toward another substrate, proved by variation in the determined kinetic parameters. A synergistically positive effect was observed and an apparently elevated detection sensitivity and a significantly lowered detection limit of glucose were achieved when integrated with both sulfhydryl and amino groups (SH-NH₂-MION). Our findings suggest that substrate-specific surface modifications are a straightforward and robust strategy to improve MION peroxidase-like activity. The high activity extends magnetic nanoparticles to wide applications other than glucose detection.
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Low energy electron catalyst: the electronic origin of catalytic strategies.
Davis, Daly; Sajeev, Y
2016-10-12
Using a low energy electron (LEE) as a catalyst, the electronic origin of the catalytic strategies corresponding to substrate selectivity, reaction specificity and reaction rate enhancement is investigated for a reversible unimolecular elementary reaction. An electronic energy complementarity between the catalyst and the substrate molecule is the origin of substrate selectivity and reaction specificity. The electronic energy complementarity is induced by tuning the electronic energy of the catalyst. The energy complementarity maximizes the binding forces between the catalyst and the molecule. Consequently, a new electronically metastable high-energy reactant state and a corresponding new low barrier reaction path are resonantly created for a specific reaction of the substrate through the formation of a catalyst-substrate transient adduct. The LEE catalysis also reveals a fundamental structure-energy correspondence in the formation of the catalyst-substrate transient adduct. Since the energy complementarities corresponding to the substrate molecules of the forward and the backward steps of the reversible reactions are not the same due to their structural differences, the LEE catalyst exhibits a unique one-way catalytic strategy, i.e., the LEE catalyst favors the reversible reaction more effectively in one direction. A characteristic stronger binding of the catalyst to the transition state of the reaction than in the initial reactant state and the final product state is the molecular origin of barrier lowering.
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A Scientist's Guide to Achieving Broader Impacts through K–12 STEM Collaboration
Komoroske, Lisa M.; Hameed, Sarah O.; Szoboszlai, Amber I.; Newsom, Amanda J.; Williams, Susan L.
2015-01-01
The National Science Foundation and other funding agencies are increasingly requiring broader impacts in grant applications to encourage US scientists to contribute to science education and society. Concurrently, national science education standards are using more inquiry-based learning (IBL) to increase students’ capacity for abstract, conceptual thinking applicable to real-world problems. Scientists are particularly well suited to engage in broader impacts via science inquiry outreach, because scientific research is inherently an inquiry-based process. We provide a practical guide to help scientists overcome obstacles that inhibit their engagement in K–12 IBL outreach and to attain the accrued benefits. Strategies to overcome these challenges include scaling outreach projects to the time available, building collaborations in which scientists’ research overlaps with curriculum, employing backward planning to target specific learning objectives, encouraging scientists to share their passion, as well as their expertise with students, and transforming institutional incentives to support scientists engaging in educational outreach. PMID:26955078
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DNA's Encounter with Ultraviolet Light: An Instinct for Self-Preservation?
Barlev, Adam; Sen, Dipankar
2018-02-20
Photochemical modification is the major class of environmental damage suffered by DNA, the genetic material of all free-living organisms. Photolyases are enzymes that carry out direct photochemical repair (photoreactivation) of covalent pyrimidine dimers formed in DNA from exposure to ultraviolet light. The discovery of catalytic RNAs in the 1980s led to the "RNA world hypothesis", which posits that early in evolution RNA or a similar polymer served both genetic and catalytic functions. Intrigued by the RNA world hypothesis, we set out to test whether a catalytic RNA (or a surrogate, a catalytic DNA) with photolyase activity could be contemplated. In vitro selection from a random-sequence DNA pool yielded two DNA enzymes (DNAzymes): Sero1C, which requires serotonin as an obligate cofactor, and UV1C, which is cofactor-independent and optimally uses light of 300-310 nm wavelength to repair cyclobutane thymine dimers within a gapped DNA substrate. Both Sero1C and UV1C show multiple turnover kinetics, and UV1C repairs its substrate with a quantum yield of ∼0.05, on the same order as the quantum yields of certain classes of photolyase enzymes. Intensive study of UV1C has revealed that its catalytic core consists of a guanine quadruplex (G-quadruplex) positioned proximally to the bound substrate's thymine dimer. We hypothesize that electron transfer from photoexcited guanines within UV1C's G-quadruplex is responsible for substrate photoreactivation, analogous to electron transfer to pyrimidine dimers within a DNA substrate from photoexcited flavin cofactors located within natural photolyase enzymes. Though the analogy to evolution is necessarily limited, a comparison of the properties of UV1C and Sero1C, which arose out of the same in vitro selection experiment, reveals that although the two DNAzymes comparably accelerate the rate of thymine dimer repair, Sero1C has a substantially broader substrate repertoire, as it can repair many more kinds of pyrimidine dimers than UV1C. Therefore, the co-opting of an amino acid-like cofactor by a nucleic acid enzyme in this case contributes functional versatility rather than a greater rate enhancement. In recent work on UV1C, we have succeeded in shifting its action spectrum from the UVB into the blue region of the spectrum and determined that although it catalyzes both repair and de novo formation of thymine dimers, UV1C is primarily a catalyst for thymine dimer repair. Our work on photolyase DNAzymes has stimulated broader questions about whether analogous, purely nucleotide-based photoreactivation also occurs in double-helical DNA, the dominant form of DNA in living cells. Recently, a number of different groups have reported that this kind of repair is indeed operational in DNA duplexes, i.e., that there exist nucleotide sequences that actively protect, by way of photoreactivation (rather than by simply preventing their formation), pyrimidine dimers located proximal to them. Nucleotide-based photoreactivation thus appears to be a salient, if unanticipated, property of DNA and RNA. The phenomenon also offers pointers in the direction of how in primordial evolution-in an RNA world-early nucleic acids may have protected themselves from structural and functional damage wrought by ultraviolet light.
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Design, Test and Demonstration of Fault Current Limiting HTS Transformer
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hazelton, Drew
The project developed new technology that enables the creation of a high temperature superconductor-based FCL power transformer. SuperPower’s research and development created new methods to bond HTS conductor to a supporting substrate, test, and insulate the resulting bonded conductor, reduce winding ac losses, ensure FCL functionality during a transformer fault and build firm superconducting joints in the transformer harnesses and cabling. The bonded conductor in this program was shown to meet the critical operating parameters of providing the superconducting transformer operation while being able to meet the target normal state resistance required for FCL operation. The bonded conductor was alsomore » shown to be able to handle the fabrication stresses associated with the manufacture of the FCL transformer while also being able to handle the high hoop stresses and axial forces during a fault transient. Much of the technology developed here is applicable to the broader applied superconductivity community. The ability to tailor the clad conductors performance characteristics gives the designer of devices utilizing HTS a broader capability to address the particular needs of an given application. SuperPower worked with its sub-recipients Waukesha Electric Systems, ORNL, Southern California Edison and University of Houston to develop the design, fabrication, installation and operational aspects of a fault current limiting transformer on the electrical grid.« less
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Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J
2011-08-10
Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.
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2011-01-01
Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316
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Fukunaga, Ryuya; Zamore, Phillip D
2014-01-01
The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225
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Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.
2013-01-01
The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247
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Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G
2013-12-27
The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.
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Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.
2016-01-01
Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245
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Mobley, E M; Pan, T
1999-01-01
Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624
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Biochemistry students' ideas about how an enzyme interacts with a substrate.
Linenberger, Kimberly J; Bretz, Stacey Lowery
2015-01-01
Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.
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Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min
2016-03-01
In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.
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Harnessing Proteasome Dynamics and Allostery in Drug Design
Osmulski, Pawel A.
2014-01-01
Abstract Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness. The non-catalytic sites of the proteasome emerge as an attractive alternative target in search of highly specific and diverse proteasome regulators. Recent Advances: Crystallographic models of the proteasome leave the false impression of fixed structures with minimal molecular dynamics lacking long-distance allosteric signaling. However, accumulating biochemical and structural observations strongly support the notion that the proteasome is regulated by precise allosteric interactions arising from protein dynamics, encouraging the active search for allosteric regulators. Here, we discuss properties of several promising compounds that affect substrate gating and processing in antechambers, and interactions of the catalytic core with regulatory proteins. Critical Issues: Given the structural complexity of proteasome assemblies, it is a painstaking process to better understand their allosteric regulation and molecular dynamics. Here, we discuss the challenges and achievements in this field. We place special emphasis on the role of atomic force microscopy imaging in probing the allostery and dynamics of the proteasome, and in dissecting the mechanisms involving small-molecule allosteric regulators. Future Directions: New small-molecule allosteric regulators may become a next generation of drugs targeting the proteasome, which is critical to the development of new therapies in cancers and other diseases. Antioxid. Redox Signal. 21, 2286–2301. PMID:24410482
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Brailo, Vlaho; Firriolo, Francis John; Tanaka, Takako Imai; Varoni, Elena; Sykes, Rosemary; McCullough, Michael; Hua, Hong; Sklavounou, Alexandra; Jensen, Siri Beier; Lockhart, Peter B; Mattsson, Ulf; Jontell, Mats
2015-08-01
To assess the current scope and status of Oral Medicine-specific software (OMSS) utilized to support clinical care, research, and education in Oral Medicine and to propose a strategy for broader implementation of OMSS within the global Oral Medicine community. An invitation letter explaining the objectives was sent to the global Oral Medicine community. Respondents were interviewed to obtain information about different aspects of OMSS functionality. Ten OMSS tools were identified. Four were being used for clinical care, one was being used for research, two were being used for education, and three were multipurpose. Clinical software was being utilized as databases developed to integrate of different type of clinical information. Research software was designed to facilitate multicenter research. Educational software represented interactive, case-orientated technology designed for clinical training in Oral Medicine. Easy access to patient data was the most commonly reported advantage. Difficulty of use and poor integration with other software was the most commonly reported disadvantage. The OMSS presented in this paper demonstrate how information technology (IT) can have an impact on the quality of patient care, research, and education in the field of Oral Medicine. A strategy for broader implementation of OMSS is proposed. Copyright © 2015 Elsevier Inc. All rights reserved.
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Li, Ling-Yun; Xiong, Xiaorui R; Ibrahim, Leena A; Yuan, Wei; Tao, Huizhong W; Zhang, Li I
2015-07-01
Cortical inhibitory circuits play important roles in shaping sensory processing. In auditory cortex, however, functional properties of genetically identified inhibitory neurons are poorly characterized. By two-photon imaging-guided recordings, we specifically targeted 2 major types of cortical inhibitory neuron, parvalbumin (PV) and somatostatin (SOM) expressing neurons, in superficial layers of mouse auditory cortex. We found that PV cells exhibited broader tonal receptive fields with lower intensity thresholds and stronger tone-evoked spike responses compared with SOM neurons. The latter exhibited similar frequency selectivity as excitatory neurons. The broader/weaker frequency tuning of PV neurons was attributed to a broader range of synaptic inputs and stronger subthreshold responses elicited, which resulted in a higher efficiency in the conversion of input to output. In addition, onsets of both the input and spike responses of SOM neurons were significantly delayed compared with PV and excitatory cells. Our results suggest that PV and SOM neurons engage in auditory cortical circuits in different manners: while PV neurons may provide broadly tuned feedforward inhibition for a rapid control of ascending inputs to excitatory neurons, the delayed and more selective inhibition from SOM neurons may provide a specific modulation of feedback inputs on their distal dendrites. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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Merlaen, Britt; De Keyser, Ellen; Van Labeke, Marie-Christine
2018-01-01
The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry ( Fragaria x ananassa ). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa , few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.
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Code of Federal Regulations, 2014 CFR
2014-07-01
... refers to the coverage of general matters in broader environmental impact statements (such as national... regional or basinwide program statements or ultimately site-specific statements) incorporating by reference..., or policy environmental impact statement to a program, plan, or policy statement or analysis of...
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Code of Federal Regulations, 2011 CFR
2011-07-01
... refers to the coverage of general matters in broader environmental impact statements (such as national... regional or basinwide program statements or ultimately site-specific statements) incorporating by reference..., or policy environmental impact statement to a program, plan, or policy statement or analysis of...
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Code of Federal Regulations, 2012 CFR
2012-07-01
... refers to the coverage of general matters in broader environmental impact statements (such as national... regional or basinwide program statements or ultimately site-specific statements) incorporating by reference..., or policy environmental impact statement to a program, plan, or policy statement or analysis of...
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Code of Federal Regulations, 2013 CFR
2013-07-01
... refers to the coverage of general matters in broader environmental impact statements (such as national... regional or basinwide program statements or ultimately site-specific statements) incorporating by reference..., or policy environmental impact statement to a program, plan, or policy statement or analysis of...
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Code of Federal Regulations, 2010 CFR
2010-07-01
... refers to the coverage of general matters in broader environmental impact statements (such as national... regional or basinwide program statements or ultimately site-specific statements) incorporating by reference..., or policy environmental impact statement to a program, plan, or policy statement or analysis of...
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The Structure of Lombricine Kinase
Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S.
2011-01-01
Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. PMID:21212263
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Biochemical profiling in silico--predicting substrate specificities of large enzyme families.
Tyagi, Sadhna; Pleiss, Juergen
2006-06-25
A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.
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Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates
Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy
2012-01-01
Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900
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Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David
2009-01-01
Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088
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Bacillus subtilis spreads by surfing on waves of surfactant
Angelini, Thomas E.; Roper, Marcus; Kolter, Roberto; Weitz, David A.; Brenner, Michael P.
2009-01-01
The bacterium Bacillus subtilis produces the molecule surfactin, which is known to enhance the spreading of multicellular colonies on nutrient substrates by lowering the surface tension of the surrounding fluid, and to aid in the formation of aerial structures. Here we present experiments and a mathematical model that demonstrate how the differential accumulation rates induced by the geometry of the bacterial film give rise to surfactant waves. The spreading flux increases with increasing biofilm viscosity. Community associations are known to protect bacterial populations from environmental challenges such as predation, heat, or chemical stresses, and enable digestion of a broader range of nutritive sources. This study provides evidence of enhanced dispersal through cooperative motility, and points to nonintuitive methods for controlling the spread of biofilms. PMID:19826092
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Spin-polarized confined states in Ag films on Fe(110)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Moras, Paolo; Bihlmayer, G.; Vescovo, Elio
Spin- and angle-resolved photoemission spectroscopy of thin Ag(111) films on ferromagnetic Fe(110) shows a series of spin-polarized peaks. These features derive from Ag sp-bands, which form quantum well states and resonances due to confinement by a spin-dependent interface potential barrier. The spin-up states are broader and located at higher binding energy than the corresponding spin-down states at Gamma, although the differences attenuate near the Fermi level. The spin-down states display multiple gap openings, which interrupt their parabolic-like dispersion. As a result, first-principles calculations attribute these findings to the symmetry- and spin-selective hybridization of the Ag states with the exchange-split bandsmore » of the substrate.« less
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Spin-polarized confined states in Ag films on Fe(110)
Moras, Paolo; Bihlmayer, G.; Vescovo, Elio; ...
2017-11-16
Spin- and angle-resolved photoemission spectroscopy of thin Ag(111) films on ferromagnetic Fe(110) shows a series of spin-polarized peaks. These features derive from Ag sp-bands, which form quantum well states and resonances due to confinement by a spin-dependent interface potential barrier. The spin-up states are broader and located at higher binding energy than the corresponding spin-down states at Gamma, although the differences attenuate near the Fermi level. The spin-down states display multiple gap openings, which interrupt their parabolic-like dispersion. As a result, first-principles calculations attribute these findings to the symmetry- and spin-selective hybridization of the Ag states with the exchange-split bandsmore » of the substrate.« less
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Hybrid silica coarse wavelength-division multiplexer transmitter optical subassembly
NASA Astrophysics Data System (ADS)
An, Jun-Ming; Zhang, Jia-Shun; Wang, Liang-Liang; Zhu, Kaiwu; Sun, Bingli; Li, Yong; Hou, Jie; Li, Jian-Guang; Wu, Yuan-Da; Wang, Yue; Yin, Xiao-Jie
2018-01-01
Based on silica arrayed waveguide grating technology, a hybrid integrated transmitter optical subassembly was developed. Four direct-modulating distributed feedback lasers and four focusing microlenses were integrated to a coarse wavelength-division multiplexer (CWDM) on a CuW substrate. The four-channel silica-on-silicon CWDM was fabricated with 1.5% refractive index difference and 20-nm wavelength spacing. The experimental results showed that the output optical power was >3 mW with 45 mA of injection current, the slope efficiency was >0.0833 W/A, and the 3-dB bandwidth was broader than 18.15 GHz. The 1-dB compress points were higher than 18 and 15.8 dBm for frequency of 10 and 18 GHz, respectively.
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Kayombo, S; Mbwette, T S A; Katima, J H Y; Jorgensen, S E
2003-07-01
This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon. The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors. From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model. The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation. The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l. The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l. The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l. The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass). The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate. Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.
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Buryska, Tomas; Babkova, Petra; Vavra, Ondrej; Damborsky, Jiri; Prokop, Zbynek
2018-01-15
The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols. Copyright © 2018 American Society for Microbiology.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Abd El-Lateef, Hany M., E-mail: Hany_shubra@yahoo.co.uk; Khalaf, Mai M., E-mail: Mai_kha1@yahoo.com
This work reports the achievement of preparing of x% zirconia (ZrO{sub 2})–titania (TiO{sub 2}) composite coatings with different ZrO{sub 2} percent on the carbon steel by dipping substrates in sol–gel solutions. The prepared coated samples were investigated by various surface techniques including X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDAX). Open-circuit potential (OCP), potentiodynamic polarization, and electrochemical impedance spectroscopy (EIS) methods were employed to investigate the corrosion resistance of the coated carbon steel substrates in 1.0 M HCl solution at 50 °C. The data showed that, the corrosion protection property ismore » not always proportional to the percent of ZrO{sub 2}. It can be inferred that there is an optimum percent (10%ZrO{sub 2}) for beneficial effects of loading ZrO{sub 2} on the protection efficiency (98.70%), while higher loading percent of ZrO{sub 2} in the sol–gel coating leads to the formation of a fragile film with poor barrier properties. EDAX/SEM suggests that the metal surface was protected through coating with ZrO{sub 2}–TiO{sub 2} composite films. - Highlights: • Sol–gel TiO{sub 2} doped with ZrO{sub 2} films deposited on carbon steel substrate • XRD measurements of x wt.% ZrO{sub 2}–TiO{sub 2} showed the (101) peaks broader than that of TiO{sub 2}. • SEM results proved that, the cracking decreases with the number of layers. • The prepared films can improve the corrosion resistance of the carbon steel substrate. • 10%ZrO{sub 2} loading is the optimal percent for useful effects on the corrosion resistance.« less
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UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.
Xie, Youming; Varshavsky, Alexander
2002-12-01
The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.
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Spectrally based mapping of riverbed composition
Legleiter, Carl; Stegman, Tobin K.; Overstreet, Brandon T.
2016-01-01
Remote sensing methods provide an efficient means of characterizing fluvial systems. This study evaluated the potential to map riverbed composition based on in situ and/or remote measurements of reflectance. Field spectra and substrate photos from the Snake River, Wyoming, USA, were used to identify different sediment facies and degrees of algal development and to quantify their optical characteristics. We hypothesized that accounting for the effects of depth and water column attenuation to isolate the reflectance of the streambed would enhance distinctions among bottom types and facilitate substrate classification. A bottom reflectance retrieval algorithm adapted from coastal research yielded realistic spectra for the 450 to 700 nm range; but bottom reflectance-based substrate classifications, generated using a random forest technique, were no more accurate than classifications derived from above-water field spectra. Additional hypothesis testing indicated that a combination of reflectance magnitude (brightness) and indices of spectral shape provided the most accurate riverbed classifications. Convolving field spectra to the response functions of a multispectral satellite and a hyperspectral imaging system did not reduce classification accuracies, implying that high spectral resolution was not essential. Supervised classifications of algal density produced from hyperspectral data and an inferred bottom reflectance image were not highly accurate, but unsupervised classification of the bottom reflectance image revealed distinct spectrally based clusters, suggesting that such an image could provide additional river information. We attribute the failure of bottom reflectance retrieval to yield more reliable substrate maps to a latent correlation between depth and bottom type. Accounting for the effects of depth might have eliminated a key distinction among substrates and thus reduced discriminatory power. Although further, more systematic study across a broader range of fluvial environments is needed to substantiate our initial results, this case study suggests that bed composition in shallow, clear-flowing rivers potentially could be mapped remotely.
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NASA Technical Reports Server (NTRS)
Woods, Lawrence M.; Kalla, Ajay; Ribelin, Rosine
2007-01-01
Thin-film photovoltaics (TFPV) on lightweight and flexible substrates offer the potential for very high solar array specific power (W/kg). ITN Energy Systems, Inc. (ITN) is developing flexible TFPV blanket technology that has potential for specific power greater than 2000 W/kg (including space coatings) that could result in solar array specific power between 150 and 500 W/kg, depending on array size, when mated with mechanical support structures specifically designed to take advantage of the lightweight and flexible substrates.(1) This level of specific power would far exceed the current state of the art for spacecraft PV power generation, and meet the needs for future spacecraft missions.(2) Furthermore the high specific power would also enable unmanned aircraft applications and balloon or high-altitude airship (HAA) applications, in addition to modular and quick deploying tents for surface assets or lunar base power, as a result of the high power density (W/sq m) and ability to be integrated into the balloon, HAA or tent fabric. ITN plans to achieve the high specific power by developing single-junction and two-terminal monolithic tandem-junction PV cells using thin-films of high-efficiency and radiation resistant CuInSe2 (CIS) partnered with bandgap-tunable CIS-alloys with Ga (CIGS) or Al (CIAS) on novel lightweight and flexible substrates. Of the various thin-film technologies, single-junction and radiation resistant CIS and associated alloys with gallium, aluminum and sulfur have achieved the highest levels of TFPV device performance, with the best efficiency reaching 19.5% under AM1.5 illumination conditions and on thick glass substrates.(3) Thus, it is anticipated that single- and tandem-junction devices with flexible substrates and based on CIS and related alloys will achieve the highest levels of thin-film space and HAA solar array performance.
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ERIC Educational Resources Information Center
Anguiano, Claudia A.; Nájera, Lourdes Gutiérrez
2015-01-01
This article examines the problematic labels of "deserving" and "undeserving" within a broader context of undocumented immigration. Specifically, we interrogate the categorization of "deservingness" that imposes distinctions between "good" versus "bad" immigrants. We demonstrate these categories…
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Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark
2017-01-24
Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.
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Kinetic modeling of lactic acid production from batch submerged fermentation of cheese whey
DOE Office of Scientific and Technical Information (OSTI.GOV)
Tango, M.S.A.; Ghaly, A.E.
1999-12-01
A kinetic model for the production of lactic acid through batch submerged fermentation of cheese whey using Lactobacillus helveticus was developed. The model accounts for the effect of substrate limitation, substrate inhibition, lactic acid inhibition, maintenance energy and cell death on the cell growth, substrate utilization, and lactic acid production during the fermentation process. The model was evaluated using experimental data from Tango and Ghaly (1999). The predicted results obtained from the model compared well with experimental (R{sup 2} = 0.92--0.98). The model was also used to investigate the effect of the initial substrate concentration on the lag period, fermentationmore » time, specific growth rate, and cell productivity during batch fermentation. The maximum specific growth rate ({micro}{sub m}), the saturation constant (K{sub S}), the substrate inhibition constant (K{sub IS}), and the lactic acid inhibition constant (K{sub IP}) were found to be 0.25h{sup {minus}1}, 0.9 g/L, 250.0 g/L, and 60.0 g/L, respectively. High initial lactose concentration in cheese whey reduced both the specific growth rate and substrate utilization rate due to the substrate inhibition phenomenon. The maximum lactic acid production occurred at about 100 g/L initial lactose concentration after 40 h of fermentation. The maximum lactic acid concentration above which Lactobacillus helveticus did not grow was found to be 80.0 g/L.« less
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Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.
2001-01-01
We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors. PMID:11533208
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Framework for ecological monitoring on lands of Alaska National Wildlife Refuges and their partners
Woodward, Andrea; Beever, Erik A.
2010-01-01
National Wildlife Refuges in Alaska and throughout the U.S. have begun developing a spatially comprehensive monitoring program to inform management decisions, and to provide data to broader research projects. In an era of unprecedented rates of climate change, monitoring is essential to detecting, understanding, communicating and mitigating climate-change effects on refuge and other resources under the protection of U.S. Fish and Wildlife Service, and requires monitoring results to address spatial scales broader than individual refuges. This document provides guidance for building a monitoring program for refuges in Alaska that meets refuge-specific management needs while also allowing synthesis and summary of ecological conditions at the ecoregional and statewide spatial scales.
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Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre
2011-02-01
A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.
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Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike
2014-09-26
Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP
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
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Begic, Sanela; Worobec, Elizabeth A
2008-05-01
Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.
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Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane
2013-01-01
The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969
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Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane
2013-01-01
The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.
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Basova, I N; Iagodina, O V
2012-01-01
Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Tremblay,L.; Dunaway-Mariano, D.; Allen, K.
2006-01-01
The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia colimore » K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.« less
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Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da
2013-01-01
Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.
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Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.
Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung
2015-06-01
N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.
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The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.
2012-05-29
Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR ofmore » arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.« less
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ERIC Educational Resources Information Center
Carr, Elizabeth
2016-01-01
Planning to deliver the new GCSE [General Certificate of Secondary Education] specifications presents a challenge and an opportunity to any history department, whatever their previous specification. The sweep of history that students will now study at GCSE is much broader than "Modern World" departments are used to; including a medieval…
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Identifying Neurobiological Markers of the Broader Autism Phenotype
2015-01-01
the BAP. Control participants matched for age, sex and IQ with no history of neurologic, psychiatric or hearing disorders have also been successfully...were then required to imitate the speaker’s emotional expression as accurately as possible. Gender -specific MAV stimuli were presented to each...Participants completed a total of 35 randomized trials, over which five gender -specific MAV stimuli were presented from each of the six emotions and
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A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues
Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.
2012-01-01
The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yao, Jianzhuang; Xu, Qin; Chen, Feng
2010-01-01
Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-l-methionine (AdoMet) to 4-hydroxybenzoatemore » in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.« less
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Bölte, Sven; Poustka, Fritz
2006-06-01
The objective of this study was to investigate the tendency for local processing style ('weak central coherence') and executive dysfunction in parents of subjects with an autism spectrum disorder (ASD) compared with parents of individuals with early onset schizophrenia (EOS) and mental retardation (MR). Sixty-two parents of subjects with ASD, 36 parents of subjects with EOS and 30 parents of subjects with MR were examined. Data on two scales indicative of local visual processing (Embedded Figures Test, Block Design) and on three executive function tests (Wisconsin Card Sorting Test, Tower of Hanoi, Trailmaking Test) were collected for all participants. Parents of subjects with ASD performed significantly faster on the Embedded Figures Test compared with both control samples. No other substantial group differences were observed. The findings indicate that an increased tendency for local processing in terms of visual disembedding could be a relatively specific core feature of the broader cognitive phenotype of autism in parents.
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Student attraction to engineering through flexibility and breadth in the curriculum
NASA Astrophysics Data System (ADS)
Alpay, E.
2013-03-01
Several European universities provide entry to general engineering studies prior to degree specialisation. The potential advantages of such entry include the provision of a broader foundation in engineering fundamentals, the option for students to defer specialisation until a greater awareness of the different engineering disciplines and the preparation of students for a more versatile career. In this paper, the attractiveness of general engineering (specifically in the first year of study) is explored through a national (UK) survey on pre-university students. Attention is given to gauging student enthusiasm for flexibility in engineering specialisation, combined degree options and exposure to other non-technical courses. The findings indicate that a general engineering programme is highly attractive to students who are currently considering an engineering degree. The programme is also attractive to some students who had previously not considered engineering. For both sets of students, the desire for education on broader topics is indicated, specifically in areas of leadership, teamwork and business skills, and more generally self-awareness and personal development.
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Masuda, Nobuhisa; Sakagawa, Eiko; Ohya, Satoshi; Gotoh, Naomasa; Tsujimoto, Hideto; Nishino, Takeshi
2000-01-01
To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously. PMID:11083635
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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.
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[Substrate specifity in Amoeba proteus].
Sopina, V A
2006-01-01
Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.
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NASA Technical Reports Server (NTRS)
Lazcano, A.; Diaz-Villagomez, E.; Mills, T.; Oro, J.
1995-01-01
The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called 'patchwork' theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.
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MnO2-Based Electrochemical Supercapacitors on Flexible Carbon Substrates
NASA Astrophysics Data System (ADS)
Tadjer, Marko J.; Mastro, Michael A.; Rojo, José M.; Mojena, Alberto Boscá; Calle, Fernando; Kub, Francis J.; Eddy, Charles R.
2014-04-01
Manganese dioxide films were grown on large area flexible carbon aerogel substrates. Characterization by x-ray diffraction confirmed α-MnO2 growth. Three types of films were compared as a function of hexamethylenetetramine (HMTA) concentration during growth. The highest concentration of HM TA produced MnO2 flower-like films, as observed by scanning electron microscopy, whose thickness and surface coverage lead to both a higher specific capacitance and higher series resistance. Specific capacitance was measured to be 64 F/g using a galvanostatic setup, compared to the 47 F/g-specific capacitance of the carbon aerogel substrate. Such supercapacitor devices can be fabricated on large area sheets of carbon aerogel to achieve high total capacitance.
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Cruz-Cárdenas, Carlos I.; Miranda-Ham, María L.; Castro-Concha, Lizbeth A.; Ku-Cauich, José R.; Vergauwen, Rudy; Reijnders, Timmy; Van den Ende, Wim; Escobedo-GraciaMedrano, Rosa M.
2015-01-01
The water soluble carbohydrates (WSC) glucose, fructose, and sucrose are well-known to the great public, but fructans represent another type of WSC that deserves more attention given their prebiotic and immunomodulatory properties in the food context. Although the occurrence of inulin-type fructo-oligosaccharides (FOS) was proposed in the fruit of some banana accessions, little or no information is available neither on the exact identity of the fructan species, nor on the fructan content in different parts of banana plants and among a broader array of banana cultivars. Here, we investigated the WSC composition in leaves, pulp of ripe fruits and rhizomes from mature banana plants of 11 accessions (I to XI), including both cultivated varieties and wild Musa species. High performance anion exchange chromatography with integrated pulsed amperometric detection (HPAEC-IPAD) showed the presence of 1-kestotriose [GF2], inulobiose [F2], inulotriose [F3], 6-kestotriose and 6G-kestotriose (neokestose) fructan species in the pulp of mature fruits of different accessions, but the absence of 1,1-nystose and 1,1,1 kestopentaose and higher degree of polymerization (DP) inulin-type fructans. This fructan fingerprint points at the presence of one or more invertases that are able to use fructose and sucrose as alternative acceptor substrates. Quantification of glucose, fructose, sucrose and 1-kestotriose and principal component analysis (PCA) identified related banana groups, based on their specific WSC profiles. These data provide new insights in the biochemical diversity of wild and cultivated bananas, and shed light on potential roles that fructans may fulfill across species, during plant development and adaptation to changing environments. Furthermore, the promiscuous behavior of banana fruit invertases (sucrose and fructose as acceptor substrates besides water) provides a new avenue to boost future work on structure-function relationships on these enzymes, potentially leading to the development of genuine banana fructosyltransferases that are able to increase fructan content in banana fruits. PMID:26106398
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NASA Astrophysics Data System (ADS)
Lu, Yu-Peng; Xiao, Gui-Yong; Li, Shi-Tong; Sun, Rui-Xue; Li, Mu-Sen
2006-01-01
The microstructural inhomogeneity in the plasma-sprayed hydroxyapatite (HA) coatings was characterized by using electron probe microanalyser (EPMA). A simple and artful method was developed to detect the interface characteristics. All the samples for observation were ground and polished along the direction parallel to the coating surfaces. The BSE images directly and clearly showed the inhomogeneity in the as-sprayed coatings with the amorphous regions being bright gray and crystalline regions being dark gray. X-ray diffractometer (XRD) patterns indicated that after immersion in deionized water for 20 days, bone-like apatite and α-Ca 2P 2O 7 precipitated on the polished surfaces of the as-sprayed HA coatings. The post-heat treatment could eliminate the microstructural inhomogeneity in the coatings. Only β-Ca 2P 2O 7 precipitated on the surfaces of the heat-treated HA coatings. The immersed samples were re-polished till tiny substrate was bared to investigate the effect of immersion on interface. It was shown that the immersion decreased the cohesive strength of the as-sprayed coatings. There were more and broader cracks in the splats that came into contact with the substrate and amorphous phase increased toward the coating-substrate interface. Post-heat treatment was proved to reduce the peeling off of coating during re-polishing operation. It was proposed that the distributions of amorphous phase and cracks in as-sprayed coatings are detrimental to coating properties and should be modified through improving the plasma spraying processing.
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Jarling, René; Kühner, Simon; Basílio Janke, Eline; Gruner, Andrea; Drozdowska, Marta; Golding, Bernard T.; Rabus, Ralf; Wilkes, Heinz
2015-01-01
Anaerobic metabolism of hydrocarbons proceeds either via addition to fumarate or by hydroxylation in various microorganisms, e.g., sulfate-reducing or denitrifying bacteria, which are specialized in utilizing n-alkanes or alkylbenzenes as growth substrates. General pathways for carbon assimilation and energy gain have been elucidated for a limited number of possible substrates. In this work the metabolic activity of 11 bacterial strains during anaerobic growth with crude oil was investigated and compared with the metabolite patterns appearing during anaerobic growth with more than 40 different hydrocarbons supplied as binary mixtures. We show that the range of co-metabolically formed alkyl- and arylalkyl-succinates is much broader in n-alkane than in alkylbenzene utilizers. The structures and stereochemistry of these products are resolved. Furthermore, we demonstrate that anaerobic hydroxylation of alkylbenzenes does not only occur in denitrifiers but also in sulfate reducers. We propose that these processes play a role in detoxification under conditions of solvent stress. The thermophilic sulfate-reducing strain TD3 is shown to produce n-alkylsuccinates, which are suggested not to derive from terminal activation of n-alkanes, but rather to represent intermediates of a metabolic pathway short-cutting fumarate regeneration by reverse action of succinate synthase. The outcomes of this study provide a basis for geochemically tracing such processes in natural habitats and contribute to an improved understanding of microbial activity in hydrocarbon-rich anoxic environments. PMID:26441848
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Measurements of Elastic Moduli of Silicone Gel Substrates with a Microfluidic Device
Gutierrez, Edgar; Groisman, Alex
2011-01-01
Thin layers of gels with mechanical properties mimicking animal tissues are widely used to study the rigidity sensing of adherent animal cells and to measure forces applied by cells to their substrate with traction force microscopy. The gels are usually based on polyacrylamide and their elastic modulus is measured with an atomic force microscope (AFM). Here we present a simple microfluidic device that generates high shear stresses in a laminar flow above a gel-coated substrate and apply the device to gels with elastic moduli in a range from 0.4 to 300 kPa that are all prepared by mixing two components of a transparent commercial silicone Sylgard 184. The elastic modulus is measured by tracking beads on the gel surface under a wide-field fluorescence microscope without any other specialized equipment. The measurements have small and simple to estimate errors and their results are confirmed by conventional tensile tests. A master curve is obtained relating the mixing ratios of the two components of Sylgard 184 with the resulting elastic moduli of the gels. The rigidity of the silicone gels is less susceptible to effects from drying, swelling, and aging than polyacrylamide gels and can be easily coated with fluorescent tracer particles and with molecules promoting cellular adhesion. This work can lead to broader use of silicone gels in the cell biology laboratory and to improved repeatability and accuracy of cell traction force microscopy and rigidity sensing experiments. PMID:21980487
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Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.
Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz
2009-08-25
Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.
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Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan
2015-12-11
Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Reactive ion etched substrates and methods of making and using
Rucker, Victor C [San Francisco, CA; Shediac, Rene [Oakland, CA; Simmons, Blake A [San Francisco, CA; Havenstrite, Karen L [New York, NY
2007-08-07
Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.
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Substrate Material for Holographic Emulsions Utilizing Fluorinated Polyimide Film
NASA Technical Reports Server (NTRS)
Gierow, Paul A. (Inventor); Clayton, William R. (Inventor); St.Clair, Anne K. (Inventor)
1999-01-01
A new holographic substrate utilizing flexible. optically transparent fluorinated polyimides. Said substrates have 0 extremely low birefringence which results in a high signal to noise ratio in subsequent holograms. Specific examples of said fluorinated polyimides include 6FDA+APB and 6FDA+4BDAF.
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Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.
Andersen, Rokhsana J; Brask, Jesper
2016-06-01
Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.
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Bateman, Thomas S.; Hess, Andrew M.
2015-01-01
Scientific journal publications, and their contributions to knowledge, can be described by their depth (specialized, domain-specific knowledge extensions) and breadth (topical scope, including spanning multiple knowledge domains). Toward generating hypotheses about how scientists’ personal dispositions would uniquely predict deeper vs. broader contributions to the literature, we assumed that conducting broader studies is generally viewed as less attractive (e.g., riskier) than conducting deeper studies. Study 1 then supported our assumptions: the scientists surveyed considered a hypothetical broader study, compared with an otherwise-comparable deeper study, to be riskier, a less-significant opportunity, and of lower potential importance; they further reported being less likely to pursue it and, in a forced choice, most chose to work on the deeper study. In Study 2, questionnaire measures of medical researchers’ personal dispositions and 10 y of PubMed data indicating their publications’ topical coverage revealed how dispositions differentially predict depth vs. breadth. Competitiveness predicted depth positively, whereas conscientiousness predicted breadth negatively. Performance goal orientation predicted depth but not breadth, and learning goal orientation contrastingly predicted breadth but not depth. Openness to experience positively predicted both depth and breadth. Exploratory work behavior (the converse of applying and exploiting one’s current knowledge) predicted breadth positively and depth negatively. Thus, this research distinguishes depth and breadth of published knowledge contributions, and provides new insights into how scientists’ personal dispositions influence research processes and products. PMID:25733900
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Bateman, Thomas S; Hess, Andrew M
2015-03-24
Scientific journal publications, and their contributions to knowledge, can be described by their depth (specialized, domain-specific knowledge extensions) and breadth (topical scope, including spanning multiple knowledge domains). Toward generating hypotheses about how scientists' personal dispositions would uniquely predict deeper vs. broader contributions to the literature, we assumed that conducting broader studies is generally viewed as less attractive (e.g., riskier) than conducting deeper studies. Study 1 then supported our assumptions: the scientists surveyed considered a hypothetical broader study, compared with an otherwise-comparable deeper study, to be riskier, a less-significant opportunity, and of lower potential importance; they further reported being less likely to pursue it and, in a forced choice, most chose to work on the deeper study. In Study 2, questionnaire measures of medical researchers' personal dispositions and 10 y of PubMed data indicating their publications' topical coverage revealed how dispositions differentially predict depth vs. breadth. Competitiveness predicted depth positively, whereas conscientiousness predicted breadth negatively. Performance goal orientation predicted depth but not breadth, and learning goal orientation contrastingly predicted breadth but not depth. Openness to experience positively predicted both depth and breadth. Exploratory work behavior (the converse of applying and exploiting one's current knowledge) predicted breadth positively and depth negatively. Thus, this research distinguishes depth and breadth of published knowledge contributions, and provides new insights into how scientists' personal dispositions influence research processes and products.
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Mizuguchi, Nobuaki; Uehara, Shintaro; Hirose, Satoshi; Yamamoto, Shinji; Naito, Eiichi
2016-01-01
Motor performance fluctuates trial by trial even in a well-trained motor skill. Here we show neural substrates underlying such behavioral fluctuation in humans. We first scanned brain activity with functional magnetic resonance imaging while healthy participants repeatedly performed a 10 s skillful sequential finger-tapping task. Before starting the experiment, the participants had completed intensive training. We evaluated task performance per trial (number of correct sequences in 10 s) and depicted brain regions where the activity changes in association with the fluctuation of the task performance across trials. We found that the activity in a broader range of frontoparietocerebellar network, including the bilateral dorsolateral prefrontal cortex (DLPFC), anterior cingulate and anterior insular cortices, and left cerebellar hemisphere, was negatively correlated with the task performance. We further showed in another transcranial direct current stimulation (tDCS) experiment that task performance deteriorated, when we applied anodal tDCS to the right DLPFC. These results indicate that fluctuation of brain activity in the nonmotor frontoparietocerebellar network may underlie trial-by-trial performance variability even in a well-trained motor skill, and its neuromodulation with tDCS may affect the task performance.
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Foot pressure distributions during walking in African elephants (Loxodonta africana)
Pataky, Todd C.; Day, Madeleine; Hensman, Michael C.; Hensman, Sean; Hutchinson, John R.; Clemente, Christofer J.
2016-01-01
Elephants, the largest living land mammals, have evolved a specialized foot morphology to help reduce locomotor pressures while supporting their large body mass. Peak pressures that could cause tissue damage are mitigated passively by the anatomy of elephants' feet, yet this mechanism does not seem to work well for some captive animals. This study tests how foot pressures vary among African and Asian elephants from habitats where natural substrates predominate but where foot care protocols differ. Variations in pressure patterns might be related to differences in husbandry, including but not limited to trimming and the substrates that elephants typically stand and move on. Both species' samples exhibited the highest concentration of peak pressures on the lateral digits of their feet (which tend to develop more disease in elephants) and lower pressures around the heel. The trajectories of the foot's centre of pressure were also similar, confirming that when walking at similar speeds, both species load their feet laterally at impact and then shift their weight medially throughout the step until toe-off. Overall, we found evidence of variations in foot pressure patterns that might be attributable to husbandry and other causes, deserving further examination using broader, more comparable samples. PMID:27853539
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Low Temperature Activation of Supported Metathesis Catalysts by Organosilicon Reducing Agents
2016-01-01
Alkene metathesis is a widely and increasingly used reaction in academia and industry because of its efficiency in terms of atom economy and its wide applicability. This reaction is notably responsible for the production of several million tons of propene annually. Such industrial processes rely on inexpensive silica-supported tungsten oxide catalysts, which operate at high temperatures (>350 °C), in contrast with the mild room temperature reaction conditions typically used with the corresponding molecular alkene metathesis homogeneous catalysts. This large difference in the temperature requirements is generally thought to arise from the difficulty in generating active sites (carbenes or metallacyclobutanes) in the classical metal oxide catalysts and prevents broader applicability, notably with functionalized substrates. We report here a low temperature activation process of well-defined metal oxo surface species using organosilicon reductants, which generate a large amount of active species at only 70 °C (0.6 active sites/W). This high activity at low temperature broadens the scope of these catalysts to functionalized substrates. This activation process can also be applied to classical industrial catalysts. We provide evidence for the formation of a metallacyclopentane intermediate and propose how the active species are formed. PMID:27610418
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Handa, Shinya; Gnanadesikan, Vijay; Matsunaga, Shigeki; Shibasaki, Masakatsu
2010-04-07
The full details of a catalytic asymmetric syn-selective nitro-Mannich reaction promoted by heterobimetallic Cu/Sm/dinucleating Schiff base complexes are described, demonstrating the effectiveness of the heterobimetallic transition metal/rare earth metal bifunctional catalysis. The first-generation system prepared from Cu(OAc)(2)/Sm(O-iPr)(3)/Schiff base 1a = 1:1:1 with an achiral phenol additive was partially successful for achieving the syn-selective catalytic asymmetric nitro-Mannich reaction. The substrate scope and limitations of the first-generation system remained problematic. After mechanistic studies on the catalyst prepared from Sm(O-iPr)(3), we reoptimized the catalyst preparation method, and a catalyst derived from Sm(5)O(O-iPr)(13) showed broader substrate generality as well as higher reactivity and stereoselectivity compared to Sm(O-iPr)(3). The optimal system with Sm(5)O(O-iPr)(13) was applicable to various aromatic, heteroaromatic, and isomerizable aliphatic N-Boc imines, giving products in 66-99% ee and syn/anti = >20:1-13:1. Catalytic asymmetric synthesis of nemonapride is also demonstrated using the catalyst derived from Sm(5)O(O-iPr)(13).
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Prueksaritanont, T; Tatosian, D A; Chu, X; Railkar, R; Evers, R; Chavez-Eng, C; Lutz, R; Zeng, W; Yabut, J; Chan, G H; Cai, X; Latham, A H; Hehman, J; Stypinski, D; Brejda, J; Zhou, C; Thornton, B; Bateman, K P; Fraser, I; Stoch, S A
2017-04-01
A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions. © 2016 American Society for Clinical Pharmacology and Therapeutics.
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Regulation of Proteolysis by Human Deubiquitinating Enzymes
Eletr, Ziad M.; Wilkinson, Keith D.
2013-01-01
The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989
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Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till
2004-01-01
The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930
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CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.
Fedoreyeva, L I; Vanyushin, B F
2011-06-01
Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.
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Shanklin, John; Cahoon, Edgar B.
2004-02-03
The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.
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The effect of substrate composition and storage time on urine specific gravity in dogs.
Steinberg, E; Drobatz, K; Aronson, L
2009-10-01
The purpose of this study is to evaluate the effects of substrate composition and storage time on urine specific gravity in dogs. A descriptive cohort study of 15 dogs. The urine specific gravity of free catch urine samples was analysed during a 5-hour time period using three separate storage methods; a closed syringe, a diaper pad and non-absorbable cat litter. The urine specific gravity increased over time in all three substrates. The syringe sample had the least change from baseline and the diaper sample had the greatest change from baseline. The urine specific gravity for the litter and diaper samples had a statistically significant increase from the 1-hour to the 5-hour time point. The urine specific gravity from canine urine stored either on a diaper or in a non-absorbable litter increased over time. Although the change was found to be statistically significant over the 5-hour study period it is unlikely to be clinically significant.
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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.
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Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi
2015-01-01
In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.
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Cvejic, Rachael C; Eagleson, Claire; Weise, Janelle; Davies, Kimberley; Hopwood, Malcolm; Jenkins, Kym; Trollor, Julian N
2018-06-01
To describe the characteristics of psychiatrists working in the area of intellectual and developmental disability mental health (IDDMH) across Australia and New Zealand. A secondary analysis of data collected by the Royal Australian and New Zealand College of Psychiatrists 2014 workforce survey. Characteristics of the IDDMH workforce ( n=146 psychiatrists) were compared with those of the broader psychiatry workforce ( n=1050 psychiatrists). The IDDMH workforce were more likely than the broader psychiatry workforce to be working across both the public and private health sectors, be engaged in outreach work, endorse specialty practice areas pertinent to IDDMH, treat younger patients and work more clinical hours per week. Part-time status and retirement plans of the IDDMH workforce matched those of the broader psychiatry workforce. While some elements of the IDDMH workforce profile suggest this workforce is tailored to the needs of the population, the potential shortage of IDDMH psychiatrists highlights the need for the development of a specific training programme and pathway in this area.
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The "Doublethink" of Data: Educational Performativity and the Field of Schooling Practices
ERIC Educational Resources Information Center
Hardy, Ian; Lewis, Steven
2017-01-01
This article provides insights into teachers' and school administrators' responses to the current "fetishisation" of school performance data in Australian schooling. Specifically, the research investigates the accountability practices that emerged in a Queensland metropolitan primary school in response to this broader focus upon…
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Key Actor Perceptions of Athletics Strategy
ERIC Educational Resources Information Center
Collins, William Thomas
2012-01-01
Athletics strategy refers to specific initiatives within the intercollegiate athletics program that are designed to meet the broader strategic goals of a post-secondary institution. This case focused on athletics strategy decisions that were enacted within the context of organizational change as Cartwright College, a pseudonym, transitioned from a…
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The Alzheimerization of Aging.
ERIC Educational Resources Information Center
Adelman, Richard C.
1995-01-01
The National Institute on Aging (NIA) invests a disproportionately large share of its resources in research on Alzheimer's Disease at the expense of other interests of the broader scientific community in gerontology. Complex social forces that continue to shape this outcome embrace discipline-specific traditions of science advocacy, as well as…
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The Faculty Role in Budgeting.
ERIC Educational Resources Information Center
Meisinger, Richard J., Jr.; Dubeck, Leroy W.
1984-01-01
Specific roles faculty members can play in their institution's budget processes are discussed, and the general ends served by budgets are identified. Each of the dimensions of institutional character (e.g., size, mission) determines the ways in which participants in budgeting will interact. For example, broader faculty participation in budgeting…
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Computer graphics testbed to simulate and test vision systems for space applications
NASA Technical Reports Server (NTRS)
Cheatham, John B.
1991-01-01
Research activity has shifted from computer graphics and vision systems to the broader scope of applying concepts of artificial intelligence to robotics. Specifically, the research is directed toward developing Artificial Neural Networks, Expert Systems, and Laser Imaging Techniques for Autonomous Space Robots.
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He, Yong; Gao, Yang; Zhang, Cuiping; Chen, Chuansheng; Bi, Suyu; Yang, Pin; Wang, Yiwen; Wang, Wenjing
2017-01-01
Chinese calligraphic handwriting (CCH) is a traditional art form that requires high levels of concentration and motor control. Previous research has linked short-term training in CCH to improvements in attention and memory. Little is known about the potential impacts of long-term CCH practice on a broader array of executive functions and their potential neural substrates. In this cross-sectional study, we recruited 36 practitioners with at least 5 years of CCH experience and 50 control subjects with no more than one month of CCH practice and investigated their differences in the three components of executive functions (i.e., shifting, updating, and inhibition). Valid resting-state fMRI data were collected from 31 CCH and 40 control participants. Compared with the controls, CCH individuals showed better updating (as measured by the Corsi Block Test) and inhibition (as measured by the Stroop Word-Color Test), but the two groups did not differ in shifting (as measured by a cue-target task). The CCH group showed stronger resting-state functional connectivity (RSFC) than the control group in brain areas involved in updating and inhibition. These results suggested that long-term CCH training may be associated with improvements in specific aspects of executive functions and strengthened neural networks in related brain regions. PMID:28129407
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Hargrove, Tatiana Y.; Friggeri, Laura; Wawrzak, Zdzislaw; Sivakumaran, Suneethi; Yazlovitskaya, Eugenia M.; Hiebert, Scott W.; Guengerich, F. Peter; Waterman, Michael R.; Lepesheva, Galina I.
2016-01-01
Rapidly multiplying cancer cells synthesize greater amounts of cholesterol to build their membranes. Cholesterol-lowering drugs (statins) are currently in clinical trials for anticancer chemotherapy. However, given at higher doses, statins cause serious side effects by inhibiting the formation of other biologically important molecules derived from mevalonate. Sterol 14α-demethylase (CYP51), which acts 10 steps downstream, is potentially a more specific drug target because this portion of the pathway is fully committed to cholesterol production. However, screening a variety of commercial and experimental inhibitors of microbial CYP51 orthologs revealed that most of them (including all clinical antifungals) weakly inhibit human CYP51 activity, even if they display high apparent spectral binding affinity. Only one relatively potent compound, (R)-N-(1-(3,4′-difluorobiphenyl-4-yl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadiazol-2-yl)benzamide (VFV), was identified. VFV has been further tested in cellular experiments and found to decrease proliferation of different cancer cell types. The crystal structures of human CYP51-VFV complexes (2.0 and 2.5 Å) both display a 2:1 inhibitor/enzyme stoichiometry, provide molecular insights regarding a broader substrate profile, faster catalysis, and weaker susceptibility of human CYP51 to inhibition, and outline directions for the development of more potent inhibitors. PMID:27313059
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Natalia, Agnes; Kristiani, Lidya; Kim, Hyung Kwoun
2014-10-01
Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was 60°C, which was 20°C higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to 40°C and in a pH range of 5.0- 10.0, whereas soluble K80 maintained its activity up to 35°C and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.
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Chen, Wen; He, Yong; Gao, Yang; Zhang, Cuiping; Chen, Chuansheng; Bi, Suyu; Yang, Pin; Wang, Yiwen; Wang, Wenjing
2017-01-01
Chinese calligraphic handwriting (CCH) is a traditional art form that requires high levels of concentration and motor control. Previous research has linked short-term training in CCH to improvements in attention and memory. Little is known about the potential impacts of long-term CCH practice on a broader array of executive functions and their potential neural substrates. In this cross-sectional study, we recruited 36 practitioners with at least 5 years of CCH experience and 50 control subjects with no more than one month of CCH practice and investigated their differences in the three components of executive functions (i.e., shifting, updating, and inhibition). Valid resting-state fMRI data were collected from 31 CCH and 40 control participants. Compared with the controls, CCH individuals showed better updating (as measured by the Corsi Block Test) and inhibition (as measured by the Stroop Word-Color Test), but the two groups did not differ in shifting (as measured by a cue-target task). The CCH group showed stronger resting-state functional connectivity (RSFC) than the control group in brain areas involved in updating and inhibition. These results suggested that long-term CCH training may be associated with improvements in specific aspects of executive functions and strengthened neural networks in related brain regions.
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Liland, Nina S.; Biancarosa, Irene; Araujo, Pedro; Biemans, Daan; Bruckner, Christian G.; Waagbø, Rune; Torstensen, Bente E.
2017-01-01
Black soldier fly (Hermetia illucens) larvae are a promising source of protein and lipid for animal feeds. The nutritional composition of the BSF larvae depend partly on the composition of the feeding medium. The BSF lipid profile in part mimics the feeding media lipid profile, and micronutrients, like minerals and vitamins, can readily accumulate in black soldier fly larvae. However, investigative studies on bioconversion and accumulation of nutrients from media to black soldier fly larvae are scarce. Here we show that inclusion of the brown algae Ascophyllum nodosum in the substrate for black soldier fly larvae can introduce valuable nutrients, commonly associated with the marine environment, into the larvae. The omega-3 fatty acid eicosapentaenoic acid (20:5n-3), iodine and vitamin E concentrations increased in the larvae when more seaweed was included in the diet. When the feeding media consisted of more than 50% seaweed, the larvae experienced poorer growth, lower nutrient retention and lower lipid levels, compared to a pure plant based feeding medium. Our results confirm the plasticity of the nutritional make-up of black soldier fly larvae, allowing it to accumulate both lipid- and water-soluble compounds. A broader understanding of the effect of the composition of the feeding media on the larvae composition can help to tailor black soldier fly larvae into a nutrient profile more suited for specific feed or food purposes. PMID:28837591
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NASA Astrophysics Data System (ADS)
Cornet, Carme; Gili, Josep-Maria
1993-11-01
The vertical distribution and migratory behaviour of hyperiid amphipods were studied in a series of tows carried out during a 48-h sampling period at an oceanic station at the northern edge of the Benguela System during a major penetration by Angola Current waters. A total of 49 species of hyperiid amphipods were collected; of these, Tetrathyrus forcipatus was the most abundant, with densities greater than two individuals per 10 m 3. Vibilia armata, Lestrigonus latissimus, L. bengalensis and Paratyphis promontorii were also highly abundant. During the sampling period most species were concentrated in the uppermost 40 m of the water column, though in other regions the vertical distribution of these same species has been reported to be broader. Only a few species were able to migrate through the thermocline. We hypothesize that both the non-migratory behaviour and the aggregation of individuals and species were caused by two primary factors: the existence of a strong thermocline, which hindered the transit of species to deeper layers, and abundant concentrations of gelatinous zooplankton above the thermocline. Hyperiids and the gelatinous zooplankton, particularly medusae and siphonophores, exhibited a close association during the sampling period, suggesting that hyperiids are able to partition their habitat by using the different medusan and siphonophoran species as specific substrates, thereby reducing interspecific competition.
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Orsolini, Laura; Papanti, Gabriele Duccio; De Berardis, Domenico; Guirguis, Amira; Corkery, John Martin; Schifano, Fabrizio
2017-01-01
Hallucinogen-persisting perception disorder (HPPD) is a syndrome characterized by prolonged or reoccurring perceptual symptoms, reminiscent of acute hallucinogen effects. HPPD was associated with a broader range of LSD (lysergic acid diethylamide)-like substances, cannabis, methylenedioxymethamphetamine (MDMA), psilocybin, mescaline, and psychostimulants. The recent emergence of novel psychoactive substances (NPS) posed a critical concern regarding the new onset of psychiatric symptoms/syndromes, including cases of HPPD. Symptomatology mainly comprises visual disorders (i.e., geometric pseudo-hallucinations, haloes, flashes of colors/lights, motion-perception deficits, afterimages, micropsia, more acute awareness of floaters, etc.), even though depressive symptoms and thought disorders may be comorbidly present. Although HPPD was first described in 1954, it was just established as a fully syndrome in 2000, with the revised fourth version of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR). HPPD neural substrates, risk factors, and aetiopathogenesys still largely remain unknown and under investigation, and many questions about its pharmacological targets remain unanswered too. A critical mini review on psychopathological bases, etiological hypothesis, and psychopharmacological approaches toward HPPD, including the association with some novel substances, are provided here, by means of a literature search on PubMed/Medline, Google Scholar, and Scopus databases without time restrictions, by using a specific set of keywords. Pharmacological and clinical issues are considered, and practical psychopharmacological recommendations and clinical guidelines are suggested.
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Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid
Hibi, Makoto; Mori, Ryosuke; Miyake, Ryoma; Kawabata, Hiroshi; Kozono, Shoko; Takahashi, Satomi
2016-01-01
Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities. PMID:26801577
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Phylogenetic trends in respiratory rhythmogenesis: insights from ectothermic vertebrates.
Kinkead, Richard
2009-08-31
Understanding the neural substrate driving breathing has puzzled physiologists for more than a century. The discovery of the pre-Bötzinger complex (preBötC) in newborn rodents as a structure with a unique physiological function in respiratory rhythm generation was an important progress in respiratory neurobiology that stimulated much research. Owing to the extensive literature describing the location, organisation, and function of the preBötC mainly in newborn rodents, this structure has become the point of reference in studies addressing respiratory rhythm generation in other mammals and various classes of vertebrates. This paper reviews recent progress made in non-mammalian vertebrates in our understanding of the location and function of the neural networks driving respiratory activity. As in newborn rodents, data from lampreys, air breathing fish, and amphibians show that the production of eupnea is the result of interactions between multiple (at least two) rhythmogenic networks. These networks are located in anatomically distinct areas and show different functional properties in terms of their ability to produce (or not) bursting activity in the absence of synaptic inputs (e.g. pacemaker neurons) and their sensitivity to specific neuromodulators such as substance P, somatostatin, and opioids. Current data indicate that respiratory rhythmogenesis is a phylogenetically ancient function that was highly conserved throughout evolution and that a comparative approach remains important to derive broader biological principles and a more comprehensive view.
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Liland, Nina S; Biancarosa, Irene; Araujo, Pedro; Biemans, Daan; Bruckner, Christian G; Waagbø, Rune; Torstensen, Bente E; Lock, Erik-Jan
2017-01-01
Black soldier fly (Hermetia illucens) larvae are a promising source of protein and lipid for animal feeds. The nutritional composition of the BSF larvae depend partly on the composition of the feeding medium. The BSF lipid profile in part mimics the feeding media lipid profile, and micronutrients, like minerals and vitamins, can readily accumulate in black soldier fly larvae. However, investigative studies on bioconversion and accumulation of nutrients from media to black soldier fly larvae are scarce. Here we show that inclusion of the brown algae Ascophyllum nodosum in the substrate for black soldier fly larvae can introduce valuable nutrients, commonly associated with the marine environment, into the larvae. The omega-3 fatty acid eicosapentaenoic acid (20:5n-3), iodine and vitamin E concentrations increased in the larvae when more seaweed was included in the diet. When the feeding media consisted of more than 50% seaweed, the larvae experienced poorer growth, lower nutrient retention and lower lipid levels, compared to a pure plant based feeding medium. Our results confirm the plasticity of the nutritional make-up of black soldier fly larvae, allowing it to accumulate both lipid- and water-soluble compounds. A broader understanding of the effect of the composition of the feeding media on the larvae composition can help to tailor black soldier fly larvae into a nutrient profile more suited for specific feed or food purposes.
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Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates
Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP
2014-01-01
Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121
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Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.
Fang, Shujun; Chang, Jie; Lee, Yong Seok; Guo, Weiliang; Choi, Yong Lark; Zhou, Yongcan
2014-01-01
A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.
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Deu, Edgar; Yang, Zhimou; Wang, Flora; Klemba, Michael; Bogyo, Matthew
2010-01-01
Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. PMID:20700487
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Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.
Chen, Sheng; Wan, Hoi Ying
2011-01-15
BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.
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Surtees, Jennifer A; Alani, Eric
2006-07-14
Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.
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Aerobic biological treatment of leachates from municipal solid waste landfill.
Andrés, P; Gutierrez, F; Arrabal, C; Cortijo, M
2004-01-01
The main objective of the study was to improve chemical oxygen demand (COD) elimination by secondary biological treatment from leachate of municipal solid waste landfill. This effluent was a supernatant liquid obtained after physicochemical processes and coagulating with Al3+ followed by ammoniacal stripping. First, respirometric assays were carried out to determine the substrate biodegradability. Specific sludge respiration rate (R(s)) vs. concentration of substrate (S), showed an increasing specific rate of assimilation of substrate (Rs), which reached the highest value, when the substrate concentration (COD) was between 75 and 200 mg O2 L(-1). Second, continuous experiments were made in an aerobic digester to test the previous respirometric data and the results showed removal efficiency of COD between 83 and 69%, and a substrate assimilation rate between 1.3 and 3.1 g COD g(-1) volatile suspended solids d(-1).
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NASA Astrophysics Data System (ADS)
Negash, Solomon; Tatek, Yergou B.; Tsige, Mesfin
2018-04-01
We have carried out atomistic (all-atom) molecular dynamics simulations to investigate the effect of tacticity on the structure and glass transition temperature (Tg) of polystyrene (PS) thin films adsorbed on two distinct types of solid substrates. The systems consist of thin films made of atactic, isotactic, and syndiotactic PS chains supported by graphite or hydroxylated α-quartz substrates, which are known to be atomically flat but chemically and structurally different. We have observed a marked dependence of the film structure on substrate type as well as on tacticity. For instance, rings' orientation near substrate surfaces depends on substrate type for atactic PS and isotactic PS films, while no such dependence is observed for syndiotactic PS films whose interfacial structure seems to result from their propensity to adopt the trans conformation rather than their specific interaction with the substrates. Moreover, our results indicate that glass transition temperatures of substrate supported polystyrene films are higher compared to those of the corresponding free-standing films. More specifically, PS films on graphite exhibit larger Tg values than those on α-quartz, and we have noticed that syndiotactic PS has the largest Tg irrespective of the substrate type. Furthermore, the local Tg in the region of the film in contact with the substrates shows a strong tacticity and substrate dependence, whereas no dependencies were found for the local Tg in the middle of the film. Substrate-film interaction energy and chains' dynamics near substrate-film interfaces were subsequently investigated in order to substantiate the obtained Tgs, and it was found that films with higher Tgs are strongly adsorbed on the substrates and/or exhibit smaller interfacial chains' dynamics essentially due to steric hindrance.
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NASA Astrophysics Data System (ADS)
Nooshabadi, Fatemeh; Yang, Hee-Jeong; Cheng, Yunfeng; Xie, Hexin; Rao, Jianghong; Cirillo, Jeffrey D.; Maitland, Kristen C.
2016-03-01
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most frequent causes of death worldwide. The slow growth rate of Mtb limits progress toward understanding tuberculosis including diagnosis of infections and evaluating therapeutic efficacy. Development of near-infrared (NIR) β-lactamase (BlaC)-specific fluorogenic substrate has made a significant breakthrough in the whole-animal imaging to detect Mtb infection. The reporter enzyme fluorescence (REF) system using a BlaC-specific fluorogenic substrate has improved the detection sensitivity in whole-animal optical imaging down to ~104 colony forming units (CFU) of bacteria, about 100-fold improvement over recombinant strains. However, improvement of detection sensitivity is strongly needed for clinical diagnosis of early stage infection at greater tissue depth. In order to improve detection sensitivity, we have integrated a fiber-based microendoscpe into a whole-animal imaging system to transmit the excitation light from the fiber bundle to the fluorescent target directly and measure fluorescent level using BlaC-specific REF substrate in the mouse lung. REF substrate, CNIR800, was delivered via aerosol route to the pulmonary infected mice with M. bovis BCG strain at 24 hours post-infection and groups of mice were imaged at 1-4 hours post-administration of the substrate using the integrated imaging system. In this study we evaluated the kinetics of CNIR800 substrate using REF technology using the integrated imaging system. Integration of these technologies has great promise for improved detection sensitivity allowing pre-clinical imaging for evaluation of new therapeutic agents.
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Junyong Zhu; G.S. Wang; X.J. Pan; Roland Gleisner
2009-01-01
Sieving methods have been almost exclusively used for feedstock size-reduction characterization in the biomass refining literature. This study demonstrates a methodology to properly characterize specific surface of biomass substrates through two dimensional measurement of each fiber of the substrate using a wet imaging technique. The methodology provides more...
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Bjerve, K S
1984-01-01
The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines. PMID:6430274
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On the promotion of human flourishing
VanderWeele, Tyler J.
2017-01-01
Many empirical studies throughout the social and biomedical sciences focus only on very narrow outcomes such as income, or a single specific disease state, or a measure of positive affect. Human well-being or flourishing, however, consists in a much broader range of states and outcomes, certainly including mental and physical health, but also encompassing happiness and life satisfaction, meaning and purpose, character and virtue, and close social relationships. The empirical literature from longitudinal, experimental, and quasiexperimental studies is reviewed in attempt to identify major determinants of human flourishing, broadly conceived. Measures of human flourishing are proposed. Discussion is given to the implications of a broader conception of human flourishing, and of the research reviewed, for policy, and for future research in the biomedical and social sciences. PMID:28705870
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A dual-catalysis approach to enantioselective [2 + 2] photocycloadditions using visible light.
Du, Juana; Skubi, Kazimer L; Schultz, Danielle M; Yoon, Tehshik P
2014-04-25
In contrast to the wealth of catalytic systems that are available to control the stereochemistry of thermally promoted cycloadditions, few similarly effective methods exist for the stereocontrol of photochemical cycloadditions. A major unsolved challenge in the design of enantioselective catalytic photocycloaddition reactions has been the difficulty of controlling racemic background reactions that occur by direct photoexcitation of substrates while unbound to catalyst. Here, we describe a strategy for eliminating the racemic background reaction in asymmetric [2 + 2] photocycloadditions of α,β-unsaturated ketones to the corresponding cyclobutanes by using a dual-catalyst system consisting of a visible light-absorbing transition-metal photocatalyst and a stereocontrolling Lewis acid cocatalyst. The independence of these two catalysts enables broader scope, greater stereochemical flexibility, and better efficiency than previously reported methods for enantioselective photochemical cycloadditions.
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Interrupting behaviour: Minimizing decision costs via temporal commitment and low-level interrupts.
Lloyd, Kevin; Dayan, Peter
2018-01-01
Ideal decision-makers should constantly assess all sources of information about opportunities and threats, and be able to redetermine their choices promptly in the face of change. However, perpetual monitoring and reassessment impose inordinate sensing and computational costs, making them impractical for animals and machines alike. The obvious alternative of committing for extended periods of time to limited sensory strategies associated with particular courses of action can be dangerous and wasteful. Here, we explore the intermediate possibility of making provisional temporal commitments whilst admitting interruption based on limited broader observation. We simulate foraging under threat of predation to elucidate the benefits of such a scheme. We relate our results to diseases of distractibility and roving attention, and consider mechanistic substrates such as noradrenergic neuromodulation.
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Interrupting behaviour: Minimizing decision costs via temporal commitment and low-level interrupts
Dayan, Peter
2018-01-01
Ideal decision-makers should constantly assess all sources of information about opportunities and threats, and be able to redetermine their choices promptly in the face of change. However, perpetual monitoring and reassessment impose inordinate sensing and computational costs, making them impractical for animals and machines alike. The obvious alternative of committing for extended periods of time to limited sensory strategies associated with particular courses of action can be dangerous and wasteful. Here, we explore the intermediate possibility of making provisional temporal commitments whilst admitting interruption based on limited broader observation. We simulate foraging under threat of predation to elucidate the benefits of such a scheme. We relate our results to diseases of distractibility and roving attention, and consider mechanistic substrates such as noradrenergic neuromodulation. PMID:29338004
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Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio
2011-04-01
Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.
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Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof
2011-01-01
Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ju, Xiaohui; Bowden, Mark E.; Engelhard, Mark H.
Three commercial cellulase preparations, Novozymes Cellic® Ctec2, Dupont Accellerase® 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulose enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulose performance. While the degree of polymerization of cellulose in the reference substrates didmore » not present a major recalcitrance factor to Novozymes Cellic® Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.« less
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Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.
Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence
2016-03-08
Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Different substrates and starter inocula govern microbial community structures in biogas reactors.
Satpathy, Preseela; Steinigeweg, Sven; Cypionka, Heribert; Engelen, Bert
2016-01-01
The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.
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Roles of s3 site residues of nattokinase on its activity and substrate specificity.
Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong
2007-09-01
Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.
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Ju, Xiaohui; Bowden, Mark; Engelhard, Mark; Zhang, Xiao
2014-05-01
Three commercial cellulase preparations, Novozymes Cellic(®) Ctec2, Dupont Accellerase(®) 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic(®) Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.
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2013-01-01
Background The widespread protozoan parasite Toxoplasma gondii interferes with host cell functions by exporting the contents of a unique apical organelle, the rhoptry. Among the mix of secreted proteins are an expanded, lineage-specific family of protein kinases termed rhoptry kinases (ROPKs), several of which have been shown to be key virulence factors, including the pseudokinase ROP5. The extent and details of the diversification of this protein family are poorly understood. Results In this study, we comprehensively catalogued the ROPK family in the genomes of Toxoplasma gondii, Neospora caninum and Eimeria tenella, as well as portions of the unfinished genome of Sarcocystis neurona, and classified the identified genes into 42 distinct subfamilies. We systematically compared the rhoptry kinase protein sequences and structures to each other and to the broader superfamily of eukaryotic protein kinases to study the patterns of diversification and neofunctionalization in the ROPK family and its subfamilies. We identified three ROPK sub-clades of particular interest: those bearing a structurally conserved N-terminal extension to the kinase domain (NTE), an E. tenella-specific expansion, and a basal cluster including ROP35 and BPK1 that we term ROPKL. Structural analysis in light of the solved structures ROP2, ROP5, ROP8 and in comparison to typical eukaryotic protein kinases revealed ROPK-specific conservation patterns in two key regions of the kinase domain, surrounding a ROPK-conserved insert in the kinase hinge region and a disulfide bridge in the kinase substrate-binding lobe. We also examined conservation patterns specific to the NTE-bearing clade. We discuss the possible functional consequences of each. Conclusions Our work sheds light on several important but previously unrecognized features shared among rhoptry kinases, as well as the essential differences between active and degenerate protein kinases. We identify the most distinctive ROPK-specific features conserved across both active kinases and pseudokinases, and discuss these in terms of sequence motifs, evolutionary context, structural impact and potential functional relevance. By characterizing the proteins that enable these parasites to invade the host cell and co-opt its signaling mechanisms, we provide guidance on potential therapeutic targets for the diseases caused by coccidian parasites. PMID:23742205
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Affinity through Mathematical Activity: Cultivating Democratic Learning Communities
ERIC Educational Resources Information Center
Sengupta-Irving, Tesha
2014-01-01
In this article, the author demonstrates how a broader view of what shapes affinity is ideologically and practically linked to creating democratic learning communities. Specifically, the author explores how a teacher employed complex instruction (an equity pedagogy) with her ethnically and racially diverse students in the "lowest track"…
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Beyond School Boundaries: New Health Imperatives, Families and Schools
ERIC Educational Resources Information Center
Rich, Emma
2012-01-01
This article draws upon research examining the impact of new health imperatives on schools in the United Kingdom. Specifically, it examines features of emerging surveillant relations, which not only speak to the changing nature of health-related practices in schools but have particular currency for broader understandings of theorisations of…
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A Coherent Approach to High School Improvement: A District and School Self-Assessment Tool
ERIC Educational Resources Information Center
Fryer, Lindsay; Johnson, Amy
2012-01-01
High school improvement initiatives often focus on specific intervention strategies, programs, or priority topics (e.g., dropout intervention, dual enrollment, freshman academies). However, research shows that systemic and sustainable improvement can be achieved only when initiatives are implemented with consideration for the broader education…
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Developing Transferrable Geospatial Skills in a Liberal Arts Context
ERIC Educational Resources Information Center
Colaianne, Blake A.; Powell, Matthew G.
2011-01-01
Geology education usually takes place within the context of a broader curriculum, but specific synergies between disciplines have rarely been explored or exploited. Here, we have assessed the spatial visualization skills of undergraduate students in a variety of disciplines to determine which are most compatible with a geology curriculum. Spatial…
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Information Literacy: Different Contexts, Different Concepts, Different Truths?
ERIC Educational Resources Information Center
Lloyd, Annemaree
2005-01-01
This article reports on doctoral research which explored the meaning and role of information literacy among a specific group of workers--firefighters. An alternative concept of information literacy and a broader definition of information literate people are introduced. The article suggests that librarians are currently bound by an educational…
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ERIC Educational Resources Information Center
Bernard van Leer Foundation Newsletter, 1994
1994-01-01
This newsletter theme issue deals with the phenomenon of mobility or transience in India, Kenya, Greece, Ireland, Malaysia, Thailand and Israel. The primary focus is on mobility's effect on young children, specifically their health and education; some of the broader concerns also addressed by the newsletter are the causes of mobility and its…
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Is Speech Learning "Gated" by the Social Brain?
ERIC Educational Resources Information Center
Kuhl, Patricia K.
2007-01-01
I advance the hypothesis that the earliest phases of language acquisition--the developmental transition from an initial universal state of language processing to one that is language-specific--requires social interaction. Relating human language learning to a broader set of neurobiological cases of communicative development, I argue that the…
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Haack, S.K.; Garchow, H.; Klug, M.J.; Forney, L.J.
1995-01-01
We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microliter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal- components analysis (PCA), and model communities with different compositions were clearly separated un the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.
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Dudek, Hanna M.; de Gonzalo, Gonzalo; Torres Pazmiño, Daniel E.; Stępniak, Piotr; Wyrwicz, Lucjan S.; Rychlewski, Leszek; Fraaije, Marco W.
2011-01-01
Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope. PMID:21724896
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Zimanyi, Christina M; Chen, Percival Yang-Ting; Kang, Gyunghoon; Funk, Michael A; Drennan, Catherine L
2016-01-01
Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell. DOI: http://dx.doi.org/10.7554/eLife.07141.001 PMID:26754917
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Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L
1990-01-01
A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.
GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less
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Butterfield, Cristina N; Tebo, Bradley M
2017-02-22
Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.
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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
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Yu, S; Ahmad, T; Kenne, L; Pedersén, M
1995-05-11
The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gracilariopsis lemaneiformis, is a single polypeptide with a molecular mass of 116,654 Da as determined by matrix-assisted laser-desorption mass spectrometry. It degraded maltose, maltosaccharides, amylose, amylopectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reducing end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occurring and synthesized substrates. This enzyme was highly specific for the alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was used as substrate, the enzyme split the substrate from the non-reducing end and released 1,5-anhydro-D-fructose successively until only one glucose unit was left. When a branched pentasaccharide of 6(2)-alpha-maltosylmaltotriose, obtained from glycogen by alpha-amylase limitation, was used as substrate, the glucose group in the 4-position of the 4,6-branched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and 1H-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack mechanism. 1H- and 13C-NMR spectroscopic studies on unlabelled and labelled amylose (1-2H, 2-2H, 1-13C) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It also indicated that the catalytic process of the lyase involved proton exchanges among C-1, C-2, C-3 and the solvent.
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METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS
DOE Office of Scientific and Technical Information (OSTI.GOV)
John J. Kilbane III
The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project will focus on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate amidase. The objective of the final phase of the project will bemore » to develop derivative CN bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. The project is on schedule and no major difficulties have been encountered. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments have resulted in the isolation of promising cultures that may be capable of cleaving C-N bonds in aromatic amides, several amidase genes have been cloned and are currently undergoing directed evolution to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. Future research will address expression of these genes in Rhodococcus erythropolis. Enrichment culture experiments and directed evolution experiments continue to be a main focus of research activity and further work is required to obtain an appropriate amidase that will selectively cleave C-N bonds in aromatic substrates. Once an appropriate amidase gene is obtained it must be combined with genes encoding an enzyme capable of converting carbazole to 2'aminobiphenyl-2,3-diol: specifically carA genes. The carA genes from two sources have been cloned and are ready for construction of C-N bond cleavage pathway. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum.« less
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Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias
2017-10-18
Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NASA Astrophysics Data System (ADS)
Kromer, R.; Danlos, Y.; Costil, S.
2018-04-01
Cold spraying enables a variety of metals dense coatings onto metal surfaces. Supersonic gas jet accelerates particles which undergo with the substrate plastic deformation. Different bonding mechanisms can be created depending on the materials. The particle-substrate contact time, contact temperature and contact area upon impact are the parameters influencing physicochemical and mechanical bonds. The resultant bonding arose from plastic deformation of the particle and substrate and temperature increasing at the interface. The objective was to create specific topography to enable metallic particle adhesion onto ceramic substrates. Ceramic did not demonstrate deformation during the impact which minimized the intimate bonds. Laser surface texturing was hence used as prior surface treatment to create specific topography and to enable mechanical anchoring. Particle compressive states were necessary to build up coating. The coating deposition efficiency and adhesion strength were evaluated. Textured surface is required to obtain strong adhesion of metallic coatings onto ceramic substrates. Consequently, cold spray coating parameters depend on the target material and a methodology was established with particle parameters (diameters, velocities, temperatures) and particle/substrate properties to adapt the surface topography. Laser surface texturing is a promising tool to increase the cold spraying applications.
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Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter
1999-03-30
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.
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Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter
1998-01-06
Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.
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Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu
2013-01-01
Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780
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Exosites in the substrate specificity of blood coagulation reactions.
Bock, P E; Panizzi, P; Verhamme, I M A
2007-07-01
The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.
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Cofactor-dependent specificity of a DEAD-box protein.
Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin
2013-07-16
DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.
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Kitchen, A D; Newham, J A
2011-05-01
Whilst some of the assays used for serological screening of post-mortem blood samples from deceased tissue donors in some countries have been specifically validated by the manufacturer for this purpose, a significant number of those currently in use globally have not. Although specificity has previously been considered a problem in the screening of such samples, we believe that ensuring sensitivity is more important. The aim of this study was to validate a broader range of assays for the screening of post-mortem blood samples from deceased tissue donors. Six microplate immunoassays currently in use within National Health Service Blood and Transplant (NHSBT) for the screening of blood, tissue and stem cell donations were included. Representative samples from confirmed positive donors were titrated in screen negative post-mortem samples in parallel with normal pooled negative serum to determine if there was any inhibition with the post-mortem samples. There were no significant differences seen (P < 0.005) between the dilution curves obtained for the positive samples diluted in post-mortem samples and normal pooled sera. Although small numbers of samples were studied, it can be surmised that the post-mortem blood samples from deceased tissue donors, collected according to United Kingdom guidelines, are a suitable substrate for the assays evaluated. No diminution of reactivity was seen when dilution with sera from deceased donors was compared to dilution using pooled serum from live donors. In the absence of genuine low titre positive post-mortem samples, the use of samples spiked with various levels of target material provides a means of qualifying serological screening assays used by NHSBT for the screening of post-mortem blood samples from deceased tissue donors.
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Cheriyan, Manoj; Chan, Siu-Hong; Perler, Francine
2014-12-12
Inteins self-catalytically cleave out of precursor proteins while ligating the surrounding extein fragments with a native peptide bond. Much attention has been lavished on these molecular marvels with the hope of understanding and harnessing their chemistry for novel biochemical transformations including coupling peptides from synthetic or biological origins and controlling protein function. Despite an abundance of powerful applications, the use of inteins is still hampered by limitations in our understanding of their specificity (defined as flanking sequences that permit splicing) and the challenge of inserting inteins into target proteins. We examined the frequently used Nostoc punctiforme Npu DnaE intein after the C-extein cysteine nucleophile (Cys+1) was mutated to serine or threonine. Previous studies demonstrated reduced rates and/or splicing yields with the Npu DnaE intein after mutation of Cys+1 to Ser+1. In this study, genetic selection identified extein sequences with Ser+1 that enabled the Npu DnaE intein to splice with only a 5-fold reduction in rate compared to the wild-type Cys+1 intein and without mutation of the intein itself to activate Ser+1 as a nucleophile. Three different proteins spliced efficiently after insertion of the intein flanked by the selected sequences. We then used this selected specificity to achieve traceless splicing in a targeted enzyme at a location predicted by primary sequence similarity to only the selected C-extein sequence. This study highlights the latent catalytic potential of the Npu DnaE intein to splice with an alternative nucleophile and enables broader intein utility by increasing insertion site choices. Copyright © 2014. Published by Elsevier Ltd.
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Helms, S. R.; Brattain, M. G.; Pretlow, T. G.; Kreisberg, J. I.
1977-01-01
Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells. PMID:560800
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Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano
2009-01-01
Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652
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Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F
DOE Office of Scientific and Technical Information (OSTI.GOV)
Agarwal, R.; Schmidt, J; Stafford, R
2009-01-01
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exositesmore » away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.« less
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Substrate Specificity of Human Protein Arginine Methyltransferase 7 (PRMT7)
Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G.
2014-01-01
Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-NG-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. PMID:25294873
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Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...
2015-09-15
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*
Meekins, David A.; Raththagala, Madushi; Auger, Kyle D.; Turner, Benjamin D.; Santelia, Diana; Kötting, Oliver; Gentry, Matthew S.; Vander Kooi, Craig W.
2015-01-01
Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. PMID:26231210
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Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.
1998-01-06
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.
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Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.
1999-03-30
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.
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Islamic ethics of saving life: a comparative perspective.
Brockop, Jonathan E
2002-01-01
Addressing specific modes of reasoning common to legal and theological discussions, this paper seeks to understand how the modern debate on euthanasia fits into a broader Islamic worldview. After an overview of contrasting religious and secular ethical theories, the paper demonstrates that Islamic theological views, and also traditions on martyrdom and suicide, continue to inform Muslim authorities today. Also included are specific suggestions on the incorporation of Islamic ideas of death into the modern hospital environment.
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Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).
Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A
2006-05-19
Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.
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Shiota, Hiroto; Kanzaki, Hiroshi; Hatanaka, Tadashi; Nitoda, Teruhiko
2013-06-28
TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases. Copyright © 2013 Elsevier Ltd. All rights reserved.
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Stratton, Christopher F; Namanja-Magliano, Hilda A; Cameron, Scott A; Schramm, Vern L
2015-10-16
Dihydropteroate synthase is a key enzyme in folate biosynthesis and is the target of the sulfonamide class of antimicrobials. Equilibrium binding isotope effects and density functional theory calculations indicate that the substrate binding sites for para-aminobenzoic acid on the dihydropteroate synthase enzymes from Staphylococcus aureus and Plasmodium falciparum present distinct chemical environments. Specifically, we show that para-aminobenzoic acid occupies a more sterically constrained vibrational environment when bound to dihydropteroate synthase from P. falciparum relative to that of S. aureus. Deletion of a nonhomologous, parasite-specific insert from the plasmodial dihydropteroate synthase abrogated the binding of para-aminobenzoic acid. The loop specific to P. falciparum is important for effective substrate binding and therefore plays a role in modulating the chemical environment at the substrate binding site.
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Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong
2015-09-01
Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta
Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less
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Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; ...
2015-08-05
Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less
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Kumar, Ish; Pratt, R F
2005-08-02
The Streptomyces R61 dd-peptidase, a functional model for penicillin-binding proteins, catalyzes the hydrolysis and aminolysis of d-alanyl-d-alanine-terminating peptides by specific amines. In vivo, this reaction completes bacterial cell wall biosynthesis. For in vitro studies of this enzyme to date, various nonspecific acyl-donor substrates have been employed. Recently, however, a peptidoglycan-mimetic peptide substrate, glycyl-l-alpha-amino-epsilon-pimelyl-d-alanyl-d-alanine, has been described that is much more specific for this enzyme. In this paper, we describe the synthesis and kinetic characterization of an analogous thiolester substrate, 3-(N-glycyl-l-cysteinyl)-propanoyl-d-alanyl-d-thiolactate, that the enzyme hydrolyzes and aminolyzes very efficiently (k(cat)/K(m) = 1.0 x 10(7) s(-)(1) M(-)(1)). Direct or indirect, by means of a thiol trap, spectrophotometric monitoring of the reactions of this substrate is readily achieved. Deacylation of the enzyme is rate-determining under substrate saturation conditions, and therefore the aminolysis reaction can be directly studied. The results show that d-amino acids and certain Gly-l-Xaa dipeptides and tripeptides may act as acyl acceptors at the active site of the enzyme. d-Phenylalanine and Gly-l-Phe were the most effective d-amino acid and dipeptide acceptors, respectively. On the basis of the dual specificity of the active site for acceptors (d-amino acids and Gly-l-Xaa peptides), "dual function" acceptors were designed and synthesized. Two of these, aminomalon-(N-ethyl)amide and aminomalon-(N-phenethyl)amide, were particularly effective. It did seem, however, that the observed rates of reaction of these very effective acceptors may be limited by some common, possibly physical, step. More extended, peptidoglycan-like, acceptors were found to be essentially unreactive. The reasons for this counterintuitive behavior are discussed.
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Crystal Structures of the Novel Cytosolic 5′-Nucleotidase IIIB Explain Its Preference for m7GMP
Monecke, Thomas; Buschmann, Juliane; Neumann, Piotr; Wahle, Elmar; Ficner, Ralf
2014-01-01
5′-nucleotidases catalyze the hydrolytic dephosphorylation of nucleoside monophosphates. As catabolic enzymes they contribute significantly to the regulation of cellular nucleotide levels; misregulation of nucleotide metabolism and nucleotidase deficiencies are associated with a number of diseases. The seven human 5′-nucleotidases differ with respect to substrate specificity and cellular localization. Recently, the novel cytosolic 5′-nucleotidase III-like protein, or cN-IIIB, has been characterized in human and Drosophila. cN-IIIB exhibits a strong substrate preference for the modified nucleotide 7-methylguanosine monophosphate but the structural reason for this preference was unknown. Here, we present crystal structures of cN-IIIB from Drosophila melanogaster bound to the reaction products 7-methylguanosine or cytidine. The structural data reveal that the cytosine- and 7-methylguanine moieties of the products are stacked between two aromatic residues in a coplanar but off-centered position. 7-methylguanosine is specifically bound through π-π interactions and distinguished from unmodified guanosine by additional cation-π coulomb interactions between the aromatic side chains and the positively charged 7-methylguanine. Notably, the base is further stabilized by T-shaped edge-to-face stacking of an additional tryptophan packing perpendicularly against the purine ring and forming, together with the other aromates, an aromatic slot. The structural data in combination with site-directed mutagenesis experiments reveal the molecular basis for the broad substrate specificity of cN-IIIB but also explain the substrate preference for 7-methylguanosine monophosphate. Analyzing the substrate specificities of cN-IIIB and the main pyrimidine 5′-nucleotidase cN-IIIA by mutagenesis studies, we show that cN-IIIA dephosphorylates the purine m7GMP as well, hence redefining its substrate spectrum. Docking calculations with cN-IIIA and m7GMP as well as biochemical data reveal that Asn69 does not generally exclude the turnover of purine substrates thus correcting previous suggestions. PMID:24603684
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Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi
2015-07-01
Cynomolgus monkeys are widely used as primate models in preclinical studies, because of their evolutionary closeness to humans. In humans, the cytochrome P450 (P450) 2C enzymes are important drug-metabolizing enzymes and highly expressed in livers. The CYP2C enzymes, including CYP2C9, are also expressed abundantly in cynomolgus monkey liver and metabolize some endogenous and exogenous substances like testosterone, S-mephenytoin, and diclofenac. However, comprehensive evaluation regarding substrate specificity of monkey CYP2C9 has not been conducted. In the present study, 89 commercially available drugs were examined to find potential monkey CYP2C9 substrates. Among the compounds screened, 20 drugs were metabolized by monkey CYP2C9 at a relatively high rates. Seventeen of these compounds were substrates or inhibitors of human CYP2C9 or CYP2C19, whereas three drugs were not, indicating that substrate specificity of monkey CYP2C9 resembled those of human CYP2C9 or CYP2C19, with some differences in substrate specificities. Although efavirenz is known as a marker substrate for human CYP2B6, efavirenz was not oxidized by CYP2B6 but by CYP2C9 in monkeys. Liquid chromatography-mass spectrometry analysis revealed that monkey CYP2C9 and human CYP2B6 formed the same mono- and di-oxidized metabolites of efavirenz at 8 and 14 positions. These results suggest that the efavirenz 8-oxidation could be one of the selective markers for cynomolgus monkey CYP2C9 among the major three CYP2C enzymes tested. Therefore, monkey CYP2C9 has the possibility of contributing to limited specific differences in drug oxidative metabolism between cynomolgus monkeys and humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
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Wang, Hualei; Sun, Huihui; Wei, Dongzhi
2013-02-18
A nitrilase-mediated pathway has significant advantages in the production of optically pure (R)-(-)-mandelic acid. However, unwanted byproduct, low enantioselectivity, and specific activity reduce its value in practical applications. An ideal nitrilase that can efficiently hydrolyze mandelonitrile to optically pure (R)-(-)-mandelic acid without the unwanted byproduct is needed. A novel nitrilase (BCJ2315) was discovered from Burkholderia cenocepacia J2315 through phylogeny-based enzymatic substrate specificity prediction (PESSP). This nitrilase is a mandelonitrile hydrolase that could efficiently hydrolyze mandelonitrile to (R)-(-)-mandelic acid, with a high enantiomeric excess of 98.4%. No byproduct was observed in this hydrolysis process. BCJ2315 showed the highest identity of 71% compared with other nitrilases in the amino acid sequence. BCJ2315 possessed the highest activity toward mandelonitrile and took mandelonitrile as the optimal substrate based on the analysis of substrate specificity. The kinetic parameters Vmax, Km, Kcat, and Kcat/Km toward mandelonitrile were 45.4 μmol/min/mg, 0.14 mM, 15.4 s(-1), and 1.1×10(5) M(-1)s(-1), respectively. The recombinant Escherichia coli M15/BCJ2315 had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100 mM) with fewer amounts of wet cells (10 mg/ml) within 1 h. PESSP is an efficient method for discovering an ideal mandelonitrile hydrolase. BCJ2315 has high affinity and catalytic efficiency toward mandelonitrile. This nitrilase has great advantages in the production of optically pure (R)-(-)-mandelic acid because of its high activity and enantioselectivity, strong substrate tolerance, and having no unwanted byproduct. Thus, BCJ2315 has great potential in the practical production of optically pure (R)-(-)-mandelic acid in the industry.
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Hou, Jing; Zheng, Heping; Tzou, Wen-Shyong; Cooper, David R; Chruszcz, Maksymilian; Chordia, Mahendra D; Kwon, Keehwan; Grabowski, Marek; Minor, Wladek
2018-06-19
Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-ACP synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most Gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH and six vcFabH2 structures determined in either apo-form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Gournas, Christos; Evangelidis, Thomas; Athanasopoulos, Alexandros; Mikros, Emmanuel; Sophianopoulou, Vicky
2015-01-01
Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly56, Thr57), TMS3 (Glu138), and TMS6 (Phe248), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle. PMID:25572393
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Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu
2014-01-01
Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904
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Substrate tunnels in enzymes: structure-function relationships and computational methodology.
Kingsley, Laura J; Lill, Markus A
2015-04-01
In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein, and, in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years because of implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with buried active sites and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. © 2015 Wiley Periodicals, Inc.
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Niland, Courtney N.; Jankowsky, Eckhard; Harris, Michael E.
2016-01-01
Quantification of the specificity of RNA binding proteins and RNA processing enzymes is essential to understanding their fundamental roles in biological processes. High Throughput Sequencing Kinetics (HTS-Kin) uses high throughput sequencing and internal competition kinetics to simultaneously monitor the processing rate constants of thousands of substrates by RNA processing enzymes. This technique has provided unprecedented insight into the substrate specificity of the tRNA processing endonuclease ribonuclease P. Here, we investigate the accuracy and robustness of measurements associated with each step of the HTS-Kin procedure. We examine the effect of substrate concentration on the observed rate constant, determine the optimal kinetic parameters, and provide guidelines for reducing error in amplification of the substrate population. Importantly, we find that high-throughput sequencing, and experimental reproducibility contribute their own sources of error, and these are the main sources of imprecision in the quantified results when otherwise optimized guidelines are followed. PMID:27296633
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NASA Astrophysics Data System (ADS)
Zhao, Qin; Wang, Chengcheng; Wang, Chengyuan; Guo, Hui; Bao, Zhihao; Zhang, Minhua; Zhang, Peng
2015-07-01
Energy-coupling factor (ECF) transporters are a new family of ABC transporters that consist of four subunits, two cytoplasmic ATPases EcfA and EcfA' and two transmembrane proteins namely EcfS for substrate-specific binding and EcfT for energy coupling. Here, we report the 3.2-Å resolution crystal structure of the EcfS protein of a folate ECF transporter from Enterococcus faecalis-EfFolT, a close homologue of FolT from Lactobacillus brevis-LbFolT. Structural and biochemical analyses reveal the residues constituting the folate-binding pocket and determining the substrate-binding specificity. Structural comparison of the folate-bound EfFolT with the folate-free LbFolT contained in the holotransporter complex discloses significant conformational change at the L1 loop, and reveals a gating mechanism of ECF transporters in which the L1 loop of EcfS acts as a gate in the substrate binding and release.
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Berrios, Julio; Flores, María-Olga; Díaz-Barrera, Alvaro; Altamirano, Claudia; Martínez, Irene; Cabrera, Zaida
2017-03-01
The production of recombinant proteins by Pichia pastoris under AOX1 promoter is usually performed using methanol together with either glycerol or sorbitol as co-substrate. Although both co-substrates have been widely used, comparative studies are scarce. In addition, these comparisons have been performed at different specific growth rate (µ) that it is well known that has an important effect on productivity. Thus, the effect of using these co-substrates on the production of Rhyzopus oryzae lipase (ROL) by P. pastoris was compared in continuous cultures growing at the same µ at either 22 or 30 °C. Results show that using glycerol as co-substrate led to higher volumetric productivities, and lower specific and volumetric methanol consumption rates. Scale-up simulation with 10-10,000 L bioreactor sizes indicated that glycerol produced the highest volumetric productivity of ROL with lower aeration requirements. Therefore, glycerol rises as a better option than sorbitol in ROL production.
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PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates
NASA Astrophysics Data System (ADS)
Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping
2014-03-01
Protein phosphorylation catalysed by kinases plays crucial regulatory roles in intracellular signal transduction. With the increasing number of kinase-specific phosphorylation sites and disease-related phosphorylation substrates that have been identified, the desire to explore the regulatory relationship between protein kinases and disease-related phosphorylation substrates is motivated. In this work, we analysed the kinases' characteristic of all disease-related phosphorylation substrates by using our developed Phosphorylation Set Enrichment Analysis (PSEA) method. We evaluated the efficiency of our method with independent test and concluded that our approach is reliable for identifying kinases responsible for phosphorylated substrates. In addition, we found that Mitogen-activated protein kinase (MAPK) and Glycogen synthase kinase (GSK) families are more associated with abnormal phosphorylation. It can be anticipated that our method might be helpful to identify the mechanism of phosphorylation and the relationship between kinase and phosphorylation related diseases. A user-friendly web interface is now freely available at http://bioinfo.ncu.edu.cn/PKPred_Home.aspx.
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Lentz, Christian S; Ordonez, Alvaro A; Kasperkiewicz, Paulina; La Greca, Florencia; O'Donoghue, Anthony J; Schulze, Christopher J; Powers, James C; Craik, Charles S; Drag, Marcin; Jain, Sanjay K; Bogyo, Matthew
2016-11-11
Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb "Hydrolase important for pathogenesis 1" (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections.
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2016-01-01
Although serine proteases are important mediators of Mycobacterium tuberculosis (Mtb) virulence, there are currently no tools to selectively block or visualize members of this family of enzymes. Selective reporter substrates or activity-based probes (ABPs) could provide a means to monitor infection and response to therapy using imaging methods. Here, we use a combination of substrate selectivity profiling and focused screening to identify optimized reporter substrates and ABPs for the Mtb “Hydrolase important for pathogenesis 1” (Hip1) serine protease. Hip1 is a cell-envelope-associated enzyme with minimal homology to host proteases, making it an ideal target for probe development. We identified substituted 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarins as irreversible inhibitor scaffolds. Furthermore, we used specificity data to generate selective reporter substrates and to further optimize a selective chloroisocoumarin inhibitor. These new reagents are potentially useful in delineating the roles of Hip1 during pathogenesis or as diagnostic imaging tools for specifically monitoring Mtb infections. PMID:27739665
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Substrate Tunnels in Enzymes: Structure-Function Relationships and Computational Methodology
Kingsley, Laura J.; Lill, Markus A.
2015-01-01
In enzymes, the active site is the location where incoming substrates are chemically converted to products. In some enzymes, this site is deeply buried within the core of the protein and in order to access the active site, substrates must pass through the body of the protein via a tunnel. In many systems, these tunnels act as filters and have been found to influence both substrate specificity and catalytic mechanism. Identifying and understanding how these tunnels exert such control has been of growing interest over the past several years due to implications in fields such as protein engineering and drug design. This growing interest has spurred the development of several computational methods to identify and analyze tunnels and how ligands migrate through these tunnels. The goal of this review is to outline how tunnels influence substrate specificity and catalytic efficiency in enzymes with tunnels and to provide a brief summary of the computational tools used to identify and evaluate these tunnels. PMID:25663659
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Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F
2015-07-24
The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.
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Knowledge-transfer learning for prediction of matrix metalloprotease substrate-cleavage sites.
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.
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Cheng, Keding; Sloan, Angela; Avery, Kristen M; Coulthart, Michael; Carpenter, Michael; Knox, J David
2014-01-01
Real-time quaking-induced conversion (RT-QuIC), a highly specific and sensitive assay able to detect low levels of the disease-inducing isoform of the prion protein (PrP(d)) in brain tissue biopsies and cerebral spinal fluid, has great potential to become a method for diagnosing prion disease ante mortem. In order to standardize the assay method for routine analysis, an understanding of how physical and chemical factors affect the stability of the recombinant prion protein (rPrP) substrate and the RT-QuIC assay's sensitivity, specificity, and reproducibility is required. In this study, using sporadic Creutzfeldt-Jakob Disease brain homogenate to seed the reactions and an in vitro-expressed recombinant prion protein, hamster rPrP, as the substrate, the following factors affecting the RT-QuIC assay were examined: salt and substrate concentrations, substrate storage, and pH. Results demonstrated that both the generation of the quality and quantities of rPrP substrate critical to the reaction, as well as the RT-QuIC reaction itself required strict adherence to specific physical and chemical conditions. Once optimized, the RT-QuIC assay was confirmed to be a very specific and sensitive assay method for sCJD detection. Findings in this study indicate that further optimization and standardization of RT-QuIC assay is required before it can be adopted as a routine diagnostic test.
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Tan, Boonfei; Fowler, S Jane; Abu Laban, Nidal; Dong, Xiaoli; Sensen, Christoph W; Foght, Julia; Gieg, Lisa M
2015-09-01
Methanogenic hydrocarbon metabolism is a key process in subsurface oil reservoirs and hydrocarbon-contaminated environments and thus warrants greater understanding to improve current technologies for fossil fuel extraction and bioremediation. In this study, three hydrocarbon-degrading methanogenic cultures established from two geographically distinct environments and incubated with different hydrocarbon substrates (added as single hydrocarbons or as mixtures) were subjected to metagenomic and 16S rRNA gene pyrosequencing to test whether these differences affect the genetic potential and composition of the communities. Enrichment of different putative hydrocarbon-degrading bacteria in each culture appeared to be substrate dependent, though all cultures contained both acetate- and H2-utilizing methanogens. Despite differing hydrocarbon substrates and inoculum sources, all three cultures harbored genes for hydrocarbon activation by fumarate addition (bssA, assA, nmsA) and carboxylation (abcA, ancA), along with those for associated downstream pathways (bbs, bcr, bam), though the cultures incubated with hydrocarbon mixtures contained a broader diversity of fumarate addition genes. A comparative metagenomic analysis of the three cultures showed that they were functionally redundant despite their enrichment backgrounds, sharing multiple features associated with syntrophic hydrocarbon conversion to methane. In addition, a comparative analysis of the culture metagenomes with those of 41 environmental samples (containing varying proportions of methanogens) showed that the three cultures were functionally most similar to each other but distinct from other environments, including hydrocarbon-impacted environments (for example, oil sands tailings ponds and oil-affected marine sediments). This study provides a basis for understanding key functions and environmental selection in methanogenic hydrocarbon-associated communities.
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Temperament and Family Environment in the Development of Anxiety Disorder: Two-Year Follow-Up
ERIC Educational Resources Information Center
Hudson, Jennifer L.; Dodd, Helen F.; Lyneham, Heidi J.; Bovopoulous, Nataly
2011-01-01
Objective: Behavioral inhibition (BI) in early childhood is associated with increased risk for anxiety. The present research examines BI alongside family environment factors, specifically maternal negativity and overinvolvement, maternal anxiety, and mother-child attachment, with a view to providing a broader understanding of the development of…
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Legislating Character: Moral Education in North Carolina's Public Schools
ERIC Educational Resources Information Center
Cooley, Aaron
2008-01-01
This article analyzes the epistemological aims and justification of character education legislation passed by the North Carolina General Assembly. I take this specific state law as representative of the broader national trends in the character education movement. I primarily use the work of Richard Rorty as the theoretical lens for the analysis…
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The Implicit Relational Assessment Procedure as a Measure of Self-Esteem
ERIC Educational Resources Information Center
Timko, C. Alix; England, Erica L.; Herbert, James D.; Forman, Evan M.
2010-01-01
Two studies were conducted to pilot the Implicit Relational Assessment Procedure (IRAP) in measuring attitudes toward the self: one related to body image specifically and another assessing the broader construct of self-esteem. Study 1 utilized the IRAP with female college students to examine self-referential beliefs regarding body image. Results…
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Love, Loss, and Everything in between: The Artwork of Bea Nettles
ERIC Educational Resources Information Center
Cress, Sarah
2012-01-01
For many artists, visual representation begins with the creative exploration of real and personal experiences. The primary challenge in creating such introspective works is maintaining the ability to connect with a broader audience. For high school students specifically, tremendous pride manifests in the creation of artistic works that represent…
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48 CFR 552.238-72 - Identification of Products That Have Environmental Attributes.
Code of Federal Regulations, 2012 CFR
2012-10-01
..., and low volatile organic compounds (VOCs). Post-consumer material means a material or finished product... life as a consumer item. Post-consumer material is part of the broader category of “recovered material... and post-consumer material content levels for the specific products designated by EPA (40 CFR part 247...
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48 CFR 552.238-72 - Identification of Products That Have Environmental Attributes.
Code of Federal Regulations, 2013 CFR
2013-10-01
..., and low volatile organic compounds (VOCs). Post-consumer material means a material or finished product... life as a consumer item. Post-consumer material is part of the broader category of “recovered material... and post-consumer material content levels for the specific products designated by EPA (40 CFR part 247...
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48 CFR 552.238-72 - Identification of Products That Have Environmental Attributes.
Code of Federal Regulations, 2011 CFR
2011-10-01
..., and low volatile organic compounds (VOCs). Post-consumer material means a material or finished product... life as a consumer item. Post-consumer material is part of the broader category of “recovered material... and post-consumer material content levels for the specific products designated by EPA (40 CFR part 247...
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48 CFR 552.238-72 - Identification of Products That Have Environmental Attributes.
Code of Federal Regulations, 2014 CFR
2014-10-01
..., and low volatile organic compounds (VOCs). Post-consumer material means a material or finished product... life as a consumer item. Post-consumer material is part of the broader category of “recovered material... and post-consumer material content levels for the specific products designated by EPA (40 CFR part 247...
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ERIC Educational Resources Information Center
Bureau of Labor Statistics (DOL), Washington, DC.
Selected from the "Occupational Outlook Handbook," the listings included in this document represent a number of occupations for which junior college or other special training is useful. Each listing, grouped under a broader occupational area, provides a specific occupational title, number employed in the occupation in 1974, qualifications and…
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What are Theories For? Concept Use throughout the Continuum of Dinosaur Expertise
ERIC Educational Resources Information Center
Johnson, Kathy E.; Scott, Paul; Mervis, Carolyn B.
2004-01-01
Although it is now well established that object concepts are situated within broader systems of theoretical knowledge, it is less clear how theories influence the use of object concepts at various points throughout the continuum of expertise. Two studies were conducted to investigate the impact of specific theories (concerning dinosaurs) and…
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ERIC Educational Resources Information Center
Healy, Margaret; McCutcheon, Maeve; Doran, John
2014-01-01
Research on assessment activities has considered student responses to specific initiatives, but broader concerns underlying these responses have not been fully explored. Using a survey methodology, this paper explores how students view assessment activities, from the perspective of their experience on a four-year undergraduate programme,…
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The Engagement of Universities in Older Adult Education in Aotearoa New Zealand
ERIC Educational Resources Information Center
Findsen, Brian
2017-01-01
This article investigates the engagement of universities in older adult education in the specific context of Aotearoa New Zealand. Initially, the broader context of the tertiary education system and the place of universities within it are explained. Not unexpectedly adult education, and particularly older adult education, exists only on the…
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Justifying The Arts: Drama and Intercultural Education
ERIC Educational Resources Information Center
Fleming, Mike
2005-01-01
This essay describes five approaches to the question of justifying the arts before examining the specific case of drama and intercultural education. Providing a list of reasons for teaching the arts is one approach but not the only one. Instead, looking for broader categories of justification within possible lists (e.g., between art and…
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ERIC Educational Resources Information Center
McNair, Delores E.
2010-01-01
Community colleges in the United States face shortages of leaders prepared to assume administrative positions in the 21st century. To respond to this shortage, graduate programs are emerging with a specific emphasis on community college leadership; other graduate programs offer broader curricula focused on educational leadership, policy, or higher…
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University Library Browsing: A Study Illustrating a Methodology.
ERIC Educational Resources Information Center
Ridley, Dennis R.; And Others
Previous studies on use of library materials based on borrowing behavior failed to address the issue of how the measurement of library book use might be expanded to a broader definition of use, specifically to include browsing behavior. The development of a non-obtrusive and accurate measurement for browsing behavior has remained a challenge.…
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Opportunity Theory and Agricultural Crime Victimization
ERIC Educational Resources Information Center
Mears, Daniel P.; Scott, Michelle L.; Bhati, Avinash S.
2007-01-01
A growing body of research lends support to opportunity theory and its variants, but has yet to focus systematically on a number of specific offenses and contexts. Typically, the more crimes and contexts to which a theory applies, the broader its scope and range, respectively, and thus generalizability. In this paper, we focus on agricultural…
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Problems with a Play Paradigm: A Reply to Dansky.
ERIC Educational Resources Information Center
Simon, Tony; Smith, Peter K.
1986-01-01
In answer to points raised by Dansky (1985) about specific inferences from Simon and Smith's study (1985) and broader issues about a single-session paradigm used in play and problem solving studies, Simon and Smith argue that their study adequately demonstrated how methodological factors in paradigm can swamp the effects of any treatment…
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ERIC Educational Resources Information Center
Ernst, Jeremy V.; Li, Songze; Williams, Thomas O.
2014-01-01
The ever-changing student population of engineering design graphics students necessitates broader sets of instructor adeptness. Specifically, preparedness to educate and provide adequate educational access to content for students with identified categorical disabilities and Limited English Proficiency (LEP) is now an essential readiness skill for…
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A Coherent Approach to High School Improvement: A School and District Needs Assessment Tool
ERIC Educational Resources Information Center
National High School Center, 2010
2010-01-01
High school improvement initiatives often focus on specific intervention strategies, programs, or priority topics (e.g., dropout intervention). However, research shows that systemic and sustainable improvement can only be achieved when initiatives are implemented with consideration for the broader education contexts in which they operate. The…
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Conversations about Science Education: A Retrospective of Science Education Research in "CJSTME"
ERIC Educational Resources Information Center
Pegg, Jerine; Wiseman, Dawn; Brown, Carol
2015-01-01
This review focuses on science education contributions to the "Canadian Journal of Science, Mathematics and Technology Education" (CJSMTE) from January 2001 through December 2014. Through a combination of content and citation analysis, we examine the journal as a location for conversations around specific themes and broader ideas related…
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User Guidelines and Best Practices for CASL VUQ Analysis Using Dakota.
DOE Office of Scientific and Technical Information (OSTI.GOV)
Adams, Brian M.; Coleman, Kayla; Hooper, Russell
2016-11-01
Sandia's Dakota software (available at http://dakota.sandia.gov) supports science and engineering transformation through advanced exploration of simulations. Specifically it manages and analyzes ensembles of simulations to provide broader and deeper perspective for analysts and decision makers. This enables them to enhance understanding of risk, improve products, and assess simulation credibility.
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Mixed Methods Research: Taking a Broader View
ERIC Educational Resources Information Center
Stewart, Tricia J.; Palermo-Biggs, Michelle
2013-01-01
For school districts, the increasing importance of using data for continuous improvement has become part of the educational landscape under accountability. In many ways, educators have become inundated with data but not always in ways that provide them with a full picture to adequately weigh decisions for their specific context. One way to use…
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DSM-IV and Culturally Sensitive Diagnosis: Some Observations for Counselors.
ERIC Educational Resources Information Center
Smart, David W.; Smart, Julie F.
1997-01-01
Describes the evolution of cultural sensitivity in the third and fourth editions of the Diagnostic and Statistical Manual of Mental Disorders (DSM). Discusses five improvements in the DSM-IV: (1) descriptions of specific cultural features; (2) glossary of culture-bound syndromes; (3) outline of cultural formulation; (4) broader definition of Axis…
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Freshwater Fisheries: Program Planning. Appropriate Technologies for Development. Manual M-1A.
ERIC Educational Resources Information Center
Gregory, Steven
This document is designed for policymakers, program planners, trainers, and coordinators who are considering the potential contribution of fresh water fisheries to the people for and with whom they work. Although based on materials generated by Peace Corps experiences, the document is intended for a broader audience. The specific topic area…
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Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Critton, D.; Tortajada, A; Stetson, G
2008-01-01
Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less
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Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal
2016-01-01
Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages. PMID:27768715
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Brief Report: Autism-like Traits are Associated With Enhanced Ability to Disembed Visual Forms.
Sabatino DiCriscio, Antoinette; Troiani, Vanessa
2017-05-01
Atypical visual perceptual skills are thought to underlie unusual visual attention in autism spectrum disorders. We assessed whether individual differences in visual processing skills scaled with quantitative traits associated with the broader autism phenotype (BAP). Visual perception was assessed using the Figure-ground subtest of the Test of visual perceptual skills-3rd Edition (TVPS). In a large adult cohort (n = 209), TVPS-Figure Ground scores were positively correlated with autistic-like social features as assessed by the Broader autism phenotype questionnaire. This relationship was gender-specific, with males showing a correspondence between visual perceptual skills and autistic-like traits. This work supports the link between atypical visual perception and autism and highlights the importance in characterizing meaningful individual differences in clinically relevant behavioral phenotypes.
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Oxidase-functionalized Fe(3)O(4) nanoparticles for fluorescence sensing of specific substrate.
Liu, Cheng-Hao; Tseng, Wei-Lung
2011-10-03
This study reports the development of a reusable, single-step system for the detection of specific substrates using oxidase-functionalized Fe(3)O(4) nanoparticles (NPs) as a bienzyme system and using amplex ultrared (AU) as a fluorogenic substrate. In the presence of H(2)O(2), the reaction pH between Fe(3)O(4) NPs and AU was similar to the reaction of oxidase and the substrate. The catalytic activity of Fe(3)O(4) NPs with AU was nearly unchanged following modification with poly(diallyldimethylammonium chloride) (PDDA). Based on these features, we prepared a composite of PDDA-modified Fe(3)O(4) NPs and oxidase for the quantification of specific substrates through the H(2)O(2)-mediated oxidation of AU. By monitoring fluorescence intensity at 587 nm of oxidized AU, the minimum detectable concentrations of glucose, galactose, and choline were found to be 3, 2, and 20 μM using glucose oxidase-Fe(3)O(4), galactose oxidase-Fe(3)O(4), and choline oxidase-Fe(3)O(4) composites, respectively. The identification of glucose in blood was selected as the model to validate the applicability of this proposed method. Copyright © 2011 Elsevier B.V. All rights reserved.
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Tatsukawa, Hideki; Otsu, Risa; Tani, Yuji; Wakita, Ryosuke; Hitomi, Kiyotaka
2018-05-09
Chronic kidney disease is characterized by prolonged decline in renal function, excessive accumulation of ECM, and progressive tissue fibrosis. Transglutaminase (TG) is a crosslinking enzyme that catalyzes the formation of covalent bonds between glutamine and lysine residues, and is involved in the induction of renal fibrosis via the stabilization of ECM and the activation of TGF-β1. Despite the accumulating evidences indicating that TG2 is a key enzyme in fibrosis, genetic knockout of TG2 reduced by only 50% the elevated protein crosslinking and fibrous protein in renal fibrosis model, whereas treatment with TG inhibitor almost completely reduced these levels. Here, we also clarified the distributions of TG isozymes and their in situ activities and identified the isozyme-specific crosslinked substrates for both TG1 and TG2 in fibrotic kidney. We found that TG1 activity was markedly enhanced in renal tubular epithelium and interstitial areas, whereas TG2 activity increased only in the extracellular space. In total, 47 and 67 possible candidates were identified as TG1 and TG2 substrates, respectively, only in fibrotic kidney. Among them, several possible substrates related to renal disease and fibrosis were identified. These findings provide novel insights into the mechanisms of renal fibrosis through the targeting of isozyme-specific TG substrates.
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Attachment dynamics of Photosystem I on nano-tailored surfaces for photovoltaic applications
NASA Astrophysics Data System (ADS)
Mukherjee, Dibyendu; Bruce, Barry D.; Khomami, Bamin
2010-03-01
Photosystem I (PSI), a biological photodiode, is a supra-molecular protein complex that charge separates upon exposure to light. Effective use of photo-electrochemical activities of PSI for hybrid photovoltaic (PV) device fabrications requires optimal encapsulation of these proteins onto organic/ inorganic substrates. Our results indicate that various experimental parameters alter the surface attachment dynamics of PSI deposited from colloidal aqueous buffer suspensions onto OH-terminated alkanethiolate/Au SAM substrates, thereby resulting in complex structural arrangements which affect the electron transfer and capture pathway of PSI. We present surface topographical, specific adsorption and polarization fluorescence characterizations of PSI/Au SAM substrates to elucidate the protein-surface interaction kinetics as well as the directional attachment dynamics of PSI. Our final goal is to enable site-specific homogeneous attachment of directionally aligned PSI onto chemically tailored nano-patterned substrates.
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Chen, Yi-Ju; Lu, Cheng-Tsung; Huang, Kai-Yao; Wu, Hsin-Yi; Chen, Yu-Ju; Lee, Tzong-Yi
2015-01-01
S-glutathionylation, the covalent attachment of a glutathione (GSH) to the sulfur atom of cysteine, is a selective and reversible protein post-translational modification (PTM) that regulates protein activity, localization, and stability. Despite its implication in the regulation of protein functions and cell signaling, the substrate specificity of cysteine S-glutathionylation remains unknown. Based on a total of 1783 experimentally identified S-glutathionylation sites from mouse macrophages, this work presents an informatics investigation on S-glutathionylation sites including structural factors such as the flanking amino acids composition and the accessible surface area (ASA). TwoSampleLogo presents that positively charged amino acids flanking the S-glutathionylated cysteine may influence the formation of S-glutathionylation in closed three-dimensional environment. A statistical method is further applied to iteratively detect the conserved substrate motifs with statistical significance. Support vector machine (SVM) is then applied to generate predictive model considering the substrate motifs. According to five-fold cross-validation, the SVMs trained with substrate motifs could achieve an enhanced sensitivity, specificity, and accuracy, and provides a promising performance in an independent test set. The effectiveness of the proposed method is demonstrated by the correct identification of previously reported S-glutathionylation sites of mouse thioredoxin (TXN) and human protein tyrosine phosphatase 1b (PTP1B). Finally, the constructed models are adopted to implement an effective web-based tool, named GSHSite (http://csb.cse.yzu.edu.tw/GSHSite/), for identifying uncharacterized GSH substrate sites on the protein sequences. PMID:25849935
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Intrinsic kinetic parameters of substrate utilization by immobilized anaerobic sludge.
Zaiat, M; Vieira, L G; Foresti, E
1997-01-20
This article presents a method for evaluating the intrinsic kinetic parameters of the specific substrate utilization rate (r) equation and discusses the results obtained for anaerobic sludge-bed samples taken from a horizontal-flow anaerobic immobilized sludge (HAIS) reactor. This method utilizes a differential reactor filled with polyurethane foam matrices containing immobilized anaerobic sludge which is subjected to a range of feeding substrate flow rates. The range of liquid superficial velocities thus obtained are used for generating data of observed specific substrate utilization rates (r(obs)) under a diversity of external mass transfer resistance conditions. The r(obs) curves are then adjusted to permit their extrapolation for the condition of no external mass transfer resistance, and the values determined are used as a test for the condition of absence of limitation of internal mass transfer. The intrinsic parameters r(max), the maximum specific substrate utilization rate, and K(s), the half-velocity coefficient, are evaluated from the r values under no external mass transfer resistance and no internal mass transfer limitation. The application of such a method for anaerobic sludge immobilized in polyurethane foam particles treating a glucose substrate at 30 degrees C resulted in intrinsic r(max) and K(s), respectively, of 0.330 mg chemical oxygen demand (COD) . mg(-1) volatile suspended solids (VSS) . h(-1) and 72 mg COD . L(-1). In comparison with the values found in the literature, intrinsic r(max) is significantly high and intrinsic K(s) is relatively low. (c) 1997 John Wiley & Sons, Inc.
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Substrate sequence selectivity of APOBEC3A implicates intra-DNA interactions.
Silvas, Tania V; Hou, Shurong; Myint, Wazo; Nalivaika, Ellen; Somasundaran, Mohan; Kelch, Brian A; Matsuo, Hiroshi; Kurt Yilmaz, Nese; Schiffer, Celia A
2018-05-14
The APOBEC3 (A3) family of human cytidine deaminases is renowned for providing a first line of defense against many exogenous and endogenous retroviruses. However, the ability of these proteins to deaminate deoxycytidines in ssDNA makes A3s a double-edged sword. When overexpressed, A3s can mutate endogenous genomic DNA resulting in a variety of cancers. Although the sequence context for mutating DNA varies among A3s, the mechanism for substrate sequence specificity is not well understood. To characterize substrate specificity of A3A, a systematic approach was used to quantify the affinity for substrate as a function of sequence context, length, secondary structure, and solution pH. We identified the A3A ssDNA binding motif as (T/C)TC(A/G), which correlated with enzymatic activity. We also validated that A3A binds RNA in a sequence specific manner. A3A bound tighter to substrate binding motif within a hairpin loop compared to linear oligonucleotide, suggesting A3A affinity is modulated by substrate structure. Based on these findings and previously published A3A-ssDNA co-crystal structures, we propose a new model with intra-DNA interactions for the molecular mechanism underlying A3A sequence preference. Overall, the sequence and structural preferences identified for A3A leads to a new paradigm for identifying A3A's involvement in mutation of endogenous or exogenous DNA.
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The Darwinian revolution: Rethinking its meaning and significance
Ruse, Michael
2009-01-01
The Darwinian revolution is generally taken to be one of the key events in the history of Western science. In recent years, however, the very notion of a scientific revolution has come under attack, and in the specific case of Charles Darwin and his Origin of Species there are serious questions about the nature of the change (if there was such) and the specifically Darwinian input. This article considers these issues by addressing these questions: Was there a Darwinian revolution? That is, was there a revolution at all? Was there a Darwinian revolution? That is, what was the specific contribution of Charles Darwin? Was there a Darwinian revolution? That is, what was the conceptual nature of what occurred on and around the publication of the Origin? I argue that there was a major change, both scientifically and in a broader metaphysical sense; that Charles Darwin was the major player in the change, although one must qualify the nature and the extent of the change, looking particularly at things in a broader historical context than just as an immediate event; and that the revolution was complex and we need the insights of rather different philosophies of scientific change to capture the whole phenomenon. In some respects, indeed, the process of analysis is still ongoing and unresolved. PMID:19528652
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Regulatory Flexibility: An Individual Differences Perspective on Coping and Emotion Regulation.
Bonanno, George A; Burton, Charles L
2013-11-01
People respond to stressful events in different ways, depending on the event and on the regulatory strategies they choose. Coping and emotion regulation theorists have proposed dynamic models in which these two factors, the person and the situation, interact over time to inform adaptation. In practice, however, researchers have tended to assume that particular regulatory strategies are consistently beneficial or maladaptive. We label this assumption the fallacy of uniform efficacy and contrast it with findings from a number of related literatures that have suggested the emergence of a broader but as yet poorly defined construct that we refer to as regulatory flexibility. In this review, we articulate this broader construct and define both its features and limitations. Specifically, we propose a heuristic individual differences framework and review research on three sequential components of flexibility for which propensities and abilities vary: sensitivity to context, availability of a diverse repertoire of regulatory strategies, and responsiveness to feedback. We consider the methodological limitations of research on each component, review questions that future research on flexibility might address, and consider how the components might relate to each other and to broader conceptualizations about stability and change across persons and situations. © The Author(s) 2013.
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Health Policy Training: A Review of the Literature
Heiman, Harry J.; Smith, L. Lerissa; McKool, Marissa; Mitchell, Denise N.; Roth Bayer, Carey
2015-01-01
The context within which health care and public health systems operate is framed by health policies. There is growing consensus about the need for increased health policy leadership and a health professional workforce prepared to assume these leadership roles. At the same time, there is strong evidence supporting the need for a broader policy lens and the need to intentionally target health disparities. We reviewed the published literature between 1983 and 2013 regarding health policy training. From 5124 articles identified, 33 met inclusion criteria. Articles varied across common themes including target audience, goal(s), health policy definition, and core curricular content. The majority of articles were directed to medical or nursing audiences. Most articles framed health policy as health care policy and only a small number adopted a broader health in all policies definition. Few articles specifically addressed vulnerable populations or health disparities. The need for more rigorous research and evaluation to inform health policy training is compelling. Providing health professionals with the knowledge and skills to engage and take leadership roles in health policy will require training programs to move beyond their limited health care-oriented health policy framework to adopt a broader health and health equity in all policies approach. PMID:26703657
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Health Policy Training: A Review of the Literature.
Heiman, Harry J; Smith, L Lerissa; McKool, Marissa; Mitchell, Denise N; Roth Bayer, Carey
2015-12-23
The context within which health care and public health systems operate is framed by health policies. There is growing consensus about the need for increased health policy leadership and a health professional workforce prepared to assume these leadership roles. At the same time, there is strong evidence supporting the need for a broader policy lens and the need to intentionally target health disparities. We reviewed the published literature between 1983 and 2013 regarding health policy training. From 5124 articles identified, 33 met inclusion criteria. Articles varied across common themes including target audience, goal(s), health policy definition, and core curricular content. The majority of articles were directed to medical or nursing audiences. Most articles framed health policy as health care policy and only a small number adopted a broader health in all policies definition. Few articles specifically addressed vulnerable populations or health disparities. The need for more rigorous research and evaluation to inform health policy training is compelling. Providing health professionals with the knowledge and skills to engage and take leadership roles in health policy will require training programs to move beyond their limited health care-oriented health policy framework to adopt a broader health and health equity in all policies approach.
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Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude
2016-04-04
Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.
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Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y
2000-12-07
Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.
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Allosteric Control of Substrate Specificity of the Escherichia coli ADP-glucose Pyrophosphorylase
NASA Astrophysics Data System (ADS)
Ebrecht, Ana C.; Solamen, Ligin; Hill, Benjamin L.; Iglesias, Alberto A.; Olsen, Kenneth W.; Ballicora, Miguel A.
2017-06-01
The substrate specificity of enzymes is crucial to control the fate of metabolites to different pathways. However, there is growing evidence that many enzymes can catalyze alternative reactions. This promiscuous behavior has important implications in protein evolution and the acquisition of new functions. The question is how the undesirable outcomes of in vivo promiscuity can be prevented. ADP-glucose pyrophosphorylase from Escherichia coli is an example of an enzyme that needs to select the correct substrate from a broad spectrum of alternatives. This selection will guide the flow of carbohydrate metabolism towards the synthesis of reserve polysaccharides. Here, we show that the allosteric activator fructose-1,6-bisphosphate plays a role in such selection by increasing the catalytic efficiency of the enzyme towards the use of ATP rather than other nucleotides. In the presence of fructose-1,6-bisphosphate, the kcat/S0.5 for ATP was near 600-fold higher that other nucleotides, whereas in the absence of activator was only 3-fold higher. We propose that the allosteric regulation of certain enzymes is an evolutionary mechanism of adaptation for the selection of specific substrates.
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Sakamoto, Yasumitsu; Suzuki, Yoshiyuki; Iizuka, Ippei; Tateoka, Chika; Roppongi, Saori; Fujimoto, Mayu; Inaka, Koji; Tanaka, Hiroaki; Yamada, Mitsugu; Ohta, Kazunori; Gouda, Hiroaki; Nonaka, Takamasa; Ogasawara, Wataru; Tanaka, Nobutada
2015-01-01
The dipeptidyl peptidase 11 from Porphyromonas gingivalis (PgDPP11) belongs to the S46 family of serine peptidases and preferentially cleaves substrates with Asp/Glu at the P1 position. The molecular mechanism underlying the substrate specificity of PgDPP11, however, is unknown. Here, we report the crystal structure of PgDPP11. The enzyme contains a catalytic domain with a typical double β-barrel fold and a recently identified regulatory α-helical domain. Crystal structure analyses, docking studies, and biochemical studies revealed that the side chain of Arg673 in the S1 subsite is essential for recognition of the Asp/Glu side chain at the P1 position of the bound substrate. Because S46 peptidases are not found in mammals and the Arg673 is conserved among DPP11s, we anticipate that DPP11s could be utilised as targets for antibiotics. In addition, the present structure analyses could be useful templates for the design of specific inhibitors of DPP11s from pathogenic organisms. PMID:26057589
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Cobb, Steven L.; Deng, Hai; McEwan, Andrew R.; Naismith, James H.; O’Hagan, David; Robinson, David A.
2012-01-01
The fluorinase enzyme from Streptomyces cattleya displays an unusual ability in biocatalysis in that it forms a C–F bond. We now report that the enzyme will accept 2′-deoxyadenosine in place of adenosine substrates, and structural evidence reveals a reorganisation in hydrogen bonding to accommodate this substrate series. It emerges from this study that the enzyme does not require a planar ribose conformation of the substrate to catalyse C–F bond formation. PMID:16604208
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Understanding Cullin-RING E3 Biology through Proteomics-based Substrate Identification*
Harper, J. Wade; Tan, Meng-Kwang Marcus
2012-01-01
Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field. PMID:22962057
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Understanding cullin-RING E3 biology through proteomics-based substrate identification.
Harper, J Wade; Tan, Meng-Kwang Marcus
2012-12-01
Protein turnover through the ubiquitin-proteasome pathway controls numerous developmental decisions and biochemical processes in eukaryotes. Central to protein ubiquitylation are ubiquitin ligases, which provide specificity in targeted ubiquitylation. With more than 600 ubiquitin ligases encoded by the human genome, many of which remain to be studied, considerable effort is being placed on the development of methods for identifying substrates of specific ubiquitin ligases. In this review, we describe proteomic technologies for the identification of ubiquitin ligase targets, with a particular focus on members of the cullin-RING E3 class of ubiquitin ligases, which use F-box proteins as substrate specific adaptor proteins. Various proteomic methods are described and are compared with genetic approaches that are available. The continued development of such methods is likely to have a substantial impact on the ubiquitin-proteasome field.
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Carbon-carbon mirrors for exoatmospheric and space applications
NASA Astrophysics Data System (ADS)
Krumweide, Duane E.; Wonacott, Gary D.; Woida, Patrick M.; Woida, Rigel Q.; Shih, Wei
2007-09-01
The cost and leadtime associated with beryllium has forced the MDA and other defense agencies to look for alternative materials with similar structural and thermal properties. The use of carbon-carbon material, specifically in optical components has been demonstrated analytically in prior SBIR work at San Diego Composites. Carbon-carbon material was chosen for its low in-plane and through-thickness CTE (athermal design), high specific stiffness, near-zero coefficient of moisture expansion, availability of material (specifically c-c honeycomb for lightweight substrates), and compatibility with silicon monoxide (SiO) and silicon dioxide (SiO II) coatings. Subsequent development work has produced shaped carbon-carbon sandwich substrates which have been ground, polished, coated and figured using traditional optical processing. Further development has also been done on machined monolithic carbon-carbon mirror substrates which have also been processed using standard optical finishing techniques.
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Woodman, Neal; Stabile, Frank A.
2015-01-01
Substrate use and locomotory behavior of mammals are typically reflected in external characteristics of the forefeet, such as the relative proportions of the digits and claws. Although skeletal anatomy of the forefeet can be more informative than external characters, skeletons remain rare in systematic collections. This is particularly true for the Myosoricinae (Eulipotyphla: Soricidae), a small clade of African shrews that includes both ambulatory forest shrews (Myosorex) and semifossorial mole shrews (Surdisorex). Most species in this subfamily have restricted distributions, and their behavior and ecology are mostly unstudied. To better understand the potential range of locomotory behavior among myosoricines, we used digital x-rays to image and facilitate measuring the forefoot skeletons of 9 species. As a gauge of potential variation, we compared them with the ambulatory talpid Uropsilus (Talpidae) and the semifossorial talpid Neurotrichus. The hand morphologies of myosoricines show a graded range of potential substrate use between ambulatory and semifossorial. Some of these shrews exhibit adaptations for increased burrowing efficiency that are similar to those seen in talpids and other mammals, such as longer, broader distal phalanges and claws and shorter, wider metacarpals and proximal and middle phalanges. They also, however, have characteristics that are distinct from talpids, such as maintenance of forefoot asymmetry and an increased emphasis of ray III.
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Ortega, Zaida; Mencía, Abraham; Pérez-Mellado, Valentín
2016-01-01
Mountain lizards are highly vulnerable to climate change, and the continuous warming of their habitats could be seriously threatening their survival. We aim to compare the thermal ecology and microhabitat selection of a mountain lizard, Iberolacerta galani, and a widely distributed lizard, Podarcis bocagei, in a montane area. Both species are currently in close syntopy in the study area, at 1,400 m above the sea level. We determined the precision, accuracy and effectiveness of thermoregulation, and the thermal quality of habitat for both species. We also compared the selection of thermal microhabitats between both species. Results show that I. galani is a cold-adapted thermal specialist with a preferred temperature range of 27.9-29.7 °C, while P. bocagei would be a thermal generalist, with a broader and higher preferred temperature range (30.1-34.5 °C). In addition, I. galani selects rocky substrates while P. bocagei selects warmer soil and leaf litter substrates. The thermal quality of the habitat is higher for P. bocagei than for I. galani. Finally, P. bocagei achieves a significantly higher effectiveness of thermoregulation (0.87) than I. galani (0.80). Therefore, these mountain habitat conditions seem currently more suitable for performance of thermophilic generalist lizards than for cold-specialist lizards.
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NASA Astrophysics Data System (ADS)
Viswanath, Changhyun Ko, B.; Yang, Zheng; Ramanathan, Shriram
2011-03-01
VO2 undergoes a sharp metal-insulator transition at ˜67 °C with several orders of change in conductivity and optical transmittance. Understanding and control of the properties of vanadium oxide layers grown on technologically relevant substrates such as Si (100) single crystals is therefore of great interest. In this work, we show tunability of metal-insulator transition temperature as well as recoverable stress in VO2 thin films grown on Si substrate by introducing nanoscale atomic layer deposited HfO2 interfacial layers with no degradation in the resistance ratio. For a confined VO2 film, the metal-insulator transition temperature is suppressed by ˜16 °C and the recoverable stress is 150 MPa, compared to 400 MPa for a bare film. These observations are further correlated with in situ variable temperature measurement of stress changes occurring during the phase transition. Structural and microstructural studies on the various samples have been carried out by x ray diffraction and cross-sectional transmission electron microscopy. The strategy of tuning the metal-insulator transition characteristics by nanoscale interfacial dielectrics is of broader relevance in design of programmable materials and integration into solid state devices for electronics.
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Mencía, Abraham; Pérez-Mellado, Valentín
2016-01-01
Mountain lizards are highly vulnerable to climate change, and the continuous warming of their habitats could be seriously threatening their survival. We aim to compare the thermal ecology and microhabitat selection of a mountain lizard, Iberolacerta galani, and a widely distributed lizard, Podarcis bocagei, in a montane area. Both species are currently in close syntopy in the study area, at 1,400 m above the sea level. We determined the precision, accuracy and effectiveness of thermoregulation, and the thermal quality of habitat for both species. We also compared the selection of thermal microhabitats between both species. Results show that I. galani is a cold-adapted thermal specialist with a preferred temperature range of 27.9–29.7 °C, while P. bocagei would be a thermal generalist, with a broader and higher preferred temperature range (30.1–34.5 °C). In addition, I. galani selects rocky substrates while P. bocagei selects warmer soil and leaf litter substrates. The thermal quality of the habitat is higher for P. bocagei than for I. galani. Finally, P. bocagei achieves a significantly higher effectiveness of thermoregulation (0.87) than I. galani (0.80). Therefore, these mountain habitat conditions seem currently more suitable for performance of thermophilic generalist lizards than for cold-specialist lizards. PMID:27280076
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Dong, Boliang; Peng, Haihui; Motika, Stephen E; Shi, Xiaodong
2017-08-16
The discovery of photoassisted diazonium activation toward gold(I) oxidation greatly extended the scope of gold redox catalysis by avoiding the use of a strong oxidant. Some practical issues that limit the application of this new type of chemistry are the relative low efficiency (long reaction time and low conversion) and the strict reaction condition control that is necessary (degassing and inert reaction environment). Herein, an alternative photofree condition has been developed through Lewis base induced diazonium activation. With this method, an unreactive Au I catalyst was used in combination with Na 2 CO 3 and diazonium salts to produce a Au III intermediate. The efficient activation of various substrates, including alkyne, alkene and allene was achieved, followed by rapid Au III reductive elimination, which yielded the C-C coupling products with good to excellent yields. Relative to the previously reported photoactivation method, our approach offered greater efficiency and versatility through faster reaction rates and broader reaction scope. Challenging substrates such as electron rich/neutral allenes, which could not be activated under the photoinitiation conditions (<5 % yield), could be activated to subsequently yield the desired coupling products in good to excellent yield. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G
2014-11-21
Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-N(G)-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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A Dual-Catalysis Approach to Enantioselective [2+2] Photocycloadditions Using Visible Light
Du, Juana; Skubi, Kazimer L.; Schultz, Danielle M.; Yoon, Tehshik P.
2015-01-01
In contrast to the wealth of catalytic systems that are available to control the stereochemistry of thermally promoted cycloadditions, few similarly effective methods exist for the stereocontrol of photochemical cycloadditions. A major unsolved challenge in the design of enantioselective catalytic photocycloaddition reactions has been the difficulty of controlling racemic background reactions that occur by direct photoexcitation of substrates while unbound to catalyst. Here we describe a strategy for eliminating the racemic background reaction in asymmetric [2+2] photocycloadditions of α,β-unsaturated ketones to the corresponding cyclobutanes by employing a dual-catalyst system consisting of a visible light-absorbing transition metal photocatalyst and a stereocontrolling Lewis acid co-catalyst. The independence of these two catalysts enables broader scope, greater stereochemical flexibility, and better efficiency than previously reported methods for enantioselective photochemical cycloadditions. PMID:24763585
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High-performance terahertz wave absorbers made of silicon-based metamaterials
DOE Office of Scientific and Technical Information (OSTI.GOV)
Yin, Sheng; Zhu, Jianfei; Jiang, Wei
2015-08-17
Electromagnetic (EM) wave absorbers with high efficiency in different frequency bands have been extensively investigated for various applications. In this paper, we propose an ultra-broadband and polarization-insensitive terahertz metamaterial absorber based on a patterned lossy silicon substrate. Experimentally, a large absorption efficiency more than 95% in a frequency range of 0.9–2.5 THz was obtained up to a wave incident angle as large as 70°. Much broader absorption bandwidth and excellent oblique incidence absorption performance are numerically demonstrated. The underlying mechanisms due to the combination of a waveguide cavity mode and impedance-matched diffraction are analyzed in terms of the field patternsmore » and the scattering features. The monolithic THz absorber proposed here may find important applications in EM energy harvesting systems such as THz barometer or biosensor.« less
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Wendlandt, Alison E; Stahl, Shannon S
2014-01-08
Copper amine oxidases are a family of enzymes with quinone cofactors that oxidize primary amines to aldehydes. The native mechanism proceeds via an iminoquinone intermediate that promotes high selectivity for reactions with primary amines, thereby constraining the scope of potential biomimetic synthetic applications. Here we report a novel bioinspired quinone catalyst system consisting of 1,10-phenanthroline-5,6-dione/ZnI2 that bypasses these constraints via an abiological pathway involving a hemiaminal intermediate. Efficient aerobic dehydrogenation of non-native secondary amine substrates, including pharmaceutically relevant nitrogen heterocycles, is demonstrated. The ZnI2 cocatalyst activates the quinone toward amine oxidation and provides a source of iodide, which plays an important redox-mediator role to promote aerobic catalytic turnover. These findings provide a valuable foundation for broader development of aerobic oxidation reactions employing quinone-based catalysts.
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NASA Astrophysics Data System (ADS)
Ahmed, Nauman; Voisey, K. T.; McCartney, D. G.
2014-02-01
Laser surface melting of thermally sprayed coatings has the potential to enhance their corrosion properties by incorporating favorable microstructural changes. Besides homogenizing the as-sprayed structure, laser melting may induce certain microstructural modifications (i.e., supplementary features) in addition to those that directly improve the corrosion performance. Such features, being a direct result of the laser treatment process, are described in this paper which is part of a broader study in which high velocity oxy-fuel sprayed Inconel 625 coatings on mild-steel substrates were treated with a diode laser and the modified microstructure characterized using optical and scanning electron microscopy and x-ray diffraction. The laser treated coating features several different zones, including a region with a microstructure in which there is a continuous columnar dendritic structure through a network of retained oxide stringers.
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Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.
Fuchs, Julian E; Huber, Roland G; Waldner, Birgit J; Kahler, Ursula; von Grafenstein, Susanne; Kramer, Christian; Liedl, Klaus R
2015-01-01
Biomolecular recognition is crucial in cellular signal transduction. Signaling is mediated through molecular interactions at protein-protein interfaces. Still, specificity and promiscuity of protein-protein interfaces cannot be explained using simplistic static binding models. Our study rationalizes specificity of the prototypic protein-protein interface between thrombin and its peptide substrates relying solely on binding site dynamics derived from molecular dynamics simulations. We find conformational selection and thus dynamic contributions to be a key player in biomolecular recognition. Arising entropic contributions complement chemical intuition primarily reflecting enthalpic interaction patterns. The paradigm "dynamics govern specificity" might provide direct guidance for the identification of specific anchor points in biomolecular recognition processes and structure-based drug design.
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In-vitro engineering of novel bioactivity in the natural enzymes
NASA Astrophysics Data System (ADS)
Tiwari, Vishvanath
2016-10-01
Enzymes catalyze various biochemical functions with high efficiency and specificity. In-vitro design of the enzyme leads to novel bioactivity in this natural biomolecule that give answers of some vital questions like crucial residues in binding with substrate, molecular evolution, cofactor specificity etc. Enzyme engineering technology involves directed evolution, rational designing, semi-rational designing and structure-based designing using chemical modifications. Similarly, combined computational and in-vitro evolution approaches together help in artificial designing of novel bioactivity in the natural enzyme. DNA shuffling, error prone PCR and staggered extension process are used to artificially redesign active site of enzyme, which can alter its efficiency and specificity. Modifications of the enzyme can lead to the discovery of new path of molecular evolution, designing of efficient enzymes, locating active sites and crucial residues, shift in substrate and cofactor specificity. The methods and thermodynamics of in-vitro designing of the enzyme are also discussed. Similarly, engineered thermophilic and psychrophilic enzymes attain substrate specificity and activity of mesophilic enzymes that may also be beneficial for industry and therapeutics.
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Wright, Courtney J; Zeeman, Heidi; Kendall, Elizabeth; Whitty, Jennifer A
2017-07-01
Despite the recent emphasis in Australian political, academic, and legislative narratives to more actively promote real housing choice for people with high healthcare and support needs, there is a lack of understanding regarding the specific housing features that might constitute better housing solutions for this population. Inclusive housing provision in Australia rightly emphasises safety and accessibility issues but often fails to incorporate factors related to broader psychosocial elements of housing such as dwelling location, neighbourhood quality, and overall design. While the importance of these broader elements appears obvious, it is not yet clear what specific housing features relate to these elements and how they might contribute to housing solutions for people with high healthcare and support needs. For individuals with complex neurological conditions such as brain injury or cerebral palsy, who require maximum support on a daily basis yet want to live independently and away from a primary care hospital or health facility, a more detailed understanding of the housing features that might influence design and development is needed. Thus, in order to clarify the broader factors related to housing solutions for this population, a systematic review was conducted to identify and synthesise the current research evidence (post-2003) and guide future housing design and development opportunities. From the included studies (n=26), 198 unique housing features were identified. From the 198 features, 142 related to housing design (i.e., internal or external characteristics of the dwelling and its land), 12 related to the dwelling's location (i.e., its proximity to available resources), and 54 related to the nature of the surrounding neighbourhood (i.e., the physical, social, and economic conditions of the area). The findings of this review contribute significantly to the literature by reporting a broader scope of relevant housing features for people with neurological disability, presenting preliminary guiding principles for housing design and development for this population, and identifying opportunities for future research. Copyright © 2017 Elsevier Ltd. All rights reserved.
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NASA Technical Reports Server (NTRS)
Kim, Jae-Woo; Choi, Sang H.; Lillehei, Peter T.; King, Glen C.; Elliott, James R.; Chu, Sang-Hyon; Park, Yeonjoon; Watt, Gerald D.
2004-01-01
Nanoparticle arrays biologically derived from an electrochemically-controlled site-specific biomineralization were fabricated on a gold substrate through the immobilization process of biomolecules. The work reported herein includes the immobilization of ferritin with various surface modifications, the electrochemical biomineralization of ferritins with different inorganic cores, the fabrication of self-assembled arrays with the immobilized ferritin, and the electrochemical characterization of various core materials. Protein immobilization on the substrate is achieved by anchoring ferritins with dithiobis-N-succinimidyl propionate (DTSP). A reconstitution process of electrochemical site-specific biomineralization with a protein cage loads ferritins with different core materials such as Pt, Co, Mn, and Ni. The ferritin acts as a nano-scale template, a biocompatible cage, and a separator between the nanoparticles. The nano-sized metalcored ferritins on a gold substrate displayed a good electrochemical activity for the electron transport and storage, which is suitable for bioelectronics applications such as biofuel cell, bionanobattery, biosensors, etc. Keywords: Ferritin, immobilization, site-specific reconstitution, biomineralization, and bioelectronics
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Chatellard, Lucile; Trably, Eric; Carrère, Hélène
2016-12-01
The impact on dark fermentation of seven carbohydrates as model substrates of lignocellulosic fractions (glucose, cellobiose, microcrystalline cellulose, arabinose, xylose, xylan and wheat straw) was investigated. Metabolic patterns and bacterial communities were characterized at the end of batch tests inoculated with manure digestate. It was found that hydrogen production was linked to the sugar type (pentose or hexose) and the degree of polymerisation. Hexoses produced less hydrogen, with a specific selection of lactate-producing bacterial community structures. Maximal hydrogen production was five times higher on pentose-based substrates, with specific bacterial community structures producing acetate and butyrate as main metabolites. Low hydrogen amounts accumulated from complex sugars (cellulose, xylan and wheat straw). A relatively high proportion of the reads was affiliated to Ruminococcaceae suggesting an efficient hydrolytic activity. Knowing that the bacterial community structure is very specific to a particular substrate offers new possibilities to design more efficient H 2 -producing biological systems. Copyright © 2016 Elsevier Ltd. All rights reserved.
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Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J
2012-05-15
Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
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Yoshida, Hiromi; Yoshihara, Akihide; Ishii, Tomohiko; Izumori, Ken; Kamitori, Shigehiro
2016-12-01
Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.
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Maeda, Hiroshi; Sakai, Daisuke; Kobayashi, Takuji; Morita, Hiroto; Okamoto, Ayako; Takeuchi, Michio; Kusumoto, Ken-Ichi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei
2016-06-01
Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.
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Grauffel, Cédric; Abboud, Angèle; Liszczak, Glen; Marmorstein, Ronen; Arnesen, Thomas; Reuter, Nathalie
2012-01-01
Nt-acetylation is among the most common protein modifications in eukaryotes. Although thought for a long time to protect proteins from degradation, the role of Nt-acetylation is still debated. It is catalyzed by enzymes called N-terminal acetyltransferases (NATs). In eukaryotes, several NATs, composed of at least one catalytic domain, target different substrates based on their N-terminal sequences. In order to better understand the substrate specificity of human NATs, we investigated in silico the enzyme-substrate interactions in four catalytic subunits of human NATs (Naa10p, Naa20p, Naa30p and Naa50p). To date hNaa50p is the only human subunit for which X-ray structures are available. We used the structure of the ternary hNaa50p/AcCoA/MLG complex and a structural model of hNaa10p as a starting point for multiple molecular dynamics simulations of hNaa50p/AcCoA/substrate (substrate = MLG, EEE, MKG), hNaa10p/AcCoA/substrate (substrate = MLG, EEE). Nine alanine point-mutants of the hNaa50p/AcCoA/MLG complex were also simulated. Homology models of hNaa20p and hNaa30p were built and compared to hNaa50p and hNaa10p. The simulations of hNaa50p/AcCoA/MLG reproduce the interactions revealed by the X-ray data. We observed strong hydrogen bonds between MLG and tyrosines 31, 138 and 139. Yet the tyrosines interacting with the substrate’s backbone suggest that their role in specificity is limited. This is confirmed by the simulations of hNaa50p/AcCoA/EEE and hNaa10p/AcCoA/MLG, where these hydrogen bonds are still observed. Moreover these tyrosines are all conserved in hNaa20p and hNaa30p. Other amino acids tune the specificity of the S1’ sites that is different for hNaa10p (acidic), hNaa20p (hydrophobic/basic), hNaa30p (basic) and hNaa50p (hydrophobic). We also observe dynamic correlation between the ligand binding site and helix that tightens under substrate binding. Finally, by comparing the four structures we propose maps of the peptide-enzyme interactions that should help rationalizing substrate-specificity and lay the ground for inhibitor design. PMID:23285125
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Støve, Svein Isungset; Magin, Robert S; Foyn, Håvard; Haug, Bengt Erik; Marmorstein, Ronen; Arnesen, Thomas
2016-07-06
N-Terminal acetylation is a common and important protein modification catalyzed by N-terminal acetyltransferases (NATs). Six human NATs (NatA-NatF) contain one catalytic subunit each, Naa10 to Naa60, respectively. In contrast to the ribosome-associated NatA to NatE, NatF/Naa60 specifically associates with Golgi membranes and acetylates transmembrane proteins. To gain insight into the molecular basis for the function of Naa60, we developed an Naa60 bisubstrate CoA-peptide conjugate inhibitor, determined its X-ray structure when bound to CoA and inhibitor, and carried out biochemical experiments. We show that Naa60 adapts an overall fold similar to that of the catalytic subunits of ribosome-associated NATs, but with the addition of two novel elongated loops that play important roles in substrate-specific binding. One of these loops mediates a dimer to monomer transition upon substrate-specific binding. Naa60 employs a catalytic mechanism most similar to Naa50. Collectively, these data reveal the molecular basis for Naa60-specific acetyltransferase activity with implications for its Golgi-specific functions. Copyright © 2016 Elsevier Ltd. All rights reserved.
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[Structure and function of the bacterial flagellar type III protein export system in Salmonella ].
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.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Khadempour, Lily; Burnum-Johnson, Kristin E.; Baker, Erin S.
Herbivores use symbiotic microbes to help gain access to energy and nutrients from plant material. Leaf-cutter ants are a paradigmatic example, having tremendous impact on their ecosystems as dominant generalist herbivores through cultivation of a fungus, Leucoagaricus gongylophorous. Here we examine how this mutualism could facilitate the flexible substrate incorporation of the ants by providing leaf-cutter ant subcolonies four substrate types: leaves, flowers, oats, and a mixture of all three. Through metaproteomic analysis of the fungus gardens, we were able to identify and quantify 1766 different fungal proteins, including 161 biomass-degrading enzymes. This analysis revealed that fungal protein profiles weremore » significantly different between subcolonies fed different substrates with the highest abundance of cellulolytic enzymes observed in the leaf and flower treatments. When the fungus garden is provided with leaves and flowers, which contain the majority of their energy in recalcitrant material, it increases its production of proteins that break down cellulose: endoglucanases, exoglucanase and β-glucosidase. Further, the complete metaproteomes for the leaves and flowers treatments were very similar, the mixed treatment closely resembled the treatment with oats alone. This suggests that when provided a mixture of substrates, the fungus garden preferentially produces enzymes necessary for breakdown of simpler, more digestible substrates. This flexible, substrate-specific response of the fungal cultivar allows the leaf-cutter ants to derive energy from a wide range of substrates, which may contribute to their ability to be dominant generalist herbivores.« less
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ERIC Educational Resources Information Center
Kimerling, Rachel; Alvarez, Jennifer; Pavao, Joanne; Mack, Katelyn P.; Smith, Mark W.; Baumrind, Nikki
2009-01-01
Prior research has demonstrated that intimate partner violence (IPV) is associated with employment instability among poor women. The current study assesses the broader relationship between IPV and women's workforce participation in a population-based sample of 6,698 California women. We examined past-year IPV by analyzing specific effects of…
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ERIC Educational Resources Information Center
Raselimo, Mohaeka; Wilmot, Di
2013-01-01
This article addresses how teachers in a specific developing world context interpreted a curriculum reform initiative. It is located within a broader interpretive study that investigated the integration of Environmental Education into the formal education system of Lesotho with particular reference to secondary school geography. More specifically…
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ERIC Educational Resources Information Center
Madore, Kevin P.; Gaesser, Brendan; Schacter, Daniel L.
2014-01-01
According to the constructive episodic simulation hypothesis (Schacter & Addis, 2007), both remembered past and imagined future events rely heavily on episodic memory. An alternative hypothesis is that observed similarities between remembering and imagining reflect the influence of broader factors such as descriptive ability, narrative style,…
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Letters from Early Career Academics: The Physical Education and Sport Pedagogy Field of Play
ERIC Educational Resources Information Center
Alfrey, Laura; Enright, Eimear; Rynne, Steven
2017-01-01
Taking our lead from Rainer Maria Rilke's (1929) "Letters to a Young Poet", our broader project aimed to create a space for dialogue and intergenerational learning between Physical Education and Sport Pedagogy (PESP) Early Career Academics (ECAs) and members of the PESP professoriate. This paper focuses specifically on the experiences of…
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ERIC Educational Resources Information Center
Dahlstedt, Magnus; Hertzberg, Fredrik
2014-01-01
The focus of this article is the growing importance of entrepreneurship in the context of Swedish education policy. Departing from Foucault's concept of governmentality, this article analyzes some of the main ideas in the discourse on entrepreneurship education in Sweden and points out its specifics, as an instance of the broader educational and…
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ERIC Educational Resources Information Center
Bolte, Sven; Poustka, Fritz
2006-01-01
Background: The objective of this study was to investigate the tendency for local processing style ("weak central coherence") and executive dysfunction in parents of subjects with an autism spectrum disorder (ASD) compared with parents of individuals with early onset schizophrenia (EOS) and mental retardation (MR). Method: Sixty-two…
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CTE's Role in Energy and Environmental Sustainability
ERIC Educational Resources Information Center
Hyslop, Alisha
2009-01-01
ACTE Issue Briefs are designed to highlight the role of career and technical education (CTE) in a broader issue of national interest. Each Brief is designed to strengthen the voice of CTE related to the specific issue and to draw more attention to CTE activities and best practices around the country. The Briefs provide background information,…
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Post Viking planetary protection requirements study
NASA Technical Reports Server (NTRS)
Wolfson, R. P.
1977-01-01
Past planetary quarantine requirements were reviewed in the light of present Viking data to determine the steps necessary to prevent contamination of the Martian surface on future missions. The currently used term planetary protection reflects a broader scope of understanding of the problems involved. Various methods of preventing contamination are discussed in relation to proposed projects, specifically the 1984 Rover Mission.
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75 FR 18413 - 2009-2010 Refuge-Specific Hunting and Sport Fishing Regulations-Additions
Federal Register 2010, 2011, 2012, 2013, 2014
2010-04-12
... near the time of acquisition. These regulations ensure that we make the determinations required by... result of this comment. Comment 2: The same commenter asked for an extension of time to further comment... maximize the time available for public review. We believe that a 30-day comment period, through the broader...
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Alexander Hamilton: Soldier-Statesmen of the Constitution. A Bicentennial Series No. 16.
ERIC Educational Resources Information Center
Army Center of Military History, Washington, DC.
Alexander Hamilton was among the most intellectually gifted of the Founding Fathers and a brilliant political theorist, but he lacked practical political experience, and his major political contributions occurred only when his specific policies were adopted and carried forward by others with broader vision. This booklet on Hamilton is one in a…
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Enabling Geotechnical Data for Broader Use by the Spatial Data Infrastructures
ERIC Educational Resources Information Center
Zand, Amir Ghasem
2011-01-01
Geotechnical data are one of the most prevalent data types in civil engineering projects. The majority of the civil engineering projects that are in use today are designed using site-specific geotechnical data. The usage of geotechnical data is not limited to construction projects. This data is used in a wide range of applications, including…
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Moral Education and Literature: On Cora Diamond and Eimear McBride
ERIC Educational Resources Information Center
Mahon, Aine
2017-01-01
I argue in this paper for the rich and subtle connections between moral philosophy and literature as they are articulated and explored in the work of the contemporary American philosopher, Cora Diamond. In its significance for broader educational debates--specifically, debates regarding the value of the arts and humanities in a context of global…
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The case for watchful waiting with Isle Royale's wolf population
Mech, L. David
2013-01-01
In "Should Isle Royale Wolves be Reintroduced? A Case Study on Wilderness Management in a Changing World," Vucetich et al. concluded with the hope that their analysis “motivates broader discussion that deepens understanding of the specifics on Isle Royale and the underlying principles” (2012: 137). This article represents an attempt to continue that discussion.
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ERIC Educational Resources Information Center
Caird, Sally; Lane, Andy
2015-01-01
Despite the widespread availability of information and communication technologies (ICTs) and some research into specific pedagogical practices using them, there has been little research on the role of these technologies in shaping broader pedagogical approaches in higher education (HE). Increased experimentation in using ICTs raises questions…
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A Case-Control Study of Personality Style and Psychopathology in Parents of Subjects with Autism
ERIC Educational Resources Information Center
Bolte, Sven; Knecht, Susan; Poustka, Fritz
2007-01-01
To probe the specificity of traits that might be conceptualised as the broader phenotype of autism, parents of subjects with autism from simplex and multiplex families as well as parents of subjects with obsessive-compulsive disorders (OCD), early onset schizophrenia (EOS) and mental retardation (MR) were assessed using the Personality Style and…