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Sample records for affect substrate specificity

  1. Specific combinations of presenilins and Aph1s affect the substrate specificity and activity of γ-secretase.

    PubMed

    Yonemura, Yoji; Futai, Eugene; Yagishita, Sosuke; Kaether, Christoph; Ishiura, Shoichi

    2016-09-30

    The γ-secretase complex comprises presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (Aph1), and presenilin enhancer 2 (Pen2). PS has two homologues, PS1 and PS2. Aph1 has two isoforms, Aph1a and Aph1b, with the former existing as two splice variants Aph1aL and Aph1aS. Each complex consists of one subunit each, resulting in six different γ-secretases. To better understand the functional differences among the γ-secretases, we reconstituted them using a yeast system and compared Notch1-cleavage and amyloid precursor protein (APP)-cleavage activities. Intriguingly, PS2/Aph1b had a clear substrate specificity: APP-Gal4, but not Notch-Gal4, was cleaved. In HEK cell lines expressing defined γ-secretase subunits, we showed that PS1/Aph1b, PS2/Aph1aL, PS2/Aph1aS and PS2/Aph1b γ-secretase produced amyloid β peptide (Aβ) with a higher Aβ42+Aβ43-to-Aβ40 (Aβ42(43)/Aβ40) ratio than the other γ-secretases. In addition, PS2/Aph1aS γ-secretase produced less Notch intracellular domain (NICD) than did the other 5 γ-secretases. Considering that the Aβ42(43)/Aβ40 ratio is relevant in the pathogenesis of Alzheimer's disease (AD), and that inhibition of Notch cleavage causes severe side effect, these results suggest that the PS2/Aph1aS γ-secretase complex is a potential therapeutic target in AD. PMID:27608597

  2. N-glycosylation affects substrate specificity of chicory fructan 1-exohydrolase: evidence for the presence of an inulin binding cleft.

    PubMed

    Le Roy, Katrien; Verhaest, Maureen; Rabijns, Anja; Clerens, Stefan; Van Laere, André; Van den Ende, Wim

    2007-01-01

    Recently, the three-dimensional structure of chicory (Cichorium intybus) fructan 1-exohydrolase (1-FEH IIa) in complex with its preferential substrate, 1-kestose, was determined. Unfortunately, no such data could be generated with high degree of polymerization (DP) inulin, despite several soaking and cocrystallization attempts. Here, site-directed mutagenesis data are presented, supporting the presence of an inulin-binding cleft between the N- and C-terminal domains of 1-FEH IIa. In general, enzymes that are unable to degrade high DP inulins contain an N-glycosylation site probably blocking the cleft. By contrast, inulin-degrading enzymes have an open cleft configuration. An 1-FEH IIa P294N mutant, introducing an N-glycosylation site near the cleft, showed highly decreased activity against higher DP inulin. The introduction of a glycosyl chain most probably blocks the cleft and prevents inulin binding and degradation. Besides cell wall invertases, fructan 6-exohydrolases (6-FEHs) also contain a glycosyl chain most probably blocking the cleft. Removal of this glycosyl chain by site-directed mutagenesis in Arabidopsis thaliana cell wall invertase 1 and Beta vulgaris 6-FEH resulted in a strong decrease of enzymatic activities of the mutant proteins. By analogy, glycosylation of 1-FEH IIa affected overall enzyme activity. These data strongly suggest that the presence or absence of a glycosyl chain in the cleft is important for the enzyme's stability and optimal conformation.

  3. Alteration of substrate specificity of alanine dehydrogenase

    PubMed Central

    Fernandes, Puja; Aldeborgh, Hannah; Carlucci, Lauren; Walsh, Lauren; Wasserman, Jordan; Zhou, Edward; Lefurgy, Scott T.; Mundorff, Emily C.

    2015-01-01

    The l-alanine dehydrogenase (AlaDH) has a natural history that suggests it would not be a promising candidate for expansion of substrate specificity by protein engineering: it is the only amino acid dehydrogenase in its fold family, it has no sequence or structural similarity to any known amino acid dehydrogenase, and it has a strong preference for l-alanine over all other substrates. By contrast, engineering of the amino acid dehydrogenase superfamily members has produced catalysts with expanded substrate specificity; yet, this enzyme family already contains members that accept a broad range of substrates. To test whether the natural history of an enzyme is a predictor of its innate evolvability, directed evolution was carried out on AlaDH. A single mutation identified through molecular modeling, F94S, introduced into the AlaDH from Mycobacterium tuberculosis (MtAlaDH) completely alters its substrate specificity pattern, enabling activity toward a range of larger amino acids. Saturation mutagenesis libraries in this mutant background additionally identified a double mutant (F94S/Y117L) showing improved activity toward hydrophobic amino acids. The catalytic efficiencies achieved in AlaDH are comparable with those that resulted from similar efforts in the amino acid dehydrogenase superfamily and demonstrate the evolvability of MtAlaDH specificity toward other amino acid substrates. PMID:25538307

  4. Substrate properties affect collective cell motion

    NASA Astrophysics Data System (ADS)

    Pegoraro, Adrian; Guo, Ming; Ehrlicher, Allen; Weitz, David

    2013-03-01

    When cells move collectively, cooperative motion, which is characterized by long range correlations in cell movement, is necessary for migration. This collective cell motion is influenced by cell-cell interactions as well as by cell-substrate coupling. Furthermore, on soft substrates it is possible for cells to mechanically couple over long distances through the substrate itself. By changing the properties of the substrate, it is possible to decouple some of these contributions and better understand the role they play in collective cell motion. We vary both the substrate stiffness and adhesion protein concentration and find changes in the collective cell motion of the cells despite only small differences in total cell density and average cell size in the confluent layers. We test these changes on polyacrylamide and PDMS substrates as well as on structured substrates made of PDMS posts that prevent mechanical coupling through the substrate while still allowing stiffness to be varied.

  5. EUVL mask substrate specifications (wafer-type)

    SciTech Connect

    Tong, W

    1999-07-01

    The Extreme Ultraviolet Lithography (EUVL) program currently is constructing an alpha-class exposure tool known as the Engineering Test Stand (ETS) that will employ 200mm wafer format masks. This report lists and explains the current specifications for the EUVL mask substrates suitable for use on the ETS. The shape and size of the mask are the same as those of a standard 200mm Si wafer. The flatness requirements are driven by the potential image placement distortion caused by the non-telecentric illumination of EUVL. The defect requirements are driven by the printable-defect size and desired yield for mask blank fabrication. Surface roughness can cause both a loss of light throughput and image speckle. The EUVL mask substrate must be made of low-thermal-expansion material because 40% of the light is absorbed by the multilayers and causes some uncorrectable thermal distortion during printing.

  6. Trichodiene synthase. Substrate specificity and inhibition.

    PubMed

    Cane, D E; Yang, G; Xue, Q; Shim, J H

    1995-02-28

    The substrate specificity of the sesquiterpene synthase trichodiene synthase was examined by determining the Vmax and Km parameters for the natural substrate, trans,trans-farnesyl diphosphate (1), its stereoisomer, cis,trans-farnesyl diphosphate, and the tertiary allylic isomer, (3R)-nerolidyl diphosphate (3), using both the native fungal and recombinant enzymes. A series of farnesyl diphosphate analogs, 15, 16, 20, 7, 8, and 9, was also tested as inhibitors of trichodiene synthase. 10-Fluorofarnesyl diphosphate (15) was the most effective competitive inhibitor, with a K1 of 16 nM compared to the Km for 1 of 87 nM, while the ether analog of farnesyl diphosphate, 8, an extremely potent inhibitor of squalene synthase, showed only modest inhibition of trichodiene synthase, with a K1/Km of 70. PMID:7873526

  7. Substrate stiffness affects skeletal myoblast differentiation in vitro

    NASA Astrophysics Data System (ADS)

    Romanazzo, Sara; Forte, Giancarlo; Ebara, Mitsuhiro; Uto, Koichiro; Pagliari, Stefania; Aoyagi, Takao; Traversa, Enrico; Taniguchi, Akiyoshi

    2012-12-01

    To maximize the therapeutic efficacy of cardiac muscle constructs produced by stem cells and tissue engineering protocols, suitable scaffolds should be designed to recapitulate all the characteristics of native muscle and mimic the microenvironment encountered by cells in vivo. Moreover, so not to interfere with cardiac contractility, the scaffold should be deformable enough to withstand muscle contraction. Recently, it was suggested that the mechanical properties of scaffolds can interfere with stem/progenitor cell functions, and thus careful consideration is required when choosing polymers for targeted applications. In this study, cross-linked poly-ɛ-caprolactone membranes having similar chemical composition and controlled stiffness in a supra-physiological range were challenged with two sources of myoblasts to evaluate the suitability of substrates with different stiffness for cell adhesion, proliferation and differentiation. Furthermore, muscle-specific and non-related feeder layers were prepared on stiff surfaces to reveal the contribution of biological and mechanical cues to skeletal muscle progenitor differentiation. We demonstrated that substrate stiffness does affect myogenic differentiation, meaning that softer substrates can promote differentiation and that a muscle-specific feeder layer can improve the degree of maturation in skeletal muscle stem cells.

  8. Identifying Occupationally Specific Affective Behaviors.

    ERIC Educational Resources Information Center

    Pucel, David J.

    1993-01-01

    Data from two groups of cosmetology instructors (n=15) and two groups of machinist instructors (n=17) validated the Occupational Affective Behavior Analysis instrument as capable of identifying affective behaviors viewed as important to success in a given occupation. (SK)

  9. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  10. Modelling substrate specificity and enantioselectivity for lipases and esterases by substrate-imprinted docking

    PubMed Central

    Juhl, P Benjamin; Trodler, Peter; Tyagi, Sadhna; Pleiss, Jürgen

    2009-01-01

    Background Previously, ways to adapt docking programs that were developed for modelling inhibitor-receptor interaction have been explored. Two main issues were discussed. First, when trying to model catalysis a reaction intermediate of the substrate is expected to provide more valid information than the ground state of the substrate. Second, the incorporation of protein flexibility is essential for reliable predictions. Results Here we present a predictive and robust method to model substrate specificity and enantioselectivity of lipases and esterases that uses reaction intermediates and incorporates protein flexibility. Substrate-imprinted docking starts with covalent docking of reaction intermediates, followed by geometry optimisation of the resulting enzyme-substrate complex. After a second round of docking the same substrate into the geometry-optimised structures, productive poses are identified by geometric filter criteria and ranked by their docking scores. Substrate-imprinted docking was applied in order to model (i) enantioselectivity of Candida antarctica lipase B and a W104A mutant, (ii) enantioselectivity and substrate specificity of Candida rugosa lipase and Burkholderia cepacia lipase, and (iii) substrate specificity of an acetyl- and a butyrylcholine esterase toward the substrates acetyl- and butyrylcholine. Conclusion The experimentally observed differences in selectivity and specificity of the enzymes were reproduced with an accuracy of 81%. The method was robust toward small differences in initial structures (different crystallisation conditions or a co-crystallised ligand), although large displacements of catalytic residues often resulted in substrate poses that did not pass the geometric filter criteria. PMID:19493341

  11. Biochar affects macronutrient leaching from a soilless substrate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Byproducts of pyrolysis, known collectively as biochar, are becoming more common and readily available as ventures into alternative energy generation are explored. Little is known about how these materials affect greenhouse substrates. The objective of this research was to determine the effect of ...

  12. Substrate specificity of the ubiquitin and Ubl proteases

    PubMed Central

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

    2016-01-01

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

  13. Composition of agarose substrate affects behavioral output of Drosophila larvae

    PubMed Central

    Apostolopoulou, Anthi A.; Hersperger, Fabian; Mazija, Lorena; Widmann, Annekathrin; Wüst, Alexander; Thum, Andreas S.

    2014-01-01

    In the last decade the Drosophila larva has evolved into a simple model organism offering the opportunity to integrate molecular genetics with systems neuroscience. This led to a detailed understanding of the neuronal networks for a number of sensory functions and behaviors including olfaction, vision, gustation and learning and memory. Typically, behavioral assays in use exploit simple Petri dish setups with either agarose or agar as a substrate. However, neither the quality nor the concentration of the substrate is generally standardized across these experiments and there is no data available on how larval behavior is affected by such different substrates. Here, we have investigated the effects of different agarose concentrations on several larval behaviors. We demonstrate that agarose concentration is an important parameter, which affects all behaviors tested: preference, feeding, learning and locomotion. Larvae can discriminate between different agarose concentrations, they feed differently on them, they can learn to associate an agarose concentration with an odor stimulus and change locomotion on a substrate of higher agarose concentration. Additionally, we have investigated the effect of agarose concentration on three quinine based behaviors: preference, feeding and learning. We show that in all cases examined the behavioral output changes in an agarose concentration-dependent manner. Our results suggest that comparisons between experiments performed on substrates differing in agarose concentration should be done with caution. It should be taken into consideration that the agarose concentration can affect the behavioral output and thereby the experimental outcomes per se potentially due to the initiation of an escape response or changes in foraging behavior on more rigid substrates. PMID:24478658

  14. Prebiotics: preferential substrates for specific germs?

    PubMed

    Roberfroid, M B

    2001-02-01

    A prebiotic is "a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon." The premise is based on the hypothesis that the large gut in humans contains bacteria that are beneficial or detrimental to health. Although this generalization probably gives too simplistic a view of gut microbiology, it is a feasible working concept. Currently, food components that seem to exert the best prebiotic effects are inulin-type fructans. In pure culture, most species of bifidobacteria are adapted to the utilization of these nondigestible oligosaccharides but many other bacteria are also capable of metabolizing them. Clearly, these studies of pure bacteria are of limited use unless their results are supported by the results of studies using mixed cultures. Indeed, as many components of the gut microbiota as possible should be measured to indicate a true prebiotic effect. Simple stimulation of bifidobacteria is insufficient to demonstrate an effect; the effects on other gut microorganisms in vivo with human volunteers is necessary. Adjustment of the composition and activities of the colonic microflora so that health-promoting activities are optimized remains key in functional food development. New methods are being applied extensively to human gut microbiology and promise the degree of reliability required to detect subtle changes in colonic microflora composition and to correlate such changes with health benefits. This is a review of the present state of knowledge concerning prebiotics, with emphasis on the criteria used for classification, mechanisms of selective growth stimulation, and physiologic effects.

  15. Characterizing Protease Specificity: How Many Substrates Do We Need?

    PubMed

    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

  16. Structural Insight into Substrate Specificity of Phosphodiesterase 10

    SciTech Connect

    Wang,H.; Liu, Y.; Hou, J.; Zheng, M.; Robinson, H.; Ke, H.

    2007-01-01

    Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity.

  17. Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity

    PubMed Central

    Kreel, Nathan E.; Tabita, F. Robert

    2015-01-01

    Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions. PMID:26381513

  18. Serine 363 of a hydrophobic region of Archaeal ribulose 1,5-bisphosphate carboxylase/oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis affects CO2/O2 substrate specificity and oxygen sensitivity

    DOE PAGES

    Kreel, Nathan E.; Tabita, F. Robert; Berg, Ivan

    2015-09-18

    Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conservedmore » in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. As a result, it is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.« less

  19. Kinetic and Structural Analysis of Substrate Specificity in Two Copper Amine Oxidases from Hansenula polymorpha

    SciTech Connect

    Chang, Cindy M.; Klema, Valerie J.; Johnson, Bryan J.; Mure, Minae; Klinman, Judith P.; Wilmot, Carrie M.

    2010-04-26

    The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 {angstrom} resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to those of HPAO-1. Despite 34% sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast, HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by k{sub cat}/K{sub m} value differences. In HPAO-1, the k{sub cat}/K{sub m} for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2, it is benzylamine that is the better substrate by 750-fold. In HPAO-2, an inflated {sup D}k{sub cat}/K{sub m}(methylamine) in relation to {sup D}k{sub cat}/K{sub m}(benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, {sup D}k{sub cat}/K{sub m}(S) changes little with the slow substrate and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is k{sub cat}/K{sub m} for the second substrate, O{sub 2}, significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are

  20. Force-driven aggregation of specific bonds on compliant substrates.

    PubMed

    Sarvestani, Alireza S

    2013-07-26

    A thermodynamic model was employed to understand the underlying physics of the force-dependent size of cell's focal adhesions. The model describes the specific adhesion of an elastic membrane to a compliant substrate such that the adhesion site is subjected to the force of a constant pulling traction. The membrane contains mobile adhesion receptors with specific energetic affinities for complimentary ligands on the substrate. The adhesion is resisted by a disjoining pressure induced by a squeezed layer of glycocalyx. The model demonstrates that the enlargement of adhesion area with increasing pulling traction is possible due to a spontaneous response of the adhesion site to attain the state of minimum free energy. The correlation between the force and the adhesion area strongly depends on the rigidity of the substrate. PMID:23764177

  1. Synthetic phospholipids as specific substrates for plasma endothelial lipase.

    PubMed

    Papillon, Julien P N; Pan, Meihui; Brousseau, Margaret E; Gilchrist, Mark A; Lou, Changgang; Singh, Alok K; Stawicki, Todd; Thompson, James E

    2016-08-01

    We designed and prepared synthetic phospholipids that generate lyso-phosphatidylcholine products with a unique mass for convenient detection by LC-MS in complex biological matrices. We demonstrated that compound 4, formulated either as a Triton X-100 emulsion or incorporated in synthetic HDL particles can serve as a substrate for plasma EL with useful specificity. PMID:27344207

  2. Substrate specificities of Penicillium simplicissimum alpha-galactosidases.

    PubMed

    Luonteri, E; Tenkanen, M; Viikari, L

    1998-02-15

    The substrate specificities of three Penicillium simplicissimum alpha-galactosidases, AGLI, AGLII, and AGLIII, were determined by using various isolated galactose-containing oligosaccharides and polymeric galacto(gluco)mannans. AGLI released galactose from melibiose and raffinose-family oligosaccharides but the amount of galactose released was decreased from 96% to 35% by the increasing chain length of the substrate from raffinose to verbascose. It was able to release galactose linked to the nonreducing end and less efficiently to the internal residues of the galactomanno-oligomers. AGLI was able to hydrolyze 60-92% of galactose from polymeric galacto(gluco)mannans alone but its action was facilitated by mannanase and beta-mannosidase. In addition, it was able to release about 10% of the galactose from softwood kraft pulp alone and about 22% in combination with mannanase. AGLII was highly specific toward small galactose-containing oligosaccharides in which the galactose is linked to the nonreducing end of the substrate. It released 90-100% of galactose present in melibiose, raffinose, stachyose, and verbascose; however, it was able to degrade polymeric substrates only in combination with mannanase and beta-mannosidase. AGLIII had only low activity toward the oligomeric substrates tested. It was able to release some galactose from the polymeric galacto(gluco)mannans alone, but its action was clearly enhanced by the backbone degrading enzymes.

  3. Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases

    SciTech Connect

    Blacklock, Brenda J. . E-mail: blacklock@chem.iupui.edu; Jaworski, Jan G.

    2006-07-28

    The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His){sub 6} fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates.

  4. Probing ADAMTS13 Substrate Specificity using Phage Display

    PubMed Central

    Desch, Karl C.; Kretz, Colin; Yee, Andrew; Gildersleeve, Robert; Metzger, Kristin; Agrawal, Nidhi; Cheng, Jane; Ginsburg, David

    2015-01-01

    Von Willebrand factor (VWF) is a large, multimeric protein that regulates hemostasis by tethering platelets to the subendothelial matrix at sites of vascular damage. The procoagulant activity of plasma VWF correlates with the length of VWF multimers, which is proteolytically controlled by the metalloprotease ADAMTS13. To probe ADAMTS13 substrate specificity, we created phage display libraries containing randomly mutated residues of a minimal ADAMTS13 substrate fragment of VWF, termed VWF73. The libraries were screened for phage particles displaying VWF73 mutant peptides that were resistant to proteolysis by ADAMTS13. These peptides exhibited the greatest mutation frequency near the ADAMTS13 scissile residues. Kinetic assays using mutant and wild-type substrates demonstrated excellent agreement between rates of cleavage for mutant phage particles and the corresponding mutant peptides. Cleavage resistance of selected mutations was tested in vivo using hydrodynamic injection of corresponding full-length expression plasmids into VWF-deficient mice. These studies confirmed the resistance to cleavage resulting from select amino acid substitutions and uncovered evidence of alternate cleavage sites and recognition by other proteases in the circulation of ADAMTS13 deficient mice. Taken together, these studies demonstrate the key role of specific amino acids residues including P3-P2’ and P11’, for substrate specificity and emphasize the importance in flowing blood of other ADAMTS13–VWF exosite interactions outside of VWF73. PMID:25849793

  5. Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

    SciTech Connect

    Eren, Elif; Vijayaraghavan, Jagamya; Liu, Jiaming; Cheneke, Belete R.; Touw, Debra S.; Lepore, Bryan W.; Indic, Mridhu; Movileanu, Liviu; van den Berg, Bert; Dutzler, Raimund

    2012-01-17

    Many Gram-negative bacteria, including human pathogens such as Pseudomonas aeruginosa, do not have large-channel porins. This results in an outer membrane (OM) that is highly impermeable to small polar molecules, making the bacteria intrinsically resistant towards many antibiotics. In such microorganisms, the majority of small molecules are taken up by members of the OprD outer membrane protein family. Here we show that OprD channels require a carboxyl group in the substrate for efficient transport, and based on this we have renamed the family Occ, for outer membrane carboxylate channels. We further show that Occ channels can be divided into two subfamilies, based on their very different substrate specificities. Our results rationalize how certain bacteria can efficiently take up a variety of substrates under nutrient-poor conditions without compromising membrane permeability. In addition, they explain how channel inactivation in response to antibiotics can cause resistance but does not lead to decreased fitness.

  6. Structural Basis of Substrate Specificity in Geobacter metallireducens SMUG1.

    PubMed

    Zhang, Zhemin; Shen, Jiemin; Yang, Ye; Li, Jing; Cao, Weiguo; Xie, Wei

    2016-06-17

    Base deamination is a common type of DNA damage that occurs in all organisms. DNA repair mechanisms are critical to maintain genome integrity, in which the base excision repair pathway plays an essential role. In the BER pathway, the uracil DNA glycosylase superfamily is responsible for removing the deaminated bases from DNA and generates apurinic/apyrimidinic (AP) sites. Geobacter metallireducens SMUG1 (GmeSMUG1) is an interesting family 3 enzyme in the UDG superfamily, with dual substrate specificities for DNA with uracil or xanthine. In contrast, the mutant G63P of GmeSMUG1 has exclusive activity for uracil, while N58D is inactive for both substrates, as we have reported previously. However, the structural bases for these substrate specificities are not well understood. In this study, we solved a series of crystal structures of WT and mutants of GmeSMUG1 at relatively high resolutions. These structures provide insight on the molecular mechanism of xanthine recognition for GmeSMUG1 and indicate that H210 plays a key role in xanthine recognition, which is in good agreement with the results of our EMSA and activity assays. More importantly, our mutant structures allow us to build models to rationalize our previous experimental observations of altered substrate activities of these mutants. PMID:27071000

  7. Mesenchymal stem cell adhesion but not plasticity is affected by high substrate stiffness

    NASA Astrophysics Data System (ADS)

    Kal Van Tam, Janice; Uto, Koichiro; Ebara, Mitsuhiro; Pagliari, Stefania; Forte, Giancarlo; Aoyagi, Takao

    2012-12-01

    The acknowledged ability of synthetic materials to induce cell-specific responses regardless of biological supplies provides tissue engineers with the opportunity to find the appropriate materials and conditions to prepare tissue-targeted scaffolds. Stem and mature cells have been shown to acquire distinct morphologies in vitro and to modify their phenotype when grown on synthetic materials with tunable mechanical properties. The stiffness of the substrate used for cell culture is likely to provide cells with mechanical cues mimicking given physiological or pathological conditions, thus affecting the biological properties of cells. The sensitivity of cells to substrate composition and mechanical properties resides in multiprotein complexes called focal adhesions, whose dynamic modification leads to cytoskeleton remodeling and changes in gene expression. In this study, the remodeling of focal adhesions in human mesenchymal stem cells in response to substrate stiffness was followed in the first phases of cell-matrix interaction, using poly-ɛ-caprolactone planar films with similar chemical composition and different elasticity. As compared to mature dermal fibroblasts, mesenchymal stem cells showed a specific response to substrate stiffness, in terms of adhesion, as a result of differential focal adhesion assembly, while their multipotency as a bulk was not significantly affected by matrix compliance. Given the sensitivity of stem cells to matrix mechanics, the mechanobiology of such cells requires further investigations before preparing tissue-specific scaffolds.

  8. Micropatterning topology on soft substrates affects myoblast proliferation and differentiation.

    PubMed

    Zatti, Susi; Zoso, Alice; Serena, Elena; Luni, Camilla; Cimetta, Elisa; Elvassore, Nicola

    2012-02-01

    Micropatterning techniques and substrate engineering are becoming useful tools to investigate several aspects of cell-cell interaction biology. In this work, we rationally study how different micropatterning geometries can affect myoblast behavior in the early stage of in vitro myogenesis. Soft hydrogels with physiological elastic modulus (E = 15 kPa) were micropatterned in parallel lanes (100, 300, and 500 μm width) resulting in different local and global myoblast densities. Proliferation and differentiation into multinucleated myotubes were evaluated for murine and human myoblasts. Wider lanes showed a decrease in murine myoblast proliferation: (69 ± 8)% in 100 μm wide lanes compared to (39 ± 7)% in 500 μm lanes. Conversely, fusion index increased in wider lanes: from (46 ± 7)% to (66 ± 7)% for murine myoblasts, and from (15 ± 3)% to (36 ± 2)% for human primary myoblasts, using a patterning width of 100 and 500 μm, respectively. These results are consistent with both computational modeling data and conditioned medium experiments, which demonstrated that wider lanes favor the accumulation of endogenous secreted factors. Interestingly, human primary myoblast proliferation is not affected by patterning width, which may be because the high serum content of their culture medium overrides the effect of secreted factors. These data highlight the role of micropatterning in shaping the cellular niche through secreted factor accumulation, and are of paramount importance in rationally understanding myogenesis in vitro for the correct design of in vitro skeletal muscle models.

  9. Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates.

    PubMed

    Perz, Veronika; Bleymaier, Klaus; Sinkel, Carsten; Kueper, Ulf; Bonnekessel, Melanie; Ribitsch, Doris; Guebitz, Georg M

    2016-03-25

    The enzymatic hydrolysis of the biodegradable polyester ecoflex and of a variety of oligomeric and polymeric ecoflex model substrates was investigated. For this purpose, substrate specificities of two enzymes of typical compost inhabitants, namely a fungal cutinase from Humicola insolens (HiC) and a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) were compared. Model substrates were systematically designed with variations of the chain length of the alcohol and the acid as well as with varying content of the aromatic constituent terephthalic acid (Ta). HPLC/MS identification and quantification of the hydrolysis products terephthalic acid (Ta), benzoic acid (Ba), adipic acid (Ada), mono(4-hydroxybutyl) terephthalate (BTa), mono-(2-hydroxyethyl) terephthalate (ETa), mono-(6-hydroxyhexyl) terephthalate (HTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated that these enzymes indeed hydrolyze the tested esters. Shorter terminal chain length acids but longer chain length alcohols in oligomeric model substrates were generally hydrolyzed more efficiently. Thc_Cut1 hydrolyzed aromatic ester bonds more efficiently than HiC resulting in up to 3-fold higher concentrations of the monomeric hydrolysis product Ta. Nevertheless, HiC exhibited a higher overall hydrolytic activity on the tested polyesters, resulting in 2-fold higher concentration of released molecules. Thermogravimetry and differential scanning calorimetry (TG-DSC) of the polymeric model substrates revealed a general trend that a lower difference between melting temperature (Tm) and the temperature at which the enzymatic degradation takes place resulted in higher susceptibility to enzymatic hydrolysis. PMID:26594021

  10. A loop of coagulation factor VIIa influencing macromolecular substrate specificity.

    PubMed

    Bjelke, Jais R; Persson, Egon; Rasmussen, Hanne B; Kragelund, Birthe B; Olsen, Ole H

    2007-01-01

    Coagulation factor VIIa (FVIIa) belongs to a family of proteases being part of the stepwise, self-amplifying blood coagulation cascade. To investigate the impact of the mutation Met(298{156})Lys in FVIIa, we replaced the Gly(283{140})-Met(298{156}) loop with the corresponding loop of factor Xa. The resulting variant exhibited increased intrinsic activity, concurrent with maturation of the active site, a less accessible N-terminus, and, interestingly, an altered macromolecular substrate specificity reflected in an increased ability to cleave factor IX (FIX) and a decreased rate of FX activation compared to that of wild-type FVIIa. In complex with tissue factor, activation of FIX, but not of FX, returned to normal. Deconvolution of the loop graft in order to identify important side chain substitutions resulted in the mutant Val(158{21})Asp/Leu(287{144})Thr/Ala(294{152})Ser/Glu(296{154}) Ile/Met(298{156})Lys-FVIIa with almost the same activity and specificity profile. We conclude that a lysine residue in position 298{156} of FVIIa requires a hydrophilic environment to be fully accommodated. This position appears critical for substrate specificity among the proteases of the blood coagulation cascade due to its prominent position in the macromolecular exosite and possibly via its interaction with the corresponding position in the substrate (i.e. FIX or FX). PMID:17182039

  11. Evolution of bacterial protein-tyrosine kinases and their relaxed specificity toward substrates.

    PubMed

    Shi, Lei; Ji, Boyang; Kolar-Znika, Lorena; Boskovic, Ana; Jadeau, Fanny; Combet, Christophe; Grangeasse, Christophe; Franjevic, Damjan; Talla, Emmanuel; Mijakovic, Ivan

    2014-04-01

    It has often been speculated that bacterial protein-tyrosine kinases (BY-kinases) evolve rapidly and maintain relaxed substrate specificity to quickly adopt new substrates when evolutionary pressure in that direction arises. Here, we report a phylogenomic and biochemical analysis of BY-kinases, and their relationship to substrates aimed to validate this hypothesis. Our results suggest that BY-kinases are ubiquitously distributed in bacterial phyla and underwent a complex evolutionary history, affected considerably by gene duplications and horizontal gene transfer events. This is consistent with the fact that the BY-kinase sequences represent a high level of substitution saturation and have a higher evolutionary rate compared with other bacterial genes. On the basis of similarity networks, we could classify BY kinases into three main groups with 14 subgroups. Extensive sequence conservation was observed only around the three canonical Walker motifs, whereas unique signatures proposed the functional speciation and diversification within some subgroups. The relationship between BY-kinases and their substrates was analyzed using a ubiquitous substrate (Ugd) and some Firmicute-specific substrates (YvyG and YjoA) from Bacillus subtilis. No evidence of coevolution between kinases and substrates at the sequence level was found. Seven BY-kinases, including well-characterized and previously uncharacterized ones, were used for experimental studies. Most of the tested kinases were able to phosphorylate substrates from B. subtilis (Ugd, YvyG, and YjoA), despite originating from very distant bacteria. Our results are consistent with the hypothesis that BY-kinases have evolved relaxed substrate specificity and are probably maintained as rapidly evolving platforms for adopting new substrates.

  12. A protein multiplex microarray substrate with high sensitivity and specificity

    PubMed Central

    Fici, Dolores A.; McCormick, William; Brown, David W.; Herrmann, John E.; Kumar, Vikram; Awdeh, Zuheir L.

    2010-01-01

    The problems that have been associated with protein multiplex microarray immunoassay substrates and existing technology platforms include: binding, sensitivity, a low signal to noise ratio, target immobilization and the optimal simultaneous detection of diverse protein targets. Current commercial substrates for planar multiplex microarrays rely on protein attachment chemistries that range from covalent attachment to affinity ligand capture, to simple adsorption. In this pilot study, experimental performance parameters for direct monoclonal mouse IgG detection were compared for available two and three dimensional slide surface coatings with a new colloidal nitrocellulose substrate. New technology multiplex microarrays were also developed and evaluated for the detection of pathogen specific antibodies in human serum and the direct detection of enteric viral antigens. Data supports the nitrocellulose colloid as an effective reagent with the capacity to immobilize sufficient diverse protein target quantities for increased specificory signal without compromising authentic protein structure. The nitrocellulose colloid reagent is compatible with the array spotters and scanners routinely used for microarray preparation and processing. More importantly, as an alternate to fluorescence, colorimetric chemistries may be used for specific and sensitive protein target detection. The advantages of the nitrocellulose colloid platform indicate that this technology may be a valuable tool for the further development and expansion of multiplex microarray immunoassays in both the clinical and research laborat environment. PMID:20974147

  13. Determination of substrate specificity of polyamine transporters in roseobacter species

    NASA Astrophysics Data System (ADS)

    Madhuri, S.; Mou, X.

    2012-12-01

    Polyamines, such as cadaverine, putrescine, spermidine, spermine and norspermine are a class of dissolved organic nitrogen (DON) that is ubiquitously found in marine environments. Intracellular polyamines are important in a variety of biological reactions, such as nucleic acid synthesis and protein synthesis. Free polyamines in seawater can be transported into bacterial cells by ABC transporter systems, each of which consists of four components including one substrate binding protein, one ATPase and two permeases. In silico analysis of marine bacterial genomes has revealed that roseobacter, a numerically and ecologically important taxa of marine bacteria, have at least two sets of polyamine transporter genes. This study was to examine the potential preference of roseobacter to different polyamine compounds and the substrate specificity of different polyamine transporters. Eleven roseobacter species, which genomes have been sequenced, were grown in defined media supplied with single polyamine compound as the sole carbon and nitrogen source. Growth assay showed a small number of roseobacter isolates to be generalist showing no preference among the tested polyamines (Ruegeria pomeroyi DSS-3, Roseovarius sp. TM1035, Roseovarius nubinhibens ISM, Jannaschia sp. CCS1 and Sagittula stellata E-37), whereas other isolates were specilists and were specific on polyamine compounds (Roseobacter sp. CCS2 and Roseobacter denitrificans OCh 114). Primers that probe poly-1 and pot-D genes, the two genes that encode common polyamine-binding genes of polyamine transporter systems were designed using net primer and primer design program. The specificity of the primers was validated by PCR followed by amplicon sequencing. Single step reverse transcription quantitative polymerase chain reactions (RT-qPCR) was performed to investigate substrate specificity of poly-1 and pot-D genes. Key-words Roseobacter, polyamine, polyamine transporter, dissolved organic nitrogen

  14. Structural determinants of tobacco vein mottling virus protease substrate specificity

    SciTech Connect

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef; Waugh, David

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  15. Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase.

    PubMed

    Ferri, S R; Toguri, T

    1997-01-15

    The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.

  16. Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates.

    PubMed

    Nagaki, Masahiko; Nakada, Minori; Musashi, Tohru; Kawakami, Jun; Ohya, Norimasa; Kurihara, Masayo; Maki, Yuji; Nishino, Tokuzo; Koyama, Tanetoshi

    2007-07-01

    To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained. PMID:17617711

  17. Structural and functional basis of protein phosphatase 5 substrate specificity

    PubMed Central

    Oberoi, Jasmeen; Dunn, Diana M.; Woodford, Mark R.; Mariotti, Laura; Schulman, Jacqualyn; Bourboulia, Dimitra; Mollapour, Mehdi

    2016-01-01

    The serine/threonine phosphatase protein phosphatase 5 (PP5) regulates hormone- and stress-induced cellular signaling by association with the molecular chaperone heat shock protein 90 (Hsp90). PP5-mediated dephosphorylation of the cochaperone Cdc37 is essential for activation of Hsp90-dependent kinases. However, the details of this mechanism remain unknown. We determined the crystal structure of a Cdc37 phosphomimetic peptide bound to the catalytic domain of PP5. The structure reveals PP5 utilization of conserved elements of phosphoprotein phosphatase (PPP) structure to bind substrate and provides a template for many PPP–substrate interactions. Our data show that, despite a highly conserved structure, elements of substrate specificity are determined within the phosphatase catalytic domain itself. Structure-based mutations in vivo reveal that PP5-mediated dephosphorylation is required for kinase and steroid hormone receptor release from the chaperone complex. Finally, our data show that hyper- or hypoactivity of PP5 mutants increases Hsp90 binding to its inhibitor, suggesting a mechanism to enhance the efficacy of Hsp90 inhibitors by regulation of PP5 activity in tumors. PMID:27466404

  18. A Detailed Analysis of the Murine TAP Transporter Substrate Specificity

    PubMed Central

    Burgevin, Anne; Saveanu, Loredana; Kim, Yohan; Barilleau, Émilie; Kotturi, Maya; Sette, Alessandro; van Endert, Peter; Peters, Bjoern

    2008-01-01

    Background The transporter associated with antigen processing (TAP) supplies cytosolic peptides into the endoplasmic reticulum for binding to major histocompatibility complex (MHC) class I molecules. Its specificity therefore influences the repertoire of peptides presented by MHC molecules. Compared to human TAP, murine TAP's binding specificity has not been characterized as well, even though murine systems are widely used for basic studies of antigen processing and presentation. Methodology/Principal Findings We performed a detailed experimental analysis of murine TAP binding specificity by measuring the binding affinities of 323 peptides. Based on this experimental data, a computational model of murine TAP specificity was constructed. The model was compared to previously generated data on human and murine TAP specificities. In addition, the murine TAP specificities for known epitopes and random peptides were predicted and compared to assess the impact of murine TAP selectivity on epitope selection. Conclusions/Significance Comparisons to a previously constructed model of human TAP specificity confirms the well-established differences for peptide substrates with positively charged C-termini. In addition these comparisons show that several residues at the N-terminus of peptides which strongly influence binding to human TAP showed little effect on binding to murine TAP, and that the overall influence of the aminoterminal residues on peptide affinity for murine TAP is much lower than for the human transporter. Murine TAP also partly prefers different hydrophobic amino acids than human TAP in the carboxyterminal position. These species-dependent differences in specificity determined in vitro are shown to correlate with the epitope repertoire recognized in vivo. The quantitative model of binding specificity of murine TAP developed herein should be useful for interpreting epitope mapping and immunogenicity data obtained in humanized mouse models. PMID:18545702

  19. Molecular Determinants of Substrate Specificity in Plant 5-Methylthioadenosine Nucleosidases

    SciTech Connect

    Siu,K.; Lee, J.; Sufrin, J.; Moffatt, B.; McMillan, M.; Cornell, K.; Isom, C.; Howell, L.

    2008-01-01

    5?-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5?-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5?-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Angstroms resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5?-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5?-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pKa of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and

  20. Distinct substrate specificities of Arabidopsis DCL3 and DCL4

    PubMed Central

    Nagano, Hideaki; Fukudome, Akihito; Hiraguri, Akihiro; Moriyama, Hiromitsu; Fukuhara, Toshiyuki

    2014-01-01

    In Arabidopsis thaliana, Dicer-like 3 (DCL3) and Dicer-like 4 (DCL4) cleave long, perfect double-stranded RNAs (dsRNAs) into 24 and 21 nucleotides (nt) small interfering RNAs, respectively, which in turn function in RNA-directed DNA methylation and RNA interference, respectively. To reveal how DCL3 and DCL4 individually recognize long perfect dsRNAs as substrates, we biochemically characterized DCL3 and DCL4 and compared their enzymatic properties. DCL3 preferentially cleaves short dsRNAs with 5′ phosphorylated adenosine or uridine and a 1 nt 3′ overhang, whereas DCL4 cleaves long dsRNAs with blunt ends or with a 1 or 2 nt 3′ overhang with similar efficiency. DCL3 produces 24 nt RNA duplexes with 2 nt 3′ overhangs by the 5′ counting rule. Inorganic phosphate, NaCl and KCl enhance DCL3 activity but inhibit DCL4 activity. These results indicate that plants use DCLs with distinct catalytic profiles to ensure each dsRNA substrate generates only a specific length of siRNAs that trigger a unique siRNA-mediated response. PMID:24214956

  1. Tocopherol Cyclases—Substrate Specificity and Phylogenetic Relations

    PubMed Central

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B.; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

  2. Tocopherol Cyclases-Substrate Specificity and Phylogenetic Relations.

    PubMed

    Dłużewska, Jolanta; Szymańska, Renata; Gabruk, Michal; Kós, Peter B; Nowicka, Beatrycze; Kruk, Jerzy

    2016-01-01

    In the present studies, we focused on substrate specificity of tocopherol cyclase, the key enzyme in the biosynthesis of the tocopherols and plastochromanol-8, the main plant lipid antioxidants, with special emphasis on the preference for tocopherols and plastochromanol-8 precursors, taking advantage of the recombinant enzyme originating from Arabidopsis thaliana and isolated plastoglobules, thylakoids and various model systems like micelles and thylakoids. Plastoglobules and triacylglycerol micelles were the most efficient reaction environment for the cyclase. In various investigated systems, synthesis of γ-tocopherol proceeded considerably faster than that of plastochromanol-8, probably mainly due to different localization of the corresponding substrates in the analyzed lipid structures. Moreover, our study was complemented by bioinformatics analysis of the phylogenetic relations of the cyclases and sequence motifs, crucial for the enzyme activity, were proposed. The analysis revealed also a group of tocopherol cyclase-like proteins in a number of heterotrophic bacterial species, with a conserved region common with photosynthetic organisms, that might be engaged in the catalytic activity of both groups of organisms. PMID:27462710

  3. Substrate specificity of allelic variants of the TAP peptide transporter.

    PubMed

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant. PMID:7895166

  4. Substrate specificity of allelic variants of the TAP peptide transporter.

    PubMed

    Heemels, M T; Ploegh, H L

    1994-12-01

    The transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum (ER). An important determinant for the specificity of translocation is the identity of the C-terminal residue of the peptide substrate. In the rat, a suitable C terminus is necessary but not always sufficient for a peptide to be selected for translocation. Here we show that sequence constraints within a peptide of optimal length (9 residues) may interfere with transport; that the transporter selectively translocates shorter derivatives of a 16-mer peptide rather than the 16-mer itself; and that the transporter cimb allele, which is most selective in the C termini it will tolerate, is more relaxed in peptide length preference than is the clma variant.

  5. Detailed characterization of the substrate specificity of mouse wax synthase.

    PubMed

    Miklaszewska, Magdalena; Kawiński, Adam; Banaś, Antoni

    2013-01-01

    Wax synthases are membrane-associated enzymes catalysing the esterification reaction between fatty acyl-CoA and a long chain fatty alcohol. In living organisms, wax esters function as storage materials or provide protection against harmful environmental influences. In industry, they are used as ingredients for the production of lubricants, pharmaceuticals, and cosmetics. Currently the biological sources of wax esters are limited to jojoba oil. In order to establish a large-scale production of desired wax esters in transgenic high-yielding oilseed plants, enzymes involved in wax esters synthesis from different biological resources should be characterized in detail taking into consideration their substrate specificity. Therefore, this study aims at determining the substrate specificity of one of such enzymes -- the mouse wax synthase. The gene encoding this enzyme was expressed heterologously in Saccharomyces cerevisiae. In the in vitro assays (using microsomal fraction from transgenic yeast), we evaluated the preferences of mouse wax synthase towards a set of combinations of 11 acyl-CoAs with 17 fatty alcohols. The highest activity was observed for 14:0-CoA, 12:0-CoA, and 16:0-CoA in combination with medium chain alcohols (up to 5.2, 3.4, and 3.3 nmol wax esters/min/mg microsomal protein, respectively). Unsaturated alcohols longer than 18°C were better utilized by the enzyme in comparison to the saturated ones. Combinations of all tested alcohols with 20:0-CoA, 22:1-CoA, or Ric-CoA were poorly utilized by the enzyme, and conjugated acyl-CoAs were not utilized at all. Apart from the wax synthase activity, mouse wax synthase also exhibited a very low acyl-CoA:diacylglycerol acyltransferase activity. However, it displayed neither acyl-CoA:monoacylglycerol acyltransferase, nor acyl-CoA:sterol acyltransferase activity.

  6. Engineering a substrate-specific cold-adapted subtilisin.

    PubMed

    Tindbaek, Nikolaj; Svendsen, Allan; Oestergaard, Peter Rahbek; Draborg, Henriette

    2004-02-01

    One region predicted to be highly flexible for a psychrophilic enzyme, TA39 subtilisin (S39), was transferred in silico to the mesophilic subtilisin, savinase (EC 3.4.21.62), from Bacillus lentus (clausii). The engineered hybrid and savinase were initially investigated by molecular dynamic simulations at 300 K to show binding region and global flexibility. The predicted S39 region consists of 12 residues, which due to homology between the subtilisins, results in a total change of eight residues. By site-directed modifications, the region was transferred to the binding region of savinase, thus a savinase-S39 hybrid, named H5, was constructed. The designed hybrid showed the same temperature optimum and pH profile as savinase, but H5 had higher specific activity on the synthetic substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (AAPF) at all temperatures measured and, at the same time, H5 showed a decrease in thermostability. The H5 hybrid showed broader substrate specificity, measured at room temperature, due to an increase in catalytic efficiency on AAPF, AAPA and FAAF compared with savinase (N-succinyl-XXXX-pNA; XXXX = AAPF, AAPA and FAAF). The H5 hybrid showed increased activity at low temperature, increased binding region and global flexibility, as investigated by molecular dynamic simulations, and global destabilization from differential scanning calorimetry measurements. These psychrophilic characteristics indicated an increase in binding site flexibility, probably due to the modifications P129S, S130G, P131E, and thus we show that it is possible to increase low temperature activity and global flexibility by engineered flexibility in the binding region. PMID:15047911

  7. Distributions of enzyme residues yielding mutants with improved substrate specificities from two different directed evolution strategies.

    PubMed

    Paramesvaran, Janahan; Hibbert, Edward G; Russell, Andrew J; Dalby, Paul A

    2009-07-01

    A previous study of random mutations, mostly introduced by error-prone PCR (EPPCR) or DNA shuffling (DS), demonstrated that those closer to the enzyme active site were more effective than distant ones at improving enzyme activity, substrate specificity or enantioselectivity. Since then, many studies have taken advantage of this observation by targeting site-directed saturation mutagenesis (SDSM) to residues closer to or within enzyme active sites. Here, we have analysed a set of SDSM studies, in parallel to a similar set from EPPCR/DS, to determine whether the greater range of amino-acid types accessible by SDSM affects the distances at which the most effective sites occur. We have also analysed the relative effectiveness for obtaining beneficial mutants of residues with different degrees of natural sequence variation, as determined by their sequence entropy which is related to sequence conservation. These analyses attempt to answer the question-how well focused have targeted mutagenesis strategies been? We also compared two different sets of active-site atoms from which to measure distances and found that the inclusion of catalytic, substrate and cofactor atoms refined the analysis compared to using a single key catalytic atom. Using this definition, we found that EPPCR/DS is not effective for altering substrate specificity at sites that are within 5 A of the active-site atoms. In contrast, SDSM is most effective when targeted to residues at <5-6 A from the catalytic, substrate or cofactor atom, and also for residues with intermediate sequence entropies. Furthermore, SDSM is capable of altering substrate specificity at highly and completely conserved residues in the active site. The results suggest ways in which directed evolution by SDSM could be improved for greater efficiency in terms of reducing the library sizes required to obtain beneficial mutations that alter substrate specificity.

  8. Factors affecting early seedling development in whole pine tree substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wood-based materials derived from pine trees, such as processed whole pine tree (WPT), can be a viable option for producers looking to offset pine bark or peatmoss usage in container substrates. Reduced root development of stem cuttings rooted in WPT compared with pine bark (PB) has been observed, b...

  9. Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

    PubMed

    Kusevic, Denis; Kudithipudi, Srikanth; Jeltsch, Albert

    2016-03-18

    Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1. PMID:26797129

  10. Improving the Specificity of the Prostate-Specific Antigen Substrate Glutaryl-Hyp-Ala-Ser-Chg-Gln as a Promoiety.

    PubMed

    Aloysius, Herve; Hu, Longqin

    2015-10-01

    To develop PSA peptide substrates with improved specificity and plasma stability from the known substrate sequence glutaryl-Hyp-Ala-Ser-Chg-Gln, systematic replacements of the N-terminal segment with D-retro-inverso-peptides were performed with the incorporation of 7-amino-4-methylcoumarin (7-AMC) after Gln for convenient fluorometric determination and ranking of the PSA substrate activity. The D-retro-inverso-peptide conjugates with P2-P5 D-amino acid substitutions were moderate but poorer PSA substrates as compared to the original peptide, suggesting that inversion of the amide bonds and/or incorporation of the additional atom as in the urea linker adversely affected PSA binding. However, P5 substitution of Hyp with Ser showed significant improvements in PSA cleavage rate; the resulting AMC conjugate, glutaryl-Ser-Ala-Ser-Chg-Gln-AMC (11), exhibited the fastest PSA cleavage rate of 351 pmol/min/100 nmol PSA. In addition, GABA←mGly-Ala-Ser-Chg-Gln-AMC (conjugate 6) was the second best PSA substrate and released 7-AMC at a rate of 225 pmol/min/100 nmol PSA as compared to 171 pmol/min/100 nmol PSA for the control conjugate glutaryl-Hyp-Ala-Ser-Chg-Gln-AMC. Incubations of selected AMC conjugates with mouse and human plasma revealed that GABA←D-Ser-ψ[NH-CO-NH]-Ala-Ser-Chg-Gln-AMC (5) and GABA←mGly-Ala-Ser-Chg-Gln-AMC (6) were most stable to non-PSA-mediated proteolysis. Our results suggest that the PSA specificity of glutaryl-Hyp-Ala-Ser-Chg-Gln is improved with Ser and mGly substitutions of Hyp at the P5.

  11. Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

    PubMed Central

    Dunn, Briana J.; Khosla, Chaitan

    2013-01-01

    Polyketide natural products act as a broad range of therapeutics, including antibiotics, immunosuppressants and anti-cancer agents. This therapeutic diversity stems from the structural diversity of these small molecules, many of which are produced in an assembly line manner by modular polyketide synthases. The acyltransferase (AT) domains of these megasynthases are responsible for selection and incorporation of simple monomeric building blocks, and are thus responsible for a large amount of the resulting polyketide structural diversity. The substrate specificity of these domains is often targeted for engineering in the generation of novel, therapeutically active natural products. This review outlines recent developments that can be used in the successful engineering of these domains, including AT sequence and structural data, mechanistic insights and the production of a diverse pool of extender units. It also provides an overview of previous AT domain engineering attempts, and concludes with proposed engineering approaches that take advantage of current knowledge. These approaches may lead to successful production of biologically active ‘unnatural’ natural products. PMID:23720536

  12. Substrate specificity of amino acid transport in sheep erythrocytes.

    PubMed Central

    Young, J D; Ellory, J C

    1977-01-01

    The specificity of amino acid transport in normal (high-glutathione) sheep erythrocytes was investigated by studying the interaction of various neutral and dibasic amino acids in both competition and exchange experiments. Apparent Ki values were obtained for amino acids as inhibitors of L-alanine influx. Amino acids previously found to be transported by high-glutathione cells at fast rates (L-cysteine, L-alpha-amino-n-butyrate) were the most effective inhibitors. D-Alanine and D-alpha-amino-n-butyrate were without effect. Of the remaining amino acids studied, only L-norvaline, L-valine, L-norleucine, L-serine and L-2,4-diamino-n-butyrate significantly inhibited L-alanine uptake. L-Alanine efflux from pre-loaded cells was markedly stimulated by extracellular L-alanine. Those amino acids that inhibited L-alanine influx also stimulated L-alanine efflux. In addition, D-alanine, D-alpha-amino-n-biutyrate, L-threonine, L-asparagine, L-alpha, beta-diaminoproprionate, L-ornithine, L-lysine and S-2-aminoethyl-L-cysteine also significantly stimulated L-alanine efflux. L-Lysine uptake was inhibited by L-alanine but not by D-alanine, and the inhibitory potency of L-alanine was not influenced by the replacement of Na+ in the incubation medium with choline. L-Lysine efflux from pre-loaded cells was stimulated by L-alanine but not by D-alanine. It is concluded that these cells possess a highly selective stero-specific amino acid-transport system. Although the optimum substrates are small neutral amino acids, this system also has a significant affinity for dibasic amino acids. PMID:849280

  13. Substrate specificity of recombinant osteoclast-specific cathepsin K from rabbits.

    PubMed

    Aibe, K; Yazawa, H; Abe, K; Teramura, K; Kumegawa, M; Kawashima, H; Honda, K

    1996-08-01

    A cDNA clone encoding the rabbit cysteine proteinase cathepsin K, which is predominantly expressed in osteoclasts and is closely related to cathepsins L (EC 3.4.22.15) and S (EC 3.4.22.27) [Tezuka K., Tezuka Y., Maejima A., Sato T., Nemoto K., Kamioka H., Hakeda Y., Kumegawa M., J. Biol. Chem., 269, 1106 (1994)], was expressed at high levels in Escherichia coli in a T7 expression system. The insoluble recombinant enzyme was solubilized in urea and refolded at an alkaline pH. Cathepsin K (37-kDa) was purified by gel filtration and its enzymatic characteristics were determined. The enzymatic activity of cathepsin K was strongly inhibited by cysteine proteinase inhibitors and its optimal pH was pH 5.5. Synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumaryl-amide, which is hydrolyzed by cathepsins L and S, was also cleaved by cathepsin K. On the other hand, benzyloxycarbonyl-Gly-Pro-Arg-7-(4-methyl)coumaryl-amide was the most suitable substrate for cathepsin K, but was hardly hydrolyzed by cathepsin L. The substrate specificity of cathepsin K, as determined using various chemogenic substrates, showed different characteristics from cathepsins L and S. PMID:8874809

  14. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    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.

  15. Mutants of the EcoRI endonuclease with promiscuous substrate specificity implicate residues involved in substrate recognition.

    PubMed Central

    Heitman, J; Model, P

    1990-01-01

    The EcoRI restriction endonuclease cleaves DNA molecules at the sequence GAATTC. We devised a genetic screen to isolate EcoRI mutants with altered or broadened substrate specificity. In vitro, the purified mutant enzymes cleave both the wild-type substrate and sites which differ from this by one nucleotide (EcoRI star sites). These mutations identify four residues involved in substrate recognition and catalysis that are different from the amino acids proposed to recognize the substrate based on the EcoRI-DNA co-crystal structure. In fact, these mutations suppress EcoRI mutants altered at some of the proposed substrate binding residues (R145, R200). We argue that these mutations permit cleavage of additional DNA sequences either by perturbing or removing direct DNA-protein interactions or by facilitating conformational changes that allosterically couple substrate binding to DNA scission. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2209548

  16. Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship?

    PubMed

    Medina, Izarne; Casal, José; Fabre, Caroline C G

    2015-01-01

    Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. Here, we study the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. We examined the intervals between each vibratory pulse, a feature that was reported to carry information for animal communication. We were unable to find evidence of periodic variations in the lengths of these intervals, as has been reported for fly acoustical signals. Because it was suggested that the genes involved in the circadian clock may also regulate shorter rhythms, we search for effects of period on the interval lengths. Males that are mutant for the period gene produced vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. Our results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. We wondered if we could alter the interpulse intervals by other means, and found that ambient temperature also had a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations. PMID:26519517

  17. Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship?

    PubMed Central

    Medina, Izarne; Casal, José; Fabre, Caroline C. G.

    2015-01-01

    ABSTRACT Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. Here, we study the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. We examined the intervals between each vibratory pulse, a feature that was reported to carry information for animal communication. We were unable to find evidence of periodic variations in the lengths of these intervals, as has been reported for fly acoustical signals. Because it was suggested that the genes involved in the circadian clock may also regulate shorter rhythms, we search for effects of period on the interval lengths. Males that are mutant for the period gene produced vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. Our results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. We wondered if we could alter the interpulse intervals by other means, and found that ambient temperature also had a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations. PMID:26519517

  18. Do circadian genes and ambient temperature affect substrate-borne signalling during Drosophila courtship?

    PubMed

    Medina, Izarne; Casal, José; Fabre, Caroline C G

    2015-01-01

    Courtship vibratory signals can be air-borne or substrate-borne. They convey distinct and species-specific information from one individual to its prospective partner. Here, we study the substrate-borne vibratory signals generated by the abdominal quivers of the Drosophila male during courtship; these vibrations travel through the ground towards courted females and coincide with female immobility. It is not known which physical parameters of the vibrations encode the information that is received by the females and induces them to pause. We examined the intervals between each vibratory pulse, a feature that was reported to carry information for animal communication. We were unable to find evidence of periodic variations in the lengths of these intervals, as has been reported for fly acoustical signals. Because it was suggested that the genes involved in the circadian clock may also regulate shorter rhythms, we search for effects of period on the interval lengths. Males that are mutant for the period gene produced vibrations with significantly altered interpulse intervals; also, treating wild type males with constant light results in similar alterations to the interpulse intervals. Our results suggest that both the clock and light/dark cycles have input into the interpulse intervals of these vibrations. We wondered if we could alter the interpulse intervals by other means, and found that ambient temperature also had a strong effect. However, behavioural analysis suggests that only extreme ambient temperatures can affect the strong correlation between female immobility and substrate-borne vibrations.

  19. Kinetic and Structural Analysis of Substrate Specificity in Two Copper Amine Oxidases from Hansenula polymorpha†,‡

    PubMed Central

    Chang, Cindy M.; Klema, Valerie J.; Johnson, Bryan J.; Mure, Minae; Klinman, Judith P.; Wilmot, Carrie M.

    2010-01-01

    The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 Å resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to HPAO-1. Despite 34 % sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side-chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by kcat/Km value differences: in HPAO-1, the kcat/Km for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2 it is benzylamine that is the better substrate by 750-fold. In HPAO-2 an inflated Dkcat/Km(methylamine) in relation to Dkcat/Km(benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, Dkcat/Km(S) changes little with the slow substrate, and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is kcat/Km for the second substrate, O2, significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the

  20. Recent advances and concepts in substrate specificity determination of proteases using tailored libraries of fluorogenic substrates with unnatural amino acids.

    PubMed

    Rut, Wioletta; Kasperkiewicz, Paulina; Byzia, Anna; Poreba, Marcin; Groborz, Katarzyna; Drag, Marcin

    2015-04-01

    Substrate specificity of proteases can be determined using several methods among which the most frequently used are positional scanning library, proteomics and phage display. Classic approaches can deliver information about preferences for natural amino acids in binding pockets of virtually all proteases. However, recent studies demonstrate the ability to obtain much more information by application of unnatural amino acids to positional scanning library approaches. This knowledge can be used for the design of more active and specific substrates, inhibitors and activity based probes. In this minireview we describe recent strategies and concepts for the design and application of fluorogenic substrates library tailored for exopeptidases and endopeptidases.

  1. Substrate specificity of human liver neutral alpha-mannosidase.

    PubMed Central

    al Daher, S; De Gasperi, R; Daniel, P; Hirani, S; Warren, C; Winchester, B

    1992-01-01

    The digestion of radiolabelled natural oligosaccharide substrates by human liver neutral alpha-mannosidase has been studied by h.p.l.c. and h.p.t.l.c. The high-mannose oligosaccharides Man9GlcNAc and Man8GlcNAc are hydrolysed by the enzyme by two distinct non-random routes to a common product of composition Man6GlcNAc, which is then slowly converted into a unique Man5GlcNAc oligosaccharide, Man alpha(1----2)Man alpha(1----2)Man alpha(1----3)[Man alpha (1----6)] Man beta(1----4)GlcNAc. These pathways are different from the processing and lysosomal catabolic pathways for these structures. In particular, the alpha(1----2)-linked mannose residues attached to the core alpha(1----3)-linked mannose residue are resistant to hydrolysis. The key processing intermediate, Man alpha(1----3)[Man alpha(1----6)]Man alpha(1----6)[Man alpha(1----3)] Man beta(1----4)GlcNAc, is not produced in the digestion of high-mannose glycans by the neutral alpha-mannosidase, but it is hydrolysed by the enzyme by a non-random route to Man beta(1----4)GlcNAc via the core structure Man alpha(1----3)[Man alpha(1----6)]Man beta(1----4)GlcNAc. In contrast with its ready hydrolysis by lysosomal alpha-mannosidase, the core alpha(1----3)-mannosidic linkage is quite resistant to hydrolysis by neutral alpha-mannosidase. The precise specificity of neutral alpha-mannosidase towards high-mannose oligosaccharides suggests that it has a role in the modification of such structures in the cytosol. Images Fig. 4. PMID:1520283

  2. MTH1 Substrate Recognition--An Example of Specific Promiscuity.

    PubMed

    Nissink, J Willem M; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  3. MTH1 Substrate Recognition—An Example of Specific Promiscuity

    PubMed Central

    Nissink, J. Willem M.; Bista, Michal; Breed, Jason; Carter, Nikki; Embrey, Kevin; Read, Jonathan; Winter-Holt, Jon J.

    2016-01-01

    MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1. PMID:26999531

  4. The response of an egg parasitoid to substrate-borne semiochemicals is affected by previous experience

    PubMed Central

    Peri, Ezio; Salerno, Gianandrea; Slimani, Takoua; Frati, Francesca; Conti, Eric; Colazza, Stefano; Cusumano, Antonino

    2016-01-01

    Animals can adjust their behaviour according to previous experience gained during foraging. In parasitoids, experience plays a key role in host location, a hierarchical process in which air-borne and substrate-borne semiochemicals are used to find hosts. In nature, chemical traces deposited by herbivore hosts when walking on the plant are adsorbed by leaf surfaces and perceived as substrate-borne semiochemicals by parasitoids. Chemical traces left on cabbage leaves by adults of the harlequin bug (Murgantia histrionica) induce an innate arrestment response in the egg parasitoid Trissolcus brochymenae characterized by an intense searching behaviour on host-contaminated areas. Here we investigated whether the T. brochymenae response to host walking traces left on leaf surfaces is affected by previous experience in the context of parasitoid foraging behaviour. We found that: 1) an unrewarded experience (successive encounters with host-contaminated areas without successful oviposition) decreased the intensity of the parasitoid response; 2) a rewarded experience (successful oviposition) acted as a reinforcing stimulus; 3) the elapsed time between two consecutive unrewarded events affected the parasitoid response in a host-gender specific manner. The ecological role of these results to the host location process of egg parasitoids is discussed. PMID:27250870

  5. Substrate elasticity affects bovine satellite cell activation kinetics in vitro.

    PubMed

    Lapin, M R; Gonzalez, J M; Johnson, S E

    2013-05-01

    Satellite cells support efficient postnatal skeletal muscle hypertrophy through fusion into the adjacent muscle fiber. Nuclear contribution allows for maintenance of the fiber myonuclear domain and proficient transcription of myogenic genes. Niche growth factors affect satellite cell biology; however, the interplay between fiber elasticity and microenvironment proteins remains largely unknown. The objective of the experiment was to examine the effects of hepatocyte growth factor (HGF) and surface elasticity on bovine satellite cell (BSC) activation kinetics in vitro. Young's elastic modulus was calculated for the semimembranosus (SM) and LM muscles of young bulls (5 d; n = 8) and adult cows (27 mo; n = 4) cattle. Results indicate that LM elasticity decreased (P < 0.05) with age; no difference in Young's modulus for the SM was noted. Bovine satellite cells were seeded atop polyacrylamide bioscaffolds with surface elasticities that mimic young bull and adult cow LM or traditional cultureware. Cells were maintained in low-serum media supplemented with 5 ng/mL HGF or vehicle only for 24 or 48 h. Activation was evaluated by proliferating cell nuclear antigen (PCNA) immunocytochemistry. Results indicate that BSC maintained on rigid surfaces were activated at 24 h and refractive to HGF supplementation. By contrast, fewer (P < 0.05) BSC had exited quiescence after 24 h of culture on surfaces reflective of either young bull (8.1 ± 1.7 kPa) or adult cow (14.6 ± 1.6 kPa) LM. Supplementation with HGF promoted activation of BSC cultured on bioscaffolds as measured by an increase (P < 0.05) in PCNA immunopositive cells. Culture on pliant surfaces affected neither activation kinetics nor numbers of Paired box 7 (Pax7) immunopositive muscle stem cells (P > 0.05). However, with increasing surface elasticity, an increase (P < 0.05) in the numbers of muscle progenitors was observed. These results confirm that biophysical and biochemical signals regulate BSC activation.

  6. A high-throughput screening for phosphatases using specific substrates.

    PubMed

    Senn, Alejandro M; Wolosiuk, Ricardo A

    2005-04-01

    A high-throughput screening was developed for the detection of phosphatase activity in bacterial colonies. Unlike other methods, the current procedure can be applied to any phosphatase because it uses physiological substrates and detects the compelled product of all phosphatase reactions, that is, orthophosphate. In this method, substrates diffuse from a filter paper across a nitrocellulose membrane to bacterial colonies situated on the opposite face, and then reaction products flow back to the paper. Finally, a colorimetric reagent discloses the presence of orthophosphate in the filter paper. We validated the performance of this assay with several substrates and experimental conditions and with different phosphatases, including a library of randomly mutagenized rapeseed chloroplast fructose-1,6-bisphosphatase. This procedure could be extended to other enzymatic activities provided that an appropriate detection of reaction products is available.

  7. Substrate specificity of small-intestinal lactase. Assessment of the role of the substrate hydroxyl groups.

    PubMed

    Rivera-Sagredo, A; Cañada, F J; Nieto, O; Jimenez-Barbero, J; Martín-Lomas, M

    1992-10-01

    Lactase-phlorizin hydrolase is a disaccharidase present in the small intestine of mammals. This enzyme has two active sites, one being responsible for the hydrolysis of lactose. Lactase activity is thought to be selective towards glycosides with a hydrophilic aglycon. In this work, we report a systematic study on the importance of each hydroxyl group in the substrate molecule for lactase activity. For this purpose, all of the monodeoxy derivatives of methyl beta-lactoside and other lactose analogues are studied as lactase substrates. With respect to the galactose moiety, it is shown here that HO-3' and HO-2' are necessary for hydrolysis of the substrates by lactase. Using these chemically modified substrates, it has been confirmed that lactase does not behave as a typical beta-galactosidase, since it does not show an absolute selectivity with respect to substitution and stereochemistry at C4' in the galactose moiety of the substrate. However, the glucose moiety, in particular the HO-6, appears to be important for substrate hydrolysis, although none of the hydroxyl groups seemed to be essential. In order to differentiate both activities of the enzyme, a new assay for the phlorizin-hydrolase activity has also been developed.

  8. Well-being and affective style: neural substrates and biobehavioural correlates.

    PubMed

    Davidson, Richard J

    2004-09-29

    One of the most salient features of emotion is the pronounced variability among individuals in their reactions to emotional incentives and in their dispositional mood. Collectively, these individual differences have been described as affective style. Recent research has begun to dissect the constituents of affective style. The search for these components is guided by the neural systems that instantiate emotion and emotion regulation. In this article, this body of research and theory is applied specifically to positive affect and well-being. The central substrates and peripheral biological correlates of well-being are described. A resilient affective style is associated with high levels of left prefrontal activation, effective modulation of activation in the amygdala and fast recovery in response to negative and stressful events. In peripheral biology, these central patterns are associated with lower levels of basal cortisol and with higher levels of antibody titres to influenza vaccine. The article concludes with a consideration of whether these patterns of central and peripheral biology can be modified by training and shifted toward a more salubrious direction. PMID:15347531

  9. Well-being and affective style: neural substrates and biobehavioural correlates.

    PubMed Central

    Davidson, Richard J

    2004-01-01

    One of the most salient features of emotion is the pronounced variability among individuals in their reactions to emotional incentives and in their dispositional mood. Collectively, these individual differences have been described as affective style. Recent research has begun to dissect the constituents of affective style. The search for these components is guided by the neural systems that instantiate emotion and emotion regulation. In this article, this body of research and theory is applied specifically to positive affect and well-being. The central substrates and peripheral biological correlates of well-being are described. A resilient affective style is associated with high levels of left prefrontal activation, effective modulation of activation in the amygdala and fast recovery in response to negative and stressful events. In peripheral biology, these central patterns are associated with lower levels of basal cortisol and with higher levels of antibody titres to influenza vaccine. The article concludes with a consideration of whether these patterns of central and peripheral biology can be modified by training and shifted toward a more salubrious direction. PMID:15347531

  10. Substrate-specific structural rearrangements of human Dicer

    PubMed Central

    Taylor, David W.; Ma, Enbo; Shigematsu, Hideki; Cianfrocco, Michael A.; Noland, Cameron L.; Nagayama, Kuniaki; Nogales, Eva; Doudna, Jennifer A.; Wang, Hong-Wei

    2013-01-01

    Dicer plays a central role in RNA interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) or microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used electron microscopy and single particle analysis of Dicer–RNA complexes to gain insight into the structural basis for human Dicer’s substrate preference. Our studies show that Dicer traps pre-siRNAs in a non-productive conformation, while interactions of Dicer with pre-miRNAs and dsRNA binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer. PMID:23624860

  11. 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…

  12. Substrate specificity of papain dynamic structures for peptides consisting of 8-10 GLY residues

    NASA Astrophysics Data System (ADS)

    Nishiyama, Katsuhiko

    2011-01-01

    We investigated the substrate specificity of papain dynamic structures for peptides of 8-10 glycine residues (8-10GLY) via molecular dynamics and docking simulations. The substrate specificity of papain for 8-10GLY fluctuated considerably with time. There were several residues that were different among those that had a significant impact on binding (RESIDUES_IMPACT) with 10GLY, 9GLY, and 8GLY. Modification of these different residues should allow for control of substrate specificity, providing a framework for modifying substrate specificity in papain and other enzymes.

  13. Universal Common Communication Substrate (UCCS) Specification; Universal Common Communication Substrate (UCCS) Implementation

    SciTech Connect

    2014-08-22

    Universal Common Communication Substrate (UCCS) is a low-level communication substrate that exposes high-performance communication primitives, while providing network interoperability. It is intended to support multiple upper layer protocol (ULPs) or programming models including SHMEM,UPC,Titanium,Co-Array Fortran,Global Arrays,MPI,GASNet, and File I/O. it provides various communication operations including one-sided and two-sided point-to-point, collectives, and remote atomic operations. In addition to operations for ULPs, it provides an out-of-band communication channel required typically required to wire-up communication libraries.

  14. High-throughput profiling of N-myristoylation substrate specificity across species including pathogens.

    PubMed

    Traverso, José A; Giglione, Carmela; Meinnel, Thierry

    2013-01-01

    One of the most critical modifications affecting the N-terminus of proteins is N-myristoylation. This irreversible modification affects the membrane-binding properties of crucial proteins involved in signal transduction cascades. This cotranslational modification, catalyzed by N-myristoyl transferase, occurs both in lower and higher eukaryotes and is a validated therapeutic target for several pathologies. However, this lipidation proves very difficult to be evidenced in vivo even with state-of-the-art proteomics approaches or bioinformatics tools. A large part of N-myristoylated proteins remains to be discovered and the rules of substrate specificity need to be established in each organism. Because the peptide substrate recognition occurs around the first eight residues, short peptides are used for modeling the reaction in vitro. Here, we provide a novel approach including a dedicated peptide array for high-throughput profiling protein N-myristoylation specificity. We show that myristoylation predictive tools need to be fine-tuned to organisms and that their poor accuracy should be significantly enhanced. This should lead to strongly improved knowledge of the number and function of myristoylated proteins occurring in any proteome.

  15. Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

    PubMed Central

    Wahlen, Bradley D.; Garner, EmmaLee; Wei, Jiashi; Seefeldt, Lance C.

    2012-01-01

    Wax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria: Marinobacter aquaeolei VT8, Acinetobacter baylyi, Rhodococcus jostii RHA1, and Psychrobacter cryohalolentis K5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. These in vitro results are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular from M. aquaeolei VT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production. PMID:22685145

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

    PubMed Central

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

    2011-01-01

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

  17. Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

    PubMed

    Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

    2016-04-12

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity. PMID:26985580

  18. Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport

    PubMed Central

    Michaelis, Martin; Rothweiler, Florian; Wurglics, Mario; Aniceto, Natália; Dittrich, Michaela; Zettl, Heiko; Wiese, Michael; Wass, Mark; Ghafourian, Taravat; Schubert-Zsilavecz, Manfred; Cinatl, Jindrich

    2016-01-01

    Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2μM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport. PMID:26887049

  19. Novel {alpha}-glucosidase from human gut microbiome : substrate specificities and their switch.

    SciTech Connect

    Tan, K.; Tesar, C.; Wilton, R.; Keigher, L.; Babnigg, G.; Joachimiak, A.; Biosciences Division

    2010-01-01

    The human intestine harbors a large number of microbes forming a complex microbial community that greatly affects the physiology and pathology of the host. In the human gut microbiome, the enrichment in certain protein gene families appears to be widespread. They include enzymes involved in carbohydrate metabolism such as glucoside hydrolases of dietary polysaccharides and glycoconjugates. We report the crystal structures (wild type, 2 mutants, and a mutant/substrate complex) and the enzymatic activity of a recombinant {alpha}-glucosidase from human gut bacterium Ruminococcus obeum. The first ever protein structures from this bacterium reveal a structural homologue to human intestinal maltase-glucoamylase with a highly conserved catalytic domain and reduced auxiliary domains. The {alpha}-glucosidase, a member of GH31 family, shows substrate preference for {alpha}(1-6) over {alpha}(1-4) glycosidic linkages and produces glucose from isomaltose as well as maltose. The preference can be switched by a single mutation at its active site, suggestive of widespread adaptation to utilization of a variety of polysaccharides by intestinal micro-organisms as energy resources. Novel {alpha}-glucosidase from human gut microbiome: substrate specificities and their switch.

  20. Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes.

    PubMed

    Poreba, Marcin; Solberg, Rigmor; Rut, Wioletta; Lunde, Ngoc Nguyen; Kasperkiewicz, Paulina; Snipas, Scott J; Mihelic, Marko; Turk, Dusan; Turk, Boris; Salvesen, Guy S; Drag, Marcin

    2016-08-18

    Legumain (AEP) is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP upregulation is linked to a number of diseases including inflammation, arteriosclerosis, and tumorigenesis. Thus this protease is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.

  1. Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes.

    PubMed

    Poreba, Marcin; Solberg, Rigmor; Rut, Wioletta; Lunde, Ngoc Nguyen; Kasperkiewicz, Paulina; Snipas, Scott J; Mihelic, Marko; Turk, Dusan; Turk, Boris; Salvesen, Guy S; Drag, Marcin

    2016-08-18

    Legumain (AEP) is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP upregulation is linked to a number of diseases including inflammation, arteriosclerosis, and tumorigenesis. Thus this protease is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling. PMID:27478158

  2. Substrate specificity of the sialic acid biosynthetic pathway

    SciTech Connect

    Jacobs, Christina L.; Goon, Scarlett; Yarema, Kevin J.; Hinderlich, Stephan; Hang, Howard C.; Chai, Diana H.; Bertozzi, Carolyn R.

    2001-07-18

    Unnatural analogs of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogs bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell-surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogs with ketone-containing N-acyl groups that varied in the lengthor steric bulk was chemically synthesized and tested for metabolic conversion to cell-surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

  3. Lipid substrate specificity of phosphatidylethanolamine N-methyltransferase of Tetrahymena

    SciTech Connect

    Smith, J.D.

    1986-05-01

    The ciliate protozoan Tetrahymena thermophila forms about 60% of its phosphatidylcholine by methylation of phosphatidylethanolamine with S-adenosylmethionine using the enzyme phosphatidylethanolamine N-methyltransferase. Analogues of ethanolamine or of ethanolamine phosphate are incorporated into the phospholipids of Tetrahymena when cells are cultured in their presence. These compounds, 3-amino-1-propanol, 2-aminoethylphosphonate, 3-aminopropylphosphonate and N,N-dimethylaminoethylphosphonate replace from 50 to 75% of the ethanolamine phosphate in phosphatidylethanolamine. However, analysis of the phospholipids of lipid-altered Tetrahymena showed that none of the phosphatidylethanolamine analogues had been converted to the corresponding phosphatidylcholine analogue. No incorration of (/sup 14/C-CH/sub 3/)methionine into the phosphatidylcholine analogues could be demonstrated in vivo, nor was label from (/sup 3/H-CH/sub 3/)S-adenosylmethionine incorporated in virto. Thus, only phosphatidylethanolamine and its monomethyl and dimethyl derivatives have been found to be substrates for the phosphatidylethanoiamine N-methyltransferase. The enzyme therefore requires a phospholipid substrate containing an ester linkage between the alkylamine and phosphorus, with the amino group required to be ..beta.. to the alcohol.

  4. Crystal Structure and Substrate Specificity of D-Galactose-6-Phosphate Isomerase Complexed with Substrates

    PubMed Central

    Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays. PMID:24015281

  5. Crystal structure and substrate specificity of D-galactose-6-phosphate isomerase complexed with substrates.

    PubMed

    Jung, Woo-Suk; Singh, Raushan Kumar; Lee, Jung-Kul; Pan, Cheol-Ho

    2013-01-01

    D-Galactose-6-phosphate isomerase from Lactobacillus rhamnosus (LacAB; EC 5.3.1.26), which is encoded by the tagatose-6-phosphate pathway gene cluster (lacABCD), catalyzes the isomerization of D-galactose-6-phosphate to D-tagatose-6-phosphate during lactose catabolism and is used to produce rare sugars as low-calorie natural sweeteners. The crystal structures of LacAB and its complex with D-tagatose-6-phosphate revealed that LacAB is a homotetramer of LacA and LacB subunits, with a structure similar to that of ribose-5-phosphate isomerase (Rpi). Structurally, LacAB belongs to the RpiB/LacAB superfamily, having a Rossmann-like αβα sandwich fold as has been identified in pentose phosphate isomerase and hexose phosphate isomerase. In contrast to other family members, the LacB subunit also has a unique α7 helix in its C-terminus. One active site is distinctly located at the interface between LacA and LacB, whereas two active sites are present in RpiB. In the structure of the product complex, the phosphate group of D-tagatose-6-phosphate is bound to three arginine residues, including Arg-39, producing a different substrate orientation than that in RpiB, where the substrate binds at Asp-43. Due to the proximity of the Arg-134 residue and backbone Cα of the α6 helix in LacA to the last Asp-172 residue of LacB with a hydrogen bond, a six-carbon sugar-phosphate can bind in the larger pocket of LacAB, compared with RpiB. His-96 in the active site is important for ring opening and substrate orientation, and Cys-65 is essential for the isomerization activity of the enzyme. Two rare sugar substrates, D-psicose and D-ribulose, show optimal binding in the LacAB-substrate complex. These findings were supported by the results of LacA activity assays.

  6. Structural basis of substrate specificity in the serine proteases.

    PubMed Central

    Perona, J. J.; Craik, C. S.

    1995-01-01

    Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system. PMID:7795518

  7. How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis

    PubMed Central

    2011-01-01

    Background In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin

  8. Emotion-specific load disrupts concomitant affective processing.

    PubMed

    Vermeulen, Nicolas; Niedenthal, Paula M; Pleyers, Gordy; Bayot, Marie; Corneille, Olivier

    2014-01-01

    Findings in the neuroimaging literature suggest that separate brain circuitries are involved when individuals perform emotional compared to nonemotional working memory (WM) tasks. Here we test this hypothesis with behavioural measures. We predicted that the conceptual processing of affect would be disrupted more by concurrent affective than nonaffective load. Participants performed a conceptual task in which they verified affective versus sensory properties of concepts, and a second, concurrent, working memory (n-back) task in which the target stimuli were facial expressions. Results revealed that storing and updating affective (as compared with identity) features of facial expressions altered performance more for affective than for sensory properties of concepts. The findings are supportive of the ideas that affective resources exist and that these resources are specifically used during the processing and representation of affective properties of objects and events.

  9. AcrB drug-binding pocket substitution confers clinically relevant resistance and altered substrate specificity

    PubMed Central

    Blair, Jessica M. A.; Bavro, Vassiliy N.; Ricci, Vito; Modi, Niraj; Cacciotto, Pierpaolo; Kleinekathӧfer, Ulrich; Ruggerone, Paolo; Vargiu, Attilio V.; Baylay, Alison J.; Smith, Helen E.; Brandon, Yvonne; Galloway, David; Piddock, Laura J. V.

    2015-01-01

    The incidence of multidrug-resistant bacterial infections is increasing globally and the need to understand the underlying mechanisms is paramount to discover new therapeutics. The efflux pumps of Gram-negative bacteria have a broad substrate range and transport antibiotics out of the bacterium, conferring intrinsic multidrug resistance (MDR). The genomes of pre- and posttherapy MDR clinical isolates of Salmonella Typhimurium from a patient that failed antibacterial therapy and died were sequenced. In the posttherapy isolate we identified a novel G288D substitution in AcrB, the resistance-nodulation division transporter in the AcrAB-TolC tripartite MDR efflux pump system. Computational structural analysis suggested that G288D in AcrB heavily affects the structure, dynamics, and hydration properties of the distal binding pocket altering specificity for antibacterial drugs. Consistent with this hypothesis, recreation of the mutation in standard Escherichia coli and Salmonella strains showed that G288D AcrB altered substrate specificity, conferring decreased susceptibility to the fluoroquinolone antibiotic ciprofloxacin by increased efflux. At the same time, the substitution increased susceptibility to other drugs by decreased efflux. Information about drug transport is vital for the discovery of new antibacterials; the finding that one amino acid change can cause resistance to some drugs, while conferring increased susceptibility to others, could provide a basis for new drug development and treatment strategies. PMID:25737552

  10. Determination of the Substrate Specificity of Protein Kinases with Peptide Micro- and Macroarrays.

    PubMed

    Lai, Shenshen; Winkler, Dirk F H; Zhang, Hong; Pelech, Steven

    2016-01-01

    Elucidation of the key determinants for the phosphorylation site specificities of protein kinases facilitates identification of their physiological substrates, and serves to better define their critical roles in the signaling networks that underlie a multitude of cellular activities. Albeit with some apparent limitations, such as the lack of contextual information for secondary substrate-binding sites, the synthetic peptide-based approach has been adopted widely for the kinase specificity profiling studies, especially when they are used in an array format, which permits the screening of large numbers of potential peptide substrates in parallel. In this chapter, we present detailed protocols for determining protein kinase substrate specificity using an approach that involves both peptide microarrays and macroarrays. In particular, SPOT synthesis on macroarrays can be used to follow up on in silico predictions of protein kinase substrate specificity with predictive algorithms. PMID:26501911

  11. Mechanism by which metal cofactors control substrate specificity in pyrophosphatase.

    PubMed Central

    Zyryanov, Anton B; Shestakov, Alexander S; Lahti, Reijo; Baykov, Alexander A

    2002-01-01

    Family I soluble pyrophosphatases (PPases) exhibit appreciable ATPase activity in the presence of a number of transition metal ions, but not the physiological cofactor Mg(2+). The results of the present study reveal a strong correlation between the catalytic efficiency of three family I PPases (from Saccharomyces cerevisiae, Escherichia coli and rat liver) and one family II PPase (from Streptococcus mutans ) in ATP and tripolyphosphate (P(3)) hydrolysis in the presence of Mg(2+), Mn(2+), Zn(2+) and Co(2+) on the one hand, and the phosphate-binding affinity of the enzyme subsite P2 that interacts with the electrophilic terminal phosphate group of ATP on the other. A similar correlation was observed in S. cerevisiae PPase variants with modified P1 and P2 subsites. The effect of the above metal ion cofactors on ATP binding to S. cerevisiae PPase paralleled their effect on phosphate binding, resulting in a low affinity of Mg-PPase to ATP. We conclude that PPase mainly binds ATP and P(3) through the terminal phosphate group that is attacked by water. Moreover, this interaction is critical in creating a reactive geometry at the P2 site with these bulky substrates, which do not otherwise fit the active site perfectly. We propose further that ATP is not hydrolysed by Mg-PPase, since its interaction with the terminal phosphate is not adequately strong for proper positioning of the nucleophile-electrophile pair. PMID:12169093

  12. CROSS-STREAM COMPARISON OF SUBSTRATE-SPECIFIC DENITRIFICATION POTENTIAL

    SciTech Connect

    Findlay, Stuart; Mulholland, Patrick J; Hamilton, Stephen; Tank, Jennifer; Bernot, Melody; Burgin, Amy; Crenshaw, Chelsea; Grimm, Nancy; McDowell, William; Potter, Jody; Sobota, Daniel

    2011-01-01

    Headwater streams have a demonstrated ability to denitrify a portion of their nitrate (NO(3) (-)) load but there has not been an extensive consideration of where in a stream this process is occurring and how various habitats contribute to total denitrification capability. As part of the Lotic Intersite Nitrogen Experiment II (LINX II) we measured denitrification potential in 65 streams spanning eight regions of the US and draining three land-use types. In each stream, potential denitrification rates were measured in common substrate types found across many streams as well as locations unique to particular streams. Overall, habitats from streams draining urban and agricultural land-uses showed higher potential rates of denitrification than reference streams draining native vegetation. This difference among streams was probably driven by higher ambient nitrate concentrations found in urban or agricultural streams. Within streams, sandy habitats and accumulations of fine benthic organic matter contributed more than half of the total denitrification capacity (mg N removed m(-2) h(-1)). A particular rate of potential denitrification per unit area could be achieved either by high activity per unit organic matter or lower activities associated with larger standing stocks of organic matter. We found that both small patches with high rates (hot spots) or more widespread but less active areas (cool matrix) contributed significantly to whole stream denitrification capacity. Denitrification estimated from scaled-up denitrification enzyme assay (DEA) potentials were not always dramatically higher than in situ rates of denitrification measured as (15)N gas generation following 24-h (15)N-NO(3) tracer additions. In general, headwater streams draining varying land-use types have significant potential to remove nitrate via denitrification and some appear to be functioning near their maximal capacity.

  13. Engineering Substrate Preference in Subtilisin: Structural and Kinetic Analysis of a Specificity Mutant

    SciTech Connect

    Ruan, Biao; London, Viktoriya; Fisher, Kathryn E.; Gallagher, D. Travis; Bryan, Philip N.

    2008-08-06

    Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k{sub cat} for the reaction. This also leads to strong product inhibition of substrate

  14. Specificity of infants' response to mothers' affective behavior.

    PubMed

    Cohn, J F; Tronick, E

    1989-03-01

    Mother-infant face-to-face interaction is central to infant socioemotional development. Little has been known about the mechanisms that mediate the mother's influence. Findings are reviewed from a series of laboratory studies that suggest the major functional components of a mother's behavior are its affective quality and its contingent relationship to her baby's behavior. Quality of mother's affective expression accounted for individual differences in the behavior of thirteen 7-month-old infants living in multiproblem families. Infants' response was specific to the type of affective expression mothers displayed. Flat, withdrawn maternal affective expression was associated with infant distress. Intrusive maternal expression was associated with increased gaze aversion. Lack of contingent responsiveness was common to all but four mothers. Findings suggest that withdrawn or intrusive maternal affective expression, together with lack of contingent responsiveness, may in part be responsible for the risk-status of infants in multiproblem families.

  15. Origin, diversification and substrate specificity in the family of NCS1/FUR transporters.

    PubMed

    Krypotou, Emilia; Evangelidis, Thomas; Bobonis, Jacob; Pittis, Alexandros A; Gabaldón, Toni; Scazzocchio, Claudio; Mikros, Emmanuel; Diallinas, George

    2015-06-01

    NCS1 proteins are H(+)/Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub-families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.

  16. Glycosylation Substrate Specificity of Pseudomonas aeruginosa 1244 Pilin*S

    PubMed Central

    Horzempa, Joseph; Comer, Jason E.; Davis, Sheila A.; Castric, Peter

    2008-01-01

    The β-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the “tail” region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur. PMID:16286455

  17. Application of fluorescent substrates to the in situ detection of prostate specific antigen.

    PubMed

    Gooch, James; Daniel, Barbara; Frascione, Nunzianda

    2014-07-01

    The forensic identification of body fluids frequently presents an important source of genetic material and investigative interpretation. However, presumptive testing techniques presently employed in the discrimination of biological fluids are subject to criticism for poor specificity, lack of fluid localisation ability and detrimental effects on DNA recovery rates. The recognition of fluid-specific biomarkers by fluorogenic substrates may provide a novel resolution to these issues but research has yet to establish any pertinent in situ fluid detection applicability. This study therefore utilises a fluorogenic substrate (Mu-HSSKLQ-AFC) specific to the seminal protein prostate specific antigen in an effort to detect human semen deposited on a number of surfaces typical to criminal investigation. The ability of fluorescent fluorogenic substrates to simultaneously identify and visualise biological fluids in situ is demonstrated for the first time, whilst the production of complete STR profiles from fluid sources is also confirmed to be completely unaffected by substrate application.

  18. High and stable substrate specificities of microorganisms in enhanced biological phosphorus removal plants.

    PubMed

    Kindaichi, Tomonori; Nierychlo, Marta; Kragelund, Caroline; Nielsen, Jeppe Lund; Nielsen, Per Halkjaer

    2013-06-01

    Microbial communities are typically characterized by conditions of nutrient limitation so the availability of the resources is likely a key factor in the niche differentiation across all species and in the regulation of the community structure. In this study we have investigated whether four species exhibit any in situ short-term changes in substrate uptake pattern when exposed to variations in substrate and growth conditions. Microautoradiography was combined with fluorescence in situ hybridization to investigate in situ cell-specific substrate uptake profiles of four probe-defined coexisting species in a wastewater treatment plant with enhanced biological phosphorus removal. These were the filamentous 'Candidatus Microthrix' and Caldilinea (type 0803), the polyphosphate-accumulating organism 'Candidatus Accumulibacter', and the denitrifying Azoarcus. The experimental conditions mimicked the conditions potentially encountered in the respective environment (starvation, high/low substrate concentration, induction with specific substrates, and single/multiple substrates). The results showed that each probe-defined species exhibited very distinct and constant substrate uptake profile in time and space, which hardly changed under any of the conditions tested. Such niche partitioning implies that a significant change in substrate composition will be reflected in a changed community structure rather than the substrate uptake response from the different species.

  19. Reprogramming Caspase-7 Specificity by Regio-Specific Mutations and Selection Provides Alternate Solutions for Substrate Recognition.

    PubMed

    Hill, Maureen E; MacPherson, Derek J; Wu, Peng; Julien, Olivier; Wells, James A; Hardy, Jeanne A

    2016-06-17

    The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. Here, 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-7 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. This approach to specificity reprogramming should also be generalizable across a wide range of proteases.

  20. Age-Specific Preferences for Infant-Directed Affective Intent

    ERIC Educational Resources Information Center

    Kitamura, Christine; Lam, Christa

    2009-01-01

    This study examined the developmental course of infants' attentional preferences for 3 types of infant-directed affective intent, which have been shown to be commonly used at particular ages in the first year of life. Specifically, Kitamura and Burnham (2003) found mothers' tone of voice in infant-directed speech is most comforting between birth…

  1. Comparison versus Contrast: Task Specifics Affect Category Acquisition

    ERIC Educational Resources Information Center

    Ankowski, Amber A.; Vlach, Haley A.; Sandhofer, Catherine M.

    2013-01-01

    A large literature has documented that comparison and contrast lead to better performance in a variety of tasks. However, studies of comparison and contrast present contradictory conclusions as to when and how these processes benefit learners. Across four studies, we examined how the specifics of the comparison and contrast task affect performance…

  2. Prediction of Membrane Transport Proteins and Their Substrate Specificities Using Primary Sequence Information

    PubMed Central

    Mishra, Nitish K.; Chang, Junil; Zhao, Patrick X.

    2014-01-01

    Background Membrane transport proteins (transporters) move hydrophilic substrates across hydrophobic membranes and play vital roles in most cellular functions. Transporters represent a diverse group of proteins that differ in topology, energy coupling mechanism, and substrate specificity as well as sequence similarity. Among the functional annotations of transporters, information about their transporting substrates is especially important. The experimental identification and characterization of transporters is currently costly and time-consuming. The development of robust bioinformatics-based methods for the prediction of membrane transport proteins and their substrate specificities is therefore an important and urgent task. Results Support vector machine (SVM)-based computational models, which comprehensively utilize integrative protein sequence features such as amino acid composition, dipeptide composition, physico-chemical composition, biochemical composition, and position-specific scoring matrices (PSSM), were developed to predict the substrate specificity of seven transporter classes: amino acid, anion, cation, electron, protein/mRNA, sugar, and other transporters. An additional model to differentiate transporters from non-transporters was also developed. Among the developed models, the biochemical composition and PSSM hybrid model outperformed other models and achieved an overall average prediction accuracy of 76.69% with a Mathews correlation coefficient (MCC) of 0.49 and a receiver operating characteristic area under the curve (AUC) of 0.833 on our main dataset. This model also achieved an overall average prediction accuracy of 78.88% and MCC of 0.41 on an independent dataset. Conclusions Our analyses suggest that evolutionary information (i.e., the PSSM) and the AAIndex are key features for the substrate specificity prediction of transport proteins. In comparison, similarity-based methods such as BLAST, PSI-BLAST, and hidden Markov models do not provide

  3. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    PubMed Central

    Xu, Qingping; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.

    2015-01-01

    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-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-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-terminal l-Ala. Their crystal structures revealed a highly conserved structure 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. PMID:26374125

  4. Broad Substrate Specificity of the Loading Didomain of the Lipomycin Polyketide Synthase

    SciTech Connect

    Yuzawa, S; Eng, CH; Katz, L; Keasling, JD

    2013-06-04

    LipPks1, a polyketide synthase subunit of the lipomycin synthase, is believed to catalyze the polyketide chain initiation reaction using isobutyryl-CoA as a substrate, followed by an elongation reaction with methylmalonyl-CoA to start the biosynthesis of antibiotic alpha-lipomycin in Streptomyces aureofaciens Tu117. Recombinant LipPks1, containing the thioesterase domain from the 6-deoxyerythronolide B synthase, was produced in Escherichia coli, and its substrate specificity was investigated in vitro. Surprisingly, several different acyl-CoAs, including isobutyryl-CoA, were accepted as the starter substrates, while no product was observed with acetyl-CoA. These results demonstrate the broad substrate specificity of LipPks1 and may be applied to producing new antibiotics.

  5. Substrate-specific modulation of CYP3A4 activity by genetic variants of cytochrome P450 oxidoreductase (POR)

    PubMed Central

    Agrawal, Vishal; Choi, Ji Ha; Giacomini, Kathleen M.; Miller, Walter L.

    2010-01-01

    Objectives CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis. Methods We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates. Results POR A287P and R457H had low activity with all substrates; Q153R had 76–94% of wild type (WT) activity with midazolam and erythromycin, but 129–150% activity with testosterone and quinidine. The A503V polymorphism reduced CYP3A4 activity to 61–77% of wild type with testosterone and midazolam, but had nearly wild type activity with quinidine and erythromycin. Conclusion POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably due to substrate-induced conformational changes in CYP3A4. PMID:20697309

  6. Subcellular localization and substrate specificity of dolichol kinase from rat liver.

    PubMed

    Keller, R K; Rottler, G D; Cafmeyer, N; Adair, W L

    1982-10-28

    When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total activity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the alpha-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (alpha-cis-isoprene) compared to synthetic alpha-trans-polyprenol-16. Taken together, the data indicate that the alpha-isoprene specificity follows the order: saturated greater than cis greater than trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required.

  7. Substrate binding and specificity of rhomboid intramembrane protease revealed by substrate–peptide complex structures

    PubMed Central

    Zoll, Sebastian; Stanchev, Stancho; Began, Jakub; Škerle, Jan; Lepšík, Martin; Peclinovská, Lucie; Majer, Pavel; Strisovsky, Kvido

    2014-01-01

    The mechanisms of intramembrane proteases are incompletely understood due to the lack of structural data on substrate complexes. To gain insight into substrate binding by rhomboid proteases, we have synthesised a series of novel peptidyl-chloromethylketone (CMK) inhibitors and analysed their interactions with Escherichia coli rhomboid GlpG enzymologically and structurally. We show that peptidyl-CMKs derived from the natural rhomboid substrate TatA from bacterium Providencia stuartii bind GlpG in a substrate-like manner, and their co-crystal structures with GlpG reveal the S1 to S4 subsites of the protease. The S1 subsite is prominent and merges into the ‘water retention site’, suggesting intimate interplay between substrate binding, specificity and catalysis. Unexpectedly, the S4 subsite is plastically formed by residues of the L1 loop, an important but hitherto enigmatic feature of the rhomboid fold. We propose that the homologous region of members of the wider rhomboid-like protein superfamily may have similar substrate or client-protein binding function. Finally, using molecular dynamics, we generate a model of the Michaelis complex of the substrate bound in the active site of GlpG. PMID:25216680

  8. Substrate Specificity and Structure of Human Aminoadipate Aminotransferase/kynurenine Aminotransferase II

    SciTech Connect

    Han,Q.; Cai, T.; Tagle, D.; Robinson, H.; Li, J.

    2008-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to a-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested a-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with a-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  9. Substrate Specificity and Structure of Human aminoadipate aminotransferase/kynurenine aminotransferase II

    SciTech Connect

    Han, Q.; Cai, T; Tagle, D; Robinson, H; Li, J

    2009-01-01

    KAT (kynurenine aminotransferase) II is a primary enzyme in the brain for catalysing the transamination of kynurenine to KYNA (kynurenic acid). KYNA is the only known endogenous antagonist of the N-methyl-D-aspartate receptor. The enzyme also catalyses the transamination of aminoadipate to alpha-oxoadipate; therefore it was initially named AADAT (aminoadipate aminotransferase). As an endotoxin, aminoadipate influences various elements of glutamatergic neurotransmission and kills primary astrocytes in the brain. A number of studies dealing with the biochemical and functional characteristics of this enzyme exist in the literature, but a systematic assessment of KAT II addressing its substrate profile and kinetic properties has not been performed. The present study examines the biochemical and structural characterization of a human KAT II/AADAT. Substrate screening of human KAT II revealed that the enzyme has a very broad substrate specificity, is capable of catalysing the transamination of 16 out of 24 tested amino acids and could utilize all 16 tested alpha-oxo acids as amino-group acceptors. Kinetic analysis of human KAT II demonstrated its catalytic efficiency for individual amino-group donors and acceptors, providing information as to its preferred substrate affinity. Structural analysis of the human KAT II complex with alpha-oxoglutaric acid revealed a conformational change of an N-terminal fraction, residues 15-33, that is able to adapt to different substrate sizes, which provides a structural basis for its broad substrate specificity.

  10. [Substrate specificity of carotenoid 3',4'-desaturase from Deinococcus radiodurans].

    PubMed

    Sun, Zongtao; Tian, Bing; Shen, Shaochuan; Hu, Yuejin

    2010-10-01

    To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBI(Eu) and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').

  11. On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways

    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.

  12. Specific Effects of Fiber Size and Fiber Swelling on Biomass Substrate Surface Area and Enzymatic Digestibility

    SciTech Connect

    Ju, Xiaohui; Grego, Courtnee; Zhang, Xiao

    2013-09-01

    To clarify the specific effect of biomass substrate surface area on its enzymatic digestibility, factors of fiber size reduction and swelling changes were investigated by using poplar substrates with controlled morphological and chemical properties after modified chemical pulping. Results showed that fiber size changes had insignificant influence on enzymatic hydrolysis, although the external surface area increased up to 41% with the reduction of fiber size. Swelling changes caused by increased biomass fiber porosities after PFI refining showed a significant influence on the efficiency of enzymatic hydrolysis. It is also found that chemical properties such as xylan and lignin content can influence the swelling effect. Xylan is confirmed to facilitate substrate hydrolysability by swelling, while lignin restricts swelling effect and thus minimizes the enzyme accessibility to substrates.

  13. The Exosome Is Recruited to RNA Substrates through Specific Adaptor Proteins.

    PubMed

    Thoms, Matthias; Thomson, Emma; Baßler, Jochen; Gnädig, Marén; Griesel, Sabine; Hurt, Ed

    2015-08-27

    The exosome regulates the processing, degradation, and surveillance of a plethora of RNA species. However, little is known about how the exosome recognizes and is recruited to its diverse substrates. We report the identification of adaptor proteins that recruit the exosome-associated helicase, Mtr4, to unique RNA substrates. Nop53, the yeast homolog of the tumor suppressor PICT1, targets Mtr4 to pre-ribosomal particles for exosome-mediated processing, while a second adaptor Utp18 recruits Mtr4 to cleaved rRNA fragments destined for degradation by the exosome. Both Nop53 and Utp18 contain the same consensus motif, through which they dock to the "arch" domain of Mtr4 and target it to specific substrates. These findings show that the exosome employs a general mechanism of recruitment to defined substrates and that this process is regulated through adaptor proteins.

  14. Flow variation and substrate type affect dislodgement of the freshwater polychaete, Manayunkia speciosa

    USGS Publications Warehouse

    Malakauskas, David M.; Wilson, Sarah J.; Wilzbach, Margaret A.; Som, Nicholas A.

    2013-01-01

    We quantified microscale flow forces and their ability to entrain the freshwater polychaete, Manayunkia speciosa, the intermediate host for 2 myxozoan parasites (Ceratomyxa shasta and Parvicapsula minibicornis) that cause substantial mortalities in salmonid fishes in the Pacific Northwest. In a laboratory flume, we measured the shear stress associated with 2 mean flow velocities and 3 substrates and quantified associated dislodgement of polychaetes, evaluated survivorship of dislodged polychaetes, and observed behavioral responses of the polychaetes in response to increased flow. We used a generalized linear mixed model to estimate the probability of polychaete dislodgement for treatment combinations of velocity (mean flow velocity  =  55 cm/s with a shear velocity  =  3 cm/s, mean flow velocity  =  140 cm/s with a shear velocity  =  5 cm/s) and substrate type (depositional sediments and analogs of rock faces and the filamentous alga, Cladophora). Few polychaetes were dislodged at shear velocities <3 cm/s on any substrate. Above this level of shear, probability of dislodgement was strongly affected by both substrate type and velocity. After accounting for substrate, odds of dislodgement were 8× greater at the higher flow. After accounting for velocity, probability of dislodgement was greatest from fine sediments, intermediate from rock faces, and negligible from Cladophora. Survivorship of dislodged polychaetes was high. Polychaetes exhibited a variety of behaviors for avoiding increases in flow, including extrusion of mucus, burrowing into sediments, and movement to lower-flow microhabitats. Our findings suggest that polychaete populations probably exhibit high resilience to flow-mediated disturbances.

  15. Effects of oil spill dispersants and drilling fluids on substrate specificity of marine bacteria

    SciTech Connect

    Okpokwasili, G.C.; Nnubia, C.

    1995-12-31

    The effects of oil spill dispersants and drilling fluids on the sizes of populations of specific heterotroph subgroups of marine bacteria were monitored in this study. The bacteria were isolated from drill cuttings recovered from Agbara--an offshore oilfield located some 100 nautical miles off the Atlantic coast of Nigeria. Numbers of cellulolytic, proteolytic, starch-hydrolyzing and lipolytic bacteria in the drill cuttings were monitored for 28 days in the presence of oil spill dispersants and drilling fluids. The percentages of these bacterial subgroups within the total heterotrophic population enumerated on tryptic soy agar (10% with 3% NaCl) fluctuated between 3.0 and 17.0%, 0.0 and 27.0%, 4.0 and 25.0% and 3.0 and 18.0% for cellulolytic, proteolytic, starch-hydrolyzing and lipolytic bacteria respectively. These results indicate that oil spill dispersants and drilling fluids affect the ability of marine bacteria to metabolize these substrates in the environment.

  16. Structural analysis of human dual-specificity phosphatase 22 complexed with a phosphotyrosine-like substrate.

    PubMed

    Lountos, George T; Cherry, Scott; Tropea, Joseph E; Waugh, David S

    2015-02-01

    4-Nitrophenyl phosphate (p-nitrophenyl phosphate, pNPP) is widely used as a small molecule phosphotyrosine-like substrate in activity assays for protein tyrosine phosphatases. It is a colorless substrate that upon hydrolysis is converted to a yellow 4-nitrophenolate ion that can be monitored by absorbance at 405 nm. Therefore, the pNPP assay has been widely adopted as a quick and simple method to assess phosphatase activity and is also commonly used in assays to screen for inhibitors. Here, the first crystal structure is presented of a dual-specificity phosphatase, human dual-specificity phosphatase 22 (DUSP22), in complex with pNPP. The structure illuminates the molecular basis for substrate binding and may also facilitate the structure-assisted development of DUSP22 inhibitors.

  17. Photo-Activatable Substrates for Site-Specific Differentiation of Stem Cells.

    PubMed

    Han, Kai; Yin, Wei-Na; Fan, Jin-Xuan; Cao, Feng-Yi; Zhang, Xian-Zheng

    2015-10-28

    In this report, a UV sensitive, PEGylated PFSSTKTC (Pro-Phe-Ser-Ser-Thr-Lys-Thr-Cys) peptide was modified on quartz substrate to investigate the spatial controlled differentiation of stem cells. This substrate could restrict the cell adhesion due to the steric hindrance of PEG shell. With UV irradiation, PFSSTKTC became exposed owing to the breakage of o-nitrobenzyl group with the detachment of PEG shell. The irradiation boundary on substrate was stable in the long term. The in vitro osteogenic differentiation results revealed that under the site-specific irradiation, the mesenchymal stem cells (MSCs) could specifically differentiate into osteoblast under the induction of PFSSTKTC peptide. This photoactivatable biomaterial shows great potential for region controllable and precise MSCs differentiation. PMID:26452046

  18. Estrogen deficiency heterogeneously affects tissue specific stem cells in mice.

    PubMed

    Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

    2015-01-01

    Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

  19. The C-terminal loop of aldehyde reductase determines the substrate and inhibitor specificity.

    PubMed

    Barski, O A; Gabbay, K H; Bohren, K M

    1996-11-12

    Human aldehyde reductase has a preference for carboxyl group-containing negatively charged substrates. It belongs to the NADPH-dependent aldo-keto reductase superfamily whose members are in part distinguished by unique C-terminal loops. To probe the role of the C-terminal loops in determining substrate specificities in these enzymes, two arginine residues, Arg308 and Arg311, located in the C-terminal loop of aldehyde reductase, and not found in any other C-terminal loop, were replaced with alanine residues. The catalytic efficiency of the R311A mutant for aldehydes containing a carboxyl group is reduced 150-250-fold in comparison to that of the wild-type enzyme, while substrates not containing a negative charge are unaffected. The R311A mutant is also significantly less sensitive to inhibition by dicarboxylic acids, indicating that Arg311 interacts with one of the carboxyl groups. The inhibition pattern indicates that the other carboxyl group binds to the anion binding site formed by Tyr49, His112, and the nicotinamide moiety of NADP+. The correlation between inhibitor potency and the length of the dicarboxylic acid molecules suggests a distance of approximately 10 A between the amino group of Arg311 and the anion binding site in the aldehyde reductase molecule. The sensitivity of inhibition of the R311A mutant by several commercially available aldose reductase inhibitors (ARIs) was variable, with tolrestat and zopolrestat becoming more potent inhibitors (30- and 5-fold, respectively), while others remained the same or became less potent. The catalytic properties, substrate specificity, and susceptibility to inhibition of the R308A mutant remained similar to that of the wild-type enzyme. The data provide direct evidence for C-terminal loop participation in determining substrate and inhibitor specificity of aldo-keto reductases and specifically identifies Arg311 as the basis for the carboxyl-containing substrate preference of aldehyde reductase. PMID:8916913

  20. Structural view and substrate specificity of papain-like protease from avian infectious bronchitis virus.

    PubMed

    Kong, Lingying; Shaw, Neil; Yan, Lingming; Lou, Zhiyong; Rao, Zihe

    2015-03-13

    Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys(48)- and Lys(63)-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys(101), His(264), and Asp(275) is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus. PMID:25609249

  1. Structural View and Substrate Specificity of Papain-like Protease from Avian Infectious Bronchitis Virus*

    PubMed Central

    Kong, Lingying; Shaw, Neil; Yan, Lingming; Lou, Zhiyong; Rao, Zihe

    2015-01-01

    Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys48- and Lys63-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys101, His264, and Asp275 is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus. PMID:25609249

  2. Molecular Mechanisms of Substrate Recognition and Specificity of New Delhi Metallo-β-Lactamase

    PubMed Central

    Chiou, Jiachi; Leung, Thomas Yun-Chung

    2014-01-01

    Carbapenems are one of the last lines of defense for Gram-negative pathogens, such as members of the Enterobacteriaceae. Despite the fact that most carbapenems are resistant to extended-spectrum β-lactamase (ESBL), emerging metallo-β-lactamases (MBLs), including New Delhi metallo-β-lactamase 1 (NDM-1), that can hydrolyze carbapenems have become prevalent and are frequently associated with the so-called “superbugs,” for which treatments are extremely limited. Crystallographic study sheds light on the modes of antibiotic binding to NDM-1, yet the mechanisms governing substrate recognition and specificity are largely unclear. This study provides a connection between crystallographic study and the functional significance of NDM-1, with an emphasis on the substrate specificity and catalysis of various β-lactams. L1 loop residues L59, V67, and W87 were important for the activity of NDM-1, most likely through maintaining the partial folding of the L1 loop or active site conformation through hydrophobic interaction with the R groups of β-lactams or the β-lactam ring. Substitution of alanine for L59 showed greater reduction of MICs to ampicillin and selected cephalosporins, whereas substitutions of alanine for V67 had more impact on the MICs of carbapenems. K224 and N233 on the L3 loop played important roles in the recognition of substrate and contributed to substrate hydrolysis. These data together with the structure comparison of the B1 and B2 subclasses of MBLs revealed that the broad substrate specificity of NDM-1 could be due to the ability of its wide active site cavity to accommodate a wide range of β-lactams. This study provides insights into the development of efficient inhibitors for NDM-1 and offers an efficient tactic with which to study the substrate specificities of other β-lactamases. PMID:24982075

  3. Computational approaches for classification and prediction of P-type ATPase substrate specificity in Arabidopsis.

    PubMed

    Zinati, Zahra; Alemzadeh, Abbas; KayvanJoo, Amir Hossein

    2016-01-01

    As an extended gamut of integral membrane (extrinsic) proteins, and based on their transporting specificities, P-type ATPases include five subfamilies in Arabidopsis, inter alia, P4ATPases (phospholipid-transporting ATPase), P3AATPases (plasma membrane H(+) pumps), P2A and P2BATPases (Ca(2+) pumps) and P1B ATPases (heavy metal pumps). Although, many different computational methods have been developed to predict substrate specificity of unknown proteins, further investigation needs to improve the efficiency and performance of the predicators. In this study, various attribute weighting and supervised clustering algorithms were employed to identify the main amino acid composition attributes, which can influence the substrate specificity of ATPase pumps, classify protein pumps and predict the substrate specificity of uncharacterized ATPase pumps. The results of this study indicate that both non-reduced coefficients pertaining to absorption and Cys extinction within 280 nm, the frequencies of hydrogen, Ala, Val, carbon, hydrophilic residues, the counts of Val, Asn, Ser, Arg, Phe, Tyr, hydrophilic residues, Phe-Phe, Ala-Ile, Phe-Leu, Val-Ala and length are specified as the most important amino acid attributes through applying the whole attribute weighting models. Here, learning algorithms engineered in a predictive machine (Naive Bays) is proposed to foresee the Q9LVV1 and O22180 substrate specificities (P-type ATPase like proteins) with 100 % prediction confidence. For the first time, our analysis demonstrated promising application of bioinformatics algorithms in classifying ATPases pumps. Moreover, we suggest the predictive systems that can assist towards the prediction of the substrate specificity of any new ATPase pumps with the maximum possible prediction confidence. PMID:27186030

  4. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    SciTech Connect

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu -Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc -André; Deacon, Ashley M.; Wilson, Ian A.

    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-A2pm (A2pm 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 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

  5. Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

    SciTech Connect

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu-Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; Lesley, Scott A.; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2015-09-15

    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-A2pm (A2pm 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 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.

    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

  6. Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

    DOE PAGES

    Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; Patin, Delphine; Farr, Carol L.; Grant, Joanna C.; Chiu, Hsiu -Ju; Jaroszewski, Lukasz; Knuth, Mark W.; Godzik, Adam; et al

    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-A2pm (A2pm 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 consisting ofmore » 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

  7. Probing polypeptide GalNAc-transferase isoform substrate specificities by in vitro analysis.

    PubMed

    Kong, Yun; Joshi, Hiren J; Schjoldager, Katrine Ter-Borch Gram; Madsen, Thomas Daugbjerg; Gerken, Thomas A; Vester-Christensen, Malene B; Wandall, Hans H; Bennett, Eric Paul; Levery, Steven B; Vakhrushev, Sergey Y; Clausen, Henrik

    2015-01-01

    N-acetylgalactosaminyltransferase (GalNAc)-type (mucin-type) O-glycosylation is an abundant and highly diverse modification of proteins. This type of O-glycosylation is initiated in the Golgi by a large family of up to 20 homologous polypeptide GalNAc-T isoenzymes that transfer GalNAc to Ser, Thr and possibly Tyr residues. These GalNAc residues are then further elongated by a large set of glycosyltransferases to build a variety of complex O-glycan structures. What determines O-glycan site occupancy is still poorly understood, although it is clear that the substrate specificities of individual isoenzymes and the repertoire of GalNAc-Ts in cells are key parameters. The GalNAc-T isoenzymes are differentially expressed in cells and tissues in principle allowing cells to produce unique O-glycoproteomes dependent on the specific subset of isoforms present. In vitro analysis of acceptor peptide substrate specificities using recombinant expressed GalNAc-Ts has been the method of choice for probing activities of individual isoforms, but these studies have been hampered by biological validation of actual O-glycosylation sites in proteins and number of substrate testable. Here, we present a systematic analysis of the activity of 10 human GalNAc-T isoenzymes with 195 peptide substrates covering known O-glycosylation sites and provide a comprehensive dataset for evaluating isoform-specific contributions to the O-glycoproteome.

  8. Shotgun isotope array for rapid, substrate-specific detection of microorganisms in a microbial community.

    PubMed

    Tobino, Tomohiro; Kurisu, Futoshi; Kasuga, Ikuro; Furumai, Hiroaki

    2011-10-01

    The shotgun isotope array method has been proposed to be an effective new tool for use in substrate-specific microbe exploration without any prior knowledge of the community composition. Proof of concept was demonstrated by detection of acetate-degrading microorganisms in activated sludge and further verified by independent stable isotope probing (SIP).

  9. Chemoenzymatic synthesis of prodigiosin analogues--exploring the substrate specificity of PigC.

    PubMed

    Chawrai, Suresh R; Williamson, Neil R; Salmond, George P C; Leeper, Finian J

    2008-04-28

    Analogues of prodigiosin, a tripyrrolic pigment produced by Serratia species with potent immunosuppressive and anticancer activities, have been produced by feeding synthetic analogues of the normal precursor MBC to mutants of Serratia sp. ATCC 39006 or to engineered strains of Escherichia coli; in this way it has been shown that the prodigiosin synthesising enzyme, PigC, has a relaxed substrate-specificity.

  10. Highly Specific, Bi-substrate-Competitive Src Inhibitors from DNA-Templated Macrocycles

    PubMed Central

    Georghiou, George; Kleiner, Ralph E.; Pulkoski-Gross, Michael

    2011-01-01

    Protein kinases are attractive therapeutic targets, but their high sequence and structural conservation complicates the development of specific inhibitors. We recently discovered from a DNA-templated macrocycle library inhibitors with unusually high selectivity among Src-family kinases. Starting from these compounds, we developed and characterized in molecular detail potent macrocyclic inhibitors of Src kinase and its cancer-associated gatekeeper mutant. We solved two co-crystal structures of macrocycles bound to Src kinase. These structures reveal the molecular basis of the combined ATP- and substrate peptide-competitive inhibitory mechanism and the remarkable kinase specificity of the compounds. The most potent compounds inhibit Src activity in cultured mammalian cells. Our work establishes that macrocycles can inhibit protein kinases through a bi-substrate competitive mechanism with high potency and exceptional specificity, reveals the precise molecular basis for their desirable properties, and provides new insights into the development of Src-specific inhibitors with potential therapeutic relevance. PMID:22344177

  11. Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria.

    PubMed

    Ito, M; Dunn, B M; Oda, K

    1996-10-01

    Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).

  12. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    PubMed

    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.

  13. Substrate Specificity of the Nonribosomal Peptide Synthetase PvdD from Pseudomonas aeruginosa

    PubMed Central

    Ackerley, David F.; Caradoc-Davies, Tom T.; Lamont, Iain L.

    2003-01-01

    Pseudomonas aeruginosa PAO1 secretes a siderophore, pyoverdinePAO, which contains a short peptide attached to a dihydroxyquinoline moiety. Synthesis of this peptide is thought to be catalyzed by nonribosomal peptide synthetases, one of which is encoded by the pvdD gene. The first module of pvdD was overexpressed in Escherichia coli, and the protein product was purified. l-Threonine, one of the amino acid residues in pyoverdinePAO, was an effective substrate for the recombinant protein in ATP-PPi exchange assays, showing that PvdD has peptide synthetase activity. Other amino acids, including d-threonine, l-serine, and l-allo-threonine, were not effective substrates, indicating that PvdD has a high degree of substrate specificity. A three-dimensional modeling approach enabled us to identify amino acids that are likely to be critical in determining the substrate specificity of PvdD and to explore the likely basis of the high substrate selectivity. The approach described here may be useful for analysis of other peptide synthetases. PMID:12700264

  14. Mammalian folylpoly-. gamma. -glutamate synthetase. 2. Substrate specificity and kinetic properties

    SciTech Connect

    Cichowicz, D.J.; Shane, B.

    1987-01-27

    The specificity of hog liver folylpolyglutamate synthetase for folate substrates and for nucleotide and L-(/sup 14/C)glutamate substrates and analogues has been investigated. The kinetic mechanism, determined by using aminopterin as the folate substrate, is ordered Ter-Ter with MgATP binding first, folate second, and glutamate last. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. Folate, dihydrofolate, and tetrahydrofolate possess the optimal configurations for catalysis while 5- and 10-position substitutions of the folate molecule impair catalysis. k/sub cat/ values decrease with increasing glutamate chain length, and the rate of decrease varies depending on the state of reduction and substitution of the folate molecule. Folate binding, as assessed by on rates, is slow. Dihydrofolate exhibits the fastest rate, and the rates are slightly reduced for tetrahydrofolate and 10-formyltetrahydrofolate and greatly reduced for 5-methyltetrahydrofolate and folic acid. Tetrahydrofolate polyglutamates are the only long glutamate chain length folates with detectable substrate activity. The specificity of the L-glutamate binding site is very narrow. L-Homocysteate and 4-threo-fluoroglutamate are alternate substrates and act as chain termination inhibitors in that their addition to the folate molecule prevents or severely retards the further addition of glutamate moieties. The K/sub m/ for glutamate is dependent on the folate substrate used. MgATP is the preferred nucleotide substrate, and ..beta..,..gamma..-methylene-ATP, ..beta..,..gamma..-imido-ATP, adenosine 5'-O-(3-thiotriphosphate), P/sup 1/,P/sup 5/-di(adenosine-5') pentaphosphate, and free ATP/sup 4 -/ are potent inhibitors of the reaction.

  15. Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa

    PubMed Central

    Modi, Niraj; Ganguly, Sonalli; Bárcena-Uribarri, Iván; Benz, Roland; van den Berg, Bert; Kleinekathöfer, Ulrich

    2015-01-01

    The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well. PMID:26445443

  16. Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa.

    PubMed

    Modi, Niraj; Ganguly, Sonalli; Bárcena-Uribarri, Iván; Benz, Roland; van den Berg, Bert; Kleinekathöfer, Ulrich

    2015-10-01

    The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.

  17. Understanding the substrate specificity of the heparan sulfate sulfotransferases by an integrated biosynthetic and crystallographic approach.

    PubMed

    Liu, Jian; Moon, Andrea F; Sheng, Juzheng; Pedersen, Lars C

    2012-10-01

    Heparan sulfates (HSs) have potential therapeutic value as anti-inflammatory and antimetastasis drugs, in addition to their current use as anticoagulants. Recent advances in chemoenzymatic synthesis of HS provide a way to conveniently produce homogenous HS with different biological properties. Crystal structures of sulfotransferases involved in this process are providing atomic detail of their substrate binding clefts and interactions with their HS substrates. In theory, the flexibility of this method can be increased by modifying the specificities of the sulfotransferases based on the structures, thereby producing a new array of products.

  18. Serine 363 of a hydrophobic region of Archaeal ribulose 1,5-bisphosphate carboxylase/oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis affects CO2/O2 substrate specificity and oxygen sensitivity

    SciTech Connect

    Kreel, Nathan E.; Tabita, F. Robert; Berg, Ivan

    2015-09-18

    Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. As a result, it is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions.

  19. Structure of Human Dual Specificity Protein Phosphatase 23, VHZ, Enzyme-Substrate/Product Complex

    SciTech Connect

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

    2008-01-01

    Protein phosphorylation plays a crucial role in mitogenic signal transduction and regulation of cell growth and differentiation. Dual specificity protein phosphatase 23 (DUSP23) or VHZ mediates dephosphorylation of phospho-tyrosyl (pTyr) and phospho-seryl/threonyl (pSer/pThr) residues in specific proteins. In vitro, it can dephosphorylate p44ERK1 but not p54SAPK-{beta} and enhance activation of c-Jun N-terminal kinase (JNK) and p38. Human VHZ, the smallest of the catalytically active protein-tyrosine phosphatases (PTP) reported to date (150 residues), is a class I Cys-based PTP and bears the distinctive active site signature motif HCXXGXXRS(T). We present the crystal structure of VHZ determined at 1.93 angstrom resolution. The polypeptide chain adopts the typical a{beta}a PTP fold, giving rise to a shallow active site cleft that supports dual phosphorylated substrate specificity. Within our crystals, the Thr-135-Tyr-136 from a symmetry-related molecule bind in the active site with a malate ion, where they mimic the phosphorylated TY motif of the MAPK activation loop in an enzyme-substrate/product complex. Analyses of intermolecular interactions between the enzyme and this pseudo substrate/product along with functional analysis of Phe-66, Leu-97, and Phe-99 residues provide insights into the mechanism of substrate binding and catalysis in VHZ.

  20. Prediction and experimental validation of enzyme substrate specificity in protein structures

    PubMed Central

    Amin, Shivas R.; Erdin, Serkan; Ward, R. Matthew; Lua, Rhonald C.; Lichtarge, Olivier

    2013-01-01

    Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase–like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. PMID:24145433

  1. Profiling substrate specificity of two series of phenethylamine analogs at monoamine oxidase A and B.

    PubMed

    Heuson, Egon; Storgaard, Morten; Huynh, Tri H V; Charmantray, Franck; Gefflaut, Thierry; Bunch, Lennart

    2014-11-21

    The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B (MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians. Despite their importance and being a prevalent target for the development of inhibitors as drugs, no systematic study of substrate specificity has been reported. In this study we present a systematic study of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs. Km and kcat values were determined for four N-alkyl analogs 2-5 and four aryl halide analogs 6-9 at MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be relevant for future drug design within the field of monoamines.

  2. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    SciTech Connect

    Sayer, Christopher; Isupov, Michail N.; Westlake, Aaron; Littlechild, Jennifer A.

    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 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.

  3. Alternative splicing determines the function of CYP4F3 by switching substrate specificity.

    PubMed

    Christmas, P; Jones, J P; Patten, C J; Rock, D A; Zheng, Y; Cheng, S M; Weber, B M; Carlesso, N; Scadden, D T; Rettie, A E; Soberman, R J

    2001-10-12

    Diversity of cytochrome P450 function is determined by the expression of multiple genes, many of which have a high degree of identity. We report that the use of alternate exons, each coding for 48 amino acids, generates isoforms of human CYP4F3 that differ in substrate specificity, tissue distribution, and biological function. Both isoforms contain a total of 520 amino acids. CYP4F3A, which incorporates exon 4, inactivates LTB4 by omega-hydroxylation (Km = 0.68 microm) but has low activity for arachidonic acid (Km = 185 microm); it is the only CYP4F isoform expressed in myeloid cells in peripheral blood and bone marrow. CYP4F3B incorporates exon 3 and is selectively expressed in liver and kidney; it is also the predominant CYP4F isoform in trachea and tissues of the gastrointestinal tract. CYP4F3B has a 30-fold higher Km for LTB4 compared with CYP4F3A, but it utilizes arachidonic acid as a substrate for omega-hydroxylation (Km = 22 microm) and generates 20-HETE, an activator of protein kinase C and Ca2+/calmodulin-dependent kinase II. Homology modeling demonstrates that the alternative exon has a position in the molecule which could enable it to contribute to substrate interactions. The results establish that tissue-specific alternative splicing of pre-mRNA can be used as a mechanism for changing substrate specificity and increasing the functional diversity of cytochrome P450 genes.

  4. A novel suicide substrate for DNA topoisomerases and site-specific recombinases.

    PubMed Central

    Burgin, A B; Huizenga, B N; Nash, H A

    1995-01-01

    DNA topoisomerases and DNA site-specific recombinases are biologically important enzymes involved in a diverse set of cellular processes. We show that replacement of a phosphodiester linkage by a 5'-bridging phosphorothioate linkage creates an efficient suicide substrate for calf thymus topoisomerase I and lambda integrase protein (Int). Although the bridging phosphorothioate linkage is cleaved by these enzymes, the 5'-sulfhydryl which is generated is not competent for subsequent ligation reactions. We use the irreversibility of Int-promoted cleavage to explore conditions and factors that contribute to various steps of lambda integrative recombination. The phosphorothioate substrates offer advantages over conventional suicide substrates, may be potent tools for inhibition of the relevant cellular enzymes and represent a unique tool for the study of many other phosphoryl transfer reactions. Images PMID:7659520

  5. Substrate specificity determinants of the methanogen homoaconitase enzyme: structure and function of small subunit residues

    SciTech Connect

    Jeyakanthan, Jeyaraman; Drevland, Randy; Gayathri, Dasara; Velmurugan, Devadasan; Shinkai, Akeo; Graham, David E

    2010-01-01

    The aconitase family of hydro-lyase enzymes includes three classes of proteins that catalyze the isomerization of -hydroxyacids to -hydroxyacids. Besides aconitase, isopropylmalate isomerase (IPMI) proteins specifically catalyze the isomerization of , -dicarboxylates with hydrophobic -chain groups, and homoaconitase (HACN) proteins catalyze the isomerization of tricarboxylates with variable chain length -carboxylate groups. These enzymes stereospecific hydro-lyase activities make them attractive catalysts to produce diastereomers from unsaturated precursors. However, sequence similarity and convergent evolution among these proteins leads to widespread misannotation and uncertainty about gene function. To find the substrate specificity determinants of homologous IPMI and HACN proteins from Methanocaldococcus jannaschii, the small-subunit HACN protein (MJ1271) was crystallized for X-ray diffraction. The structural model showed characteristic residues in a flexible loop region between 2 and 3 that distinguish HACN from IPMI and aconitase proteins. Site-directed mutagenesis of MJ1271 produced loop-region variant proteins that were reconstituted with wild-type MJ1003 large-subunit protein. The heteromers formed promiscuous hydro-lyases with reduced activity but broader substrate specificity. Both R26K and R26V variants formed relatively efficient IPMI enzymes, while the T27A variant had uniformly lower specificity constants for both IPMI and HACN substrates. The R26V T27Y variant resembles the MJ1277 IPMI small subunit in its flexible loop sequence, but demonstrated the broad substrate specificity of the R26V variant. These mutations may reverse the evolution of HACN activity from an ancestral IPMI gene, demonstrating the evolutionary potential for promiscuity in hydro-lyase enzymes. Understanding these specificity determinants enables the functional reannotation of paralogous HACN and IPMI genes in numerous genome sequences. These structural and kinetic results will

  6. Quantitative analysis of substrate specificity of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26.

    PubMed

    Kmunícek, Jan; Hynková, Kamila; Jedlicka, Tomás; Nagata, Yuji; Negri, Ana; Gago, Federico; Wade, Rebecca C; Damborský, Jirí

    2005-03-01

    Haloalkane dehalogenases are microbial enzymes that cleave a carbon-halogen bond in halogenated compounds. The haloalkane dehalogenase LinB, isolated from Sphingomonas paucimobilis UT26, is a broad-specificity enzyme. Fifty-five halogenated aliphatic and cyclic hydrocarbons were tested for dehalogenation with the LinB enzyme. The compounds for testing were systematically selected using a statistical experimental design. Steady-state kinetic constants K(m) and k(cat) were determined for 25 substrates that showed detectable cleavage by the enzyme and low abiotic hydrolysis. Classical quantitative structure-activity relationships (QSARs) were used to correlate the kinetic constants with molecular descriptors and resulted in a model that explained 94% of the experimental data variability. The binding affinity of the tested substrates for this haloalkane dehalogenase correlated with hydrophobicity, molecular surface, dipole moment, and volume:surface ratio. Binding of the substrate molecules in the active site pocket of LinB depends nonlinearly on the size of the molecules. Binding affinity increases with increasing substrate size up to a chain length of six carbon atoms and then decreases. Comparative binding energy (COMBINE) analysis was then used to identify amino acid residues in LinB that modulate its substrate specificity. A model with three statistically significant principal components explained 95% of the experimental data variability. van der Waals interactions between substrate molecules and the enzyme dominated the COMBINE model, in agreement with the importance of substrate size in the classical QSAR model. Only a limited number of protein residues (6-8%) contribute significantly to the explanation of variability in binding affinities. The amino acid residues important for explaining variability in binding affinities are as follows: (i) first-shell residues Asn38, Asp108, Trp109, Glu132, Ile134, Phe143, Phe151, Phe169, Val173, Trp207, Pro208, Ile211, Leu248

  7. Investigation of the dimer interface and substrate specificity of prolyl dipeptidase DPP8.

    PubMed

    Lee, Hong-Jen; Chen, Yuan-Shou; Chou, Chi-Yuan; Chien, Chia-Hui; Lin, Chun-Hung; Chang, Gu-Gang; Chen, Xin

    2006-12-15

    DPP8 belongs to the family of prolyl dipeptidases, which are capable of cleaving the peptide bond after a penultimate proline residue. Unlike DPP-IV, a drug target for type II diabetes, no information is available on the crystal structure of DPP8, the regulation of its enzymatic activity, or its substrate specificity. In this study, using analytical ultracentrifugation and native gel electrophoresis, we show that the DPP8 protein is predominantly dimeric when purified or in the cell extracts. Four conserved residues in the C-terminal loop of DPP8 (Phe(822), Val(833), Tyr(844), and His(859)), corresponding to those located at the dimer interface of DPP-IV, were individually mutated to Ala. Surprisingly, unlike DPP-IV, these single-site mutations abolished the enzymatic activity of DPP8 without disrupting its quaternary structure, indicating that dimerization itself is not sufficient for the optimal enzymatic activity of DPP8. Moreover, these mutations not only decreased k(cat), as did the corresponding DPP-IV mutations, but also dramatically increased K(m). We further show that the K(m) effect is independent of the substrate assayed. Finally, we identified the distinctive and strict substrate selectivity of DPP8 for hydrophobic or basic residues at the P2 site, which is in sharp contrast to the much less discriminative substrate specificity of DPP-IV. Our study has identified the residues absolutely required for the optimal activity of DPP8 and its unique substrate specificity. This study extends the functional importance of the C-terminal loop to the whole family of prolyl dipeptidases.

  8. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs.

    PubMed

    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

  9. Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

    PubMed Central

    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

  10. Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

    PubMed Central

    Kalupov, Timofey; Brillard-Bourdet, Michèle; Dadé, Sébastien; Serrano, Hélène; Wartelle, Julien; Guyot, Nicolas; Juliano, Luiz; Moreau, Thierry; Belaaouaj, Azzaq; Gauthier, Francis

    2009-01-01

    It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases. PMID:19833730

  11. Investigating Commercial Cellulase Performances Toward Specific Biomass Recalcitrance Factors Using Reference Substrates

    SciTech Connect

    Ju, Xiaohui; Bowden, Mark E.; Engelhard, Mark H.; Zhang, Xiao

    2014-04-01

    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 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 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.

  12. A protease substrate profiling method that links site-specific proteolysis with antibiotic resistance.

    PubMed

    Sandersjöö, Lisa; Kostallas, George; Löfblom, John; Samuelson, Patrik

    2014-01-01

    Proteases are involved in many biological processes and have become important tools in biomedical research and industry. Technologies for engineering and characterization of, for example, proteolytic activity and specificity are essential in protease research. Here, we present a novel method for assessment of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that, upon processing by a co-expressed protease, confer antibiotic resistance to bacteria in proportion to the cleavage efficiency. We have demonstrated that cells co-expressing cleavable reporters together with tobacco etch virus protease (TEVp) could be discriminated from cells with non-cleavable reporters by growth in selective media. Importantly, the resistance to antibiotics proved to correlate with the substrate processing efficiency. Thus, by applying competitive growth of a mock library in antibiotic-containing medium, we could show that the substrate preferred by TEVp was enriched relative to less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases and protease recognition sequences towards improved or even new functionality.

  13. Foliar Substrate Affects Cuticular Hydrocarbon Profiles and Intraspecific Aggression in the Leafcutter Ant Atta sexdens

    PubMed Central

    Valadares, Lohan; Nascimento, Daniela; Nascimento, Fabio S.

    2015-01-01

    Cuticular hydrocarbons (CHCs) are traditionally considered to be one of the most important chemical cues used in the nestmate recognition process of social hymenopterans. However, it has been suggested that in the leafcutter ant genus Atta, it is not the CHCs, but the alarm pheromone that is involved in the nestmate recognition process. In this study we used a laboratory population of Atta sexdens to explore the association between their CHC profile variation and intraspecific aggression. In the first part of the experiment, four colonies were divided into two groups with distinct diets to stimulate differentiation of their CHC profiles. In the second part of the experiment, all colonies received the same diet to examine resemblance of chemical profiles. At the end of each part of the experiment we extracted the CHCs from workers. The results demonstrated that colonies that shared the same food resource had similar cuticular hydrocarbon profiles. Furthermore, colonies were significantly more aggressive towards conspecifics that used a different foliar substrate and consequently had greater differences in their cuticular chemical composition. This study suggests that the CHC profiles of A. sexdens can be affected by the foliar substrates used, and that the CHCs are used in the nestmate recognition process of this species. PMID:26463072

  14. Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity.

    PubMed

    Ip, Laura Ren Huey; Gewinner, Christina Anja

    2016-01-01

    Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.

  15. Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

    PubMed

    Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

    2016-04-01

    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. PMID:26785049

  16. Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer.

    PubMed

    Noda-García, Lianet; Camacho-Zarco, Aldo R; Medina-Ruíz, Sofía; Gaytán, Paul; Carrillo-Tripp, Mauricio; Fülöp, Vilmos; Barona-Gómez, Francisco

    2013-09-01

    Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole l-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in l-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA's substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in l-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism. PMID:23800623

  17. Crystal structure of Trypanosoma cruzi tyrosine aminotransferase: substrate specificity is influenced by cofactor binding mode.

    PubMed Central

    Blankenfeldt, W.; Nowicki, C.; Montemartini-Kalisz, M.; Kalisz, H. M.; Hecht, H. J.

    1999-01-01

    The crystal structure of tyrosine aminotransferase (TAT) from the parasitic protozoan Trypanosoma cruzi, which belongs to the aminotransferase subfamily Igamma, has been determined at 2.5 A resolution with the R-value R = 15.1%. T. cruzi TAT shares less than 15% sequence identity with aminotransferases of subfamily Ialpha but shows only two larger topological differences to the aspartate aminotransferases (AspATs). First, TAT contains a loop protruding from the enzyme surface in the larger cofactor-binding domain, where the AspATs have a kinked alpha-helix. Second, in the smaller substrate-binding domain, TAT has a four-stranded antiparallel beta-sheet instead of the two-stranded beta-sheet in the AspATs. The position of the aromatic ring of the pyridoxal-5'-phosphate cofactor is very similar to the AspATs but the phosphate group, in contrast, is closer to the substrate-binding site with one of its oxygen atoms pointing toward the substrate. Differences in substrate specificities of T. cruzi TAT and subfamily Ialpha aminotransferases can be attributed by modeling of substrate complexes mainly to this different position of the cofactor-phosphate group. Absence of the arginine, which in the AspATs fixes the substrate side-chain carboxylate group by a salt bridge, contributes to the inability of T. cruzi TAT to transaminate acidic amino acids. The preference of TAT for tyrosine is probably related to the ability of Asn17 in TAT to form a hydrogen bond to the tyrosine side-chain hydroxyl group. PMID:10595543

  18. Mechanism of substrate specificity in 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidases.

    PubMed

    Siu, Karen K W; Asmus, Kyle; Zhang, Allison N; Horvatin, Cathy; Li, Sheng; Liu, Tong; Moffatt, Barbara; Woods, Virgil L; Howell, P Lynne

    2011-01-01

    5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/SAH) nucleosidase (MTAN) plays a key role in the methionine-recycling pathway of bacteria and plants. Despite extensive structural and biochemical studies, the molecular mechanism of substrate specificity for MTAN remains an outstanding question. Bacterial MTANs show comparable efficiency in hydrolyzing MTA and SAH, while the plant enzymes select preferentially for MTA, with either no or significantly reduced activity towards SAH. Bacterial and plant MTANs show significant conservation in the overall structure, and the adenine- and ribose-binding sites. The observation of a more constricted 5'-alkylthio binding site in Arabidopsis thalianaAtMTAN1 and AtMTAN2, two plant MTAN homologues, led to the hypothesis that steric hindrance may play a role in substrate selection in plant MTANs. We show using isothermal titration calorimetry that SAH binds to both Escherichia coli MTAN (EcMTAN) and AtMTAN1 with comparable micromolar affinity. To understand why AtMTAN1 can bind but not hydrolyze SAH, we determined the structure of the protein-SAH complex at 2.2Å resolution. The lack of catalytic activity appears to be related to the enzyme's inability to bind the substrate in a catalytically competent manner. The role of dynamics in substrate selection was also examined by probing the amide proton exchange rates of EcMTAN and AtMTAN1 via deuterium-hydrogen exchange coupled mass spectrometry. These results correlate with the B factors of available structures and the thermodynamic parameters associated with substrate binding, and suggest a higher level of conformational flexibility in the active site of EcMTAN. Our results implicate dynamics as an important factor in substrate selection in MTAN.

  19. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases.

    PubMed

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

    2016-01-21

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design.

  20. Quantitative Correlation of Conformational Binding Enthalpy with Substrate Specificity of Serine Proteases

    PubMed Central

    2015-01-01

    Members of the same protease family show different substrate specificity, even if they share identical folds, depending on the physiological processes they are part of. Here, we investigate the key factors for subpocket and global specificity of factor Xa, elastase, and granzyme B which despite all being serine proteases and sharing the chymotrypsin-fold show distinct substrate specificity profiles. We determined subpocket interaction potentials with GRID for static X-ray structures and an in silico generated ensemble of conformations. Subpocket interaction potentials determined for static X-ray structures turned out to be insufficient to explain serine protease specificity for all subpockets. Therefore, we generated conformational ensembles using molecular dynamics simulations. We identified representative binding site conformations using distance-based hierarchical agglomerative clustering and determined subpocket interaction potentials for each representative conformation of the binding site. Considering the differences in subpocket interaction potentials for these representative conformations as well as their abundance allowed us to quantitatively explain subpocket specificity for the nonprime side for all three example proteases on a molecular level. The methods to identify key regions determining subpocket specificity introduced in this study are directly applicable to other serine proteases, and the results provide starting points for new strategies in rational drug design. PMID:26709959

  1. A short peptide is a protein kinase C (PKC) alpha-specific substrate.

    PubMed

    Kang, Jeong-Hun; Asai, Daisuke; Yamada, Satoshi; Toita, Riki; Oishi, Jun; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki

    2008-05-01

    The purpose of this study was to find protein kinase C (PKC) isozyme-specific peptides. A peptide library containing 1772 sequences was designed using Scansite and screened by MALDI-TOF MS and kinase activity assays for PKC isozyme-specificity. A peptide (Alphatomega; H-FKKQGSFAKKK-NH(2)) with high specificity for PKC alpha relative to other isozymes was identified. The peptide was phosphorylated to a greater extent by tissue lysates from B16 melanoma, HepG2, and human breast cancer, which had higher levels of activated PKC alpha, when compared to normal skin, liver, and human breast tissue lysates, respectively. Moreover, addition of Ro-31-7549, an inhibitor with great specificity for PKC alpha, to the phosphorylation reaction caused a dose-dependent reduction in phosphorylation, but no inhibition was identified with the addition of rottlerin and H-89. These results show that this peptide has great potential as a PKC alpha-specific substrate.

  2. Novel substrate specificity of glutathione synthesis enzymes from Streptococcus agalactiae and Clostridium acetobutylicum

    SciTech Connect

    Kino, Kuniki . E-mail: kkino@waseda.jp; Kuratsu, Shoko; Noguchi, Atsushi; Kokubo, Masahiro; Nakazawa, Yuji; Arai, Toshinobu; Yagasaki, Makoto; Kirimura, Kohtaro

    2007-01-12

    Glutathione (GSH) is synthesized by {gamma}-glutamylcysteine synthetase ({gamma}-GCS) and glutathione synthetase (GS) in living organisms. Recently, bifunctional fusion protein, termed {gamma}-GCS-GS catalyzing both {gamma}-GCS and GS reactions from gram-positive firmicutes Streptococcus agalactiae, has been reported. We revealed that in the {gamma}-GCS activity, S. agalactiae {gamma}-GCS-GS had different substrate specificities from those of Escherichia coli {gamma}-GCS. Furthermore, S. agalactiae {gamma}-GCS-GS synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-X{sub aa}-Gly, from free three amino acids. In Clostridium acetobutylicum, the genes encoding {gamma}-GCS and putative GS were found to be immediately adjacent by BLAST search, and had amino acid sequence homology with S. agalactiae {gamma}-GCS-GS, respectively. We confirmed that the proteins expressed from each gene showed {gamma}-GCS and GS activity, respectively. C. acetobutylicum GS had broad substrate specificities and synthesized several kinds of {gamma}-glutamyltripeptide, {gamma}-Glu-Cys-X{sub aa}. Whereas the substrate specificities of {gamma}-GCS domain protein and GS domain protein of S. agalactiae {gamma}-GCS-GS were the same as those of S. agalactiae {gamma}-GCS-GS.

  3. A novel member of glycoside hydrolase family 30 subfamily 8 with altered substrate specificity

    PubMed Central

    St John, Franz J.; Dietrich, Diane; Crooks, Casey; Pozharski, Edwin; González, Javier M.; Bales, Elizabeth; Smith, Kennon; Hurlbert, Jason C.

    2014-01-01

    Endoxylanases classified into glycoside hydrolase family 30 subfamily 8 (GH30-8) are known to hydrolyze the hemicellulosic polysaccharide glucuronoxylan (GX) but not arabinoxylan or neutral xylooligosaccharides. This is owing to the specificity of these enzymes for the α-1,2-linked glucuronate (GA) appendage of GX. Limit hydrolysis of this substrate produces a series of aldouronates each containing a single GA substituted on the xylose penultimate to the reducing terminus. In this work, the structural and biochemical characterization of xylanase 30A from Clostridium papyro­solvens (CpXyn30A) is presented. This xylanase possesses a high degree of amino-acid identity to the canonical GH30-8 enzymes, but lacks the hallmark β8–α8 loop region which in part defines the function of this GH30 subfamily and its role in GA recognition. CpXyn30A is shown to have a similarly low activity on all xylan substrates, while hydrolysis of xylohexaose revealed a competing transglycosylation reaction. These findings are directly compared with the model GH30-8 enzyme from Bacillus subtilis, XynC. Despite its high sequence identity to the GH30-8 enzymes, CpXyn30A does not have any apparent specificity for the GA appendage. These findings confirm that the typically conserved β8–α8 loop region of these enzymes influences xylan substrate specificity but not necessarily β-1,4-xylanase function. PMID:25372685

  4. Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity.

    PubMed

    Welin, Martin; Wang, Liya; Eriksson, Staffan; Eklund, Hans

    2007-03-01

    Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.

  5. Substrate specificity of plant and fungi pectin methylesterases: Identification of novel inhibitors of PMEs.

    PubMed

    L'Enfant, Mélanie; Domon, Jean-Marc; Rayon, Catherine; Desnos, Thierry; Ralet, Marie-Christine; Bonnin, Estelle; Pelloux, Jérôme; Pau-Roblot, Corinne

    2015-11-01

    Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of ∼3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity. PMID:26342461

  6. Substrate specificity of plant and fungi pectin methylesterases: Identification of novel inhibitors of PMEs.

    PubMed

    L'Enfant, Mélanie; Domon, Jean-Marc; Rayon, Catherine; Desnos, Thierry; Ralet, Marie-Christine; Bonnin, Estelle; Pelloux, Jérôme; Pau-Roblot, Corinne

    2015-11-01

    Pectin methylesterases (PMEs) play a central role in pectin remodeling during plant development. They are also present in phytopathogens such as bacteria and fungi. We investigated the substrate specificity and pH dependence of plant and fungi PMEs using tailor-made pectic substrates. For this purpose, we used two plant PMEs (from orange peel: Citrus sinensis and from Arabidopsis thaliana) and one fungal PME (from Botrytis cinerea). We showed that plant and fungi PMEs differed in their substrate specificity and pH dependence, and that there were some differences between plant PMEs. We further investigated the inhibition of these enzyme activities using characterized polyphenols such as catechins and tannic acid. We showed that PMEs differed in their sensitivity to chemical compounds. In particular, fungal PME was not sensitive to inhibition. Finally, we screened for novel chemical inhibitors of PMEs using a chemical library of ∼3600 compounds. We identified a hundred new inhibitors of plant PMEs, but none had an effect on the fungal enzyme. This study sheds new light on the specificity of pectin methylesterases and provides new tools to modulate their activity.

  7. Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

    PubMed

    Modak, Joyanta K; Rut, Wioletta; Wijeyewickrema, Lakshmi C; Pike, Robert N; Drag, Marcin; Roujeinikova, Anna

    2016-02-01

    The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases.

  8. Structural basis for substrate specificity of Helicobacter pylori M17 aminopeptidase.

    PubMed

    Modak, Joyanta K; Rut, Wioletta; Wijeyewickrema, Lakshmi C; Pike, Robert N; Drag, Marcin; Roujeinikova, Anna

    2016-02-01

    The M17 aminopeptidase from the carcinogenic gastric bacterium Helicobacter pylori (HpM17AP) is an important housekeeping enzyme involved in catabolism of endogenous and exogenous peptides. It is implicated in H. pylori defence against the human innate immune response and in the mechanism of metronidazole resistance. Bestatin inhibits HpM17AP and suppresses H. pylori growth. To address the structural basis of catalysis and inhibition of this enzyme, we have established its specificity towards the N-terminal amino acid of peptide substrates and determined the crystal structures of HpM17AP and its complex with bestatin. The position of the D-phenylalanine moiety of the inhibitor with respect to the active-site metal ions, bicarbonate ion and with respect to other M17 aminopeptidases suggested that this residue binds to the S1 subsite of HpM17AP. In contrast to most characterized M17 aminopeptidases, HpM17AP displays preference for L-Arg over L-Leu residues in peptide substrates. Compared to very similar homologues from other bacteria, a distinguishing feature of HpM17AP is a hydrophilic pocket at the end of the S1 subsite that is likely to accommodate the charged head group of the L-Arg residue of the substrate. The pocket is flanked by a sodium ion (not present in M17 aminopeptidases that show preference for L-Leu) and its coordinating water molecules. In addition, the structure suggests that variable loops at the entrance to, and in the middle of, the substrate-binding channel are important determinants of substrate specificity of M17 aminopeptidases. PMID:26616008

  9. Dissecting APOBEC3G Substrate Specificity by Nucleoside Analog Interference*S⃞

    PubMed Central

    Rausch, Jason W.; Chelico, Linda; Goodman, Myron F.; Le Grice, Stuart F. J.

    2009-01-01

    The apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminase genes encode a set of enzymes including APOBEC1 (A1), APOBEC2 (A2), APOBEC4 (A4), and APOBEC3A-H (A3A-H). Although each possesses one or more zinc binding motifs conserved among enzymes catalyzing C → U conversion, the functions and substrate specificities of these gene products vary considerably. For example, although two closely related enzymes, A3F and A3G, both restrict HIV-1 infection in strains deficient in virus infectivity factor (vif), A3F selectively deaminates cytosine within 5′-TTCA-3′ motifs in single stranded DNA, whereas A3G targets 5′-CCCA-3′ sequences. In the present study we have used nucleoside analog interference mapping to probe A3G-DNA interactions throughout the enzyme-substrate complex as well as to determine which DNA structural features determine substrate specificity. Our results indicate that multiple components of nucleosides within the consensus sequence are important for substrate recognition by A3G (with base moieties being most critical), whereas deamination interference by analog substitution outside this region is minimal. Furthermore, exocyclic groups in pyrimidines 1–2 nucleotides 5′ of the target cytosine were shown to dictate substrate recognition by A3G, with chemical composition at ring positions 3 and 4 found to be more important than at ring position 5. Taken together, these results provide insights into how the enzyme selects A3G hotspot motifs for deamination as well as which approaches might be best suited for forming a stable, catalytically competent cross-linked A3G-DNA complex for future structural studies. PMID:19136562

  10. GSHSite: Exploiting an Iteratively Statistical Method to Identify S-Glutathionylation Sites with Substrate Specificity

    PubMed Central

    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

  11. Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.

    PubMed Central

    Zhang, R; Durkin, J; Windsor, W T; McNemar, C; Ramanathan, L; Le, H V

    1997-01-01

    We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position. PMID:9223519

  12. Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

    SciTech Connect

    Han, Q.; Ding, H; Robinson, H; Christensen, B; Li, J

    2010-01-01

    3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.

  13. 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.

  14. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates.

    PubMed

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    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. PMID:24681538

  15. Secreted lipases from Malassezia globosa: recombinant expression and determination of their substrate specificities.

    PubMed

    Sommer, Bettina; Overy, David P; Haltli, Bradley; Kerr, Russell G

    2016-07-01

    Malassezia globosa, which is associated with skin conditions such as dandruff and seborrhoeic dermatitis, possesses 13 secreted lipases, but only MgLip1, MgMDL2 and MgLip2 have been characterized. To understand the substrate preferences of these lipases and by extension their potential role in colonizing human skin, we expressed all 13 predicted secreted lipases in Pichia pastoris and evaluated their ability to utilize mono-, di- and triolein substrates. The M. globosa family class 3 lipases were shown to be specific for mono- and diacylglycerols, but exhibited no regio-selective production of diacylglycerols, which are of special interest for industrial applications. Lipases belonging to the Lip family utilized all substrates. In a further step, five lipases previously demonstrated to be expressed on human skin were tested against the eight most common di- and triacylglycerols in human sebum. All lipases liberated free fatty acids from three to eight of these substrates, proving their ability to hydrolyse key components of human sebum. Again, only Lip family lipases showed activity on triacylglycerides. Based on the demonstrated activity and expression levels of MgLip2 in M. globosa, the Lip lipase family appears to have the highest impact for the pathogenicity of M. globosa. PMID:27130210

  16. PSEA: Kinase-specific prediction and analysis of human phosphorylation substrates

    PubMed Central

    Suo, Sheng-Bao; Qiu, Jian-Ding; Shi, Shao-Ping; Chen, Xiang; Liang, Ru-Ping

    2014-01-01

    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. PMID:24681538

  17. Transmembrane aromatic amino acid distribution in P-glycoprotein. A functional role in broad substrate specificity.

    PubMed

    Pawagi, A B; Wang, J; Silverman, M; Reithmeier, R A; Deber, C M

    1994-01-14

    Multidrug resistance (MDR) in cancer cells is associated with overexpression of P-glycoprotein (Pgp), a membrane protein which interacts with structurally diverse hydrophobic molecules of high membrane affinity. In an analysis of the molecular basis for this broad range of substrate specificity, we found that the transmembrane (TM) regions of Pgp are rich in highly conserved aromatic amino acid residues. Computer-generated three-dimensional model structures showed that a typical substrate, rhodamine 123, can intercalate between three to four phenylalanine side-chains in any of several Pgp TM helices with minimal protrusion of the drug into bulk lipid, and that five to six (of the 12 Pgp putative TM segments) helices can facilitate transport through creation of a sterically compatible pore. In contrast to the case for proteins involved in the transport of membrane-impermeable, relatively polar substrates, the "transport path" for Pgp substrates need not be polar, and may involve either an internal channel occupied largely by aromatic side-chains, or external gaps along TM helix-lipid interfaces. Weakly polar interactions between drug cationic sites and Pgp aromatic residues contribute additionally to overall protein/drug binding. The ability of Pgp to recognize and efflux structurally diverse molecules suggests that rather than a unique structure, the Pgp channel may maintain the intrinsic capacity to undergo wide-ranging drug-dependent dynamic reorganization. PMID:7904655

  18. System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveals Substrate Specificity of Protein Acetyltransferases

    SciTech Connect

    Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory {Greg} B; Escalante-Semerena, Jorge C

    2012-01-01

    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.

  19. The yeast mitochondrial citrate transport protein: molecular determinants of its substrate specificity.

    PubMed

    Aluvila, Sreevidya; Kotaria, Rusudan; Sun, Jiakang; Mayor, June A; Walters, D Eric; Harrison, David H T; Kaplan, Ronald S

    2010-08-27

    The objective of this study was to identify the role of individual amino acid residues in determining the substrate specificity of the yeast mitochondrial citrate transport protein (CTP). Previously, we showed that the CTP contains at least two substrate-binding sites. In this study, utilizing the overexpressed, single-Cys CTP-binding site variants that were functionally reconstituted in liposomes, we examined CTP specificity from both its external and internal surfaces. Upon mutation of residues comprising the more external site, the CTP becomes less selective for citrate with numerous external anions able to effectively inhibit [(14)C]citrate/citrate exchange. Thus, the site 1 variants assume the binding characteristics of a nonspecific anion carrier. Comparison of [(14)C]citrate uptake in the presence of various internal anions versus water revealed that, with the exception of the R189C mutant, the other site 1 variants showed substantial uniport activity relative to exchange. Upon mutation of residues comprising site 2, we observed two types of effects. The K37C mutant displayed a markedly enhanced selectivity for external citrate. In contrast, the other site 2 mutants displayed varying degrees of relaxed selectivity for external citrate. Examination of internal substrates revealed that, in contrast to the control transporter, the R181C variant exclusively functioned as a uniporter. This study provides the first functional information on the role of specific binding site residues in determining mitochondrial transporter substrate selectivity. We interpret our findings in the context of our homology-modeled CTP as it cycles between the outward-facing, occluded, and inward-facing states.

  20. Identification of Threonine 348 as a Residue Involved in Aminopeptidase A Substrate Specificity*

    PubMed Central

    Claperon, Cédric; Banegas-Font, Inmaculada; Iturrioz, Xavier; Rozenfeld, Raphael; Maigret, Bernard; Llorens-Cortes, Catherine

    2009-01-01

    Aminopeptidase A (APA; EC 3.4.11.7) 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 central control of blood pressure in hypertensive animals. We docked the specific APA inhibitor, glutamate phosphonate, in the three-dimensional model of the mouse APA ectodomain in the presence of Ca2+. In the S1 subsite of this model, the Ca2+ atom was coordinated with Asp-213, Asp-218,y and Glu-215 and three water molecules, one of which formed a hydrogen bond with the carboxylate side chain of the inhibitor. We report here that the carboxylate side chain of glutamate phosphonate also formed a hydrogen bond with the alcohol side chain of Thr-348. Mutagenic replacement of Thr-348 with an aspartate, tyrosine, or serine residue led to a modification of the hydrolysis velocity, with no change in the affinity of the recombinant enzymes for the substrate GluNA, either in the absence or presence of Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA, which was nevertheless restored by the addition of Ca2+. An analysis of three-dimensional models of the corresponding Thr-348 mutants revealed that the interaction between this residue and the inhibitor was abolished or disturbed, leading to a change in the position of the inhibitor in the active site. These findings demonstrate a key role of Thr-348 in substrate specificity of APA for N-terminal acidic amino acids by insuring the optimal positioning of the substrate during catalysis. PMID:19228697

  1. Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

    PubMed Central

    Talesa, V; Grauso, M; Giovannini, E; Rosi, G; Toutant, J P

    1995-01-01

    Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M. Images Figure 2 Figure 3 Figure 4 PMID:7702560

  2. Dynamics Govern Specificity of a Protein-Protein Interface: Substrate Recognition by Thrombin.

    PubMed

    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. PMID:26496636

  3. Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

    PubMed

    Pati, Sarah G; Kohler, Hans-Peter E; Pabis, Anna; Paneth, Piotr; Parales, Rebecca E; Hofstetter, Thomas B

    2016-07-01

    Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

  4. A Polysaccharide Lyase from Stenotrophomonas maltophilia with a Unique, pH-regulated Substrate Specificity*

    PubMed Central

    MacDonald, Logan C.; Berger, Bryan W.

    2014-01-01

    Polysaccharide lyases (PLs) catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. PLs also play important roles in microbial pathogenesis, participating in bacterial invasion and toxin spread into the host tissue via degradation of the host extracellular matrix, or in microbial biofilm formation often associated with enhanced drug resistance. Stenotrophomonas maltophilia is a Gram-negative bacterium that is among the emerging multidrug-resistant organisms associated with chronic lung infections as well as with cystic fibrosis patients. A putative alginate lyase (Smlt1473) from S. maltophilia was heterologously expressed in Escherichia coli, purified in a one-step fashion via affinity chromatography, and activity as well as specificity determined for a range of polysaccharides. Interestingly, Smlt1473 catalyzed the degradation of not only alginate, but poly-β-d-glucuronic acid and hyaluronic acid as well. Furthermore, the pH optimum for enzymatic activity is substrate-dependent, with optimal hyaluronic acid degradation at pH 5, poly-β-d-glucuronic acid degradation at pH 7, and alginate degradation at pH 9. Analysis of the degradation products revealed that each substrate was cleaved endolytically into oligomers comprised predominantly of even numbers of sugar groups, with lower accumulation of trimers and pentamers. Collectively, these results imply that Smlt1473 is a multifunctional PL that exhibits broad substrate specificity, but utilizes pH as a mechanism to achieve selectivity. PMID:24257754

  5. Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

    PubMed Central

    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

  6. Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

    PubMed

    Pati, Sarah G; Kohler, Hans-Peter E; Pabis, Anna; Paneth, Piotr; Parales, Rebecca E; Hofstetter, Thomas B

    2016-07-01

    Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants. PMID:26895026

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  10. Substrate Specificity of the Citrate Transporter CitP of Lactococcus lactis

    PubMed Central

    Pudlik, Agata M.

    2012-01-01

    The citrate transporter CitP of lactic acid bacteria catalyzes electrogenic precursor-product exchange of citrate versus l-lactate during citrate-glucose cometabolism. In the absence of sugar, l-lactate is replaced by the metabolic intermediates/end products pyruvate, α-acetolactate, and acetate. In this study, the binding and translocation properties of CitP were analyzed systematically for a wide variety of mono- and dicarboxylates of the form X-CR2-COO−, where X represents OH (2-hydroxy acid), O (2-keto acid), or H (acid) and R groups differ in size, hydrophobicity, and composition. It follows that CitP is a very promiscuous carboxylate transporter. A carboxylate group is both essential and sufficient for recognition by the transporter. A C-2 atom is not essential, formate is a substrate, and C-2 may be part of a ring structure, as in benzoate. The R group may be as bulky as an indole ring structure. For all monocarboxylates of the form X-CHR-COO−, the hydroxy (X = OH) analogs were the preferred substrates. The preference for keto (X = O) or acid (X = H) analogs was dependent on the bulkiness of the R group, such that the acid was preferred for small R groups and the 2-ketoacid was preferred for more bulky R groups. The C4 to C6 dicarboxylates succinate, glutarate, and adipate were also substrates of CitP. The broad substrate specificity is discussed in the context of a model of the binding site of CitP. Many of the substrates of CitP are intermediates or products of amino acid metabolism, suggesting that CitP may have a broader physiological function than its role in citrate fermentation alone. PMID:22563050

  11. Family shuffling of expandase genes to enhance substrate specificity for penicillin G.

    PubMed

    Hsu, Jyh-Shing; Yang, Yunn-Bor; Deng, Chan-Hui; Wei, Chia-Li; Liaw, Shwu-Huey; Tsai, Ying-Chieh

    2004-10-01

    Deacetoxycephalosporin C synthase (expandase) from Streptomyces clavuligerus, encoded by cefE, is an important industrial enzyme for the production of 7-aminodeacetoxycephalosporanic acid from penicillin G. To improve the substrate specificity for penicillin G, eight cefE-homologous genes were directly evolved by using the DNA shuffling technique. After the first round of shuffling and screening, using an Escherichia coli ESS bioassay, four chimeras with higher activity were subjected to a second round. Subsequently, 20 clones were found with significantly enhanced activity. The kinetic parameters of two isolates that lack substrate inhibition showed 8.5- and 118-fold increases in the k(cat)/K(m) ratio compared to the S. clavuligerus expandase. The evolved enzyme with the 118-fold increase is the most active obtained to date anywhere. Our shuffling results also indicate the remarkable plasticity of the expandase, suggesting that more-active chimeras might be achievable with further rounds. PMID:15466573

  12. Substrate specificities of bacterial polyhydroxyalkanoate depolymerases and lipases: bacterial lipases hydrolyze poly(omega-hydroxyalkanoates).

    PubMed Central

    Jaeger, K E; Steinbüchel, A; Jendrossek, D

    1995-01-01

    The substrate specificities of extracellular lipases purified from Bacillus subtilis, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Pseudomonas fluorescens, and Burkholderia cepacia (former Pseudomonas cepacia) and of extracellular polyhydroxyalkanoate (PHA) depolymerases purified from Comamonas sp., Pseudomonas lemoignei, and P. fluorescens GK13, as well as that of an esterase purified from P. fluorescens GK 13, to various polyesters and to lipase substrates were analyzed. All lipases and the esterase of P. fluorescens GK13 but none of the PHA depolymerases tested hydrolyzed triolein, thereby confirming a functional difference between lipases and PHA depolymerases. However, most lipases were able to hydrolyze polyesters consisting of an omega-hydroxyalkanoic acid such as poly(6-hydroxyhedxanoate) or poly(4-hydroxybutyrate). The dimeric ester of hydroxyhexanoate was the main product of enzymatic hydrolysis of polycaprolactone by P. aeruginosa lipase. Polyesters containing side chains in the polymer backbone such as poly (3-hydroxybutyrate) and other poly(3-hydroxyalkanoates) were not or were only slightly hydrolyzed by the lipases tested. PMID:7487042

  13. No significant difference in antigenicity or tissue transglutaminase substrate specificity of Irish and US wheat gliadins.

    PubMed

    Keaveny, A P; Offner, G D; Bootle, E; Nunes, D P

    2000-04-01

    The prevalence of clinical celiac disease has been shown to vary both across time and between genetically similar populations. Differences in wheat antigenicity and transglutaminase substrate properties are a possible explanation for these differences. This study assessed the antigenicity and transglutaminase substrate specificities of gliadins from regions of high and low celiac disease prevalence. Gliadin was extracted from three commercial US wheat sources and two Irish sources. SDS-PAGE and western blotting revealed minor, but significant variations in the gliadin extracts. However, ELISA showed no difference in the antigenicity of these gliadins. Transglutaminase pretreatment of gliadin resulted in no significant change in gliadin antigenicity and kinetic studies showed that the Kms of the various gliadins were very similar. Purified IgA and IgG had no effect on transglutaminase activity. In summary, minor variations in wheat gliadins are unlikely to explain the observed differences in disease expression across genetically similar populations. PMID:10759247

  14. EGF receptor specificity for phosphotyrosine-primed substrates provides signal integration with Src

    PubMed Central

    Begley, Michael J; Yun, Cai-hong; Gewinner, Christina A; Asara, John M; Johnson, Jared L; Coyle, Anthony J; Eck, Michael J; Apostolou, Irina; Cantley, Lewis C

    2016-01-01

    Aberrant activation of the EGF receptor (EGFR) contributes to many human cancers by activating the Ras-MAPK and other pathways. EGFR signaling is augmented by Src-family kinases, but the mechanism is poorly understood. Here, we show that human EGFR preferentially phosphorylates peptide substrates that are primed by a prior phosphorylation. Utilizing peptides based on the sequence of the adaptor protein Shc1, we show that Src mediates the priming phosphorylation, promoting subsequent phosphorylation by EGFR. Importantly, the doubly phosphorylated Shc1 peptide binds more tightly to the Ras activator Grb2, a key step in activating the Ras-MAPK pathway, than singly phosphorylated peptides. Finally, a crystal structure of EGFR in complex with a primed Shc1 peptide reveals the structural basis for EGFR substrate specificity. These results provide a molecular explanation for the integration of Src and EGFR signaling with downstream effectors such as Ras. PMID:26551075

  15. Enzymatic Properties and Substrate Specificity of the Trehalose Phosphorylase from Caldanaerobacter subterraneus▿

    PubMed Central

    Van der Borght, Jef; Chen, Chao; Hoflack, Lieve; Van Renterghem, Lucas; Desmet, Tom; Soetaert, Wim

    2011-01-01

    A putative glycoside phosphorylase from Caldanaerobacter subterraneus subsp. pacificus was recombinantly expressed in Escherichia coli, after codon optimization and chemical synthesis of the encoding gene. The enzyme was purified by His tag chromatography and was found to be specifically active toward trehalose, with an optimal temperature of 80°C. In addition, no loss of activity could be detected after 1 h of incubation at 65°C, which means that it is the most stable trehalose phosphorylase reported so far. The substrate specificity was investigated in detail by measuring the relative activity on a range of alternative acceptors, applied in the reverse synthetic reaction, and determining the kinetic parameters for the best acceptors. These results were rationalized based on the enzyme-substrate interactions observed in a homology model with a docked ligand. The specificity for the orientation of the acceptor's hydroxyl groups was found to decrease in the following order: C-3 > C-2 > C-4. This results in a particularly high activity on the monosaccharides d-fucose, d-xylose, l-arabinose, and d-galactose, as well as on l-fucose. However, determination of the kinetic parameters revealed that these acceptors bind less tightly in the active site than the natural acceptor d-glucose, resulting in drastically increased Km values. Nevertheless, the enzyme's high thermostability and broad acceptor specificity make it a valuable candidate for industrial disaccharide synthesis. PMID:21803886

  16. Steel slag affects pH and Si content of container substrates

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A substrate representing a typical greenhouse potting mix was prepared using 85% sphagnum peat and 15% perlite. The substrate was filled into 10 cm wide containers. A pulverized steel slag (SS) from a basic oxygen furnace, and dolomitic limestone (DL) were amended to the base substrate at a rate o...

  17. Characterization and identification of protein O-GlcNAcylation sites with substrate specificity

    PubMed Central

    2014-01-01

    Background Protein O-GlcNAcylation, involving the attachment of single N-acetylglucosamine (GlcNAc) to the hydroxyl group of serine or threonine residues. Elucidation of O-GlcNAcylation sites on proteins is required in order to decipher its crucial roles in regulating cellular processes and aid in drug design. With an increasing number of O-GlcNAcylation sites identified by mass spectrometry (MS)-based proteomics, several methods have been proposed for the computational identification of O-GlcNAcylation sites. However, no development that focuses on the investigation of O-GlcNAcylated substrate motifs has existed. Thus, we were motivated to design a new method for the identification of protein O-GlcNAcylation sites with the consideration of substrate site specificity. Results In this study, 375 experimentally verified O-GlcNAcylation sites were collected from dbOGAP, which is an integrated resource for protein O-GlcNAcylation. Due to the difficulty in characterizing the substrate motifs by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. To construct the predictive models learned from the identified substrate motifs, we adopted Support Vector Machines (SVMs). A five-fold cross validation was used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 0.76, 0.80, and 0.78, respectively. Additionally, an independent testing set, which was really blind to the training data of predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (0.94) and outperform three other O-GlcNAcylation site prediction tools. Conclusion This work proposed a computational method to identify informative substrate motifs for O-GlcNAcylation sites. The evaluation of cross validation and independent testing indicated that the identified motifs were effective in the identification of O-GlcNAcylation sites. A case study demonstrated that the

  18. Substrate Specificity of Thiamine Pyrophosphate-Dependent 2-Oxo-Acid Decarboxylases in Saccharomyces cerevisiae

    PubMed Central

    Romagnoli, Gabriele; Luttik, Marijke A. H.; Kötter, Peter; Pronk, Jack T.

    2012-01-01

    Fusel alcohols are precursors and contributors to flavor and aroma compounds in fermented beverages, and some are under investigation as biofuels. The decarboxylation of 2-oxo acids is a key step in the Ehrlich pathway for fusel alcohol production. In Saccharomyces cerevisiae, five genes share sequence similarity with genes encoding thiamine pyrophosphate-dependent 2-oxo-acid decarboxylases (2ODCs). PDC1, PDC5, and PDC6 encode differentially regulated pyruvate decarboxylase isoenzymes; ARO10 encodes a 2-oxo-acid decarboxylase with broad substrate specificity, and THI3 has not yet been shown to encode an active decarboxylase. Despite the importance of fusel alcohol production in S. cerevisiae, the substrate specificities of these five 2ODCs have not been systematically compared. When the five 2ODCs were individually overexpressed in a pdc1Δ pdc5Δ pdc6Δ aro10Δ thi3Δ strain, only Pdc1, Pdc5, and Pdc6 catalyzed the decarboxylation of the linear-chain 2-oxo acids pyruvate, 2-oxo-butanoate, and 2-oxo-pentanoate in cell extracts. The presence of a Pdc isoenzyme was also required for the production of n-propanol and n-butanol in cultures grown on threonine and norvaline, respectively, as nitrogen sources. These results demonstrate the importance of pyruvate decarboxylases in the natural production of n-propanol and n-butanol by S. cerevisiae. No decarboxylation activity was found for Thi3 with any of the substrates tested. Only Aro10 and Pdc5 catalyzed the decarboxylation of the aromatic substrate phenylpyruvate, with Aro10 showing superior kinetic properties. Aro10, Pdc1, Pdc5, and Pdc6 exhibited activity with all branched-chain and sulfur-containing 2-oxo acids tested but with markedly different decarboxylation kinetics. The high affinity of Aro10 identified it as a key contributor to the production of branched-chain and sulfur-containing fusel alcohols. PMID:22904058

  19. Dynamic culture substrate that captures a specific extracellular matrix protein in response to light

    NASA Astrophysics Data System (ADS)

    Nakanishi, Jun; Nakayama, Hidekazu; Yamaguchi, Kazuo; Garcia, Andres J.; Horiike, Yasuhiro

    2011-08-01

    The development of methods for the off-on switching of immobilization or presentation of cell-adhesive peptides and proteins during cell culture is important because such surfaces are useful for the analysis of the dynamic processes of cell adhesion and migration. This paper describes a chemically functionalized gold substrate that captures a genetically tagged extracellular matrix protein in response to light. The substrate was composed of mixed self-assembled monolayers (SAMs) of three disulfide compounds containing (i) a photocleavable poly(ethylene glycol) (PEG), (ii) nitrilotriacetic acid (NTA) and (iii) hepta(ethylene glycol) (EG7). Although the NTA group has an intrinsic high affinity for oligohistidine tag (His-tag) sequences in its Ni2+-ion complex, the interaction was suppressed by the steric hindrance of coexisting PEG on the substrate surface. Upon photoirradiation of the substrate to release the PEG chain from the surface, this interaction became possible and hence the protein was captured at the irradiated regions, while keeping the non-specific adsorption of non-His-tagged proteins blocked by the EG7 underbrush. In this way, we selectively immobilized a His-tagged fibronectin fragment (FNIII7-10) to the irradiated regions. In contrast, when bovine serum albumin—a major serum protein—was added as a non-His-tagged protein, the surface did not permit its capture, with or without irradiation. In agreement with these results, cells were selectively attached to the irradiated patterns only when a His-tagged FNIII7-10 was added to the medium. These results indicate that the present method is useful for studying the cellular behavior on the specific extracellular matrix protein in cell-culturing environments.

  20. Affective responses in tamarins elicited by species-specific music

    PubMed Central

    Snowdon, Charles T.; Teie, David

    2010-01-01

    Theories of music evolution agree that human music has an affective influence on listeners. Tests of non-humans provided little evidence of preferences for human music. However, prosodic features of speech (‘motherese’) influence affective behaviour of non-verbal infants as well as domestic animals, suggesting that features of music can influence the behaviour of non-human species. We incorporated acoustical characteristics of tamarin affiliation vocalizations and tamarin threat vocalizations into corresponding pieces of music. We compared music composed for tamarins with that composed for humans. Tamarins were generally indifferent to playbacks of human music, but responded with increased arousal to tamarin threat vocalization based music, and with decreased activity and increased calm behaviour to tamarin affective vocalization based music. Affective components in human music may have evolutionary origins in the structure of calls of non-human animals. In addition, animal signals may have evolved to manage the behaviour of listeners by influencing their affective state. PMID:19726444

  1. Affective responses in tamarins elicited by species-specific music.

    PubMed

    Snowdon, Charles T; Teie, David

    2010-02-23

    Theories of music evolution agree that human music has an affective influence on listeners. Tests of non-humans provided little evidence of preferences for human music. However, prosodic features of speech ('motherese') influence affective behaviour of non-verbal infants as well as domestic animals, suggesting that features of music can influence the behaviour of non-human species. We incorporated acoustical characteristics of tamarin affiliation vocalizations and tamarin threat vocalizations into corresponding pieces of music. We compared music composed for tamarins with that composed for humans. Tamarins were generally indifferent to playbacks of human music, but responded with increased arousal to tamarin threat vocalization based music, and with decreased activity and increased calm behaviour to tamarin affective vocalization based music. Affective components in human music may have evolutionary origins in the structure of calls of non-human animals. In addition, animal signals may have evolved to manage the behaviour of listeners by influencing their affective state.

  2. Purification of glutamate dehydrogenase isoenzymes and characterization of their substrate specificities.

    PubMed

    Osuji, Godson O; Braithwaite, Cleantis; Fordjour, Kusi; Madu, Wenceslaus C; Beyene, Asefa; Roberts, Paul S; Wright, Victor

    2003-02-01

    Glutamate dehydrogenase (GDH) isoenzymes were purified from control, and ribonucleoside triphosphate (NTP)-treated peanut seedlings. GDH purification was by preparative-scale, free solution isoelectric focusing, followed by native PAGE, and the cryoelectrophoretic elution of the isoenzymes from the gel. SDS-PAGE of the purified GDH isoenzymes, followed by either silver staining of the gel, or western analysis using anti-GDH antibody, gave identical GDH polypeptide (a, alpha, and b) bands, thus, confirming the purity of the isoenzymes. The substrate specificities in the aminating activity of the GDH isoenzymes, or disaggregated polypeptides were determined by photometry, but the substrate specificities in the RNA synthesis activity were determined in cocktails containing 0.06-0.8 mM of each of UTP, ATP, GTP, and CTP, 0-100.0 mM NH4Cl, 0-50.0 mM alpha-ketoglutaratr (alpha-KG), 0-0.2 mM NADH, 0-10.0 mM CaCl2 5 units of DNase 1, antibiotics, and approximately 5 microg pure GDH isoenzymes or polypeptides at pH 8.0, and overnight at 16 degrees C. The GDH polypeptides were active only in amination reaction, but the GDH isoenzymes were active in both amination and RNA synthesis. Whereas, NADH, NH4Cl and alpha-KG served as the substrates for the amination reaction, and as modulators in the RNA synthetic reaction, ATP, GTP, UTP, and CTP served as substrates for theisoenzymes in RNA synthesis reaction. The product RNA was up to 2 microg microg(-1) GDH, and consisted of RNA species in the size ranges of 26, 16, and 5 S rRNAs. DNAse 1 in the assay cocktail ruled out transcription as the mechanism of the RNA synthesis. Addition of [alpha-32P] NTP led to the production of labeled RNA, thus confirming the specificity of NTPs as substrates, and that the RNA was not pre-existing in the reaction cocktail.

  3. Novel HSAN1 mutation in serine palmitoyltransferase resides at a putative phosphorylation site that is involved in regulating substrate specificity.

    PubMed

    Ernst, Daniela; Murphy, Sinéad M; Sathiyanadan, Karthik; Wei, Yu; Othman, Alaa; Laurá, Matilde; Liu, Yo-Tsen; Penno, Anke; Blake, Julian; Donaghy, Michael; Houlden, Henry; Reilly, Mary M; Hornemann, Thorsten

    2015-03-01

    1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position.

  4. Novel HSAN1 mutation in serine palmitoyltransferase resides at a putative phosphorylation site that is involved in regulating substrate specificity.

    PubMed

    Ernst, Daniela; Murphy, Sinéad M; Sathiyanadan, Karthik; Wei, Yu; Othman, Alaa; Laurá, Matilde; Liu, Yo-Tsen; Penno, Anke; Blake, Julian; Donaghy, Michael; Houlden, Henry; Reilly, Mary M; Hornemann, Thorsten

    2015-03-01

    1-Deoxysphingolipids (1-deoxySL) are atypical sphingolipids that are formed by the enzyme serine palmitoyltransferase (SPT) due to a promiscuous use of L-alanine over its canonical substrate L-serine. Several mutations in SPT are associated with the hereditary sensory and autonomic neuropathy type I (HSAN1). The current hypothesis is that these mutations induce a permanent shift in the affinity from L-serine toward L-alanine which results in a pathologically increased 1-deoxySL formation in HSAN1 patients. Also, wild-type SPT forms 1-deoxySL under certain conditions, and elevated levels were found in individuals with the metabolic syndrome and diabetes. However, the molecular mechanisms which control the substrate shift of the wild-type enzyme are not understood. Here, we report a novel SPTLC2-S384F variant in two unrelated HSAN1 families. Affected patients showed elevated plasma 1-deoxySL levels and expression of the S384F mutant in HEK293 cells increased 1-deoxySL formation. Previously, S384 has been reported as one of the two (S384 and Y387) putative phosphorylation sites in SPTLC2. The phosphorylation of wild-type SPTLC2 was confirmed by isoelectric focusing. The impact of an S384 phosphorylation on SPT activity was tested by creating mutants mimicking either a constitutively phosphorylated (S384D, S384E) or non-phosphorylated (S384A, Y387F, Y387F+S384A) protein. The S384D but not the S384E variant was associated with increased 1-deoxySL formation. The other mutations had no influence on activity and substrate affinity. In summary, our data show that S384F is a novel mutation in HSAN1 and that the substrate specificity of wild-type SPT might by dynamically regulated by a phosphorylation at this position. PMID:25567748

  5. Aldo-keto reductases in retinoid metabolism: search for substrate specificity and inhibitor selectivity.

    PubMed

    Porté, Sergio; Xavier Ruiz, F; Giménez, Joan; Molist, Iago; Alvarez, Susana; Domínguez, Marta; Alvarez, Rosana; de Lera, Angel R; Parés, Xavier; Farrés, Jaume

    2013-02-25

    Biological activity of natural retinoids requires the oxidation of retinol to retinoic acid (RA) and its binding to specific nuclear receptors in target tissues. The first step of this pathway, the reversible oxidoreduction of retinol to retinaldehyde, is essential to control RA levels. The enzymes of retinol oxidation are NAD-dependent dehydrogenases of the cytosolic medium-chain (MDR) and the membrane-bound short-chain (SDR) dehydrogenases/reductases. Retinaldehyde reduction can be performed by SDR and aldo-keto reductases (AKR), while its oxidation to RA is carried out by aldehyde dehydrogenases (ALDH). In contrast to SDR, AKR and ALDH are cytosolic. A common property of these enzymes is that they only use free retinoid, but not retinoid bound to cellular retinol binding protein (CRBP). The relative contribution of each enzyme type in retinoid metabolism is discussed in terms of the different subcellular localization, topology of membrane-bound enzymes, kinetic constants, binding affinity of CRBP for retinol and retinaldehyde, and partition of retinoid pools between membranes and cytoplasm. The development of selective inhibitors for AKR enzymes 1B1 and 1B10, of clinical relevance in diabetes and cancer, granted the investigation of some structure-activity relationships. Kinetics with the 4-methyl derivatives of retinaldehyde isomers was performed to identify structural features for substrate specificity. Hydrophilic derivatives were better substrates than the more hydrophobic compounds. We also explored the inhibitory properties of some synthetic retinoids, known for binding to retinoic acid receptors (RAR) and retinoid X receptors (RXR). Consistent with its substrate specificity towards retinaldehyde, AKR1B10 was more effectively inhibited by synthetic retinoids than AKR1B1. A RARβ/γ agonist (UVI2008) inhibited AKR1B10 with the highest potency and selectivity, and docking simulations predicted that its carboxyl group binds to the anion-binding pocket. PMID

  6. Substrate Specificity of Purified Recombinant Chicken β-Carotene 9',10'-Oxygenase (BCO2).

    PubMed

    Dela Seña, Carlo; Sun, Jian; Narayanasamy, Sureshbabu; Riedl, Kenneth M; Yuan, Yan; Curley, Robert W; Schwartz, Steven J; Harrison, Earl H

    2016-07-01

    Provitamin A carotenoids are oxidatively cleaved by β-carotene 15,15'-dioxygenase (BCO1) at the central 15-15' double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9',10'-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9'-10' bond to yield an ionone and an apo-10'-carotenoid. Previously published substrate specificity studies of BCO2 were conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in Escherichia coli were unsuccessful. We have expressed recombinant chicken BCO2 in the strain E. coli BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 catalyzes the oxidative cleavage of the provitamin A carotenoids β-carotene, α-carotene, and β-cryptoxanthin. Its catalytic activity with β-carotene as substrate is at least 10-fold lower than that of BCO1. In further contrast to BCO1, purified recombinant chicken BCO2 also catalyzes the oxidative cleavage of 9-cis-β-carotene and the non-provitamin A carotenoids zeaxanthin and lutein, and is inactive with all-trans-lycopene and β-apocarotenoids. Apo-10'-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3-hydroxy-β-apo-8'-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9'-10' bond to produce apo-10'-carotenoids. BCO2 has been shown to function in preventing the excessive accumulation of carotenoids, and its broad substrate specificity is consistent with this. PMID:27143479

  7. Substrate specificity, kinetic properties and inhibition by fumonisin B1 of ceramide synthase isoforms from Arabidopsis.

    PubMed

    Luttgeharm, Kyle D; Cahoon, Edgar B; Markham, Jonathan E

    2016-03-01

    Ceramide makes up the acyl-backbone of sphingolipids and plays a central role in determining the function of these essential membrane lipids. In Arabidopsis, the varied chemical composition of ceramide is determined by the specificity of three different isoforms of ceramide synthase, denoted LAG one homologue 1, -2 and -3 (LOH1, LOH2 and LOH3), for a range of long-chain base (LCB) and acyl-CoA substrates. The contribution of each of these isoforms to the synthesis of ceramide was investigated by in vitro ceramide synthase assays. The plant LCB phytosphingosine was efficiently used by the LOH1 and LOH3 isoforms, with LOH1 having the lowest Km for the LCB substrate of the three isoforms. In contrast, sphinganine was used efficiently only by the LOH2 isoform. Acyl-CoA specificity was also distinguished between the three isoforms with LOH2 almost completely specific for palmitoyl-CoA whereas the LOH1 isoform showed greatest activity with lignoceroyl- and hexacosanoyl-CoAs. Interestingly, unsaturated acyl-CoAs were not used efficiently by any isoform whereas unsaturated LCB substrates were preferred by LOH2 and 3. Fumonisin B1 (FB1) is a general inhibitor of ceramide synthases but LOH1 was found to have a much lower Ki than the other isoforms pointing towards the origin of FB1 sensitivity in plants. Overall, the data suggest distinct roles and modes of regulation for each of the ceramide synthases in Arabidopsis sphingolipid metabolism. PMID:26635357

  8. The substrate specificity of the human ADP/ATP carrier AAC1.

    PubMed

    Mifsud, John; Ravaud, Stéphanie; Krammer, Eva-Maria; Chipot, Chris; Kunji, Edmund R S; Pebay-Peyroula, Eva; Dehez, Francois

    2013-03-01

    The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

  9. Archaeal Mo-Containing Glyceraldehyde Oxidoreductase Isozymes Exhibit Diverse Substrate Specificities through Unique Subunit Assemblies

    PubMed Central

    Miyake, Masayuki; Fushinobu, Shinya

    2016-01-01

    Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1–3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1–3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system. PMID:26808202

  10. The unique substrate specificity of human AOC2, a semicarbazide-sensitive amine oxidase.

    PubMed

    Kaitaniemi, Sam; Elovaara, Heli; Grön, Kirsi; Kidron, Heidi; Liukkonen, Janne; Salminen, Tiina; Salmi, Marko; Jalkanen, Sirpa; Elima, Kati

    2009-08-01

    Semicarbazide-sensitive amine oxidases (SSAOs) catalyze oxidative deamination of primary amines, but the true physiological function of these enzymes is still poorly understood. Here, we have studied the functional and structural characteristics of a human cell-surface SSAO, AOC2, which is homologous to the better characterized family member, AOC3. The preferred in vitro substrates of AOC2 were found to be 2-phenylethylamine, tryptamine and p-tyramine instead of methylamine and benzylamine, the favored substrates of AOC3. Molecular modeling suggested structural differences between AOC2 and AOC3, which provide AOC2 with the capability to use the larger monoamines as substrates. Even though AOC2 mRNA was expressed in many tissues, the only tissues with detectable AOC2-like enzyme activity were found in the eye. Characterization of AOC2 will help in evaluating the contribution of this enzyme to the pathological processes attributed to the SSAO activity and in designing specific inhibitors for the individual members of the SSAO family. PMID:19588076

  11. G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

    PubMed Central

    Chen, Ruming; Rato, Cláudia; Yan, Yahui; Crespillo-Casado, Ana; Clarke, Hanna J; Harding, Heather P; Marciniak, Stefan J; Read, Randy J; Ron, David

    2015-01-01

    Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex. In crystal structures of the non-selective PPP1R15B-PP1G complex, the functional core of PPP1R15 made multiple surface contacts with PP1G, but at a distance from the active site, whereas in the substrate-selective ternary complex, actin contributes to one face of a platform encompassing the active site. Computational docking of the N-terminal lobe of eIF2a at this platform placed phosphorylated serine 51 near the active site. Mutagenesis of predicted surface-contacting residues enfeebled dephosphorylation, suggesting that avidity for the substrate plays an important role in imparting specificity on the PPP1R15B-PP1G-actin ternary complex. DOI: http://dx.doi.org/10.7554/eLife.04871.001 PMID:25774600

  12. Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

    PubMed

    Marcuschi, Marina; Espósito, Talita S; Machado, Maurício F M; Hirata, Izaura Y; Machado, Marcelo F M; Silva, Márcia V; Carvalho, Luiz B; Oliveira, Vitor; Bezerra, Ranilson S

    2010-06-01

    An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry. PMID:20438707

  13. Substrate specificity of human granzyme 3: analyses of the P3-P2-P1 triplet using fluorescence resonance energy transfer substrate libraries.

    PubMed

    Hirata, Yukiyo; Inagaki, Hirofumi; Shimizu, Takako; Kawada, Tomoyuki

    2014-04-01

    Granzyme 3 (Gr3) is known as a tryptase-type member of the granzyme family and exists in the granules of immunocompetent cells. Granule proteases including granzymes, are transported into the cytoplasm of tumor cells or virus-infected cells by perforin function, degrade cytoplasmic or nuclear proteins and subsequently cause the death of the target cells. Recently, although several substrates of Gr3 in vivo have been reported, these hydrolyzed sites were unclear or lacked consistency. Our previous study investigated the optimal amino acid triplet (P3-P2-P1) as a substrate for Gr3 using a limited combination of amino acids at the P2 and P3 positions. In the present study, new fluorescence resonance energy transfer (FRET) substrate libraries to screen P2 and P3 positions were synthesized, respectively. Using these substrate libraries, the optimal amino acid triplet was shown to be Tyr-Phe-Arg as a substrate for human Gr3. Moreover, kinetic analyses also showed that the synthetic substrate FRETS-YFR had the lowest Km value for human Gr3. A substantial number of membrane proteins possessed the triplet Tyr-Phe-Arg and some of them might be in vivo substrates for Gr3. The results might also be a great help for preparing specific inhibitors to manipulate Gr3 activity both in vitro and in vivo.

  14. Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

    PubMed Central

    Byrne, Robert T.; Jenkins, Huw T.; Peters, Daniel T.; Whelan, Fiona; Stowell, James; Aziz, Naveed; Kasatsky, Pavel; Rodnina, Marina V.; Koonin, Eugene V.; Konevega, Andrey L.; Antson, Alfred A.

    2015-01-01

    The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold. PMID:25902496

  15. Live substrate positively affects root growth and stolon direction in the woodland strawberry, Fragaria vesca

    PubMed Central

    Waters, Erica M.; Watson, Maxine A.

    2015-01-01

    Studies of clonal plant foraging generally focus on growth responses to patch quality once rooted. Here we explore the possibility of true plant foraging; the ability to detect and respond to patch resource status prior to rooting. Two greenhouse experiments were conducted to investigate the morphological changes that occur when individual daughter ramets of Fragaria vesca (woodland strawberry) were exposed to air above live (non-sterilized) or dead (sterilized) substrates. Contact between daughter ramets and substrate was prohibited. Daughter ramet root biomass was significantly larger over live versus dead substrate. Root:shoot ratio also increased over live substrate, a morphological response we interpret as indicative of active nutrient foraging. Daughter ramet root biomass was positively correlated with mother ramet size over live but not dead substrate. Given the choice between a live versus a dead substrate, primary stolons extended preferentially toward live substrates. We conclude that exposure to live substrate drives positive nutrient foraging responses in F. vesca. We propose that volatiles emitted from the substrates might be effecting the morphological changes that occur during true nutrient foraging. PMID:26483826

  16. Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase.

    PubMed

    Gao, Xinxing; Cui, Wenjing; Ding, Ning; Liu, Zhongmei; Tian, Yaping; Zhou, Zhemin

    2013-01-01

    Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry.

  17. Structure-based approach to alter the substrate specificity of Bacillus subtilis aminopeptidase

    PubMed Central

    Gao, Xinxing; Cui, Wenjing; Ding, Ning; Liu, Zhongmei; Tian, Yaping; Zhou, Zhemin

    2013-01-01

    Aminopeptidases can selectively catalyze the cleavage of the N-terminal amino acid residues from peptides and proteins. Bacillus subtilis aminopeptidase (BSAP) is most active toward p-nitroanilides (pNAs) derivatives of Leu, Arg, and Lys. The BSAP with broad substrate specificity is expected to improve its application. Based on an analysis of the predicted structure of BSAP, four residues (Leu 370, Asn 385, Ile 387, and Val 396) located in the substrate binding region were selected for saturation mutagenesis. The hydrolytic activity toward different aminoacyl-pNAs of each mutant BSAP in the culture supernatant was measured. Although the mutations resulted in a decrease of hydrolytic activity toward Leu-pNA, N385L BSAP exhibited higher hydrolytic activities toward Lys-pNA (2.2-fold) and Ile-pNA (9.1-fold) than wild-type BSAP. Three mutant enzymes (I387A, I387C and I387S BSAPs) specially hydrolyzed Phe-pNA, which was undetectable in wild-type BSAP. Among these mutant BSAPs, N385L and I387A BSAPs were selected for further characterized and used for protein hydrolysis application. Both of N385L and I387A BSAPs showed higher hydrolysis efficiency than the wild-type BASP and a combination of the wild-type and N385L and I387A BSAPs exhibited the highest hydrolysis efficiency for protein hydrolysis. This study will greatly facilitate studies aimed on change the substrate specificity and our results obtained here should be useful for BSAP application in food industry. PMID:23787698

  18. Ara12 subtilisin-like protease from Arabidopsis thaliana: purification, substrate specificity and tissue localization.

    PubMed Central

    Hamilton, John M U; Simpson, David J; Hyman, Stefan C; Ndimba, Bongani K; Slabas, Antoni R

    2003-01-01

    A C-terminal portion of Ara12 subtilisin-like protease (residues 542-757) was expressed in Escherichia coli cells as a fusion protein bound to maltose binding protein. Polyclonal antisera raised against the expressed protein were used to examine the tissue specificity and subcellular localization of Ara12. The protease was found predominantly in the silique and stem of plants, but was hardly detectable in leaf and not seen in root tissue. The distribution observed using immunological techniques is different from that seen by an RNA analysis study, which demonstrated similar mRNA abundance in the stem and leaves. Using immunogold labelling, Ara12 was shown to have an extracellular localization and was found in the intercellular spaces in stem tissue. Ara12 protease was purified to homogeneity from Arabidopsis thaliana cell suspension cultures by anion exchange and hydrophobic interaction chromatography. Proteolytic activity of Ara12 was inhibited by a number of serine protease inhibitors, but was almost unaffected by inhibitors of other catalytic classes of proteases. Optimal proteolytic activity was displayed under acidic conditions (pH 5.0). Ara12 activity was relatively thermostable and was stimulated in the presence of Ca2+ ions. Substrate specificity studies were conducted using a series of internally quenched fluorogenic peptide substrates. At the P1 position of substrates, hydrophobic residues, such as Phe and Ala, were preferred to Arg, whilst at the P1' position, Asp, Leu and Ala were most favoured. Possible functions of Ara12 are discussed in the light of the involvement of a number of plant subtilisin-like proteases in morphogenesis. PMID:12413398

  19. The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II.

    PubMed

    Sivaraman, Sharada; Kirsch, Jack F

    2006-05-01

    Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.

  20. Substrate Stereo-specificity in Tryptophan dioxygenase and Indoleamine 2,3- dioxygenase

    PubMed Central

    Capece, L.; Arrar, M.; Roitberg, A. E.; Yeh, Syun-Ru; Marti, M. A.; Estrin, D. A.

    2010-01-01

    The first and rate-limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N-formylkynurenine is catalyzed by two heme-containing proteins, Indoleamine 2,3-dioxygenase (IDO) and Tryptophan 2,3-dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small-molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work we have used a combination of classical molecular dynamics (MD) and hybrid quantum-classical (QM/MM) methodologies to establish the structural basis that determine the differences in a) the interactions of TDO and IDO with small ligands (CO/O2) and b) the substrate stereo-specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo-specificity of TDO is achieved by the perfect fit of L-Trp, but not D-Trp, which exhibits weaker interactions with the protein matrix-. For hIDO, the presence of multiple stable binding conformations for L/D-Trp reveal the presence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO-selective inhibitors. PMID:20715188

  1. The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization.

    PubMed

    Ageitos, Jose Manuel; Yazawa, Kenjiro; Tateishi, Ayaka; Tsuchiya, Kousuke; Numata, Keiji

    2016-01-11

    The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease.

  2. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities.

    PubMed

    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke

    2014-06-15

    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po(lpo)) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po(lpo)-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po(lpo) allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.

  3. The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities

    PubMed Central

    Marelja, Zvonimir; Dambowsky, Miriam; Bolis, Marco; Georgiou, Marina L.; Garattini, Enrico; Missirlis, Fanis; Leimkühler, Silke

    2014-01-01

    In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1–4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Polpo) and aldehyde oxidase-1 (Aldox-1n1) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Polpo-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Polpo allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1n1 phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays. PMID:24737760

  4. 2,4-Dichlorophenol hydroxylase for chlorophenol removal: Substrate specificity and catalytic activity.

    PubMed

    Ren, Hejun; Li, Qingchao; Zhan, Yang; Fang, Xuexun; Yu, Dahai

    2016-01-01

    Chlorophenols (CPs) are common environmental pollutants. As such, different treatments have been assessed to facilitate their removal. In this study, 2,4-dichlorophenol (2,4-DCP) hydroxylase was used to systematically investigate the activity and removal ability of 19CP congeners at 25 and 0 °C. Results demonstrated that 2,4-DCP hydroxylase exhibited a broad substrate specificity to CPs. The activities of 2,4-DCP hydroxylase against specific CP congeners, including 3-CP, 2,3,6-trichlorophenol, 2-CP, and 2,3-DCP, were higher than those against 2,4-DCP, which is the preferred substrate of previously reported 2,4-DCP hydroxylase. To verify whether cofactors are necessary to promote hydroxylase activity against CP congeners, we added FAD and found that the added FAD induced a 1.33-fold to 5.13-fold significant increase in hydroxylase activity against different CP congeners. The metabolic pathways of the CP degradation in the enzymatic hydroxylation step were preliminarily proposed on the basis of the analyses of the enzymatic activities against 19CP congeners. We found that the high activity and removal rate of 2,4-DCP hydroxylase against CPs at 0 °C enhance the low-temperature-adaptability of this enzyme to the CP congeners; as such, the proposed removal process may be applied to biochemical, bioremediation, and industrial processes, particularly in cold environments.

  5. Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase*

    PubMed Central

    Liu, Chunhui; Sheng, Juzheng; Krahn, Juno M.; Perera, Lalith; Xu, Yongmei; Hsieh, Po-Hung; Dou, Wenfang; Liu, Jian; Pedersen, Lars C.

    2014-01-01

    Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway. PMID:24652287

  6. The Fanconi anemia associated protein FAAP24 uses two substrate specific binding surfaces for DNA recognition.

    PubMed

    Wienk, Hans; Slootweg, Jack C; Speerstra, Sietske; Kaptein, Robert; Boelens, Rolf; Folkers, Gert E

    2013-07-01

    To maintain the integrity of the genome, multiple DNA repair systems exist to repair damaged DNA. Recognition of altered DNA, including bulky adducts, pyrimidine dimers and interstrand crosslinks (ICL), partially depends on proteins containing helix-hairpin-helix (HhH) domains. To understand how ICL is specifically recognized by the Fanconi anemia proteins FANCM and FAAP24, we determined the structure of the HhH domain of FAAP24. Although it resembles other HhH domains, the FAAP24 domain contains a canonical hairpin motif followed by distorted motif. The HhH domain can bind various DNA substrates; using nuclear magnetic resonance titration experiments, we demonstrate that the canonical HhH motif is required for double-stranded DNA (dsDNA) binding, whereas the unstructured N-terminus can interact with single-stranded DNA. Both DNA binding surfaces are used for binding to ICL-like single/double-strand junction-containing DNA substrates. A structural model for FAAP24 bound to dsDNA has been made based on homology with the translesion polymerase iota. Site-directed mutagenesis, sequence conservation and charge distribution support the dsDNA-binding model. Analogous to other HhH domain-containing proteins, we suggest that multiple FAAP24 regions together contribute to binding to single/double-strand junction, which could contribute to specificity in ICL DNA recognition. PMID:23661679

  7. Analyses of substrate specificity of the desulfurizing bacterium Mycobacterium sp. G3.

    PubMed

    Okada, Hideki; Nomura, Nobuhiko; Nakahara, Tadaatsu; Maruhashi, Kenji

    2002-01-01

    The substrate specificity of Mycobacterium sp. G3 with desulfurization activity against dibenzothiophene (DBT) was investigated. Desulfurization reactions were carried out using a concentrated cell suspension of G3. The conversion from 4,6-dipropyl DBT, one of the sulfur-containing compounds that is difficult to desulfurize in diesel oil, to 2-hydroxy-3,3'-dipropylbiphenyl as an end -product of the desulfurization reaction was found in the water reaction system and in the oil/water two-phase reaction system. 4,6-Dibutyl DBT and 4,6-dipentyl DBT were metabolized to the hydroxybiphenyl form via the sulfone form in the water reaction system. These results indicate that G3 has high membrane permeability and superior substrate specificity for high molecular weight alkyl DBTs, which are represented by 4,6-dipentyl DBT as C10-DBT. Furthermore, G3 could desulfurize diesel oil, and the sulfur concentration was decreased from 116 mg l(-1) to 48 mg l(-1) within 24 h.

  8. Carotenoids in Rhodoplanes species: variation of compositions and substrate specificity of predicted carotenogenesis enzymes.

    PubMed

    Takaichi, Shinichi; Sasikala, Ch; Ramana, Ch V; Okamura, Keiko; Hiraishi, Akira

    2012-08-01

    Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.

  9. Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

    PubMed Central

    Jones, Stephen K.; Clarke, Starlynn C.; Craik, Charles S.

    2015-01-01

    ABSTRACT Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a “barrier” and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target. PMID:26604258

  10. Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

    PubMed Central

    Tcherkez, Guillaume G. B.; Farquhar, Graham D.; Andrews, T. John

    2006-01-01

    The cornerstone of autotrophy, the CO2-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO2 and O2 as substrates, an “abominably perplexing” puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO2 molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO2/O2 specificity, in response to reducing atmospheric CO2:O2 ratios, has resulted in a transition state for CO2 addition in which the CO2 moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO2, O2, and thermal conditions in their subcellular environments, optimizing this compromise between CO2/O2 specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO2/O2 specificity and 12C/13C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature. PMID:16641091

  11. Characterization of 2-aminoisobutyric acid transport in Neurospora crassa: a general amino acid permease-specific substrate.

    PubMed Central

    Ogilvie-Villa, S; DeBusk, R M; DeBusk, A G

    1981-01-01

    We report the characterization of an amino acid 2-aminoisobutyric acid was transported solely by the general amino acid permease and not by the neutral amino acid permease. Furthermore, this substrate was not metabolized after transport. The potential for a system-specific nonmetabolizable substrate as a tool in the analysis of amino acid transport and its regulation is discussed. PMID:6456264

  12. Toward a biaxial model of "bipolar" affective disorders: further exploration of genetic, molecular and cellular substrates.

    PubMed

    Askland, Kathleen

    2006-08-01

    Current epidemiologic and genetic evidence strongly supports the heritability of bipolar disease. Inconsistencies across linkage and association analyses have been primarily interpreted as suggesting polygenic, nonMendelian and variably-penetrant inheritance (i.e., in terms of interacting disease models). An equally-likely explanation for this genetic complexity is that trait, locus and allelic heterogeneities (i.e., a heterogeneous disease model) are primarily responsible for observed variability at the population level. The two models of genetic complexity are not mutually-exclusive, and are in fact likely to co-exist both in trait determination and disease expression. However, the current model proposes that, while both types of complex genetics are likely central to observable affective trait spectra, inheritance patterns, gross phenotypic categories and treatment-responsiveness in affective disease (as well as the widespread inconsistencies across such studies) may be primarily explained in terms of a heterogeneous disease model. Gene-gene, gene-protein and protein-protein interactions, then, are most likely to serve as trait determinants and 'phenotypic modifiers' rather than as primary pathogenic determinants. Moreover, while locus heterogeneity indicates the presence of multiple susceptibility genes at the population level, it does not necessitate polygenic inheritance at the individual or pedigree level. Rather, it is compatible with the possibility of mono- or bigenic determination of disease susceptibility within individuals/pedigrees. More specifically, the biaxial model proposes that integration of specific findings from genetic linkage and association studies, ion channels research as well as pharmacologic mechanism, phenotypic specificity and effectiveness studies suggests that each gene of potential etiologic significance in primary affective illness might be categorized into one of two classes, according to their primary role in neuronal

  13. Ground substrate affects activity budgets and hair loss in outdoor captive groups of rhesus macaques (Macaca mulatta).

    PubMed

    Beisner, Brianne A; Isbell, Lynne A

    2008-12-01

    How the captive environment influences the behavior of animals is relevant to the well-being of captive animals. Captivity diverges from the natural environment in many ways, and one goal of enrichment practices is to encourage species-typical behavior in these unnatural environments. This study investigated the influence of grass vs. gravel substrate on activity budgets and degree of hair loss in seven groups of captive rhesus macaques housed in outdoor enclosures at the California National Primate Research Center. Groups having grass substrate spent a greater proportion of their time foraging and a smaller proportion of time grooming compared with groups having gravel substrate. Increased time spent grooming in gravel enclosures may have contributed to significantly greater hair loss in those enclosures. A causal relationship between ground substrate on foraging and grooming, and therefore hair loss, is strengthened by similar changes in activity budgets and hair loss in a single group that was moved from gravel to grass substrate halfway through the study. These results add to growing evidence that substrate type in captivity is important to consider because it affects animal well-being. In particular, these results reveal that grass substrate is more effective than gravel in stimulating foraging and reducing allo-grooming to levels that are comparable to wild populations, and enable animals to maintain healthier coats.

  14. Specific Previous Experience Affects Perception of Harmony and Meter

    ERIC Educational Resources Information Center

    Creel, Sarah C.

    2011-01-01

    Prior knowledge shapes our experiences, but which prior knowledge shapes which experiences? This question is addressed in the domain of music perception. Three experiments were used to determine whether listeners activate specific musical memories during music listening. Each experiment provided listeners with one of two musical contexts that was…

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

    PubMed

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

    2014-11-01

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

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

    PubMed

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

    2014-11-01

    A new, acyclic NAD-analog, acycloNAD(+) has been synthesized where the nicotinamide ribosyl moiety has been replaced by the nicotinamide (2-hydroxyethoxy)methyl moiety. The chemical properties of this analog are comparable to those of β-NAD(+) with a redox potential of -324mV and a 341nm λmax for the reduced form. Both yeast alcohol dehydrogenase (YADH) and horse liver alcohol dehydrogenase (HLADH) catalyze the reduction of acycloNAD(+) by primary alcohols. With HLADH 1-butanol has the highest Vmax at 49% that of β-NAD(+). The primary deuterium kinetic isotope effect is greater than 3 indicating a significant contribution to the rate limiting step from cleavage of the carbon-hydrogen bond. The stereochemistry of the hydride transfer in the oxidation of stereospecifically deuterium labeled n-butanol is identical to that for the reaction with β-NAD(+). In contrast to the activity toward primary alcohols there is no detectable reduction of acycloNAD(+) by secondary alcohols with HLADH although these alcohols serve as competitive inhibitors. The net effect is that acycloNAD(+) has converted horse liver ADH from a broad spectrum alcohol dehydrogenase, capable of utilizing either primary or secondary alcohols, into an exclusively primary alcohol dehydrogenase. This is the first example of an NAD analog that alters the substrate specificity of a dehydrogenase and, like site-directed mutagenesis of proteins, establishes that modifications of the coenzyme distance from the active site can be used to alter enzyme function and substrate specificity. These and other results, including the activity with α-NADH, clearly demonstrate the promiscuity of the binding interactions between dehydrogenases and the riboside phosphate of the nicotinamide moiety, thus greatly expanding the possibilities for the design of analogs and inhibitors of specific dehydrogenases. PMID:25280628

  17. Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members.

    PubMed

    Rigoldi, Federica; Spero, Ludovica; Dalle Vedove, Andrea; Redaelli, Alberto; Parisini, Emilio; Gautieri, Alfonso

    2016-07-19

    Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels. PMID:27327839

  18. Polypeptide substrate specificity of PsLSMT. A set domain protein methyltransferase.

    PubMed

    Magnani, Roberta; Nayak, Nihar R; Mazarei, Mitra; Dirk, Lynnette M A; Houtz, Robert L

    2007-09-21

    Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.

  19. Studies on T4-head maturation. 2. Substrate specificity of gene-49-controlled endonuclease.

    PubMed

    Kemper, B; Garabett, M; Courage, U

    1981-03-16

    The substrate specificity of 49+-enzyme was investigated in vitro. The enzyme showed a marked preference for rapidly sedimenting T4 DNA (greater than 1000 S) when helix-destabilizing proteins from Escherichia coli or phage T4 were added to the reaction. Regular replicative T4 DNA (200-S DNA) or denatured T4 DNA was not cleaved by the enzyme in the presence of these proteins but if they were omitted from the reaction both DNAs become good substrates for the enzyme. 200-S DNA was cleaved at its natural sites of single strandedness which occur at one-genome intervals. Gaps in T4 DNA which were constructed by treatment of a nicked DNA with exonuclease III were also cleaved by 49+-enzyme in the absence of helix-destabilizing proteins. Single-stranded T4 DNA was extensively degraded and up to 50% of the material was found to be acid-soluble in a limit digest. The degradation products were predominantly oligonucleotides of random size. No preference for a 5'-terminal nucleotide was observed in material from a limit digest with M13 DNA. Double-stranded DNA was nicked upon exposure to 49+-enzyme and double-strand breakage finally occurred by an accumulation of single-strand interruptions. No acid-soluble material was produced from native T4 DNA. The introduction of nicks in native DNA did not improve its properties as a substrate for the enzyme. Double-stranded DNA was about 100-fold less sensitive to the enzyme than single-stranded DNA. PMID:6262078

  20. SIRT3 substrate specificity determined by peptide arrays and machine learning.

    PubMed

    Smith, Brian C; Settles, Burr; Hallows, William C; Craven, Mark W; Denu, John M

    2011-02-18

    Accumulating evidence suggests that reversible protein acetylation may be a major regulatory mechanism that rivals phosphorylation. With the recent cataloging of thousands of acetylation sites on hundreds of proteins comes the challenge of identifying the acetyltransferases and deacetylases that regulate acetylation levels. Sirtuins are a conserved family of NAD(+)-dependent protein deacetylases that are implicated in genome maintenance, metabolism, cell survival, and lifespan. SIRT3 is the dominant protein deacetylase in mitochondria, and emerging evidence suggests that SIRT3 may control major pathways by deacetylation of central metabolic enzymes. Here, to identify potential SIRT3 substrates, we have developed an unbiased screening strategy that involves a novel acetyl-lysine analogue (thiotrifluoroacetyl-lysine), SPOT-peptide libraries, machine learning, and kinetic validation. SPOT peptide libraries based on known and potential mitochondrial acetyl-lysine sites were screened for SIRT3 binding and then analyzed using machine learning to establish binding trends. These trends were then applied to the mitochondrial proteome as a whole to predict binding affinity of all lysine sites within human mitochondria. Machine learning prediction of SIRT3 binding correlated with steady-state kinetic k(cat)/K(m) values for 24 acetyl-lysine peptides that possessed a broad range of predicted binding. Thus, SPOT peptide-binding screens and machine learning prediction provides an accurate and efficient method to evaluate sirtuin substrate specificity from a relatively small learning set. These analyses suggest potential SIRT3 substrates involved in several metabolic pathways such as the urea cycle, ATP synthesis, and fatty acid oxidation. PMID:20945913

  1. Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

    PubMed Central

    Selvig, Kyla; Ballou, Elizabeth R.; Nichols, Connie B.

    2013-01-01

    Proper cellular localization is required for the function of many proteins. The CaaX prenyltransferases (where CaaX indicates a cysteine followed by two aliphatic amino acids and a variable amino acid) direct the subcellular localization of a large group of proteins by catalyzing the attachment of hydrophobic isoprenoid moieties onto C-terminal CaaX motifs, thus facilitating membrane association. This group of enzymes includes farnesyltransferase (Ftase) and geranylgeranyltransferase-I (Ggtase-1). Classically, the variable (X) amino acid determines whether a protein will be an Ftase or Ggtase-I substrate, with Ggtase-I substrates often containing CaaL motifs. In this study, we identify the gene encoding the β subunit of Ggtase-I (CDC43) and demonstrate that Ggtase-mediated activity is not essential. However, Cryptococcus neoformans CDC43 is important for thermotolerance, morphogenesis, and virulence. We find that Ggtase-I function is required for full membrane localization of Rho10 and the two Cdc42 paralogs (Cdc42 and Cdc420). Interestingly, the related Rac and Ras proteins are not mislocalized in the cdc43Δ mutant even though they contain similar CaaL motifs. Additionally, the membrane localization of each of these GTPases is dependent on the prenylation of the CaaX cysteine. These results indicate that C. neoformans CaaX prenyltransferases may recognize their substrates in a unique manner from existing models of prenyltransferase specificity. It also suggests that the C. neoformans Ftase, which has been shown to be more important for C. neoformans proliferation and viability, may be the primary prenyltransferase for proteins that are typically geranylgeranylated in other species. PMID:24014765

  2. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  3. A structural account of substrate and inhibitor specificity differences between two Naphthol reductases

    SciTech Connect

    Liao, D.-I.; Thompson, J.E.; Fahnestock, S.; Valent, B.; Jordan, D.B.

    2010-03-08

    Two short chain dehydrogenase/reductases mediate naphthol reduction reactions in fungal melanin biosynthesis. An X-ray structure of 1,3,6,8-tetrahydroxynaphthalene reductase (4HNR) complexed with NADPH and pyroquilon was determined for examining substrate and inhibitor specificities that differ from those of 1,3,8-trihydroxynaphthalene reductase (3HNR). The 1.5 {angstrom} resolution structure allows for comparisons with the 1.7 {angstrom} resolution structure of 3HNR complexed with the same ligands. The sequences of the two proteins are 46% identical, and they have the same fold. The 30-fold lower affinity of the 4HNR-NADPH complex for pyroquilon (a commercial fungicide that targets 3HNR) in comparison to that of the 3HNR-NADPH complex can be explained by unfavorable interactions between the anionic carboxyl group of the C-terminal Ile282 of 4HNR and CH and CH{sub 2} groups of the inhibitor that are countered by favorable inhibitor interactions with 3HNR. 1,3,8-Trihydroxynaphthalene (3HN) and 1,3,6,8-tetrahydroxynaphthalene (4HN) were modeled onto the cyclic structure of pyroquilon in the 4HNR-NADPH-pyroquilon complex to examine the 300-fold preference of the enzyme for 4HN over 3HN. The models suggest that the C-terminal carboxyl group of Ile282 has a favorable hydrogen bonding interaction with the C6 hydroxyl group of 4HN and an unfavorable interaction with the C6 CH group of 3HN. Models of 3HN and 4HN in the 3HNR active site suggest a favorable interaction of the sulfur atom of the C-terminal Met283 with the C6 CH group of 3HN and an unfavorable one with the C6 hydroxyl group of 4HN, accounting for the 4-fold difference in substrate specificities. Thus, the C-terminal residues of the two naphthol reductase are determinants of inhibitor and substrate specificities.

  4. Analysis of main parameters affecting substrate/mortar contact area through tridimensional laser scanner.

    PubMed

    Stolz, Carina M; Masuero, Angela B

    2015-10-01

    This study assesses the influence of the granulometric composition of sand, application energy and the superficial tension of substrates on the contact area of rendering mortars. Three substrates with distinct wetting behaviors were selected and mortars were prepared with different sand compositions. Characterization tests were performed on fresh and hardened mortars, as well as the rheological characterization. Mortars were applied to substrates with two different energies. The interfacial area was then digitized with 3D scanner. Results show that variables are all of influence on the interfacial contact in the development area. Furthermore, 3D laser scanning proved to be a good method to contact area measurement.

  5. [Microbial alpha-amylases: physicochemical properties, substrate specificity and domain structure].

    PubMed

    Avdiiuk, K V; Varbanets', L D

    2013-01-01

    The current literature data on producers, physico-chemical properties and substrate specificity of a-amylases produced by microbes from different taxonomic groups such as bacteria, fungi and yeasts are discussed in the survey. Synthesis of alpha-amylase majority is an inducible process which is stimulated in the presence of starch or products of its hydrolysis. It is possible to increase enzymes activity level by optimization of cultivation conditions of strains-producers. alpha-Amylases, isolated from different sources are distinguished in their physico-chemical properties, particularly in their molecular weights, pH- and thermooptimums, inhibitors and activators. The enzymes hydrolyse soluble starch, amylose, amylopectin, glycogen, maltodextrins, alpha- and beta3-cyclodextrins and other carbohydrate substrates. It is well known that alpha-amylases belong to GH-13 family of glycosyl-hydrolases, which contain the catalytic domain A as (beta/alpha)8-barrel. In addition to domain A, alpha-amylases contain two other domains: B and C, which are localized approximately on opposite sides of (beta/alpha)8-barrel. Most of the known alpha-amylases contain calcium ion, which is located on the surface between domains A and B and plays an important role in stability and activity of the enzyme.

  6. Substrate recognition and specificity of double-stranded RNA binding proteins.

    PubMed

    Vuković, Lela; Koh, Hye Ran; Myong, Sua; Schulten, Klaus

    2014-06-01

    Recognition of double-stranded (ds) RNA is an important part of many cellular pathways, including RNA silencing, viral recognition, RNA editing, processing, and transport. dsRNA recognition is often achieved by dsRNA binding domains (dsRBDs). We use atomistic molecular dynamics simulations to examine the binding interface of the transactivation response RNA binding protein (TRBP) dsRBDs to dsRNA substrates. Our results explain the exclusive selectivity of dsRBDs toward dsRNA and against DNA-RNA hybrid and dsDNA duplexes. We also provide corresponding experimental evidence. The dsRNA duplex is recognized by dsRBDs through the A-form of three duplex grooves and by the chemical properties of RNA bases, which have 2'-hydroxyl groups on their sugar rings. Our simulations show that TRBP dsRBD discriminates dsRNA- from DNA-containing duplexes primarily through interactions at two duplex grooves. The simulations also reveal that the conformation of the DNA-RNA duplex can be altered by dsRBD proteins, resulting in a weak binding of dsRBDs to DNA-RNA hybrids. Our study reveals the structural and molecular basis of protein-RNA interaction that gives rise to the observed substrate specificity of dsRNA binding proteins. PMID:24801449

  7. High specific surface gold electrode on polystyrene substrate: Characterization and application as DNA biosensor.

    PubMed

    Yang, Zhiliu; Liu, Yichen; Lu, Wei; Yuan, Qingpan; Wang, Wei; Pu, Qiaosheng; Yao, Bo

    2016-05-15

    In the past decades, many efforts have been made to improve the sensitivity and specificity of electrochemical DNA biosensors. However, it is still strongly required to develop disposable and reliable DNA biosensors for wide and practical application. In this article, we reported superior electrochemical properties of an integrated plastic-gold electrode (PGE) fabricated in-house by chemical plating on polystyrene substrate. PGEs were found having extremely high capacity of DNA immobilization compared with gold electrodes fabricated by standard sputtering based photolithography. Unique nano-structured surface was observed on PGEs through morphology techniques, which would to some extend give an explanation to higher capacity of DNA immobilization on PGEs. A probable mechanism of carboxylic acid produced on polystyrene substrate after exposure to UV irradiation was proposed and discussed for the first time. This biosensor was applied to detection and manipulate of DNA hybridization. Detection limit of 7.2×10(-11) M and 1-500 nM of linearity range was obtained.

  8. Neutrophil myeloperoxidase and its substrates: formation of specific markers and reactive compounds during inflammation

    PubMed Central

    Kato, Yoji

    2016-01-01

    Myeloperoxidase is an inflammatory enzyme that generates reactive hypochlorous acid in the presence of hydrogen peroxide and chloride ion. However, this enzyme also uses bromide ion or thiocyanate as a substrate to form hypobromous or hypothiocyanous acid, respectively. These species play important roles in host defense against the invasion of microorganisms. In contrast, these enzyme products modify biomolecules in hosts during excess inflammation, indicating that the action of myeloperoxidase is both beneficial and harmful. Myeloperoxidase uses other endogenous compounds, such as serotonin, urate, and l-tyrosine, as substrates. This broad-range specificity may have some biological implications. Target molecules of this enzyme and its products vary, including low-molecular weight thiols, proteins, nucleic acids, and lipids. The modified products represent biomarkers of myeloperoxidase action. Moderate inhibition of this enzyme might be critical for the prevention/modulation of excess, uncontrolled inflammatory events. Some phytochemicals inhibit myeloperoxidase, which might explain the reductive effect caused by the intake of vegetables and fruits on cardiovascular diseases. PMID:27013775

  9. O-GlcNAc transferase and O-GlcNAcase: achieving target substrate specificity

    PubMed Central

    Nagel, Alexis K.

    2015-01-01

    O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) catalyze the dynamic cycling of intracellular, post-translational O-GlcNAc modification on thousands of Ser/Thr residues of cytosolic, nuclear, and mitochondrial signaling proteins. The identification of O-GlcNAc modified substrates has revealed a functionally diverse set of proteins, and the extent of O-GlcNAcylation fluctuates in response to nutrients and cellular stress. As a result, OGT and OGA are implicated in widespread, nutrient-responsive regulation of numerous signaling pathways and transcriptional programs. These enzymes are required for normal embryonic development and are dysregulated in metabolic and age-related disease states. While a recent surge of interest in the field has contributed to understanding the functional impacts of protein O-GlcNAcylation, little is known about the upstream mechanisms which modulate OGT and OGA substrate targeting. This review focuses on elements of enzyme structure among splice variants, post-translational modification, localization, and regulatory protein interactions which drive the specificity of OGT and OGA toward different subsets of the cellular proteome. Ongoing efforts in this rapidly advancing field are aimed at revealing mechanisms of OGT and OGA regulation to harness the potential therapeutic benefit of manipulating these enzymes’ activities. PMID:25173736

  10. High-throughput neuraminidase substrate specificity study of human and avian influenza A viruses

    PubMed Central

    Li, Yanhong; Cao, Hongzhi; Dao, Nguyet; Luo, Zheng; Yu, Hai; Chen, Yi; Baumgarth, Nicole; Cardona, Carol; Chen, Xi

    2011-01-01

    Despite the importance of neuraminidase (NA) activity in effective infection by influenza A viruses, limited information exists about the differences of substrate preferences of viral neuraminidases from different hosts or from different strains. Using a high-throughput screening format and a library of twenty α2–3- or α2–6-linked para-nitrophenol-tagged sialylgalactosides, substrate specificity of NAs on thirty-seven strains of human and avian influenza A viruses was studied using intact viral particles. Neuraminidases of all viruses tested cleaved both α2–3- and α2–6-linked sialosides but preferred α2–3-linked ones and the activity was dependent on the terminal sialic acid structure. In contrast to NAs of other subtypes of influenza A viruses which did not cleave 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) or 5-deoxy Kdn (5d–Kdn), NAs of all N7 subtype viruses tested had noticeable hydrolytic activities on α2–3-linked sialosides containing Kdn or 5d–Kdn. Additionally, group 1 NAs showed efficient activity in cleaving N-azidoacetylneuraminic acid from α2 –3-linked sialoside. PMID:21501853

  11. Structural Insight Into the Altered Substrate Specificity of Human Cytochrome P450 2a6 Mutants

    SciTech Connect

    Sansen, S.; Hsu, M.-H.; Stout, C.David.; Johnson, E.F.

    2007-07-12

    Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.

  12. Specific previous experience affects perception of harmony and meter.

    PubMed

    Creel, Sarah C

    2011-10-01

    Prior knowledge shapes our experiences, but which prior knowledge shapes which experiences? This question is addressed in the domain of music perception. Three experiments were used to determine whether listeners activate specific musical memories during music listening. Each experiment provided listeners with one of two musical contexts that was presented simultaneously with a melody. After a listener was familiarized with melodies embedded in contexts, the listener heard melodies in isolation and judged the fit of a final harmonic or metrical probe event. The probe event matched either the familiar (but absent) context or an unfamiliar context. For both harmonic (Experiments 1 and 3) and metrical (Experiment 2) information, exposure to context shifted listeners' preferences toward a probe matching the context that they had been familiarized with. This suggests that listeners rapidly form specific musical memories without explicit instruction, which are then activated during music listening. These data pose an interesting challenge for models of music perception which implicitly assume that the listener's knowledge base is predominantly schematic or abstract. PMID:21553992

  13. Bacillus anthracis Edema Factor Substrate Specificity: Evidence for New Modes of Action

    PubMed Central

    Göttle, Martin; Dove, Stefan; Seifert, Roland

    2012-01-01

    Since the isolation of Bacillus anthracis exotoxins in the 1960s, the detrimental activity of edema factor (EF) was considered as adenylyl cyclase activity only. Yet the catalytic site of EF was recently shown to accomplish cyclization of cytidine 5′-triphosphate, uridine 5′-triphosphate and inosine 5′-triphosphate, in addition to adenosine 5′-triphosphate. This review discusses the broad EF substrate specificity and possible implications of intracellular accumulation of cyclic cytidine 3′:5′-monophosphate, cyclic uridine 3′:5′-monophosphate and cyclic inosine 3′:5′-monophosphate on cellular functions vital for host defense. In particular, cAMP-independent mechanisms of action of EF on host cell signaling via protein kinase A, protein kinase G, phosphodiesterases and CNG channels are discussed. PMID:22852066

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

    PubMed

    Salmon, Melissa; Thimmappa, Ramesha B; Minto, Robert E; Melton, Rachel E; Hughes, Richard K; O'Maille, Paul E; Hemmings, Andrew M; Osbourn, Anne

    2016-07-26

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

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

    PubMed Central

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

    2016-01-01

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

  16. Analysis of substrate specificity of Schizosaccharomyces pombe Mag1 alkylpurine DNA glycosylase

    SciTech Connect

    Adhikary, Suraj; Eichman, Brandt F.

    2014-10-02

    DNA glycosylases specialized for the repair of alkylation damage must identify, with fine specificity, a diverse array of subtle modifications within DNA. The current mechanism involves damage sensing through interrogation of the DNA duplex, followed by more specific recognition of the target base inside the active site pocket. To better understand the physical basis for alkylpurine detection, we determined the crystal structure of Schizosaccharomyces pombe Mag1 (spMag1) in complex with DNA and performed a mutational analysis of spMag1 and the close homologue from Saccharomyces cerevisiae (scMag). Despite strong homology, spMag1 and scMag differ in substrate specificity and cellular alkylation sensitivity, although the enzymological basis for their functional differences is unknown. We show that Mag preference for 1,N{sup 6}-ethenoadenine ({var_epsilon}A) is influenced by a minor groove-interrogating residue more than the composition of the nucleobase-binding pocket. Exchanging this residue between Mag proteins swapped their {var_epsilon}A activities, providing evidence that residues outside the extrahelical base-binding pocket have a role in identification of a particular modification in addition to sensing damage.

  17. Specific estrogen sulfotransferase (SULT1E1) substrates and molecular imaging probe candidates

    PubMed Central

    Cole, Graham B.; Keum, Gyochang; Liu, Jie; Small, Gary W.; Satyamurthy, Nagichettiar; Kepe, Vladimir; Barrio, Jorge R.

    2010-01-01

    This work focuses on the development of specific substrates for estrogen sulfotransferase (SULT1E1) to produce molecular imaging probes for this enzyme. SULT1E1 is a key enzyme in estrogen homeostasis, playing a central role in the prevention and development of human disease. In vitro sulfation assays showed alkyl and aryl substitutions to a fused heterocyclic system modeled after β-naphthol (βN), based on compounds that interact with the estrogen receptor, rendered several molecules with enhanced specificity for SULT1E1 over SULT1A1*1, SULT1A1*2, SULT1A3, and SULT2A1. Several 6-hydroxy-2-arylbenzothiazoles tested demonstrated excellent affinity—Vmax/Km ratios—and specificity for SULT1E1. Km values ranged from 0.12–2.36 μM. A strong correlation was observed between polarity of the 4′-sustituent on the 2-aryl moiety (Hammett σp) and the log(Vmax/Km) (r = 0.964). Substrate sensitivity is influenced by the acidity of the 6-phenolic group demonstrated by correlating its 1H NMR chemical shift (δOH) with the log(Vmax/Km) (r = 0.963). Acidity is mediated by the electron withdrawing capacity of the 4′-substituent outlined by the correlation of the C-2 13C NMR chemical shift (δC2) with the log(Vmax/Km) (r = 0.987). 2-[4-(Methylamino)phenyl]-6-hydroxybenzothiazole (2b) was radiolabeled with carbon-11 (11C-(2b)) and used in vivo for microPET scanning and tissue metabolite identification. High PET signal was paralleled with the presence of radiolabeled 11C-(2b)-6-O-sulfate and the SULT1E1 protein detected by western blot. Because this and other members of this family presenting specificity for SULT1E1 can be labeled with carbon-11 or fluorine-18, in vivo assays of SULT1E1 functional activity are now feasible in humans. PMID:20304798

  18. Peptidase specificity from the substrate cleavage collection in the MEROPS database and a tool to measure cleavage site conservation

    PubMed Central

    Rawlings, Neil D.

    2016-01-01

    One peptidase can usually be distinguished from another biochemically by its action on proteins, peptides and synthetic substrates. Since 1996, the MEROPS database (http://merops.sanger.ac.uk) has accumulated a collection of cleavages in substrates that now amounts to 66,615 cleavages. The total number of peptidases for which at least one cleavage is known is 1700 out of a total of 2457 different peptidases. This paper describes how the cleavages are obtained from the scientific literature, how they are annotated and how cleavages in peptides and proteins are cross-referenced to entries in the UniProt protein sequence database. The specificity profiles of 556 peptidases are shown for which ten or more substrate cleavages are known. However, it has been proposed that at least 40 cleavages in disparate proteins are required for specificity analysis to be meaningful, and only 163 peptidases (6.6%) fulfil this criterion. Also described are the various displays shown on the website to aid with the understanding of peptidase specificity, which are derived from the substrate cleavage collection. These displays include a logo, distribution matrix, and tables to summarize which amino acids or groups of amino acids are acceptable (or not acceptable) in each substrate binding pocket. For each protein substrate, there is a display to show how it is processed and degraded. Also described are tools on the website to help with the assessment of the physiological relevance of cleavages in a substrate. These tools rely on the hypothesis that a cleavage site that is conserved in orthologues is likely to be physiologically relevant, and alignments of substrate protein sequences are made utilizing the UniRef50 database, in which in each entry sequences are 50% or more identical. Conservation in this case means substitutions are permitted only if the amino acid is known to occupy the same substrate binding pocket from at least one other substrate cleaved by the same peptidase. PMID

  19. Resident microbiota affect Bordetella pertussis infectious dose and host specificity.

    PubMed

    Weyrich, Laura S; Feaga, Heather A; Park, Jihye; Muse, Sarah J; Safi, Chetan Y; Rolin, Olivier Y; Young, Sarah E; Harvill, Eric T

    2014-03-01

    Before contacting host tissues, invading pathogens directly or indirectly interact with host microbiota, but the effects of such interactions on the initial stages of infection are poorly understood. Bordetella pertussis is highly infectious among humans but requires large doses to colonize rodents, unlike a closely related zoonotic pathogen, Bordetella bronchiseptica, raising important questions about the contributions of bacterial competition to initial colonization and host selection. We observed that <100 colony-forming units (CFU) of B. bronchiseptica efficiently infected mice and displaced culturable host microbiota, whereas 10 000 CFU of B. pertussis were required to colonize murine nasal cavities and did not displace host microorganisms. Bacteria isolated from murine nasal cavities but not those from the human lower respiratory tract limited B. pertussis growth in vitro, indicating that interspecies competition may limit B. pertussis colonization of mice. Further, a broad-spectrum antibiotic treatment delivered before B. pertussis inoculation reduced the infectious dose to <100 CFU, and reintroduction of single Staphylococcus or Klebsiella species was sufficient to inhibit B. pertussis colonization of antibiotic-treated mice. Together, these results reveal that resident microorganisms can prevent B. pertussis colonization and influence host specificity, and they provide rationale for manipulating microbiomes to create more-accurate animal models of infectious diseases.

  20. Crystal structures of Mycobacterium tuberculosis HspAT and ArAT reveal structural basis of their distinct substrate specificities

    PubMed Central

    Nasir, Nazia; Anant, Avishek; Vyas, Rajan; Biswal, Bichitra Kumar

    2016-01-01

    Aminotransferases of subfamily Iβ, which include histidinol phosphate aminotransferases (HspATs) and aromatic amino acid aminotransferases (ArATs), are structurally similar but possess distinct substrate specificities. This study, encompassing structural and biochemical characterisation of HspAT and ArAT from Mycobacterium tuberculosis demonstrates that the residues lining the substrate binding pocket and N-terminal lid are the primary determinants of their substrate specificities. In mHspAT, hydrophilic residues in the substrate binding pocket and N-terminal lid allow the entry and binding of its preferential substrate, Hsp. On the other hand, the hydrophobic nature of both the substrate binding pocket and the N-terminal lid of mArAT is responsible for the discrimination of a polar substrate such as Hsp, while facilitating the binding of Phe and other aromatic residues such as Tyr and Trp. In addition, the present study delineates the ligand induced conformational rearrangements, providing insights into the plasticity of aminotransferases. Furthermore, the study also demonstrates that the adventitiously bound ligand 2-(N-morpholino)ethanesulfonic acid (MES) is indeed a specific inhibitor of HspAT. These results suggest that previously untapped morpholine-ring scaffold compounds could be explored for the design of new anti-TB agents. PMID:26738801

  1. Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum.

    PubMed

    Liu, Yuhao; Lü, Fan; Shao, Liming; He, Pinjing

    2016-10-01

    The objective of the study was to investigate whether the ratio of ethanol to acetate affects yield and product structure in chain elongation initiated by unacclimatized mixed cultures. The effect of varying the substrate concentration, while maintaining the same ratio of alcohol to acid, was also investigated. With a high substrate concentration, an alcohol to acid ratio >2:1 provided sufficient electron donor capacity for the chain elongation reaction. With an ethanol to acetate ratio of 3:1 (300mM total carbon), the highest n-caproate concentration (3033±98mg/L) was achieved during the stable phase of the reaction. A lower substrate concentration (150mM total carbon) gave a lower yield of products and led to reduced carbon transformation efficiency compared with other reaction conditions. The use of unacclimatized inoculum in chain elongation can produce significant amounts of odd-carbon-number carboxylates as a result of protein hydrolysis. PMID:27469095

  2. Alcohol-to-acid ratio and substrate concentration affect product structure in chain elongation reactions initiated by unacclimatized inoculum.

    PubMed

    Liu, Yuhao; Lü, Fan; Shao, Liming; He, Pinjing

    2016-10-01

    The objective of the study was to investigate whether the ratio of ethanol to acetate affects yield and product structure in chain elongation initiated by unacclimatized mixed cultures. The effect of varying the substrate concentration, while maintaining the same ratio of alcohol to acid, was also investigated. With a high substrate concentration, an alcohol to acid ratio >2:1 provided sufficient electron donor capacity for the chain elongation reaction. With an ethanol to acetate ratio of 3:1 (300mM total carbon), the highest n-caproate concentration (3033±98mg/L) was achieved during the stable phase of the reaction. A lower substrate concentration (150mM total carbon) gave a lower yield of products and led to reduced carbon transformation efficiency compared with other reaction conditions. The use of unacclimatized inoculum in chain elongation can produce significant amounts of odd-carbon-number carboxylates as a result of protein hydrolysis.

  3. Human skin fibroblast stromelysin: structure, glycosylation, substrate specificity, and differential expression in normal and tumorigenic cells

    SciTech Connect

    Wilhelm, S.M.; Collier, I.E.; Kronberger, A.; Eisen, A.Z.; Marmer, B.L.; Grant, G.A.; Bauer, E.A.; Goldberg, G.I.

    1987-10-01

    The authors have purified and determined the complete primary structure of human stromelysin, a secreted metalloprotease with a wide range of substrate specificities. Human stromelysin is synthesized in a preproenzyme form with a calculated size of 53,977 Da and a 17-amino acid long signal peptide. Prostromelysin is secreted in two forms, with apparent molecular masses on NaDodSO/sub 4//PAGE of 60 and 57 kDa. Human stromelysin is capable of degrading proteoglycan, fibronectin, laminin, and type IV collagen but not interstitial type I collagen. The enzyme is not capable of activating purified human fibroblast procollagenase. Analysis of its primary structure shows that stromelysin is in all likelihood the human analog of rat transin, which is an oncogene transformation-induced protease. The pattern of enzyme expression in normal and tumorigenic cells revealed that human skin fibroblasts in vitro secrete stromelysin constitutively. Human fetal lung fibroblasts transformed with simian virus 40, human bronchial epithelial cells transformed with the ras oncogene, fibrosarcoma cells (HT-1080), and a melanoma cell strain (A 2058), do not express this protease nor can the enzyme be induced in these cells by treatment with phorbol 12-myristate 13-acetate. The data indicate that the expression and the possible involvement of secreted metalloproteases in tumorigenesis result from a specific interaction between the transforming factor and the target cell, which may vary in different species.

  4. Substrate specificity of king cobra (Ophiophagus hannah) venom L-amino acid oxidase.

    PubMed

    Tan, N H; Saifuddin, M N

    1991-01-01

    1. Substrate specificity of purified king cobra (Ophiophagus hannah) venom L-amino acid oxidase was investigated. 2. The enzyme was highly specific for the L-enantiomer of amino acid. Effective oxidation of L-amino acid by the enzyme requires the presence of a free primary alpha-amino group but the alpha-carboxylate group is not as critical for the catalysis. 3. The enzyme was very active against L-Lys, L-Phe, L-Leu, L-Tyr, L-Tryp, L-Arg, L-Met, L-ornithine, L-norleucine and L-norvaline and moderately active against L-His, L-cystine and L-Ileu. Other L-amino acids were oxidized slowly or not oxidized. 4. The data suggest the presence of a side chain binding site in the enzyme, and that the binding site comprises at least five 'subsites': the hydrophobic subsites a, b and c; and the two 'amino' binding subsites d and e. Subsite b appears to be able to accommodate two methylene/methyl carbons.

  5. Enzymatic activity and substrate specificity of the recombinant tomato β-galactosidase 1.

    PubMed

    Eda, Masahiro; Ishimaru, Megumi; Tada, Toshiji; Sakamoto, Tatsuji; Kotake, Toshihisa; Tsumuraya, Yoichi; Mort, Andrew J; Gross, Kenneth C

    2014-10-15

    The open reading frame of tomato β-galactosidase 1 was expressed in yeast, and the enzymatic properties and substrate specificity were investigated. The enzyme had peak activity at pH 5.0 and 40-50°C. TBG1 was active on β-(1,3)- and β-(1,6)-galactobiose and lactose. TBG1 released galactose from lupin galactan, tomato fruit alkali soluble pectin, arabinogalactan, gum arabic and methyl β-(1,6)-galactohexaoside, but not from labeled β-(1,4)-galactoheptaose. TBG1 was assessed for its ability to degrade three galactosyl-containing cell wall fractions purified from different development and ripening stages of tomato fruit. TBG1 released galactose from all of the fractions from all of the stages tested. TBG1 activity was highest on the hemicellulose fraction at the 10 and 20d after pollination stage. This result is not correlated the with TBG1 expression pattern. TBG1 might act on a small but specific set of polysaccharide containing galactose.

  6. Substrate specificity and transport mechanism of amino-acid transceptor Slimfast from Aedes aegypti

    PubMed Central

    Boudko, Dmitri Y.; Tsujimoto, Hitoshi; Rodriguez, Stacy D.; Meleshkevitch, Ella A.; Price, David P.; Drake, Lisa L.; Hansen, Immo A.

    2015-01-01

    Anautogenous mosquitoes depend on vertebrate blood as nutrient source for their eggs. A highly efficient set of membrane transporters mediates the massive movement of nutrient amino acids between mosquito tissues after a blood meal. Here we report the characterization of the amino-acid transporter Slimfast (Slif) from the yellow-fever mosquito Aedes aegypti using codon-optimized heterologous expression. Slif is a well-known component of the target-of-rapamycin signalling pathway and fat body nutrient sensor, but its substrate specificity and transport mechanism were unknown. We found that Slif transports essential cationic and neutral amino acids with preference for arginine. It has an unusual dual-affinity mechanism with only the high affinity being Na+ dependent. Tissue-specific expression and blood meal-dependent regulation of Slif are consistent with conveyance of essential amino acids from gut to fat body. Slif represents a novel transport system and type of transceptor for sensing and transporting essential amino acids during mosquito reproduction. PMID:26449545

  7. Substrate specificities and expression patterns reflect the evolutionary divergence of maltose ABC transporters in Thermotoga maritima.

    PubMed

    Nanavati, Dhaval M; Nguyen, Tu N; Noll, Kenneth M

    2005-03-01

    Duplication of transporter genes is apparent in the genome sequence of the hyperthermophilic bacterium Thermotoga maritima. The physiological impacts of these duplications are not well understood, so we used the bacterium's two putative maltose transporters to begin a study of the evolutionary relationship between a transporter's function and the control of expression of its genes. We show that the substrate binding proteins encoded by these operons, MalE1 and MalE2, have different substrate specificities and affinities and that they are expressed under different growth conditions. MalE1 binds maltose (dissociation constant [KD], 24 +/- 1 microM), maltotriose (KD, 8 +/- 0.5 nM), and beta-(1-->4)-mannotetraose (KD, 38 +/- 1 microM). In contrast, MalE2 binds maltose (KD, 8.4 +/- 1 microM), maltotriose (KD, 11.5 +/- 1.5 microM), and trehalose (KD, 9.5 +/- 1.0 microM) confirming the findings of Wassenberg et al. (J. Mol. Biol. 295:279-288, 2000). Neither protein binds lactose. We examined the expression of these operons at both the transcriptional and translational levels and found that MalE1 is expressed in cells grown on lactose or guar gum and that MalE2 is highly expressed in starch- and trehalose-grown cells. Evidence is provided that malE1, malF1, and perhaps malG1 are cotranscribed and so constitute an operon. An open reading frame encoding a putative transcriptional regulatory protein adjacent to this operon (TM1200) is also up-regulated in response to growth on lactose. These evolutionarily related transporter operons have diverged both in function and expression to assume apparently different physiological roles. PMID:15743948

  8. Structures of 5-Methylthioribose Kinase Reveal Substrate Specificity and Unusual Mode of Nucleotide Binding

    SciTech Connect

    Ku,S.; Yip, P.; Cornell, K.; Riscoe, M.; Behr, J.; Guillerm, G.; Howell, P.

    2007-01-01

    The methionine salvage pathway is ubiquitous in all organisms, but metabolic variations exist between bacteria and mammals. 5-Methylthioribose (MTR) kinase is a key enzyme in methionine salvage in bacteria and the absence of a mammalian homolog suggests that it is a good target for the design of novel antibiotics. The structures of the apo-form of Bacillus subtilis MTR kinase, as well as its ADP, ADP-PO4, AMPPCP, and AMPPCP-MTR complexes have been determined. MTR kinase has a bilobal eukaryotic protein kinase fold but exhibits a number of unique features. The protein lacks the DFG motif typically found at the beginning of the activation loop and instead coordinates magnesium via a DXE motif (Asp{sup 250}-Glu{sup 252}). In addition, the glycine-rich loop of the protein, analogous to the 'Gly triad' in protein kinases, does not interact extensively with the nucleotide. The MTR substrate-binding site consists of Asp{sup 233} of the catalytic HGD motif, a novel twin arginine motif (Arg{sup 340}/Arg{sup 341}), and a semi-conserved W-loop, which appears to regulate MTR binding specificity. No lobe closure is observed for MTR kinase upon substrate binding. This is probably because the enzyme lacks the lobe closure/inducing interactions between the C-lobe of the protein and the ribosyl moiety of the nucleotide that are typically responsible for lobe closure in protein kinases. The current structures suggest that MTR kinase has a dissociative mechanism.

  9. Modification of the substrate specificity of porcine pepsin for the enzymatic production of bovine hide gelatin.

    PubMed Central

    Galea, C. A.; Dalrymple, B. P.; Kuypers, R.; Blakeley, R.

    2000-01-01

    The substrate specificity of porcine pepsin has been altered by site-directed mutagenesis in an attempt to selectively cleave bovine hide collagen at only a few sites, similar to cathepsin D, for the production of high quality gelatin. Kinetic parameters were determined using chromogenic peptide substrates based on the sequence Lys-Pro-Xaa-Yaa-Phe*Nph-Arg-Leu (where Xaa is Ile or Pro, Yaa is Glu. Leu, Gln or Lys, Nph is p-nitrophenylalanine, and * is the site of cleavage). Substitution of Thr222 and Glu287 within the S2 subsite of pepsin by Val and Met, respectively, produced a double mutant with a two- to fourfold higher kcat/Km, compared with wild-type pepsin, for the chromogenic peptides with residues Leu, Gln, and Glu at position P2 (Yaa). The results suggest that the functional group of the P2 side chain may be exposed to solvent, while the aliphatic portion interacts with hydrophobic residues comprising S2. Wild-type pepsin cleaved a peptide corresponding to the carboxy-terminal telopeptide region of bovine type I collagen alpha1 chain, SGGYDLSFLPQPPQE, predominantly at three sites (Asp-Leu, Leu-Ser, and Phe-Leu) and at a significantly lower rate at Ser-Phe. However, Thr222Val/Glu287Met cleaved site Ser-Phe at a rate 20-fold higher than the wild-type. Significantly, enzymes containing the double substitution Phe111Thr/Leu112Phe cleaved this peptide predominantly at one site Leu-Ser (similar to cathepsin D) and at a rate 23-fold higher than the wild-type. These mutants can potentially enhance the rate of solubilization of bovine hide collagen under conditions mild enough to maintain the triple helix structure and hence minimize the rate of subsequent denaturation and proteolytic cleavage. PMID:11106168

  10. Predicting Emotional Responses to Horror Films from Cue-Specific Affect.

    ERIC Educational Resources Information Center

    Neuendorf, Kimberly A.; Sparks, Glenn G.

    1988-01-01

    Assesses individuals' fear and enjoyment reactions to horror films, applying theories of cognition and affect that predict emotional responses to a stimulus on the basis of prior affect toward specific cues included in that stimulus. (MM)

  11. Monitoring of PQQ-dependent glucose dehydrogenase substrate specificity for its potential use in biocatalysis and bioanalysis.

    PubMed

    Streďanský, Miroslav; Monošík, Rastislav; Mastihuba, Vladimír; Sturdík, Ernest

    2013-10-01

    Substrate specificity of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase was investigated in biosensor arrangement for understanding the suitability and the limitations of its use in bioanalysis and bioproduction of chemicals. The study demonstrated a very broad substrate specificity of biosensor utilising soluble form of PQQ-dependent glucose dehydrogenase. Nineteen saccharides out of 31 were oxidised by the sensor. Investigation confirmed strong importance of hydroxyl configuration in the positions 2 and 5 of oxidised saccharides. The broad specificity suggests that the PQQ-dependent glucose dehydrogenase could be utilised for analysis of other sugars than glucose in food samples for various production processes and for biofuel cells. In addition, the results showed that the substrate specificity of enzymes can be effectively and generally studied by biosensor arrangement for research purposes. This layout utilising immobilised enzyme allowed performing comprehensive study using a small amount of enzymes and thus saving the costs and time.

  12. Universal Labeling of 5′-Triphosphate RNAs by Artificial RNA Ligase Enzyme with Broad Substrate Specificity

    PubMed Central

    Haugner, John C.; Seelig, Burckhard

    2013-01-01

    An artificial RNA ligase specific to RNA with a 5′-triphosphate (PPP-RNA) exhibits broad sequence specificity on model substrates and secondary siRNAs with direct applications in the identification of PPP-RNAs through sequencing. PMID:23851643

  13. Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease N{sup pro}

    SciTech Connect

    Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

    2014-03-15

    Pestivirus N{sup pro} is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N{sup pro} blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N{sup pro'}s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N{sup pro}-GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N{sup pro} proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N{sup pro'}s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N{sup pro} does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N{sup pro'}s autoproteolysis is studied using N{sup pro}-GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N{sup pro} prefers small amino acids with non-branched beta carbons at the P1 position.

  14. SepM, a Streptococcal Protease Involved in Quorum Sensing, Displays Strict Substrate Specificity

    PubMed Central

    Biswas, Saswati; Cao, Luyang; Kim, Albert

    2015-01-01

    ABSTRACT Streptococcus mutans, a causative agent of dental caries, relies on multiple quorum-sensing (QS) pathways that coordinate the expression of factors needed for colonization in the oral cavity. S. mutans uses small peptides as QS signaling molecules that typically are secreted into the outside milieu. Competence-stimulating peptide (CSP) is one such QS signaling molecule that functions through the ComDE two-component signal transduction pathway. CSP is secreted through NlmTE, a dedicated ABC transporter that cleaves off the N-terminal leader peptide to generate a mature peptide that is 21 residues long (CSP-21). We recently identified a surface-localized protease, SepM, which further cleaves the CSP-21 peptide at the C-terminal end and removes the last 3 residues to generate CSP-18. CSP-18 is the active QS molecule that interacts with the ComD sensor kinase to activate the QS pathway. In this study, we show that SepM specifically cleaves CSP-21 between the Ala18 and Leu19 residues. We also show that SepM recognizes only Ala at position 18 and Leu at position 19, although some CSP-18 variants with a substitution at position 18 can function equally as well as the QS peptide. Furthermore, we demonstrate that SepM homologs from other streptococci are capable of processing CSP-21 to generate functional CSP-18. IMPORTANCE SepM is a membrane-associated streptococcal protease that processes competence-stimulating peptide (CSP) to generate an active quorum-sensing molecule in S. mutans. SepM belongs to the S16 family of serine proteases, and in this study, we found that SepM behaves as an endopeptidase. SepM displays strict substrate specificity and cleaves the peptide bond between the Ala and Leu residues. This is the first report of an endopeptidase that specifically cleaves these two residues. PMID:26553848

  15. A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

    PubMed Central

    Alperi, Anabel; Larrea, Delfina; Fernández-González, Esther; Dehio, Christoph; Zechner, Ellen L.

    2013-01-01

    Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs. PMID:23995644

  16. Structural basis of substrate specificity and regiochemistry in the MycF/TylF family of sugar O-methyltransferases.

    PubMed

    Bernard, Steffen M; Akey, David L; Tripathi, Ashootosh; Park, Sung Ryeol; Konwerski, Jamie R; Anzai, Yojiro; Li, Shengying; Kato, Fumio; Sherman, David H; Smith, Janet L

    2015-05-15

    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, 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 homologue, 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. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes. PMID:25692963

  17. Structural Analysis of Saccharomyces cerevisiae α-Galactosidase and Its Complexes with Natural Substrates Reveals New Insights into Substrate Specificity of GH27 Glycosidases*

    PubMed Central

    Fernández-Leiro, Rafael; Pereira-Rodríguez, Ángel; Cerdán, M. Esperanza; Becerra, Manuel; Sanz-Aparicio, Juliana

    2010-01-01

    α-Galactosidases catalyze the hydrolysis of terminal α-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae α-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 Å resolution. The monomer folds into a catalytic (α/β)8 barrel and a C-terminal β-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the β-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions. PMID:20592022

  18. Structural analysis of Saccharomyces cerevisiae alpha-galactosidase and its complexes with natural substrates reveals new insights into substrate specificity of GH27 glycosidases.

    PubMed

    Fernández-Leiro, Rafael; Pereira-Rodríguez, Angel; Cerdán, M Esperanza; Becerra, Manuel; Sanz-Aparicio, Juliana

    2010-09-01

    Alpha-galactosidases catalyze the hydrolysis of terminal alpha-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae alpha-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 A resolution. The monomer folds into a catalytic (alpha/beta)(8) barrel and a C-terminal beta-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the beta-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.

  19. Structure and Substrate Specificity of a Eukaryotic Fucosidase from Fusarium graminearum*

    PubMed Central

    Cao, Hongnan; Walton, Jonathan D.; Brumm, Phil; Phillips, George N.

    2014-01-01

    The secreted glycoside hydrolase family 29 (GH29) α-l-fucosidase from plant pathogenic fungus Fusarium graminearum (FgFCO1) actively releases fucose from the xyloglucan fragment. We solved crystal structures of two active-site conformations, i.e. open and closed, of apoFgFCO1 and an open complex with product fucose at atomic resolution. The closed conformation supports catalysis by orienting the conserved general acid/base Glu-288 nearest the predicted glycosidic position, whereas the open conformation possibly represents an unreactive state with Glu-288 positioned away from the catalytic center. A flexible loop near the substrate binding site containing a non-conserved GGSFT sequence is ordered in the closed but not the open form. We also identified a novel C-terminal βγ-crystallin domain in FgFCO1 devoid of calcium binding motif whose homologous sequences are present in various glycoside hydrolase families. N-Glycosylated FgFCO1 adopts a monomeric state as verified by solution small angle x-ray scattering in contrast to reported multimeric fucosidases. Steady-state kinetics shows that FgFCO1 prefers α1,2 over α1,3/4 linkages and displays minimal activity with p-nitrophenyl fucoside with an acidic pH optimum of 4.6. Despite a retaining GH29 family fold, the overall specificity of FgFCO1 most closely resembles inverting GH95 α-fucosidase, which displays the highest specificity with two natural substrates harboring the Fucα1-2Gal glycosidic linkage, a xyloglucan-derived nonasaccharide, and 2′-fucosyllactose. Furthermore, FgFCO1 hydrolyzes H-disaccharide (lacking a +2 subsite sugar) at a rate 103-fold slower than 2′-fucosyllactose. We demonstrated the structurally dynamic active site of FgFCO1 with flexible general acid/base Glu, a common feature shared by several bacterial GH29 fucosidases to various extents. PMID:25086049

  20. Substrate specificity in enzymatic fluorination. The fluorinase from Streptomyces cattleya accepts 2′-deoxyadenosine substrates†

    PubMed Central

    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

  1. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  2. Comparative modeling and molecular docking of orphan human CYP4V2 protein with fatty acid substrates: Insights into substrate specificity

    PubMed Central

    Kumar, Suresh

    2011-01-01

    Cytochromes P450 (CYPs) are a super family of heme-containing enzymes well-known for their monooxgenase reaction. There are 57 CYP isoenzymes found in human which exhibit specific physiological functions. Thirteen members of this super family are classified as “orphan” CYP because of their unknown enzymatic functions. CYP4V2 is found to be a potential drug target for Bietti crystalline corneoretinal dystrophy (BCD). However, three-dimensional structure, the active site topology and substrate binding modes of CYP4V2 remain unclear. In this study, the three-dimensional model of CYP4V2 was constructed using the homology modeling method. Four possible fatty acid substrates namely, caprylic, lauric, myrisitc and palmitic acids were optimized and evaluated for drug likeness using Lipinski's rule of five. Further, these substrates were docked into active sites of CYP4V2 and several key residues responsible for substrate binding were identified. These findings will be helpful for the structure-based drug design and detailed characterization of the biological roles of CYP4V2. PMID:22355237

  3. Evaluation of normalized energy recovery (NER) in microbial fuel cells affected by reactor dimensions and substrates.

    PubMed

    Xiao, Li; Ge, Zheng; Kelly, Patrick; Zhang, Fei; He, Zhen

    2014-04-01

    The objective of this study is to provide an initial evaluation of normalized energy recovery (NER - a new parameter for presenting energy performance) in microbial fuel cells (MFCs) through investigation of the effects of reactor dimensions and anode substrates. Although the larger-size MFCs generally have lower maximum power densities, their maximum NER is comparable to that of the smaller MFCs at the same anolyte flow rate. The mixed messages obtained from the MFC size tests suggest that MFCs can be further scaled up without decreasing energy recovery under certain conditions. The low-strength substrates seem to be more suitable for MFC treatment of wastewater, in terms of both energy recovery and organic removal. However, because the MFCs could not achieve the maximum NER and the maximum organic removal efficiency at the same time, one must determine a major goal for MFCs treating wastewater between energy recovery and contaminant removal.

  4. Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass.

    PubMed

    Eichorst, Stephanie A; Joshua, Chijioke; Sathitsuksanoh, Noppadon; Singh, Seema; Simmons, Blake A; Singer, Steven W

    2014-12-01

    Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction. PMID:25261509

  5. Complexes of Thermotoga maritima S-adenosylmethionine decarboxylase provide insights into substrate specificity

    SciTech Connect

    Bale, Shridhar; Baba, Kavita; McCloskey, Diane E.; Pegg, Anthony E.; Ealick, Steven E.

    2010-06-25

    The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5{prime}-deoxy-5{prime}-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

  6. Altering the substrate specificity of methyl parathion hydrolase with directed evolution.

    PubMed

    Ng, Tee-Kheang; Gahan, Lawrence R; Schenk, Gerhard; Ollis, David L

    2015-05-01

    Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system. PMID:25797441

  7. N-terminal modifications of cellular proteins: The enzymes involved, their substrate specificities and biological effects

    PubMed Central

    Varland, Sylvia; Osberg, Camilla; Arnesen, Thomas

    2015-01-01

    The vast majority of eukaryotic proteins are N-terminally modified by one or more processing enzymes. Enzymes acting on the very first amino acid of a polypeptide include different peptidases, transferases, and ligases. Methionine aminopeptidases excise the initiator methionine leaving the nascent polypeptide with a newly exposed amino acid that may be further modified. N-terminal acetyl-, methyl-, myristoyl-, and palmitoyltransferases may attach an acetyl, methyl, myristoyl, or palmitoyl group, respectively, to the α-amino group of the target protein N-terminus. With the action of ubiquitin ligases, one or several ubiquitin molecules are transferred, and hence, constitute the N-terminal modification. Modifications at protein N-termini represent an important contribution to proteomic diversity and complexity, and are essential for protein regulation and cellular signaling. Consequently, dysregulation of the N-terminal modifying enzymes is implicated in human diseases. We here review the different protein N-terminal modifications occurring co- or post-translationally with emphasis on the responsible enzymes and their substrate specificities. PMID:25914051

  8. Substrate-Specific Gene Expression in Batrachochytrium dendrobatidis, the Chytrid Pathogen of Amphibians

    PubMed Central

    Rosenblum, Erica Bree; Poorten, Thomas J.; Joneson, Suzanne; Settles, Matthew

    2012-01-01

    Determining the mechanisms of host-pathogen interaction is critical for understanding and mitigating infectious disease. Mechanisms of fungal pathogenicity are of particular interest given the recent outbreaks of fungal diseases in wildlife populations. Our study focuses on Batrachochytrium dendrobatidis (Bd), the chytrid pathogen responsible for amphibian declines around the world. Previous studies have hypothesized a role for several specific families of secreted proteases as pathogenicity factors in Bd, but the expression of these genes has only been evaluated in laboratory growth conditions. Here we conduct a genome-wide study of Bd gene expression under two different nutrient conditions. We compare Bd gene expression profiles in standard laboratory growth media and in pulverized host tissue (i.e., frog skin). A large proportion of genes in the Bd genome show increased expression when grown in host tissue, indicating the importance of studying pathogens on host substrate. A number of gene classes show particularly high levels of expression in host tissue, including three families of secreted proteases (metallo-, serine- and aspartyl-proteases), adhesion genes, lipase-3 encoding genes, and a group of phylogenetically unusual crinkler-like effectors. We discuss the roles of these different genes as putative pathogenicity factors and discuss what they can teach us about Bd’s metabolic targets, host invasion, and pathogenesis. PMID:23185485

  9. Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass

    PubMed Central

    Eichorst, Stephanie A.; Joshua, Chijioke; Sathitsuksanoh, Noppadon; Singh, Seema; Simmons, Blake A.

    2014-01-01

    Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction. PMID:25261509

  10. Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores.

    PubMed

    Gupta, Nirupama; Farinas, Edgardo T

    2010-08-01

    Directed evolution is an effective strategy to engineer and optimize protein properties, and microbial cell-surface display is a successful method to screen protein libraries. Protein surface display on Bacillus subtilis spores is demonstrated as a tool for screening protein libraries for the first time. Spore display offers advantages over more commonly utilized microbe cell-surface display systems, which include gram-negative bacteria, phage and yeast. For instance, protein-folding problems associated with the expressed recombinant polypeptide crossing membranes are avoided. Hence, a different region of protein space can be explored that previously was not accessible. In addition, spores tolerate many physical/chemical extremes; hence, the displayed proteins are "preimmobilized" on the inherently inert spore surface. Immobilized proteins have several advantages when used in industrial processes. The protein stability is increased and separations are simplified. Finally, immobilized proteins can be used in a wide array of simple device applications and configurations. The substrate specificity of the enzyme CotA is narrowed. CotA is a laccase and it occurs naturally on the outer coat of B. subtilis spores. A library of CotA genes were expressed in the spore coat, and it was screened for activity toward ABTS [diammonium 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate)] over SGZ (4-hydroxy-3,5-dimethoxy-benzaldehyde azine). A mutant CotA was found to be 120-fold more specific for ABTS. This research demonstrates that B. subtilis spores can be a useful platform for screen protein libraries.

  11. Biochemical evidence for relaxed substrate specificity of Nα-acetyltransferase (Rv3420c/rimI) of Mycobacterium tuberculosis

    PubMed Central

    Pathak, Deepika; Bhat, Aadil Hussain; Sapehia, Vandana; Rai, Jagdish; Rao, Alka

    2016-01-01

    Nα-acetylation is a naturally occurring irreversible modification of N-termini of proteins catalyzed by Nα-acetyltransferases (NATs). Although present in all three domains of life, it is little understood in bacteria. The functional grouping of NATs into six types NatA - NatF, in eukaryotes is based on subunit requirements and stringent substrate specificities. Bacterial orthologs are phylogenetically divergent from eukaryotic NATs, and only a couple of them are characterized biochemically. Accordingly, not much is known about their substrate specificities. Rv3420c of Mycobacterium tuberculosis is a NAT ortholog coding for RimIMtb. Using in vitro peptide-based enzyme assays and mass-spectrometry methods, we provide evidence that RimIMtb is a protein Nα-acetyltransferase of relaxed substrate specificity mimicking substrate specificities of eukaryotic NatA, NatC and most competently that of NatE. Also, hitherto unknown acetylation of residues namely, Asp, Glu, Tyr and Leu by a bacterial NAT (RimIMtb) is elucidated, in vitro. Based on in vivo acetylation status, in vitro assay results and genetic context, a plausible cellular substrate for RimIMtb is proposed. PMID:27353550

  12. Substrate Specificity of Lymphoid-specific Tyrosine Phosphatase (Lyp) and Identification of Src Kinase-associated Protein of 55 kDa Homolog (SKAP-HOM) as a Lyp Substrate

    SciTech Connect

    Yu, Xiao; Chen, Ming; Zhang, Sheng; Yu, Zhi-Hong; Sun, Jin-Peng; Wang, Lina; Liu, Sijiu; Imasaki, Tsuyoshi; Takagi, Yuichiro; Zhang, Zhong-Yin

    2012-02-08

    A missense single-nucleotide polymorphism in the gene encoding the lymphoid-specific tyrosine phosphatase (Lyp) has been identified as a causal factor in a wide spectrum of autoimmune diseases. Interestingly, the autoimmune-predisposing variant of Lyp appears to represent a gain-of-function mutation, implicating Lyp as an attractive target for the development of effective strategies for the treatment of many autoimmune disorders. Unfortunately, the precise biological functions of Lyp in signaling cascades and cellular physiology are poorly understood. Identification and characterization of Lyp substrates will help define the chain of molecular events coupling Lyp dysfunction to diseases. In the current study, we identified consensus sequence motifs for Lyp substrate recognition using an 'inverse alanine scanning' combinatorial library approach. The intrinsic sequence specificity data led to the discovery and characterization of SKAP-HOM, a cytosolic adaptor protein required for proper activation of the immune system, as a bona fide Lyp substrate. To determine the molecular basis for Lyp substrate recognition, we solved crystal structures of Lyp in complex with the consensus peptide as well as the phosphopeptide derived from SKAP-HOM. Together with the biochemical data, the structures define the molecular determinants for Lyp substrate specificity and provide a solid foundation upon which novel therapeutics targeting Lyp can be developed for multiple autoimmune diseases.

  13. Enhancing Phospholipid Fatty Acid Profiling of Soil Bacterial Communities via Substrate- Specific 13C-labelling

    NASA Astrophysics Data System (ADS)

    Evershed, R. P.; Maxfield, P. J.; Bingham, E. M.; Dildar, N.; Brennand, E. L.; Hornibrook, E.

    2008-12-01

    A range of culture-independent methods, has recently emerged to study environmental microorganisms in situ[1]. One such method is phospholipid fatty acid (PLFA) analysis, wherein these ubiquitous membrane lipids provide a powerful tool for the study of unculturable soil microorganisms. PLFA analyses have been used to investigate the impacts of a wide range of environmental factors on the soil microbial community. An acknowledged shortcoming of the PLFAs approach is the lack the chemotaxonoic specificity, which restricts the ability of the method to probe the activities of specific functional groups of the microbial community selectively. However, the selectivity of PLFAs analyses can be enhanced by incubating soils with 13C- labelled substrates followed by gas chromatography-combustion-isotope ratio mass spectrometry to reveal the specific PLFAs incorporating the 13C-label. The application of this approach will be demonstrated through our recent work on methanotrophic bacteria in soils. We applied this approach initially to mineral soils[2] and then extended chemotaxonomic assessments by using a combination of 13C-labelled PLFAs and hopanoids [3]. We have used this approach to explore the properties of high affinity methanotrophs in a range of environments, investigating the relationship between methane oxidation rates and the nature and magnitude of the methanotrophic community for the first time[4,5] More recently we extended the technique using a novel time series 13C-labelling of PLFAs[6] to estimate the rate and progression of 13C- label incorporation and turnover of methanotrophic populations. This modified approach has been used to investigate the impacts of various environmental variables, e.g. soil type, vegetation cover and land use, on the methanotrophic biomass[7.8]. The unique nature of the 13CH4 as a gaseous substate/carbon source means that can be readily introduced into soils via a specific subset of the soil microbial biomass, thereby offering many

  14. Proteomic Analyses Reveal an Acidic Prime Side Specificity for the Astacin Metalloprotease Family Reflected by Physiological Substrates*

    PubMed Central

    Becker-Pauly, Christoph; Barré, Olivier; Schilling, Oliver; auf dem Keller, Ulrich; Ohler, Anke; Broder, Claudia; Schütte, André; Kappelhoff, Reinhild; Stöcker, Walter; Overall, Christopher M.

    2011-01-01

    Astacins are secreted and membrane-bound metalloproteases with clear associations to many important pathological and physiological processes. Yet with only a few substrates described their biological roles are enigmatic. Moreover, the lack of knowledge of astacin cleavage site specificities hampers assay and drug development. Using PICS (proteomic identification of protease cleavage site specificity) and TAILS (terminal amine isotopic labeling of substrates) degradomics approaches >3000 cleavage sites were proteomically identified for five different astacins. Such broad coverage enables family-wide determination of specificities N- and C-terminal to the scissile peptide bond. Remarkably, meprin α, meprin β, and LAST_MAM proteases exhibit a strong preference for aspartate in the peptide (P)1′ position because of a conserved positively charged residue in the active cleft subsite (S)1′. This unparalleled specificity has not been found for other families of extracellular proteases. Interestingly, cleavage specificity is also strongly influenced by proline in P2′ or P3′ leading to a rare example of subsite cooperativity. This specificity characterizes the astacins as unique contributors to extracellular proteolysis that is corroborated by known cleavage sites in procollagen I+III, VEGF (vascular endothelial growth factor)-A, IL (interleukin)-1β, and pro-kallikrein 7. Indeed, cleavage sites in VEGF-A and pro-kallikrein 7 identified by terminal amine isotopic labeling of substrates matched those reported by Edman degradation. Moreover, the novel substrate FGF-19 was validated biochemically and shown to exhibit altered biological activity after meprin processing. PMID:21693781

  15. Identification of Amino Acids Conferring Chain Length Substrate Specificities on Fatty Alcohol-forming Reductases FAR5 and FAR8 from Arabidopsis thaliana*

    PubMed Central

    Chacón, Micaëla G.; Fournier, Ashley E.; Tran, Frances; Dittrich-Domergue, Franziska; Pulsifer, Ian P.; Domergue, Frédéric; Rowland, Owen

    2013-01-01

    Fatty alcohols play a variety of biological roles in all kingdoms of life. Fatty acyl reductase (FAR) enzymes catalyze the reduction of fatty acyl-coenzyme A (CoA) or fatty acyl-acyl carrier protein substrates to primary fatty alcohols. FAR enzymes have distinct substrate specificities with regard to chain length and degree of saturation. FAR5 (At3g44550) and FAR8 (At3g44560) from Arabidopsis thaliana are 85% identical at the amino acid level and are of equal length, but they possess distinct specificities for 18:0 or 16:0 acyl chain length, respectively. We used Saccharomyces cerevisiae as a heterologous expression system to assess FAR substrate specificity determinants. We identified individual amino acids that affect protein levels or 16:0-CoA versus 18:0-CoA specificity by expressing in yeast FAR5 and FAR8 domain-swap chimeras and site-specific mutants. We found that a threonine at position 347 and a serine at position 363 were important for high FAR5 and FAR8 protein accumulation in yeast and thus are likely important for protein folding and stability. Amino acids at positions 355 and 377 were important for dictating 16:0-CoA versus 18:0-CoA chain length specificity. Simultaneously converting alanine 355 and valine 377 of FAR5 to the corresponding FAR8 residues, leucine and methionine, respectively, almost fully converted FAR5 specificity from 18:0-CoA to 16:0-CoA. The reciprocal amino acid conversions, L355A and M377V, made in the active FAR8-S363P mutant background converted its specificity from 16:0-CoA to 18:0-CoA. This study is an important advancement in the engineering of highly active FAR proteins with desired specificities for the production of fatty alcohols with industrial value. PMID:24005667

  16. Substrate-specific modifications on magnetic iron oxide nanoparticles as an artificial peroxidase for improving sensitivity in glucose detection.

    PubMed

    Liu, Yanping; Yu, Faquan

    2011-04-01

    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. PMID:21368352

  17. [Use of kenaf fibre in the elaboration of specific substrates for Pleurotus ostreatus (Jacq. ex Fr.) Kummer cultivation].

    PubMed

    Pardo Giménez, Arturo; Perona Zamora, Ma Aquilina; Pardo Núñez, José

    2008-03-01

    In this study, the viability of the kenaf fibre use, alone or combined with cereal straw, vine shoots and olive mill dried waste, in the elaboration of specific substrates for the cultivation of Pleurotus ostreatus (Jacq. ex Fr.) Kummer, second mushroom in importance cultivated in Spain, is described. Furthermore, three different methods of preparation of the substrate have been considered in order to obtain selectivity for the growth and later fruiting of Pleurotus sporophore. As for the production parameters, the best results have been provided by the substrates that combined kenaf with straw and with vine shoots, being unfavourable the substrates based in just kenaf or combined with olive mill dried waste. As for the treatment applied to the materials, the immersion in water alone and subsequent pasteurization and thermophilic conditioning, together with the semi-anaerobic fermentation, has been favoured in front of the immersion in water with fungicide and later pasteurization.

  18. A gatekeeper helix determines the substrate specificity of Sjögren–Larsson Syndrome enzyme fatty aldehyde dehydrogenase

    PubMed Central

    Keller, Markus A.; Zander, Ulrich; Fuchs, Julian E.; Kreutz, Christoph; Watschinger, Katrin; Mueller, Thomas; Golderer, Georg; Liedl, Klaus R.; Ralser, Markus; Kräutler, Bernhard; Werner, Ernst R.; Marquez, Jose A.

    2014-01-01

    Mutations in the gene coding for membrane-bound fatty aldehyde dehydrogenase (FALDH) lead to toxic accumulation of lipid species and development of the Sjögren–Larsson Syndrome (SLS), a rare disorder characterized by skin defects and mental retardation. Here, we present the crystallographic structure of human FALDH, the first model of a membrane-associated aldehyde dehydrogenase. The dimeric FALDH displays a previously unrecognized element in its C-terminal region, a ‘gatekeeper’ helix, which extends over the adjacent subunit, controlling the access to the substrate cavity and helping orientate both substrate cavities towards the membrane surface for efficient substrate transit between membranes and catalytic site. Activity assays demonstrate that the gatekeeper helix is important for directing the substrate specificity of FALDH towards long-chain fatty aldehydes. The gatekeeper feature is conserved across membrane-associated aldehyde dehydrogenases. Finally, we provide insight into the previously elusive molecular basis of SLS-causing mutations. PMID:25047030

  19. Impact of remote mutations on metallo-β-lactamase substrate specificity: Implications for the evolution of antibiotic resistance

    PubMed Central

    Oelschlaeger, Peter; Mayo, Stephen L.; Pleiss, Juergen

    2005-01-01

    Metallo-β-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of β-lactam antibiotics. The G262S point mutation distinguishing the metallo-β-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally. PMID:15722450

  20. Surface loops of extracellular phospholipase A1 determine both substrate specificity and preference for lysophospholipids[S

    PubMed Central

    Arima, Naoaki; Inoue, Asuka; Makide, Kumiko; Nonaka, Takamasa; Aoki, Junken

    2012-01-01

    Members of the pancreatic lipase family exhibit both lipase activity toward triacylglycerol and/or phospholipase A1 (PLA1) activity toward certain phospholipids. Some members of the pancreatic lipase family exhibit lysophospholipase activity in addition to their lipase and PLA1 activities. Two such enzymes, phosphatidylserine (PS)-specific PLA1 (PS-PLA1) and phosphatidic acid (PA)-selective PLA1α (PA-PLA1α, also known as LIPH) specifically hydrolyze PS and PA, respectively. However, little is known about the mechanisms that determine their substrate specificities. Crystal structures of lipases and mutagenesis studies have suggested that three surface loops, namely, β5, β9, and lid, have roles in determining substrate specificity. To determine roles of these loop structures in the substrate recognition of these PLA1 enzymes, we constructed a number of PS-PLA1 mutants in which the three surface loops are replaced with those of PA-PLA1α. The results indicate that the surface loops, especially the β5 loop, of PA-PLA1α play important roles in the recognition of PA, whereas other structure(s) in PS-PLA1 is responsible for PS preference. In addition, β5 loop of PS-PLA1 has a crucial role in lysophospholipase activity toward lysophosphatidylserine. The present study revealed the critical role of lipase surface loops, especially the β5 loop, in determining substrate specificities of PLA1 enzymes. PMID:22172514

  1. Substrate diameter and compliance affect the gripping strategies and locomotor mode of climbing boa constrictors.

    PubMed

    Byrnes, Greg; Jayne, Bruce C

    2010-12-15

    Arboreal habitats pose unique challenges for locomotion as a result of their narrow cylindrical surfaces and discontinuities between branches. Decreased diameter of branches increases compliance, which can pose additional challenges, including effects on stability and energy damping. However, the combined effects of substrate diameter and compliance are poorly understood for any animal. We quantified performance, kinematics and substrate deformation while boa constrictors (Boa constrictor) climbed vertical ropes with three diameters (3, 6 and 9 mm) and four tensions (0.5, 1.0, 1.5 and 2.0 body weights). Mean forward velocity decreased significantly with both decreased diameter and increased compliance. Both diameter and compliance had numerous effects on locomotor kinematics, but diameter had larger and more pervasive effects than compliance. Locomotion on the largest diameter had a larger forward excursion per cycle, and the locomotor mode and gripping strategy differed from that on the smaller diameters. On larger diameters, snakes primarily applied opposing forces at the same location on the rope to grip. By contrast, on smaller diameters forces were applied in opposite directions at different locations along the rope, resulting in increased rope deformation. Although energy is likely to be lost during deformation, snakes might use increased surface deformation as a strategy to enhance their ability to grip.

  2. Investigating the substrate specificity and oligomerisation of the leader protease of foot and mouth disease virus using NMR.

    PubMed

    Cencic, Regina; Mayer, Christina; Juliano, Maria A; Juliano, Luiz; Konrat, Robert; Kontaxis, Georg; Skern, Tim

    2007-11-01

    The leader protease (Lbpro) of foot-and-mouth disease virus frees itself during translation from the viral polyprotein by cleavage between its own C terminus and the N terminus of the subsequent protein, VP4. Lbpro also specifically cleaves the host proteins eukaryotic initiation factor (eIF) 4GI and 4GII, thus disabling host cell protein synthesis. We used NMR to study full-length Lbpro as well as a shortened species lacking six C-terminal amino acid residues (sLbpro) to examine the mechanism of self-processing, the quaternary structure and the substrate specificity. Both Lbpro forms have the same structure in solution as in the crystal. In the solution structure of sLbpro, the 12 residue C-terminal extension was flexible and disordered. In contrast, the 18 residue C-terminal extension of full-length Lbpro was bound by the substrate-binding site of a neighbouring molecule, resulting in the formation of a stable dimer in solution. The Lbpro dimer could not be dissociated by increasing the ionic strength or by dilution. Furthermore, titration with model peptides mimicking the substrates destabilised the dimer interface without dissociating the dimer. The peptides were, however, bound by sLbpro in the canonical substrate binding site. Peptide binding gave rise to chemical shifts of residues around the sLbpro substrate binding site. Shifts of Asn146 and Glu147 indicated that these residues might form the enzyme's S1' site and interact with the P1' arginine residue of the eIF4GI cleavage site. Furthermore, differences in substrate specificity between sLbpro and Lbpro observed with an in vitro translated protein indicate some involvement of the C terminus in substrate recognition.

  3. Microbial genome analyses: global comparisons of transport capabilities based on phylogenies, bioenergetics and substrate specificities.

    PubMed

    Paulsen, I T; Sliwinski, M K; Saier, M H

    1998-04-01

    We have conducted genome sequence analyses of seven prokaryotic microorganisms for which completely sequenced genomes are available (Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Bacillus subtilis, Mycoplasma genitalium, Synechocystis PCC6803 and Methanococcus jannaschii). We report the distribution of encoded known and putative polytopic cytoplasmic membrane transport proteins within these genomes. Transport systems for each organism were classified according to (1) putative membrane topology, (2) protein family, (3) bioenergetics, and (4) substrate specificities. The overall transport capabilities of each organism were thereby estimated. Probable function was assigned to greater than 90% of the putative transport proteins identified. The results show the following: (1) Numbers of transport systems in eubacteria are approximately proportional to genome size and correspond to 9.7 to 10.8% of the total encoded genes except for H. pylori (5.4%), Synechocystis (4.7%) and M. jannaschii (3.5%) which exhibit substantially lower proportions. (2) The distribution of topological types is similar in all seven organisms. (3) Transport systems belonging to 67 families were identified within the genomes of these organisms, and about half of these families are also found in eukaryotes. (4) 12% of these families are found exclusively in Gram-negative bacteria, but none is found exclusively in Gram-positive bacteria, cyanobacteria or archaea. (5) Two superfamilies, the ATP-binding cassette (ABC) and major facilitator (MF) superfamilies account for nearly 50% of all transporters in each organism, but the relative representation of these two transporter types varies over a tenfold range, depending on the organism. (6) Secondary, pmf-dependent carriers are 1.5 to threefold more prevalent than primary ATP-dependent carriers in E. coli, H. influenzae, H. pylori and B. subtilis while primary carriers are about twofold more prevalent in M. genitalium and Synechocystis. M

  4. Characterization of a Naphthalene Dioxygenase Endowed with an Exceptionally Broad Substrate Specificity Toward Polycyclic Aromatic Hydrocarbons

    SciTech Connect

    Jouanneau,Y.; Meyer, C.; Jakoncic, J.; Stojanoff, V.; Gaillard, J.

    2006-01-01

    In Sphingomonas CHY-1, a single ring-hydroxylating dioxygenase is responsible for the initial attack of a range of polycyclic aromatic hydrocarbons (PAHs) composed of up to five rings. The components of this enzyme were separately purified and characterized. The oxygenase component (ht-PhnI) was shown to contain one Rieske-type [2Fe-2S] cluster and one mononuclear Fe center per {alpha} subunit, based on EPR measurements and iron assay. Steady-state kinetic measurements revealed that the enzyme had a relatively low apparent Michaelis constant for naphthalene (K{sub m} = 0.92 {+-} 0.15 {mu}M) and an apparent specificity constant of 2.0 {+-} 0.3 M{sup -1} s{sup -1}. Naphthalene was converted to the corresponding 1,2-dihydrodiol with stoichiometric oxidation of NADH. On the other hand, the oxidation of eight other PAHs occurred at slower rates and with coupling efficiencies that decreased with the enzyme reaction rate. Uncoupling was associated with hydrogen peroxide formation, which is potentially deleterious to cells and might inhibit PAH degradation. In single turnover reactions, ht-PhnI alone catalyzed PAH hydroxylation at a faster rate in the presence of organic solvent, suggesting that the transfer of substrate to the active site is a limiting factor. The four-ring PAHs chrysene and benz[a]anthracene were subjected to a double ring-dihydroxylation, giving rise to the formation of a significant proportion of bis-cis-dihydrodiols. In addition, the dihydroxylation of benz[a]anthracene yielded three dihydrodiols, the enzyme showing a preference for carbons in positions 1,2 and 10,11. This is the first characterization of a dioxygenase able to dihydroxylate PAHs made up of four and five rings.

  5. Comparison of Substrate Specificity of Escherichia Coli p-Aminobenzoyl-Glutamate Hydrolase with Pseudomonas Carboxypeptidase G

    PubMed Central

    Larimer, Cassandra M.; Slavnic, Dejan; Pitstick, Lenore D.; Green, Jacalyn M.

    2016-01-01

    Reduced folic acid derivatives support biosynthesis of DNA, RNA and amino acids in bacteria as well as in eukaryotes, including humans. While the genes and steps for bacterial folic acid synthesis are known, those associated with folic acid catabolism are not well understood. A folate catabolite found in both humans and bacteria is p-aminobenzoyl-glutamate (PABA-GLU). The enzyme p-aminobenzoyl-glutamate hydrolase (PGH) breaks down PABA-GLU and is part of an apparent operon, the abg region, in E. coli. The subunits of PGH possess sequence and catalytic similarities to carboxypeptidase enzymes from Pseudomonas species. A comparison of the subunit sequences and activity of PGH, relative to carboxypeptidase enzymes, may lead to a better understanding of bacterial physiology and pathway evolution. We first compared the amino acid sequences of AbgA, AbgB and carboxypeptidase G2 from Pseudomonas sp. RS-16, which has been crystallized. Then we compared the enzyme activities of E. coli PGH and commercially available Pseudomonas carboxypeptidase G using spectrophotometric assays measuring cleavage of PABA-GLU, folate, aminopterin, methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate. The Km and Vmax values for the folate and anti-folate substrates of PGH could not be determined, because the instrument reached its limit before the enzyme was saturated. Therefore, activity of PGH was compared to the activity of CPG, or normalized to PABA-GLU (nmole/min/µg). Relative to its activity with 10 µM PABA-GLU (100%), PGH cleaved glutamate from methotrexate (48%), aminopterin (45%) and folate (9%). Reduced folates leucovorin (5-formyltetrahydrofolate) and 5-methyltetrahydrofolate were not cleaved by PGH. Our data suggest that E. coli PGH is specific for PABA-GLU as its activity with natural folates (folate, 5-methyltetrahydrofolate, and leucovorin) was very poor. It does, however, have some ability to cleave anti-folates which may have clinical applications in

  6. Poly‐3‐hydroxyalkanoate synthases from Pseudomonas putida U: substrate specificity and ultrastructural studies

    PubMed Central

    Arias, Sagrario; Sandoval, Angel; Arcos, Mario; Cañedo, Librada M.; Maestro, Beatriz; Sanz, Jesús M.; Naharro, Germán; Luengo, José M.

    2008-01-01

    Summary The substrate specificity of the two polymerases (PhaC1 and PhaC2) involved in the biosynthesis of medium‐chain‐length poly‐hydroxyalkanoates (mcl PHAs) in Pseudomonas putida U has been studied in vivo. For these kind of experiments, two recombinant strains derived from a genetically engineered mutant in which the whole pha locus had been deleted (P. putida U Δpha) were employed. These bacteria, which expresses only phaC1 (P. putida U Δpha pMC‐phaC1)or only phaC2(P. putida U Δpha pMC‐phaC2), accumulated different PHAs in function of the precursor supplemented to the culture broth. Thus, the P. putida U Δpha pMC‐phaC1strain was able to synthesize several aliphatic and aromatic PHAs when hexanoic, heptanoic, octanoic decanoic, 5‐phenylvaleric, 6‐phenylhexanoic, 7‐phenylheptanoic, 8‐phenyloctanoic or 9‐phenylnonanoic acid were used as precursors; the highest accumulation of polymers was observed when the precursor used were decanoic acid (aliphatic PHAs) or 6‐phenylhexanoic acid (aromatic PHAs). However, although it synthesizes similar aliphatic PHAs (the highest accumulation was observed when hexanoic acid was the precursor) the other recombinant strain (P. putida U Δpha pMC‐phaC2) only accumulated aromatic PHAs when the monomer to be polymerized was 3‐hydroxy‐5‐phenylvaleryl‐CoA. The possible influence of the putative three‐dimensional structures on the different catalytic behaviour of PhaC1 and PhaC2 is discussed. PMID:21261834

  7. Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

    PubMed

    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. PMID:25111069

  8. Binding of Macrolide Antibiotics Leads to Ribosomal Selection against Specific Substrates Based on Their Charge and Size.

    PubMed

    Sothiselvam, Shanmugapriya; Neuner, Sandro; Rigger, Lukas; Klepacki, Dorota; Micura, Ronald; Vázquez-Laslop, Nora; Mankin, Alexander S

    2016-08-16

    Macrolide antibiotic binding to the ribosome inhibits catalysis of peptide bond formation between specific donor and acceptor substrates. Why particular reactions are problematic for the macrolide-bound ribosome remains unclear. Using comprehensive mutational analysis and biochemical experiments with synthetic substrate analogs, we find that the positive charge of these specific residues and the length of their side chains underlie inefficient peptide bond formation in the macrolide-bound ribosome. Even in the absence of antibiotic, peptide bond formation between these particular donors and acceptors is rather inefficient, suggesting that macrolides magnify a problem present for intrinsically difficult substrates. Our findings emphasize the existence of functional interactions between the nascent protein and the catalytic site of the ribosomal peptidyl transferase center. PMID:27498876

  9. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

    PubMed

    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.

  10. Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

    PubMed

    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. PMID:21067917

  11. Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes.

    PubMed

    Spite, Matthew; Baba, Shahid P; Ahmed, Yonis; Barski, Oleg A; Nijhawan, Kanchan; Petrash, J Mark; Bhatnagar, Aruni; Srivastava, Sanjay

    2007-07-01

    efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  13. Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity*

    PubMed Central

    Borisova, Anna S.; Isaksen, Trine; Dimarogona, Maria; Kognole, Abhishek A.; Mathiesen, Geir; Várnai, Anikó; Røhr, Åsmund K.; Payne, Christina M.; Sørlie, Morten; Sandgren, Mats; Eijsink, Vincent G. H.

    2015-01-01

    The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation. PMID:26178376

  14. Neural substrates for the processing of cognitive and affective aspects of taste in the brain.

    PubMed

    Yamamoto, Takashi

    2006-12-01

    Taste is unique among the sensory systems in that, besides its recognition of quality, it is innately associated with hedonic aspects of reward and aversion. This review of the literature will show how taste information is conveyed through the central gustatory pathways to the cortical gustatory area and is processed in terms of qualitative and quantitative aspects. Taste information is also sent to the reward system and feeding center via several brain sites including the prefrontal cortex, insular cortex, and amygdala. The reward system contains the ventral tegmental area, nucleus accumbens, and ventral pallidum; it finally sends information to the lateral hypothalamic area, the feeding center. The dopamine system originating from the ventral tegmental area mediates the motivation to consume palatable food. The actual ingestive behavior is promoted by the orexigenic neuropeptides from the hypothalamus. In the last section, the neural substrate of learning and memory of taste is introduced and the biological mechanisms are elucidated. PMID:17287579

  15. An examination of referential and affect specificity with five emotions in infancy.

    PubMed

    Martin, Nicole G; Maza, Lindey; McGrath, Savannah J; Phelps, Amber E

    2014-08-01

    Referential specificity and affect specificity were examined in 12- to 14-month-olds (n=20), and 16- to 18-month-olds (n=20). Infants were presented with a televised social referencing paradigm involving an actress who emoted a simple descriptive message to one of two objects appearing on the video. The actress altered her affective message using a neutral baseline first, followed by 5 discrete emotions (anger, fear, sadness, happiness, surprise). Infants were given 30s to interact with the objects after watching the affective episode. Older infants demonstrated referential and affect specificity, as evidenced by their differential treatment of the target and distracter toy in response to messages of anger, fear, surprise, and happiness. In contrast, the younger infants did not show evidence of either referential or affect specificity, as evidenced by the lack of differentiation in their treatment of the target and distracter toy in response to positive and negative emotional messages across all emotional episodes. PMID:24813588

  16. Structural insights into substrate specificity and solvent tolerance in alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541.

    PubMed

    Karabec, Martin; Łyskowski, Andrzej; Tauber, Katharina C; Steinkellner, Georg; Kroutil, Wolfgang; Grogan, Gideon; Gruber, Karl

    2010-09-14

    The structure of the alcohol dehydrogenase ADH-'A' from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

  17. Substrate and/or substrate-driven changes in the abundance of methanogenic archaea cause seasonal variation of methane production potential in species-specific freshwater wetlands.

    PubMed

    Liu, Deyan; Ding, Weixin; Yuan, Junji; Xiang, Jian; Lin, Yongxin

    2014-05-01

    There are large temporal and spatial variations of methane (CH4) emissions from natural wetlands. To understand temporal changes of CH4 production potential (MPP), soil samples were collected from a permanently inundated Carex lasiocarpa marsh and a summer inundated Calamagrostis angustifolia marsh over the period from June to October of 2011. MPP, dissolved organic carbon (DOC) concentration, abundance and community structure of methanogenic archaea were assessed. In the C. lasiocarpa marsh, DOC concentration, MPP and the methanogen population showed similar seasonal variations and maximal values in September. MPP and DOC in the C. angustifolia marsh exhibited seasonal variations and values peaked during August, while the methanogen population decreased with plant growth. Methanogen abundance correlated significantly (P = 0.02) with DOC only for the C. lasiocarpa marsh. During the sampling period, the dominant methanogens were the Methanosaetaceae and Zoige cluster I (ZC-Ι) in the C. angustifolia marsh, and Methanomicrobiales and ZC-Ι in the C. lasiocarpa marsh. MPP correlated significantly (P = 0.04) with DOC and methanogen population in the C. lasiocarpa marsh but only with DOC in the C. angustifolia marsh. Addition of C. lasiocarpa litter enhanced MPP more effectively than addition of C. angustifolia litter, indicating that temporal variation of substrates is controlled by litter deposition in the C. lasiocarpa marsh while living plant matter is more important in the C. angustifolia marsh. This study indicated that there was no apparent shift in the dominant types of methanogen during the growth season in the species-specific freshwater wetlands. Temporal variation of MPP is controlled by substrates and substrate-driven changes in the abundance of methanogenic archaea in the C. lasiocarpa marsh, while MPP depends only on substrate availability derived from root exudates or soil organic matter in the C. angustifolia marsh.

  18. Engineering the substrate specificity of rhizopuspepsin: the role of Asp 77 of fungal aspartic proteinases in facilitating the cleavage of oligopeptide substrates with lysine in P1.

    PubMed Central

    Lowther, W. T.; Majer, P.; Dunn, B. M.

    1995-01-01

    Rhizopuspepsin and other fungal aspartic proteinases are distinct from the mammalian enzymes in that they are able to cleave substrates with lysine in the P1 position. Sequence and structural comparisons suggest that two aspartic acid residues, Asp 30 and Asp 77 (pig pepsin numbering), may be responsible for generating this unique specificity. Asp 30 and Asp 77 were changed to the corresponding residues in porcine pepsin, Ile 30 and Thr 77, to create single and double mutants. The zymogen forms of the wild-type and mutant enzymes were overexpressed in Escherichia coli as inclusion bodies. Following solubilization, denaturation, refolding, activation, and purification to homogeneity, structural and kinetic comparisons were made. The mutant enzymes exhibited a high degree of structural similarity to the wild-type recombinant protein and a native isozyme. The catalytic activities of the recombinant proteins were analyzed with chromogenic substrates containing lysine in the P1, P2, or P3 positions. Mutation of Asp 77 resulted in a loss of 7 kcal mol-1 of transition-state stabilization energy in the hydrolysis of the substrate containing lysine in P1. An inhibitor containing the positively charged P1-lysine side chain inhibited only the enzymes containing Asp 77. Inhibition of the Asp 77 mutants of rhizopuspepsin and several mammalian enzymes was restored upon acetylation of the lysine side chain. These results suggest that an exploitation of the specific electrostatic interaction of Asp 77 in the active site of fungal enzymes may lead to the design of compounds that preferentially inhibit a variety of related Candida proteinases in immunocompromised patients. PMID:7613467

  19. Structural basis for substrate specificity of an amino acid ABC transporter.

    PubMed

    Yu, Jie; Ge, Jingpeng; Heuveling, Johanna; Schneider, Erwin; Yang, Maojun

    2015-04-21

    ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate a variety of substrates, ranging from ions to macromolecules, either out of or into the cytosol (hence defined as importers or exporters, respectively). It has been demonstrated that ABC exporters and importers function through a common mechanism involving conformational switches between inward-facing and outward-facing states; however, the mechanism underlying their functions, particularly substrate recognition, remains elusive. Here we report the structures of an amino acid ABC importer Art(QN)2 from Thermoanaerobacter tengcongensis composed of homodimers each of the transmembrane domain ArtQ and the nucleotide-binding domain ArtN, either in its apo form or in complex with substrates (Arg, His) and/or ATPs. The structures reveal that the straddling of the TMDs around the twofold axis forms a substrate translocation pathway across the membrane. Interestingly, each TMD has a negatively charged pocket that together create a negatively charged internal tunnel allowing amino acids carrying positively charged groups to pass through. Our structural and functional studies provide a better understanding of how ABC transporters select and translocate their substrates.

  20. Structural Insight into the Mechanism of Substrate Specificity of Aedes Kynurenine Aminotransferase

    SciTech Connect

    Han,Q.; Gao, Y.; Robinson, H.; Li, J.

    2008-01-01

    Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant a-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate {beta}-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and {alpha}7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.

  1. More intense experiences, less intense forecasts: why people overweight probability specifications in affective forecasts.

    PubMed

    Buechel, Eva C; Zhang, Jiao; Morewedge, Carey K; Vosgerau, Joachim

    2014-01-01

    We propose that affective forecasters overestimate the extent to which experienced hedonic responses to an outcome are influenced by the probability of its occurrence. The experience of an outcome (e.g., winning a gamble) is typically more affectively intense than the simulation of that outcome (e.g., imagining winning a gamble) upon which the affective forecast for it is based. We suggest that, as a result, experiencers allocate a larger share of their attention toward the outcome (e.g., winning the gamble) and less to its probability specifications than do affective forecasters. Consequently, hedonic responses to an outcome are less sensitive to its probability specifications than are affective forecasts for that outcome. The results of 6 experiments provide support for our theory. Affective forecasters overestimated how sensitive experiencers would be to the probability of positive and negative outcomes (Experiments 1 and 2). Consistent with our attentional account, differences in sensitivity to probability specifications disappeared when the attention of forecasters was diverted from probability specifications (Experiment 3) or when the attention of experiencers was drawn toward probability specifications (Experiment 4). Finally, differences in sensitivity to probability specifications between forecasters and experiencers were diminished when the forecasted outcome was more affectively intense (Experiments 5 and 6).

  2. More intense experiences, less intense forecasts: why people overweight probability specifications in affective forecasts.

    PubMed

    Buechel, Eva C; Zhang, Jiao; Morewedge, Carey K; Vosgerau, Joachim

    2014-01-01

    We propose that affective forecasters overestimate the extent to which experienced hedonic responses to an outcome are influenced by the probability of its occurrence. The experience of an outcome (e.g., winning a gamble) is typically more affectively intense than the simulation of that outcome (e.g., imagining winning a gamble) upon which the affective forecast for it is based. We suggest that, as a result, experiencers allocate a larger share of their attention toward the outcome (e.g., winning the gamble) and less to its probability specifications than do affective forecasters. Consequently, hedonic responses to an outcome are less sensitive to its probability specifications than are affective forecasts for that outcome. The results of 6 experiments provide support for our theory. Affective forecasters overestimated how sensitive experiencers would be to the probability of positive and negative outcomes (Experiments 1 and 2). Consistent with our attentional account, differences in sensitivity to probability specifications disappeared when the attention of forecasters was diverted from probability specifications (Experiment 3) or when the attention of experiencers was drawn toward probability specifications (Experiment 4). Finally, differences in sensitivity to probability specifications between forecasters and experiencers were diminished when the forecasted outcome was more affectively intense (Experiments 5 and 6). PMID:24128184

  3. Alteration of Substrate Specificity: The Variable N-Terminal Domain of Tobacco Ca2+-Dependent Protein Kinase Is Important for Substrate Recognition[W

    PubMed Central

    Ito, Takeshi; Nakata, Masaru; Fukazawa, Jutarou; Ishida, Sarahmi; Takahashi, Yohsuke

    2010-01-01

    Protein kinases are major signaling molecules that are involved in a variety of cellular processes. However, the molecular mechanisms whereby protein kinases discriminate specific substrates are still largely unknown. Ca2+-dependent protein kinases (CDPKs) play central roles in Ca2+ signaling in plants. Previously, we found that a tobacco (Nicotiana tabacum) CDPK1 negatively regulated the transcription factor REPRESSION OF SHOOT GROWTH (RSG), which is involved in gibberellin feedback regulation. Here, we found that the variable N-terminal domain of CDPK1 is necessary for the recognition of RSG. A mutation (R10A) in the variable N-terminal domain of CDPK1 reduced both RSG binding and RSG phosphorylation while leaving kinase activity intact. Furthermore, the R10A mutation suppressed the in vivo function of CDPK1. The substitution of the variable N-terminal domain of an Arabidopsis thaliana CDPK, At CPK9, with that of Nt CDPK1 conferred RSG kinase activities. This chimeric CDPK behaved according to the identity of the variable N-terminal domain in transgenic plants. Our results open the possibility of engineering the substrate specificity of CDPK by manipulation of the variable N-terminal domain, enabling a rational rewiring of cellular signaling pathways. PMID:20442373

  4. Detection of specific DNA using a microfluidic device featuring tethered poly(N-isopropylacrylamide) on a silicon substrate

    NASA Astrophysics Data System (ADS)

    Chen, Jem-Kun; Li, Jun-Yan

    2010-08-01

    In this study, we grafted thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm) onto a Si substrate as the medium in a microfluidic device to detect specific DNA molecules [human genomic DNA (hgDNA528), 528 bp] at extremely low concentrations (down to 2 ng/μl). After using the polymerase chain reaction to amplify the released human gDNA signal from the tethered PNIPAAm on the substrate, the amplified human gDNA molecules were characterized through agarose gel electrophoresis. The tethered PNIPAAm in the fluid device allowed the precise detection of the human gDNA.

  5. Caffeine Affects Time to Exhaustion and Substrate Oxidation during Cycling at Maximal Lactate Steady State.

    PubMed

    Cruz, Rogério Santos de Oliveira; de Aguiar, Rafael Alves; Turnes, Tiago; Guglielmo, Luiz Guilherme Antonacci; Beneke, Ralph; Caputo, Fabrizio

    2015-06-30

    This study analyzed the effects of caffeine intake on whole-body substrate metabolism and exercise tolerance during cycling by using a more individualized intensity for merging the subjects into homogeneous metabolic responses (the workload associated with the maximal lactate steady state-MLSS). MLSS was firstly determined in eight active males (25 ± 4 years, 176 ± 7 cm, 77 ± 11 kg) using from two to four constant-load tests of 30 min. On two following occasions, participants performed a test until exhaustion at the MLSS workload 1 h after taking either 6 mg/kg of body mass of caffeine or placebo (dextrose), in a randomized, double-blinded manner. Respiratory exchange ratio was calculated from gas exchange measurements. There was an improvement of 22.7% in time to exhaustion at MLSS workload following caffeine ingestion (95% confidence limits of ±10.3%, p = 0.002), which was accompanied by decrease in respiratory exchange ratio (p = 0.001). These results reinforce findings indicating that sparing of the endogenous carbohydrate stores could be one of the several physiological effects of caffeine during submaximal performance around 1 h.

  6. The second-shell metal ligands of human arginase affect coordination of the nucleophile and substrate.

    PubMed

    Stone, Everett M; Chantranupong, Lynne; Georgiou, George

    2010-12-14

    The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of l-arginine and the subsequent production of urea and l-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of >2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution. PMID:21053939

  7. Caffeine Affects Time to Exhaustion and Substrate Oxidation during Cycling at Maximal Lactate Steady State.

    PubMed

    Cruz, Rogério Santos de Oliveira; de Aguiar, Rafael Alves; Turnes, Tiago; Guglielmo, Luiz Guilherme Antonacci; Beneke, Ralph; Caputo, Fabrizio

    2015-07-01

    This study analyzed the effects of caffeine intake on whole-body substrate metabolism and exercise tolerance during cycling by using a more individualized intensity for merging the subjects into homogeneous metabolic responses (the workload associated with the maximal lactate steady state-MLSS). MLSS was firstly determined in eight active males (25 ± 4 years, 176 ± 7 cm, 77 ± 11 kg) using from two to four constant-load tests of 30 min. On two following occasions, participants performed a test until exhaustion at the MLSS workload 1 h after taking either 6 mg/kg of body mass of caffeine or placebo (dextrose), in a randomized, double-blinded manner. Respiratory exchange ratio was calculated from gas exchange measurements. There was an improvement of 22.7% in time to exhaustion at MLSS workload following caffeine ingestion (95% confidence limits of ±10.3%, p = 0.002), which was accompanied by decrease in respiratory exchange ratio (p = 0.001). These results reinforce findings indicating that sparing of the endogenous carbohydrate stores could be one of the several physiological effects of caffeine during submaximal performance around 1 h. PMID:26133971

  8. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

    PubMed

    Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

    2015-09-01

    Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

  9. Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis.

    PubMed

    Abeldenov, Sailau; Talhaoui, Ibtissam; Zharkov, Dmitry O; Ishchenko, Alexander A; Ramanculov, Erlan; Saparbaev, Murat; Khassenov, Bekbolat

    2015-09-01

    Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3'→5' exonuclease activities. However, it remains unclear whether these enzymes hold 3'-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3'-repair phosphodiesterase and 3'-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5mM MnCl2 at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37 °C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3'-blocking sugar-phosphate and 3'-phosphate moieties at DNA strand breaks with an extremely high efficiency (kcat/KM=440 and 1280 μM(-1)∙min(-1), respectively), while MtbNfo exhibits much lower 3'-repair activities (kcat/KM=0.26 and 0.65 μM(-1)∙min(-1), respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H2O2 to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role

  10. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations.

    PubMed

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-20

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed.

  11. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations.

    PubMed

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-01

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed. PMID:25603968

  12. Plant performance on Mediterranean green roofs: interaction of species-specific hydraulic strategies and substrate water relations

    PubMed Central

    Raimondo, Fabio; Trifilò, Patrizia; Lo Gullo, Maria A.; Andri, Sergio; Savi, Tadeja; Nardini, Andrea

    2015-01-01

    Recent studies have highlighted the ecological, economic and social benefits assured by green roof technology to urban areas. However, green roofs are very hostile environments for plant growth because of shallow substrate depths, high temperatures and irradiance and wind exposure. This study provides experimental evidence for the importance of accurate selection of plant species and substrates for implementing green roofs in hot and arid regions, like the Mediterranean area. Experiments were performed on two shrub species (Arbutus unedo L. and Salvia officinalis L.) grown in green roof experimental modules with two substrates slightly differing in their water retention properties, as derived from moisture release curves. Physiological measurements were performed on both well-watered and drought-stressed plants. Gas exchange, leaf and xylem water potential and also plant hydraulic conductance were measured at different time intervals following the last irrigation. The substrate type significantly affected water status. Arbutus unedo and S. officinalis showed different hydraulic responses to drought stress, with the former species being substantially isohydric and the latter one anisohydric. Both A. unedo and S. officinalis were found to be suitable species for green roofs in the Mediterranean area. However, our data suggest that appropriate choice of substrate is key to the success of green roof installations in arid environments, especially if anisohydric species are employed. PMID:25603968

  13. Timing of APC/C substrate degradation is determined by fzy/fzr specificity of destruction boxes

    PubMed Central

    Zur, Amit; Brandeis, Michael

    2002-01-01

    The anaphase promoting complex/cyclosome (APC/C), activated by fzy and fzr, degrades cell cycle proteins that carry RXXL or KEN destruction boxes (d-boxes). APC/C substrates regulate sequential events and must be degraded in the correct order during mitosis and G1. We studied how d-boxes determine APC/Cfzy/APC/Cfzr specificity and degradation timing. Cyclin B1 has an RXXL box and is degraded by both APC/Cfzy and APC/Cfzr; fzy has a KEN box and is degraded by APC/Cfzr only. We characterized the degradation of substrates with swapped d-boxes. Cyclin B1 with KEN was degraded by APC/Cfzr only. Fzy with RXXL could be degraded by APC/Cfzy and APC/Cfzr. Interestingly, APC/Cfzy- but not APC/Cfzr-specific degradation is highly dependent on the location of RXXL. We studied degradation of tagged substrates in real time and observed that APC/Cfzr is activated in early G1. These observations demonstrate how d-box specificities of APC/Cfzy and APC/Cfzr, and the successive activation of APC/C by fzy and fzr, establish the temporal degradation pattern. Our observations can explain further why some endogenous RXXL substrates are degraded by APC/Cfzy, while others are restricted to APC/Cfzr. PMID:12198152

  14. Specificity of Processing α-Glucosidase I Is Guided by the Substrate Conformation

    PubMed Central

    Barker, Megan K.; Rose, David R.

    2013-01-01

    Processing α-glucosidase I (GluI) is a key member of the eukaryotic N-glycosylation processing pathway, selectively catalyzing the first glycoprotein trimming step in the endoplasmic reticulum. Inhibition of GluI activity impacts the infectivity of enveloped viruses; however, despite interest in this protein from a structural, enzymatic, and therapeutic standpoint, little is known about its structure and enzymatic mechanism in catalysis of the unique glycan substrate Glc3Man9GlcNAc2. The first structural model of eukaryotic GluI is here presented at 2-Å resolution. Two catalytic residues are proposed, mutations of which result in catalytically inactive, properly folded protein. Using Autodocking methods with the known substrate and inhibitors as ligands, including a novel inhibitor characterized in this work, the active site of GluI was mapped. From these results, a model of substrate binding has been formulated, which is most likely conserved in mammalian GluI. PMID:23536181

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

    PubMed Central

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

    2016-01-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface. PMID:26955973

  16. Substrate specificity of proteolytic activity in the testes fluid and seminal plasma of the common carp Cyprinus carpio.

    PubMed

    Cejko, B I; Słowińska, M; Judycka, S; Kowalski, R K

    2016-05-01

    Substrate specificity in the seminal plasma and testes fluids of the common carp Cyprinus carpio was determined using gelatin, casein, albumin and haemoglobin. Proteolytic profiles of the testes and seminal plasma were compared. Different ranges of pH (5·5-9·5) and temperature (4-37° C) were used during incubations of seminal plasma proteinases. Differences in proteolytic activity between testes and seminal plasma may reflect specific functions of the testes and sperm ducts in semen production. Seminal plasma metalloproteinases were characterized by higher substrate specificity than were serine proteinases. Zymography optimization for seminal plasma indicated that pH 7·5 and 22° C were the optimal conditions for gel incubations. PMID:27001550

  17. Distinct Pools of Non-Glycolytic Substrates Differentiate Brain Regions and Prime Region-Specific Responses of Mitochondria

    PubMed Central

    Platt, Virginia; Budworth, Helen; Canaria, Christie A.; McMurray, Cynthia T.

    2013-01-01

    Many hereditary diseases are characterized by region-specific toxicity, despite the fact that disease-linked proteins are generally ubiquitously expressed. The underlying basis of the region-specific vulnerability remains enigmatic. Here, we evaluate the fundamental features of mitochondrial and glucose metabolism in synaptosomes from four brain regions in basal and stressed states. Although the brain has an absolute need for glucose in vivo, we find that synaptosomes prefer to respire on non-glycolytic substrates, even when glucose is present. Moreover, glucose is metabolized differently in each brain region, resulting in region-specific “signature” pools of non-glycolytic substrates. The use of non-glycolytic resources increases and dominates during energy crisis, and triggers a marked region-specific metabolic response. We envision that disease-linked proteins confer stress on all relevant brain cells, but region-specific susceptibility stems from metabolism of non-glycolytic substrates, which limits how and to what extent neurons respond to the stress. PMID:23874783

  18. PATIENT-SPECIFIC AND SURGERY-SPECIFIC FACTORS THAT AFFECT RETURN TO SPORT AFTER ACL RECONSTRUCTION

    PubMed Central

    Lynch, Andrew; Rabuck, Stephen; Lynch, Brittany; Davin, Sarah; Irrgang, James

    2016-01-01

    Context Anterior cruciate ligament (ACL) reconstruction is frequently performed to allow individuals to return to their pre-injury levels of sports participation, however, return to pre-injury level of sport is poor and re-injury rates are unacceptably high. Re-injury is likely associated with the timeframe and guidelines for return to sport (RTS). It is imperative for clinicians to recognize risk factors for re-injury and to ensure that modifiable risk factors are addressed prior to RTS. The purpose of this commentary is to summarize the current literature on the outcomes following return to sport after ACL reconstruction and to outline the biologic and patient-specific factors that should be considered when counseling an athlete on their progression through rehabilitation. Evidence Acquisition A comprehensive literature search was performed to identify RTS criteria and RTS rates after ACL reconstruction with consideration paid to graft healing, anatomic reconstruction, and risk factors for re-injury and revision. Results were screened for relevant original research articles and review articles, from which results were summarized. Study Design Clinical Review of the Literature Results Variable RTS rates are presented in the literature due to variable definitions of RTS ranging from a high threshold (return to competition) to low threshold (physician clearance for return to play). Re-injury and contralateral injury rates are greater than the risk for primary ACL injury, which may be related to insufficient RTS guidelines based on time from surgery, which do not allow for proper healing or resolution of post-operative impairments and elimination of risk factors associated with both primary and secondary ACL injuries. Conclusions RTS rates to pre-injury level of activity after ACLR are poor and the risk for graft injury or contralateral injury requiring an additional surgery is substantial. Resolving impairments while eliminating movement patterns associated with

  19. Cysteine is not a substrate but a specific modulator of human ASCT2 (SLC1A5) transporter.

    PubMed

    Scalise, Mariafrancesca; Pochini, Lorena; Pingitore, Piero; Hedfalk, Kristina; Indiveri, Cesare

    2015-11-30

    The Alanine Serine Cysteine Transporter 2 (ASCT2) is involved in balancing the intracellular amino acid pool. This function is allowed by the antiport mechanism and the asymmetric specificity towards different neutral amino acids, distinctive of this transporter. In the present work, the interaction of the putative substrate Cys with the human ASCT2 has been studied using the recombinant hASCT2 over-produced in Pichia pastoris and the native ASCT2 extracted from HeLa in both proteoliposomes and intact cells. It was found that Cys is a potent competitive inhibitor of hASCT2 but is not a substrate. Moreover, Cys binding to a second site, different from that of substrate, triggers a protein-mediated unidirectional Gln efflux.

  20. The Development of Affect Specificity in Infants' Use of Emotion Cues

    ERIC Educational Resources Information Center

    Martin, Nicole Gendler; Witherington, David C.; Edwards, Alison

    2008-01-01

    This study examined the emergence of affect specificity in infancy. In this study, infants received verbal and facial signals of 2 different, negatively valenced emotions (fear and sadness) as well as neutral affect via a television monitor to determine if they could make qualitative distinctions among emotions of the same valence. Twenty 12- to…

  1. Comparison of mesophilic and thermophilic feruloyl esterases: characterization of their substrate specificity for methyl phenylalkanoates.

    PubMed

    Topakas, Evangelos; Christakopoulos, Paul; Faulds, Craig B

    2005-02-23

    The active sites of feruloyl esterases from mesophilic and thermophilic sources were probed using methyl esters of phenylalkanoic acids. Only 13 out of 26 substrates tested were significant substrates for all the enzymes. Lengthening or shortening the aliphatic side chain while maintaining the same aromatic substitutions completely abolished activity for both enzymes, which demonstrates the importance of the correct distance between the aromatic group and the ester bond. Maintaining the phenylpropanoate structure but altering the substitutions of the aromatic ring demonstrated that the type-A esterase from the mesophilic fungus Fusarium oxysporum (FoFaeA) showed a preference for methoxylated substrates, in contrast to the type-B esterase from the same source (FoFaeB) and the thermophilic type-B (StFaeB) and type-C (StFaeC) from Sporotrichum thermophile, which preferred hydroxylated substrates. All four esterases hydrolyzed short chain aliphatic acid (C2-C4) esters of p-nitrophenol, but not the C12 ester of laurate. All the feruloyl esterases were able to release ferulic acid from the plant cell wall material in conjunction with a xylanase, but only the type-A esterase FoFaeA was effective in releasing the 5,5' form of diferulic acid. The thermophilic type-B esterase had a lower catalytic efficiency than its mesophilic counterpart, but released more ferulic acid from plant cell walls.

  2. Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

    PubMed Central

    Dias, Marcio V. B.; Huang, Fanglu; Chirgadze, Dimitri Y.; Tosin, Manuela; Spiteller, Dieter; Dry, Emily F. V.; Leadlay, Peter F.; Spencer, Jonathan B.; Blundell, Tom L.

    2010-01-01

    The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr42, His76, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg120 located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity. PMID:20430898

  3. In Vitro Comparison of the Activity Requirements and Substrate Specificity of Human and Triboleum castaneum PINK1 Orthologues

    PubMed Central

    Aerts, Liesbeth; Craessaerts, Katleen; De Strooper, Bart; Morais, Vanessa A.

    2016-01-01

    Mutations in the gene encoding the mitochondrial kinase PINK1 cause early-onset familial Parkinson’s disease. To understand the biological function of PINK1 and its role in the pathogenesis of Parkinson’s disease, it is useful to study its kinase activity towards substrates both in vivo and in vitro. For in vitro kinase assays, a purified Triboleum castaneum PINK1 insect orthologue is often employed, because it displays higher levels of activity when compared to human PINK1. We show, however, that the activity requirements, and more importantly the substrate specificity, differ between both orthologues. While Triboleum castaneum PINK1 readily phosphorylates the PINKtide peptide and Histone H1 in vitro, neither of these non-physiological substrates is phosphorylated by human PINK1. Nonetheless, both Tc and human PINK1 phosphorylate Parkin and Ubiquitin, two physiological substrates of PINK1. Our results show that the substrate selectivity differs among PINK1 orthologues, an important consideration that should be taken into account when extrapolating findings back to human PINK1. PMID:26784449

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

    PubMed Central

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

    2002-01-01

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

  5. Prediction of substrate specificity and preliminary kinetic characterization of the hypothetical protein PVX_123945 from Plasmodium vivax.

    PubMed

    Srinivasan, Bharath; Kempaiah Nagappa, Lakshmeesha; Shukla, Arpit; Balaram, Hemalatha

    2015-01-01

    Members of the haloacid dehalogenase (HAD) superfamily are emerging as an important group of enzymes by virtue of their role in diverse chemical reactions. In different Plasmodium species their number varies from 16 to 21. One of the HAD superfamily members, PVX_123945, a hypothetical protein from Plasmodium vivax, was selected for examining its substrate specificity. Based on distant homology searches and structure comparisons, it was predicted to be a phosphatase. Thirty-eight metabolites were screened to identify potential substrates. Further, to validate the prediction, biochemical and kinetic studies were carried out that showed that the protein was a monomer with high catalytic efficiency for β-glycerophosphate followed by pyridoxal 5'-phosphate. The enzyme also exhibited moderate catalytic efficiencies for α-glycerophosphate, xanthosine 5'-monophosphate and adenosine 5'-monophosphate. It also hydrolyzed the artificial substrate p-nitrophenyl phosphate (pNPP). Mg(2+) was the most preferred divalent cation and phosphate inhibited the enzyme activity. The study is the first attempt at understanding the substrate specificity of a hypothetical protein belonging to HAD superfamily from the malarial parasite P. vivax. PMID:25655405

  6. Replacement of serine 237 in class A beta-lactamase of Proteus vulgaris modifies its unique substrate specificity.

    PubMed

    Tamaki, M; Nukaga, M; Sawai, T

    1994-08-23

    The chromosomal beta-lactamase gene of Proteus vulgaris K1 was cloned and sequenced. The gene comprises 813 nucleotides and codes for the mature enzyme of 29,655 Da, comprising 271 amino acids. The K1 beta-lactamase showed 30-70% similarity, in the overall amino acid sequence, to class A beta-lactamases of Gram-negative bacteria. However, the K1 beta-lactamase differs from most class A enzymes in having a unique substrate specificity as a cephalosporinase, its spectrum extending to even oxyiminocephalosporins. To clarify the relationship between its unique substrate specificity and specific amino acid residues, alignment of the amino acid sequence of the K1 beta-lactamase with those of class A beta-lactamases was performed, and Ala104 and Ser237 were found to be candidates. Ala104 and Ser237 were replaced with glutamic acid and alanine, respectively, which are commonly found in other class A beta-lactamases. The substitution at position 104 had no effect on the enzyme activity or the substrate specificity. The amino acid replacement at position 237, however, reduced the kcat/Km value for an oxyiminocephalosporin (cefuroxime) to 17% of that in the case of the wild-type enzyme, whereas the mutant enzyme showed a higher kcat/Km value for benzylpenicillin, 3 times, than that of the wild-type enzyme. These results indicated that Ser237 is one of the residues responsible for the unique substrate specificity of the P. vulgaris beta-lactamase.

  7. Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member

    SciTech Connect

    Lim, Kap; Pullalarevu, Sadhana; Surabian, Karen Talin; Howard, Andrew; Suzuki, Tomohiko; Moult, John; Herzberg, Osnat

    2010-03-12

    a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely {alpha} or solely {beta} chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same closure motion that clasps the transition state analogue, in contrast to the transition state analogue complexes of CK, where the corresponding transition state analogue occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants of substrate specificity. Despite the equivalent binding in both active sites of the GK dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not supported in the case of GK.

  8. Structural basis of substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

    PubMed Central

    Yates, Susan P.; Edwards, Thomas E.; Bryan, Cassie M.; Stein, Adam J.; Van Voorhis, Wesley C.; Myler, Peter J.; Stewart, Lance J.; Zheng, Jimin; Jia, Zongchao

    2012-01-01

    Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). Based on the recent complex structure of AceK-ICDH from E. coli, we have classified the structures of homodimeric NADP+-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH which exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria. PMID:21870819

  9. Probing Steroidal Substrate Specificity of Cytochrome P450 BM3 Variants.

    PubMed

    Liu, Xing; Wang, Zhi-Biao; Wang, Ya-Nan; Kong, Jian-Qiang

    2016-01-01

    M01A82W, M11A82W and M01A82WS72I are three cytochrome P450 BM3 (CYP102A1) variants. They can catalyze the hydroxylation of testosterone (TES) and norethisterone at different positions, thereby making them promising biocatalysts for steroid hydroxylation. With the aim of obtaining more hydroxylated steroid precursors it is necessary to probe the steroidal substrate diversity of these BM3 variants. Here, three purified BM3 variants were first incubated with eight steroids, including testosterone (TES), methyltestosterone (MT), cholesterol, β-sitosterol, dehydroepiandrosterone (DHEA), diosgenin, pregnenolone and ergosterol. The results indicated that the two 3-keto-Δ⁴-steroids TES and MT can be hydroxylated at various positions by the three BM3 mutants, respectively. On the contrary, the three enzymes displayed no any activity toward the remaining six 3-hydroxy-Δ⁵-steroids. This result indicates that the BM3 mutants prefer 3-keto-Δ⁴-steroids as hydroxylation substrates. To further verify this notion, five other substrates, including two 3-hydroxy-Δ⁵-steroids and three 3-keto-Δ⁴-steroids, were carefully selected to incubate with the three BM3 variants. The results indicated the three 3-keto-Δ⁴-steroids can be metabolized to form hydroxysteroids by the three BM3 variants. On the other hand, the two 3-hydroxy-Δ⁵-steroids cannot be hydroxylated at any position by the BM3 mutants. These results further support the above conclusion, therefore demonstrating the 3-keto-Δ⁴-steroid substrate preference of BM3 mutants, and laying a foundation for microbial production of more hydroxylated steroid intermediates using BM3 variants. PMID:27294908

  10. Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity.

    PubMed

    Altmann, F; Schweiszer, S; Weber, C

    1995-02-01

    The initial velocities of hydrolysis of nineteen glycopeptides by peptide: N-glycosidase F and A were determined. Substrates were prepared from bovine fetuin, hen ovalbumin, pineapple stem bromelain, bovine fibrin and taka-amylase. From these glycopeptides, several variants with regard to peptide and carbohydrate structure were prepared and derivatized with dabsyl chloride, dansyl chloride or activated resorufin. Tyrosine containing glycopeptides were also used without an additional chromophore. Enzymatic hydrolysis of glycopeptides was quantified by narrow bore, reversed phase HPLC with turnaround cycle times of down to 6 min, but usually 15 min. KM values ranging from 30 to 64 microM and from 4 to 36 microM were found for N-glycosidase F and A, respectively. Relative velocities of hydrolysis of the different substrates by each enzyme varied considerably. Little, if any, similarity of the performance of N-glycosidase F and A with the different substrates was observed. The minimal carbohydrate structure released by peptide: N-glycosidase F was a di-N-acetylchitobiose. N-glycosidase A could release even a single N-acetylglucosamine, albeit 3000 times slower than a di-N-acetylchitobiose or larger glycans. In general the structure of the intact glycan had little effect on activity, and with both enzymes the rate of hydrolysis appeared to be primarily governed by peptide structure and length. However, N-glycosidase F did not release glycans alpha 1,3-fucosylated at the asparagine linked N-acetylglucosamine irrespective of the presence of xylose in the substrate.

  11. Structural insights into substrate specificity of Feruloyl-CoA 6’-Hydroxylase from Arabidopsis thaliana

    DOE PAGES

    Sun, Xinxiao; Zhou, Dayong; Kandavelu, Palani; Zhang, Hua; Yuan, Qipeng; Wang, Bi -Cheng; Rose, John; Yan, Yajun

    2015-05-20

    Coumarins belong to an important class of plant secondary metabolites. Feruloyl-CoA 6’-hydroxylase (F6’H), a 2-oxoglutarate dependent dioxygenase (2OGD), catalyzes a pivotal step in the biosynthesis of a simple coumarin scopoletin. In this study, we determined the 3-dimensional structure of the F6’H1 apo enzyme by X-ray crystallography. It is the first reported structure of a 2OGD enzyme involved in coumarin biosynthesis and closely resembles the structure of Arabidopsis thaliana anthocyanidin synthase. To better understand the mechanism of enzyme catalysis and substrate specificity, we also generated a homology model of a related ortho-hydroxylase (C2’H) from sweet potato. By comparing these two structures,more » we targeted two amino acid residues and verified their roles in substrate binding and specificity by site-directed mutagenesis.« less

  12. The catalytic efficiency of yeast ribonuclease III depends on substrate specific product release rate

    PubMed Central

    Comeau, Marc-Andre; Lafontaine, Daniel A.; Abou Elela, Sherif

    2016-01-01

    Members of the ribonuclease III (RNase III) family regulate gene expression by triggering the degradation of double stranded RNA (dsRNA). Hundreds of RNase III cleavage targets have been identified and their impact on RNA maturation and stability is now established. However, the mechanism defining substrates’ reactivity remains unclear. In this study, we developed a real-time FRET assay for the detection of dsRNA degradation by yeast RNase III (Rnt1p) and characterized the kinetic bottlenecks controlling the reactivity of different substrates. Surprisingly, the results indicate that Rnt1p cleavage reaction is not only limited by the rate of catalysis but can also depend on base-pairing of product termini. Cleavage products terminating with paired nucleotides, like the degradation signals found in coding mRNA sequence, were less reactive and more prone to inhibition than products having unpaired nucleotides found in non-coding RNA substrates. Mutational analysis of U5 snRNA and Mig2 mRNA confirms the pairing of the cleavage site as a major determinant for the difference between cleavage rates of coding and non-coding RNA. Together the data indicate that the base-pairing of Rnt1p substrates encodes reactivity determinants that permit both constitutive processing of non-coding RNA while limiting the rate of mRNA degradation. PMID:27257067

  13. Insights into Substrate Specificity and Metal Activation of Mammalian Tetrahedral Aspartyl Aminopeptidase

    SciTech Connect

    Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.

    2012-07-11

    Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases.

  14. Fatty Acid Methyl Ester (FAME) Succession in Different Substrates as Affected by the Co-Application of Three Pesticides

    PubMed Central

    Cardinali, Alessandra; Pizzeghello, Diego; Zanin, Giuseppe

    2015-01-01

    Introduction In intensive agriculture areas the use of pesticides can alter soil properties and microbial community structure with the risk of reducing soil quality. Materials and Methods In this study the fatty acid methyl esters (FAMEs) evolution has been studied in a factorial lab experiment combining five substrates (a soil, two aged composts and their mixtures) treated with a co-application of three pesticides (azoxystrobin, chlorotoluron and epoxiconazole), with two extraction methods, and two incubation times (0 and 58 days). FAMEs extraction followed the microbial identification system (MIDI) and ester-linked method (EL). Results and Discussion The pesticides showed high persistence, as revealed by half-life (t1/2) values ranging from 168 to 298 days, which confirms their recalcitrance to degradation. However, t1/2 values were affected by substrate and compost age down to 8 days for chlorotoluron in S and up to 453 days for epoxiconazole in 12M. Fifty-six FAMEs were detected. Analysis of variance (ANOVA) showed that the EL method detected a higher number of FAMEs and unique FAMEs than the MIDI one, whereas principal component analysis (PCA) highlighted that the monosaturated 18:1ω9c and cyclopropane 19:0ω10c/19ω6 were the most significant FAMEs grouping by extraction method. The cyclopropyl to monoenoic acids ratio evidenced higher stress conditions when pesticides were applied to compost and compost+soil than solely soil, as well as with final time. Conclusion Overall, FAMEs profiles showed the importance of the extraction method for both substrate and incubation time, the t1/2 values highlighted the effectiveness of solely soil and the less mature compost in reducing the persistence of pesticides. PMID:26694029

  15. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity

    PubMed Central

    Herod, Morgan R.; Prince, Cynthia A.; Skilton, Rachel J.; Ward, Vernon K.; Cooper, Jonathan B.; Clarke, Ian N.

    2014-01-01

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  16. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.

    PubMed

    Herod, Morgan R; Prince, Cynthia A; Skilton, Rachel J; Ward, Vernon K; Cooper, Jonathan B; Clarke, Ian N

    2014-12-15

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  17. The role of substrate specificity and metal binding in defining the activity and structure of an intracellular subtilisin.

    PubMed

    Gamble, Michael; Künze, Georg; Brancale, Andrea; Wilson, Keith S; Jones, D Dafydd

    2012-01-01

    The dimeric intracellular subtilisin proteases (ISPs) found throughout Gram-positive bacteria are a structurally distinct class of the subtilase family. Unlike the vast majority of subtilisin-like proteases, the ISPs function exclusively within the cell, contributing the majority of observed cellular proteolytic activity. Given that they are active within the cell, little is known about substrate specificity and the role of stress signals such as divalent metal ions in modulating ISP function. We demonstrate that both play roles in defining the proteolytic activity of Bacillus clausii ISP and propose the molecular basis of their effects. Enzyme kinetics reveal that one particular synthetic tetrapeptide substrate, Phe-Ala-Ala-Phe-pNA, is hydrolysed with a catalytic efficiency ∼100-fold higher than any other tested. Heat-denatured whole proteins were found to be better substrates for ISP than the native forms. Substrate binding simulations suggest that the S1, S2 and S4 sites form defined binding pockets. The deep S1 cavity and wide S4 site are fully occupied by the hydrophobic aromatic side-chains of Phe. Divalent metal ions, probably Ca(2+), are proposed to be important for ISP activity through structural changes. The presence of >0.01 mM EDTA inactivates ISP, with CD and SEC suggesting that the protein becomes less structured and potentially monomeric. Removal of Ca(2+) at sites close to the dimer interface and the S1 pocket are thought to be responsible for the effect. These studies provide a new insight into the potential physiological function of ISPs, by reconciling substrate specificity and divalent metal binding to associate ISP with the unfolded protein response under stress conditions. PMID:23650602

  18. Nitrogen form affects pH and EC of whole pine tree substrate and growth of petunia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Wood-based substrates are potential alternatives or amendments to traditional peat-based and pine bark substrates. Undesirable changes in substrate pH may result from the application of supplemental fertilizer required by some crops grown in wood-based substrates. Experiments were conducted to evalu...

  19. Frequency-modulated 50 kHz ultrasonic vocalizations: a tool for uncovering the molecular substrates of positive affect.

    PubMed

    Burgdorf, Jeffrey; Panksepp, Jaak; Moskal, Joseph R

    2011-10-01

    The evidence that frequency modulated (FM) 50 kHz ultrasonic vocalizations (USVs) reflect a positive emotional state in rats is reviewed. Positive emotional states in humans are measured by facial-vocal displays (e.g., Duchenne smiling and laughter), approach behavior, and subjective self-report of feeling states. In laboratory animals, only facial-vocal displays, along with approach behavior can be measured. FM 50 kHz USVs are uniquely elevated by hedonic stimuli and suppressed by aversive stimuli. Rates of FM 50 kHz USVs are positively correlated to the rewarding value of the eliciting stimulus. Additionally, playbacks of these vocalizations are rewarding. The neural and pharmacological substrates of 50 kHz USVs are consistent with those of human positive affective states. By experimentally eliciting FM 50 kHz USVs, the novel molecular underpinning of positive affect can be elucidated and may be similar to those in humans. In humans, positive emotional states confer resilience to depression and anxiety, as well as promote overall health. Using rough-and-tumble play induced hedonic USVs, we have identified insulin like growth factor I and the NR2B subunit of the NMDA receptor as playing a functional role in positive affective states. From this research, we have developed a promising new class of antidepressants that is entering phase II clinical trials for the treatment of depression. PMID:21144859

  20. The impact of dispositional cynicism on job-specific affect and work intentions.

    PubMed

    Roberts, Taylor Peyton; Zigarmi, Drea

    2014-10-01

    Working from the Employee Work Passion Appraisal (EWPA) model, this article examines the relationship between employee dispositional cynicism, job-specific affect (i.e. positive and negative) and work intentions including intent to use discretionary effort, intent to perform, intent to endorse, intent to stay and intent to be an organisational citizen. An online survey generated participation from 747 current and potential clients of an international consulting company. To evaluate the fit of the data in accordance with the EWPA framework, structural equation modeling was conducted to test the overall fit of the proposed model and to examine the hypothesised relationships between constructs. Analyses confirmed correlations between dispositional cynicism and job-specific affect, supported notable relationships between positive job-specific affect and all work intentions, provided evidence for job-specific affect's mediation of cynicism and work intentions and uncovered a direct negative relationship between cynicism and intent to use organisational citizenship behaviour. Results suggest that state-specific workplace emotions are important for understanding the degree to which employee dispositional cynicism will ultimately influence most performance-related work intentions. However, independent of affect, employee cynicism may directly result in somewhat lower intentions to help others at work. Study limitations and practical implications for employee selection and training are considered.

  1. The impact of dispositional cynicism on job-specific affect and work intentions.

    PubMed

    Roberts, Taylor Peyton; Zigarmi, Drea

    2014-10-01

    Working from the Employee Work Passion Appraisal (EWPA) model, this article examines the relationship between employee dispositional cynicism, job-specific affect (i.e. positive and negative) and work intentions including intent to use discretionary effort, intent to perform, intent to endorse, intent to stay and intent to be an organisational citizen. An online survey generated participation from 747 current and potential clients of an international consulting company. To evaluate the fit of the data in accordance with the EWPA framework, structural equation modeling was conducted to test the overall fit of the proposed model and to examine the hypothesised relationships between constructs. Analyses confirmed correlations between dispositional cynicism and job-specific affect, supported notable relationships between positive job-specific affect and all work intentions, provided evidence for job-specific affect's mediation of cynicism and work intentions and uncovered a direct negative relationship between cynicism and intent to use organisational citizenship behaviour. Results suggest that state-specific workplace emotions are important for understanding the degree to which employee dispositional cynicism will ultimately influence most performance-related work intentions. However, independent of affect, employee cynicism may directly result in somewhat lower intentions to help others at work. Study limitations and practical implications for employee selection and training are considered. PMID:25178960

  2. Substrate specificity of mammalian folylpolyglutamate synthetase for 5,10-dideazatetrahydrofolate analogs.

    PubMed

    Habeck, L L; Mendelsohn, L G; Shih, C; Taylor, E C; Colman, P D; Gossett, L S; Leitner, T A; Schultz, R M; Andis, S L; Moran, R G

    1995-08-01

    The metabolism of 5,10-dideazatetrahydrofolate (DDATHF [lometrexol]) to polyglutamate derivatives by folylpoly-gamma-glutamate synthetase (FPGS) plays a central role in the activity of this compound as an antineoplastic agent. The availability of a series of DDATHF derivatives differing in structure throughout the molecule has allowed a study of the structural requirements for substrate activity with mouse liver and hog liver FPGS. Kinetics of the polyglutamation reaction in vitro have been related to the potency of these compounds as inhibitors of the growth of human CEM leukemic cells. The structure-activity relationships for enzyme from both sources were nearly identical. FPGS from both species showed a broad acceptance for structural changes in the pyridopyrimidine ring, in the phenyl group, and in the intermediate bridge region, with structural changes in these regions being reflected in changes in Km for FPGS but much more modest alterations in Vmax. The data suggested that the phenyl ring was not contributing to any pi-pi hydrophobic interactions. It appeared to function primarily in maintaining a favorable distance between the pyridopyrimidine ring and the glutamate side chain. The lowest Km values were found for DDATHF analogs in which there were small alterations at the 10 position, e.g., 5-deazatetrahydrofolate, 10-methyl-DDATHF, and 10-formyl-5-deazatetrahydrofolate; the first-order rate constants for these substrates were the highest in this series, an indication of the efficiency of polyglutamation at low substrate concentrations. After correction for the intrinsic inhibitory activity of the parent DDATHF analog as an inhibitor of the target enzyme, the first-order rate constants for FPGS were found to be predictive of the potency of tumor cell growth inhibition for most of the compounds in this structural series.

  3. Molecular Determinants of Substrate Specificity in Sodium-coupled Glutamate Transporters.

    PubMed

    Silverstein, Nechama; Ewers, David; Forrest, Lucy R; Fahlke, Christoph; Kanner, Baruch I

    2015-11-27

    Crystal structures of the archaeal homologue GltPh have provided important insights into the molecular mechanism of transport of the excitatory neurotransmitter glutamate. Whereas mammalian glutamate transporters can translocate both glutamate and aspartate, GltPh is only one capable of aspartate transport. Most of the amino acid residues that surround the aspartate substrate in the binding pocket of GltPh are highly conserved. However, in the brain transporters, Thr-352 and Met-362 of the reentrant hairpin loop 2 are replaced by the smaller Ala and Thr, respectively. Therefore, we have studied the effects of T352A and M362T on binding and transport of aspartate and glutamate by GltPh. Substrate-dependent intrinsic fluorescence changes were monitored in transporter constructs containing the L130W mutation. GltPh-L130W/T352A exhibited an ~15-fold higher apparent affinity for l-glutamate than the wild type transporter, and the M362T mutation resulted in an increased affinity of ~40-fold. An even larger increase of the apparent affinity for l-glutamate, around 130-fold higher than that of wild type, was observed with the T352A/M362T double mutant. Radioactive uptake experiments show that GltPh-T352A not only transports aspartate but also l-glutamate. Remarkably, GltPh-M362T exhibited l-aspartate but not l-glutamate transport. The double mutant retained the ability to transport l-glutamate, but its kinetic parameters were very similar to those of GltPh-T352A alone. The differential impact of mutation on binding and transport of glutamate suggests that hairpin loop 2 not only plays a role in the selection of the substrate but also in its translocation.

  4. Evaluation of Factor Xa-Specific Chromogenic Substrate Assays and the Determination of Pharmacokinetics of Fondaparinux.

    PubMed

    Yuri, Maiko; Tabe, Yoko; Tsuchiya, Koji; Sadatsuki, Ryo; Aoki, Jun; Horii, Takashi; Iba, Toshiaki; Ohsaka, Akimichi

    2016-07-01

    Fondaparinux (FPX), a synthesized factor Xa inhibitor, is one of the most popular anticoagulants for the prevention of postoperative venous thromboembolism (VTE). Although routine monitoring is not required, the bleeding adverse events cannot be neglected, and the measurement of anti-Xa activity is expected to be monitored. The primary purpose of this study is to evaluate the performances of 2 chromogenic assays for the detection of anti-Xa activity. Furthermore, the pharmacokinetics of FPX was examined using chromogenic assays. Anti-Xa activity was measured using 2 FPX-based chromogenic substrates (S2222 and STA-Liquid Anti-Xa). The reproducibility, detection limits, linearity, and correlations between the substrates were examined using normal plasma doped with low and high concentrations of FPX formulation. In addition, anti-Xa activity in 235 clinical samples from 164 cases treated was measured, and the pharmacokinetics of FPX was evaluated. Both of the tested substrates were capable of accurately measuring the anti-Xa activity of FPX, with a lower limit of 0.05 μg/mL and a coefficient of variation of less than 10%. The repeated administration of FPX induced a gradual but significant increase in the anti-Xa activity, which was negatively correlated with body weight and estimated glomerular filtration rate. No significant correlation between the anti-Xa activity and the occurrence of postoperative VTE or bleeding event was observed. Anti-Xa activity can be successfully determined using 2 chromogenic assays and automated biochemical analyzers. The clinical significance of anti-Xa activity monitoring should be examined in the future study. PMID:26177660

  5. Enzymatic activities and DNA substrate specificity of Mycobacterium tuberculosis DNA helicase XPB.

    PubMed

    Balasingham, Seetha V; Zegeye, Ephrem Debebe; Homberset, Håvard; Rossi, Marie L; Laerdahl, Jon K; Bohr, Vilhelm A; Tønjum, Tone

    2012-01-01

    XPB, also known as ERCC3 and RAD25, is a 3' → 5' DNA repair helicase belonging to the superfamily 2 of helicases. XPB is an essential core subunit of the eukaryotic basal transcription factor complex TFIIH. It has two well-established functions: in the context of damaged DNA, XPB facilitates nucleotide excision repair by unwinding double stranded DNA (dsDNA) surrounding a DNA lesion; while in the context of actively transcribing genes, XPB facilitates initiation of RNA polymerase II transcription at gene promoters. Human and other eukaryotic XPB homologs are relatively well characterized compared to conserved homologs found in mycobacteria and archaea. However, more insight into the function of bacterial helicases is central to understanding the mechanism of DNA metabolism and pathogenesis in general. Here, we characterized Mycobacterium tuberculosis XPB (Mtb XPB), a 3'→5' DNA helicase with DNA-dependent ATPase activity. Mtb XPB efficiently catalyzed DNA unwinding in the presence of significant excess of enzyme. The unwinding activity was fueled by ATP or dATP in the presence of Mg(2+)/Mn(2+). Consistent with the 3'→5' polarity of this bacterial XPB helicase, the enzyme required a DNA substrate with a 3' overhang of 15 nucleotides or more. Although Mtb XPB efficiently unwound DNA model substrates with a 3' DNA tail, it was not active on substrates containing a 3' RNA tail. We also found that Mtb XPB efficiently catalyzed ATP-independent annealing of complementary DNA strands. These observations significantly enhance our understanding of the biological roles of Mtb XPB.

  6. The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana.

    PubMed

    Caparrós-Martín, José A; McCarthy-Suárez, Iva; Culiáñez-Macià, Francisco A

    2014-09-01

    The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943-954, 2013). In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous DL-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for DL-glycerol-3-phosphate. PMID:24915748

  7. The kinetic analysis of the substrate specificity of motif 5 in a HAD hydrolase-type phosphosugar phosphatase of Arabidopsis thaliana.

    PubMed

    Caparrós-Martín, José A; McCarthy-Suárez, Iva; Culiáñez-Macià, Francisco A

    2014-09-01

    The Arabidopsis thaliana gene AtSgpp (locus tag At2g38740), encodes a protein whose sequence motifs and expected structure reveal that it belongs to the HAD hydrolases subfamily I, with the C1-type cap domain (Caparrós-Martín et al. in Planta 237:943-954, 2013). In the presence of Mg(2+) ions, the enzyme has a phosphatase activity over a wide range of phosphosugar substrates. AtSgpp promiscuity is preferentially detectable on D-ribose-5-phosphate, 2-deoxy-D-ribose-5-phosphate, 2-deoxy-D-glucose-6-phosphate, D-mannose-6-phosphate, D-fructose-1-phosphate, D-glucose-6-phosphate, DL-glycerol-3-phosphate, and D-fructose-6-phosphate. Site-directed mutagenesis analysis of the putative signature sequence motif-5 (IAGKH), which defines its specific chemistry, brings to light the active-site residues Ala-69 and His-72. Mutation A69M, changes the pH dependence of AtSgpp catalysis, and mutant protein AtSgpp-H72K was inactive in phosphomonoester dephosphorylation. It was also observed that substitutions I68M and K71R slightly affect the substrate specificity, while the replacement of the entire motif for that of homologous DL-glycerol-3-phosphatase AtGpp (MMGRK) does not switch AtSgpp activity to the specific targeting for DL-glycerol-3-phosphate.

  8. Role of aspartate 143 in Escherichia coli tRNA-guanine transglycosylase: alteration of heterocyclic substrate specificity.

    PubMed

    Todorov, Katherine Abold; Garcia, George A

    2006-01-17

    tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the post-transcriptional modification of certain tRNAs in their anticodon wobble positions with queuine. To maintain the correct Watson-Crick base pairing properties of the wobble base (and hence proper translation of the genetic code), TGT must recognize its heterocyclic substrate with high specificity. The X-ray crystal structure of a eubacterial TGT bound to preQ1 [Romier, C., et al. (1996) EMBO J. 15, 2850-2857] suggested that aspartate 143 (Escherichia coli TGT numbering) was involved in heterocyclic substrate recognition. Subsequent mutagenic and computational modeling studies from our lab [Todorov, K. A., et al. (2005) Biophys. J. 89 (3), 1965-1977] provided experimental evidence supporting this hypothesis. Herein, we report further studies probing the differential heterocyclic substrate recognition properties of the aspartate 143 mutant TGTs. Our results are consistent with one of the mutants exhibiting an inversion of substrate recognition preference (xanthine vs guanine) relative to that of the wild type, as evidenced by Km values. This confirms the key role of aspartate 143 in maintaining the anticodon identities of the queuine-containing tRNAs and suggests that TGT mutants could be developed that would alter the tRNA wobble base base pairing properties.

  9. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme.

    PubMed

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P-Y; Wang, Steven S-S

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer's disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12-16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12-16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12-16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1-7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1-7)C and qf-Aβ(12-16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells.

  10. Design of Peptide Substrate for Sensitively and Specifically Detecting Two Aβ-Degrading Enzymes: Neprilysin and Angiotensin-Converting Enzyme

    PubMed Central

    Chen, Po-Ting; Chen, Chao-Long; Lin, Lilian Tsai-Wei; Lo, Chun-Hsien; Hu, Chaur-Jong; Chen, Rita P.-Y.; Wang, Steven S.-S.

    2016-01-01

    Upregulation of neprilysin (NEP) to reduce Aβ accumulation in the brain is a promising strategy for the prevention of Alzheimer’s disease (AD). This report describes the design and synthesis of a quenched fluorogenic peptide substrate qf-Aβ(12–16)AAC (with the sequence VHHQKAAC), which has a fluorophore, Alexa-350, linked to the side-chain of its C-terminal cysteine and a quencher, Dabcyl, linked to its N-terminus. This peptide emitted strong fluorescence upon cleavage. Our results showed that qf-Aβ(12–16)AAC is more sensitive to NEP than the previously reported peptide substrates, so that concentrations of NEP as low as 0.03 nM could be detected at peptide concentration of 2 μM. Moreover, qf-Aβ(12–16)AAC had superior enzymatic specificity for both NEP and angiotensin-converting enzyme (ACE), but was inert with other Aβ-degrading enzymes. This peptide, used in conjunction with a previously reported peptide substrate qf-Aβ(1–7)C [which is sensitive to NEP and insulin-degrading enzyme (IDE)], could be used for high-throughput screening of compounds that only upregulate NEP. The experimental results of cell-based activity assays using both qf-Aβ(1–7)C and qf-Aβ(12–16)AAC as the substrates confirm that somatostatin treatment most likely upregulates IDE, but not NEP, in neuroblastoma cells. PMID:27096746

  11. Probing the substrate specificity of the bacterial Pnkp/Hen1 RNA repair system using synthetic RNAs.

    PubMed

    Zhang, Can; Chan, Chio Mui; Wang, Pei; Huang, Raven H

    2012-02-01

    Ribotoxins cleave essential RNAs involved in protein synthesis as a strategy for cell killing. RNA repair systems exist in nature to counteract the lethal actions of ribotoxins, as first demonstrated by the RNA repair system from bacteriophage T4 25 yr ago. Recently, we found that two bacterial proteins, named Pnkp and Hen1, form a stable complex and are able to repair ribotoxin-cleaved tRNAs in vitro. However, unlike the well-studied T4 RNA repair system, the natural RNA substrates of the bacterial Pnkp/Hen1 RNA repair system are unknown. Here we present comprehensive RNA repair assays with the recombinant Pnkp/Hen1 proteins from Anabaena variabilis using a total of 33 different RNAs as substrates that might mimic various damaged forms of RNAs present in living cells. We found that unlike the RNA repair system from bacteriophage T4, the bacterial Pnkp/Hen1 RNA repair system exhibits broad substrate specificity. Based on the experimental data presented here, a model of preferred RNA substrates of the Pnkp/Hen1 repair system is proposed.

  12. The Structure of Allophanate Hydrolase from Granulibacter bethesdensis Provides Insights into Substrate Specificity in the Amidase Signature Family

    SciTech Connect

    Lin, Yi; Maurice, Martin

    2013-01-02

    Allophanate hydrolase (AH) catalyzes the hydrolysis of allophanate, an intermediate in atrazine degradation and urea catabolism pathways, to NH3 and CO2. AH belongs to the amidase signature family, which is characterized by a conserved block of 130 amino acids rich in Gly and Ser and a Ser-cis-Ser-Lys catalytic triad. In this study, the first structures of AH fromGranulibacter bethesdensis were determined, with and without the substrate analogue malonate, to 2.2 and 2.8 Å, respectively. The structures confirm the identity of the catalytic triad residues and reveal an altered dimerization interface that is not conserved in the amidase signature family. The structures also provide insights into previously unrecognized substrate specificity determinants in AH. Two residues, Tyr299 and Arg307, are within hydrogen bonding distance of a carboxylate moiety of malonate. Both Tyr299 and Arg307 were mutated, and the resulting modified enzymes revealed >3 order of magnitude reductions in both catalytic efficiency and substrate stringency. It is proposed that Tyr299 and Arg307 serve to anchor and orient the substrate for attack by the catalytic nucleophile, Ser172. The structure further suggests the presence of a unique C-terminal domain in AH. While this domain is conserved, it does not contribute to catalysis or to the structural integrity of the core domain, suggesting that it may play a role in mediating transient and specific interactions with the urea carboxylase component of urea amidolyase. Analysis of the AH active site architecture offers new insights into common determinants of catalysis and specificity among divergent members of the amidase signature family.

  13. On the substrate specificity of bacterial DD-peptidases: evidence from two series of peptidoglycan-mimetic peptides.

    PubMed Central

    Anderson, John W; Adediran, Suara A; Charlier, Paulette; Nguyen-Distèche, Martine; Frère, Jean-Marie; Nicholas, Robert A; Pratt, Rex F

    2003-01-01

    The reactions between bacterial DD-peptidases and beta-lactam antibiotics have been studied for many years. Less well understood are the interactions between these enzymes and their natural substrates, presumably the peptide moieties of peptidoglycan. In general, remarkably little activity has previously been demonstrated in vitro against potential peptide substrates, although in many cases the peptides employed were non-specific and not homologous with the relevant peptidoglycan. In this paper, the specificity of a panel of DD-peptidases against elements of species-specific D-alanyl-D-alanine peptides has been assessed. In two cases, those of soluble, low-molecular-mass DD-peptidases, high activity against the relevant peptides has been demonstrated. In these cases, the high specificity is towards the free N-terminus of the peptidoglycan fragment. With a number of other enzymes, particularly high-molecular-mass DD-peptidases, little or no activity against these peptides was observed. In separate experiments, the reactivity of the enzymes against the central, largely invariant, peptide stem was examined. None of the enzymes surveyed showed high activity against this structural element although weak specificity in the expected direction towards the one structural variable (D-gammaGln versus D-gammaGlu) was observed. The current state of understanding of the activity of these enzymes in vitro is discussed. PMID:12723972

  14. Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.

    PubMed

    Torres, Leticia L; Cantero, Angel; del Valle, Mercedes; Marina, Anabel; López-Gallego, Fernando; Guisán, José M; Berenguer, José; Hidalgo, Aurelio

    2013-03-01

    A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or β24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

  15. A corpora allata farnesyl diphosphate synthase in mosquitoes displaying a metal ion dependent substrate specificity

    PubMed Central

    Rivera-Perez, Crisalejandra; Nyati, Pratik; Noriega, Fernando G.

    2015-01-01

    Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis, it catalyzes the head-to-tail condensation of dimethylallyl diphosphate (DMAPP) with two molecules of isopentenyl diphosphate (IPP) to generate farnesyl diphosphate (FPP), a precursor of juvenile hormone (JH). In this study, we functionally characterized an Aedes aegypti FPPS (AaFPPS) expressed in the corpora allata. AaFPPS is the only FPPS gene present in the genome of the yellow fever mosquito, it encodes a 49.6 kDa protein exhibiting all the characteristic conserved sequence domains on prenyltransferases. AaFPPS displays its activity in the presence of metal cofactors; and the product condensation is dependent of the divalent cation. Mg2+ ions lead to the production of FPP, while the presence of Co2+ ions lead to geranyl diphosphate (GPP) production. In the presence of Mg2+ the AaFPPS affinity for allylic substrates is GPP>DMAPP>IPP. These results suggest that AaFPPS displays “catalytic promiscuity”, changing the type and ratio of products released (GPP or FPP) depending on allylic substrate concentrations and the presence of different metal cofactors. This metal ion-dependent regulatory mechanism allows a single enzyme to selectively control the metabolites it produces, thus potentially altering the flow of carbon into separate metabolic pathways. PMID:26188328

  16. Comparative Analysis of the Substrate Specificity of trans- versus cis-Acyltransferases of Assembly Line Polyketide Synthases

    PubMed Central

    2015-01-01

    Due to their pivotal role in extender unit selection during polyketide biosynthesis, acyltransferase (AT) domains are important engineering targets. A subset of assembly line polyketide synthases (PKSs) are serviced by discrete, trans-acting ATs. Theoretically, these trans-ATs can complement an inactivated cis-AT, promoting introduction of a noncognate extender unit. This approach requires a better understanding of the substrate specificity and catalytic mechanism of naturally occurring trans-ATs. We kinetically analyzed trans-ATs from the disorazole and kirromycin synthases and compared them to a representative cis-AT from the 6-deoxyerythronolide B synthase (DEBS). During transacylation, the disorazole AT favored malonyl-CoA over methylmalonyl-CoA by >40000-fold, whereas the kirromycin AT favored ethylmalonyl-CoA over methylmalonyl-CoA by 20-fold. Conversely, the disorazole AT had broader specificity than its kirromycin counterpart for acyl carrier protein (ACP) substrates. The presence of the ACP had little effect on the specificity (kcat/KM) of the cis-AT domain for carboxyacyl-CoA substrates but had a marked influence on the corresponding specificity parameters for the trans-ATs, suggesting that these enzymes do not act strictly by a canonical ping-pong mechanism. To investigate the relevance of the kinetic analysis of isolated ATs in the context of intact PKSs, we complemented an in vitro AT-null DEBS assembly line with either trans-AT. Whereas the disorazole AT efficiently complemented the mutant PKS at substoichiometric protein ratios, the kirromycin AT was considerably less effective. Our findings suggest that knowledge of both carboxyacyl-CoA and ACP specificity is critical to the choice of a trans-AT in combination with a mutant PKS to generate novel polyketides. PMID:24871074

  17. Particle geometry affects differentially substrate composition and enzyme profiles by Pleurotus ostreatus growing on sugar cane bagasse.

    PubMed

    Membrillo, Isabel; Sánchez, Carmen; Meneses, Marcos; Favela, Ernesto; Loera, Octavio

    2011-01-01

    The growth of Pleurotus ostreatus was analyzed on three particle sizes of sugar cane bagasse: 0.92 mm and 1.68 mm in diameter, in addition to heterogeneous fibers (average 2.9 mm in diameter). Specific growth rate on heterogeneous particles was lower (μ=0.043 h(-1)), although soluble protein production was maximal (809 μg/g dry wt). Higher μ values were reached on the other two particles sizes (0.049-0.05 h(-1)) with less soluble protein (500 μg/g dry wt). Xylanases and laccases were favored in heterogeneous particles; while the highest selectivity for xylanases over cellulases was observed in 1.68 mm particles, corresponding with the maximal hemicellulose breakdown. Lignin and cellulose were preferentially degraded in smallest particles. This study shows that the geometrical ratio, shape and size of sugar cane bagasse fibers strongly influence packing density for SSF substrate, with an impact in the production of extracellular enzymes, growth rates and composition changes in substrate.

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

    PubMed Central

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

    2016-01-01

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

  19. Anomeric specificity and protein-substrate interactions support the 3D model for the hemagglutinin-neuraminidase from sendai virus.

    PubMed

    Bellini, T; Pasti, C; Manfrinato, M C; Tomasi, M; Dallocchio, F

    1999-08-27

    The 3D structure of paramyxovirus hemagglutinin-neuraminidase has not yet been resolved; however, a theoretical model has been built by using influenza virus and bacterial neuraminidases as template [V. C. Epa (1997) Proteins Struct. Funct. Gen. 29, 264-281]. Two common features of the catalytic mechanism of the neuraminidases of known 3D structure are the anomeric specificity and the involvement of a tyrosine residue in the stabilization of the transition state. These key features have been investigated on the water-soluble ectodomain of the hemagglutinin-neuraminidase from Sendai virus (cHN). The anomeric specificity of the hydrolysis of the substrate by cHN has been investigated by NMR spectroscopy. The immediate product of the reaction was the alpha-anomer, meaning that cHN belongs between glycohydrolases retaining anomeric configuration like influenza virus neuraminidase. Measurements of the UV difference spectrum upon binding of the substrate analogue 2,3-dehydro 2-deossi N-acetyl neuraminic acid indicate the ionization of a tyrosine residue and decreased polarity in the environment of a tryptophan residue. Functional significance of the spectral data was derived from the known structure of influenza neuraminidase, where a tyrosinate ion is involved in the stabilization of the transition-state carbonium ion, and a tryptophan residue is involved in the binding of the acetyl moiety of the substrate. The data give experimental support to the 3D model of paramyxovirus neuraminidase.

  20. Structural and mutational analyses of dipeptidyl peptidase 11 from Porphyromonas gingivalis reveal the molecular basis for strict substrate specificity

    PubMed Central

    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

  1. From specificity to sensitivity: affective states modulate visual working memory for emotional expressive faces

    PubMed Central

    Maran, Thomas; Sachse, Pierre; Furtner, Marco

    2015-01-01

    Previous findings suggest that visual working memory (VWM) preferentially remembers angry looking faces. However, the meaning of facial actions is construed in relation to context. To date, there are no studies investigating the role of perceiver-based context when processing emotional cues in VWM. To explore the influence of affective context on VWM for faces, we conducted two experiments using both a VWM task for emotionally expressive faces and a mood induction procedure. Affective context was manipulated by unpleasant (Experiment 1) and pleasant (Experiment 2) IAPS pictures in order to induce an affect high in motivational intensity (defensive or appetitive, respectively) compared to a low arousal control condition. Results indicated specifically increased sensitivity of VWM for angry looking faces in the neutral condition. Enhanced VWM for angry faces was prevented by inducing affects of high motivational intensity. In both experiments, affective states led to a switch from specific enhancement of angry expressions in VWM to an equally sensitive representation of all emotional expressions. Our findings demonstrate that emotional expressions are of different behavioral relevance for the receiver depending on the affective context, supporting a functional organization of VWM along with flexible resource allocation. In VWM, stimulus processing adjusts to situational requirements and transitions from a specifically prioritizing default mode in predictable environments to a sensitive, hypervigilant mode in exposure to emotional events. PMID:26379609

  2. The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

    PubMed

    Bijelic, Aleksandar; Pretzler, Matthias; Molitor, Christian; Zekiri, Florime; Rompel, Annette

    2015-12-01

    Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed. PMID:26473311

  3. The Structure of a Plant Tyrosinase from Walnut Leaves Reveals the Importance of "Substrate-Guiding Residues" for Enzymatic Specificity.

    PubMed

    Bijelic, Aleksandar; Pretzler, Matthias; Molitor, Christian; Zekiri, Florime; Rompel, Annette

    2015-12-01

    Tyrosinases and catechol oxidases are members of the class of type III copper enzymes. While tyrosinases accept both mono- and o-diphenols as substrates, only the latter substrate is converted by catechol oxidases. Researchers have been working for decades to elucidate the monophenolase/diphenolase specificity on a structural level and have introduced an early hypothesis that states that the reason for the lack of monophenolase activity in catechol oxidases may be its structurally restricted active site. However, recent structural and biochemical studies of this enzyme class have raised doubts about this theory. Herein, the first crystal structure of a plant tyrosinase (from Juglans regia) is presented. The structure reveals that the distinction between mono- and diphenolase activity does not depend on the degree of restriction of the active site, and thus a more important role for amino acid residues located at the entrance to and in the second shell of the active site is proposed.

  4. Substrate specificity screening of oat (Avena sativa) seeds aminopeptidase demonstrate unusually broad tolerance in S1 pocket.

    PubMed

    Gajda, Anna D; Pawełczak, Małgorzata; Drag, Marcin

    2012-05-01

    Aminopeptidases are proteolytic enzymes that remove one amino acid at a time from N-terminus of peptidic substrates. In plants, inhibitors of aminopeptidases can find potential applications in agriculture as herbicides. In this report we have used a library of fluorogenic derivatives of natural and unnatural amino acids for substrate specificity profiling of oat (Avena sativa) aminopeptidase. Interestingly, we have found that this enzyme recognizes effectively among the natural amino acids basic residues like Arg and Lys, hydrophobic Phe, Leu and Met, but also to some extent acidic residues Asp and Glu. In the case of unnatural amino acids hydrophobic residues (hPhe and hCha) and basic hArg were preferentially recognized.

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

    PubMed Central

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

    2010-01-01

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

  6. Understanding the molecular basis of substrate binding specificity of PTB domains

    PubMed Central

    Sain, Neetu; Tiwari, Garima; Mohanty, Debasisa

    2016-01-01

    Protein-protein interactions mediated by phosphotyrosine binding (PTB) domains play a crucial role in various cellular processes. In order to understand the structural basis of substrate recognition by PTB domains, multiple explicit solvent atomistic simulations of 100ns duration have been carried out on 6 PTB-peptide complexes with known binding affinities. MM/PBSA binding energy values calculated from these MD trajectories and residue based statistical pair potential score show good correlation with the experimental dissociation constants. Our analysis also shows that the modeled structures of PTB domains can be used to develop less compute intensive residue level statistical pair potential based approaches for predicting interaction partners of PTB domains. PMID:27526776

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

    PubMed

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

    2008-09-01

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

  8. Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.

    PubMed

    Maddock, Danielle J; Patrick, Wayne M; Gerth, Monica L

    2015-08-01

    Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.

  9. Toward an evolutionary perspective on conceptual representation: species-specific calls activate visual and affective processing systems in the macaque.

    PubMed

    Gil-da-Costa, Ricardo; Braun, Allen; Lopes, Marco; Hauser, Marc D; Carson, Richard E; Herscovitch, Peter; Martin, Alex

    2004-12-14

    Non-human primates produce a diverse repertoire of species-specific calls and have rich conceptual systems. Some of their calls are designed to convey information about concepts such as predators, food, and social relationships, as well as the affective state of the caller. Little is known about the neural architecture of these calls, and much of what we do know is based on single-cell physiology from anesthetized subjects. By using positron emission tomography in awake rhesus macaques, we found that conspecific vocalizations elicited activity in higher-order visual areas, including regions in the temporal lobe associated with the visual perception of object form (TE/TEO) and motion (superior temporal sulcus) and storing visual object information into long-term memory (TE), as well as in limbic (the amygdala and hippocampus) and paralimbic regions (ventromedial prefrontal cortex) associated with the interpretation and memory-encoding of highly salient and affective material. This neural circuitry strongly corresponds to the network shown to support representation of conspecifics and affective information in humans. These findings shed light on the evolutionary precursors of conceptual representation in humans, suggesting that monkeys and humans have a common neural substrate for representing object concepts. PMID:15583132

  10. A Chlorogenic Acid Esterase with a Unique Substrate Specificity from Ustilago maydis

    PubMed Central

    Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G.

    2014-01-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM−1 s−1, 7.63 mM−1 s−1, 3.83 mM−1 s−1 and 3.75 mM−1 s−1, respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme. PMID:25548041

  11. A chlorogenic acid esterase with a unique substrate specificity from Ustilago maydis.

    PubMed

    Nieter, Annabel; Haase-Aschoff, Paul; Kelle, Sebastian; Linke, Diana; Krings, Ulrich; Popper, Lutz; Berger, Ralf G

    2015-03-01

    An extracellular chlorogenic acid esterase from Ustilago maydis (UmChlE) was purified to homogeneity by using three separation steps, including anion-exchange chromatography on a Q Sepharose FF column, preparative isoelectric focusing (IEF), and, finally, a combination of affinity chromatography and hydrophobic interaction chromatography on polyamide. SDS-PAGE analysis suggested a monomeric protein of ∼71 kDa. The purified enzyme showed maximal activity at pH 7.5 and at 37°C and was active over a wide pH range (3.5 to 9.5). Previously described chlorogenic acid esterases exhibited a comparable affinity for chlorogenic acid, but the enzyme from Ustilago was also active on typical feruloyl esterase substrates. Kinetic constants for chlorogenic acid, methyl p-coumarate, methyl caffeate, and methyl ferulate were as follows: Km values of 19.6 μM, 64.1 μM, 72.5 μM, and 101.8 μM, respectively, and kcat/Km values of 25.83 mM(-1) s(-1), 7.63 mM(-1) s(-1), 3.83 mM(-1) s(-1) and 3.75 mM(-1) s(-1), respectively. UmChlE released ferulic, p-coumaric, and caffeic acids from natural substrates such as destarched wheat bran (DSWB) and coffee pulp (CP), confirming activity on complex plant biomass. The full-length gene encoding UmChlE consisted of 1,758 bp, corresponding to a protein of 585 amino acids, and was functionally produced in Pichia pastoris GS115. Sequence alignments with annotated chlorogenic acid and feruloyl esterases underlined the uniqueness of this enzyme.

  12. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  13. Substrate specificity and mapping of residues critical for transport in the high-affinity glutathione transporter Hgt1p.

    PubMed

    Zulkifli, Mohammad; Yadav, Shambhu; Thakur, Anil; Singla, Shiffalli; Sharma, Monika; Bachhawat, Anand Kumar

    2016-08-01

    The high-affinity glutathione transporter Hgt1p of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter (OPT) family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher affinity for reduced glutathione (GSH), whereas it transported oxidized glutathione (GSSG) and other glutathione conjugates with lower affinity. To identify the residues of Hgt1p critical for substrate binding and translocation, all amino acid residues of the 13 predicted transmembrane domains (TMDs) have been subjected to mutagenesis. Functional evaluation of these 269 mutants by growth and biochemical assay followed by kinetic analysis of the severely defective mutants including previous mutagenic studies on this transporter have led to the identification of N124 (TMD1), V185 (TMD3), Q222, G225 and Y226 (TMD4), P292 (TMD5), Y374 (TMD6), L429 (TMD7) and F523 and Q526 (TMD9) as critical for substrate binding with at least 3-fold increase in Km upon mutagenesis to alanine. In addition residues Y226 and Y374 appeared to be important for differential substrate specificity. An ab initio model of Hgt1p was built and refined using these mutagenic data that yielded a helical arrangement that includes TMD3, TMD4, TMD5, TMD6, TMD7, TMD9 and TMD13 as pore-lining helices with the functionally important residues in a channel-facing orientation. Taken together the results of this study provides the first mechanistic insights into glutathione transport by a eukaryotic high-affinity glutathione transporter. PMID:27252386

  14. Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase

    PubMed Central

    2016-01-01

    Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to

  15. The protection of individuals affected with Specific Learning Disorders in the Italian Legislation.

    PubMed

    Feola, A; Marino, V; Masullo, A; Trabucco Aurilio, M; Marsella, L T

    2015-01-01

    Specific Learning Disorders (SLDs) affect specific abilities in individuals with an otherwise normal academic development. Among Italian School population, their reported prevalence is between 2.5% and 3.5%. Dysfunctions at the base of these disorders interfere with the normal acquisition process of reading, writing and/or mathematical abilities, leading to various degrees of adjustment difficulties in the affected individuals. The aim of this study was to assess the support that Italian Government offers to its citizens affected with SLDs, with a particular focus on assistance during the school-age years, particularly through the introduction of the Law 170/2010 and successive guidelines, supplementing the existing regulations to offer more efficient means and legal instruments aimed at achieving earlier diagnoses. PMID:26152629

  16. Subunit Composition and Substrate Specificity of a MOF-containing Histone Acetyltransferase Distinct from the Male-specific Lethal (MSL) Complex*

    PubMed Central

    Cai, Yong; Jin, Jingji; Swanson, Selene K.; Cole, Michael D.; Choi, Seung Hyuk; Florens, Laurence; Washburn, Michael P.; Conaway, Joan W.; Conaway, Ronald C.

    2010-01-01

    Human MOF (MYST1), a member of the MYST (Moz-Ybf2/Sas3-Sas2-Tip60) family of histone acetyltransferases (HATs), is the human ortholog of the Drosophila males absent on the first (MOF) protein. MOF is the catalytic subunit of the male-specific lethal (MSL) HAT complex, which plays a key role in dosage compensation in the fly and is responsible for a large fraction of histone H4 lysine 16 (H4K16) acetylation in vivo. MOF was recently reported to be a component of a second HAT complex, designated the non-specific lethal (NSL) complex (Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen, M., Buscaino, A., Duncan, K., Mueller, J., Wilm, M., Stunnenberg, H. G., Saumweber, H., and Akhtar, A. (2006) Mol. Cell 21, 811–823). Here we report an analysis of the subunit composition and substrate specificity of the NSL complex. Proteomic analyses of complexes purified through multiple candidate subunits reveal that NSL is composed of nine subunits. Two of its subunits, WD repeat domain 5 (WDR5) and host cell factor 1 (HCF1), are shared with members of the MLL/SET family of histone H3 lysine 4 (H3K4) methyltransferase complexes, and a third subunit, MCRS1, is shared with the human INO80 chromatin-remodeling complex. In addition, we show that assembly of the MOF HAT into MSL or NSL complexes controls its substrate specificity. Although MSL-associated MOF acetylates nucleosomal histone H4 almost exclusively on lysine 16, NSL-associated MOF exhibits a relaxed specificity and also acetylates nucleosomal histone H4 on lysines 5 and 8. PMID:20018852

  17. Altering the substrate specificity of polyhydroxyalkanoate synthase 1 derived from Pseudomonas putida GPo1 by localized semirandom mutagenesis.

    PubMed

    Sheu, Der-Shyan; Lee, Chia-Yin

    2004-07-01

    The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1(Pp), class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA- as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB(-)4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB(-)4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB(-)4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum. PMID:15205419

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

    PubMed

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

    2016-04-01

    Diacylglycerol acyltransferases (DGATs) catalyze a rate-limiting step of triacylglycerol (TAG) biosynthesis in higher plants and yeast. The genome of the green alga Chlamydomonas reinhardtii has multiple genes encoding type 2 DGATs (DGTTs). Here we present detailed functional and biochemical analyses of Chlamydomonas DGTTs. In vitro enzyme analysis using a radiolabel-free assay revealed distinct substrate specificities of three DGTTs: CrDGTT1 preferred polyunsaturated acyl CoAs, CrDGTT2 preferred monounsaturated acyl CoAs, and CrDGTT3 preferred C16 CoAs. When diacylglycerol was used as the substrate, CrDGTT1 preferred C16 over C18 in the sn-2 position of the glycerol backbone, but CrDGTT2 and CrDGTT3 preferred C18 over C16. In vivo knockdown of CrDGTT1, CrDGTT2 or CrDGTT3 resulted in 20-35% decreases in TAG content and a reduction of specific TAG fatty acids, in agreement with the findings of the in vitro assay and fatty acid feeding test. These results demonstrate that CrDGTT1, CrDGTT2 and CrDGTT3 possess distinct specificities toward acyl CoAs and diacylglycerols, and may work in concert spatially and temporally to synthesize diverse TAG species in C. reinhardtii. CrDGTT1 was shown to prefer prokaryotic lipid substrates and probably resides in both the endoplasmic reticulum and chloroplast envelope, indicating its role in prokaryotic and eukaryotic TAG biosynthesis. Based on these findings, we propose a working model for the role of CrDGTT1 in TAG biosynthesis. This work provides insight into TAG biosynthesis in C. reinhardtii, and paves the way for engineering microalgae for production of biofuels and high-value bioproducts. PMID:26919811

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

    PubMed

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

    2016-04-01

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

  20. Cloning and substrate specificity of a human phenol UDP-glucuronosyltransferase expressed in COS-7 cells.

    PubMed Central

    Harding, D; Fournel-Gigleux, S; Jackson, M R; Burchell, B

    1988-01-01

    A rat kidney phenol UDP-glucuronosyltransferase cDNA was used to isolate a human liver phenol UDP-glucuronosyltransferase cDNA by screening of a human liver cDNA library in the expression vector lambda gt11. The 2.4-kilobase cDNA contained an open reading frame of 1593 base pairs coding for a protein of 531 residues. The human liver cDNA was subcloned into the vector pKCRH2. Transfection of this recombinant plasmid into COS-7 cells allowed the expression of a protein of approximately 55 kDa. The enzyme synthesized was a glycoprotein, as indicated by a reduction in molecular mass of approximately 3 kDa after biosynthesis in the presence of tunicamycin. The expressed enzyme rapidly catalyzed the glucuronidation of 1-naphthol, 4-methylumbelliferone, and 4-nitrophenol. The use of a related series of simple phenols provided an outline description of the substituent restrictions imposed upon the phenolic structures accepted as substrates. The glucuronidation of testosterone, androsterone, and estrone was not catalyzed by this cloned UDP-glucuronosyltransferase. Images PMID:3141926

  1. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    SciTech Connect

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A.; Lees-Miller, S.P.; Lintott, L.G.; Sakaguchi, Kazuyasu; Appella, E.

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  2. Pathways and substrate specificity of DMSP catabolism in marine bacteria of the Roseobacter clade.

    PubMed

    Dickschat, Jeroen S; Zell, Claudia; Brock, Nelson L

    2010-02-15

    The volatiles released by Phaeobacter gallaeciensis, Oceanibulbus indolifex and Dinoroseobacter shibae have been investigated by GC-MS, and several MeSH-derived sulfur volatiles have been identified. An important sulfur source in the oceans is the algal metabolite dimethylsulfoniopropionate (DMSP). Labelled [2H6]DMSP was fed to the bacteria to investigate the production of volatiles from this compound through the lysis pathway to [2H6]dimethylsulfide or the demethylation pathway to [2H3]-3-(methylmercapto)propionic acid and lysis to [2H3]MeSH. [2H6]DMSP was efficiently converted to [2H3]MeSH by all three species. Several DMSP derivatives were synthesised and used in feeding experiments. Strong dealkylation activity was observed for the methylated ethyl methyl sulfoniopropionate and dimethylseleniopropionate, as indicated by the formation of EtSH- and MeSeH-derived volatiles, whereas no volatiles were formed from dimethyltelluriopropionate. In contrast, the dealkylation activity for diethylsulfoniopropionate was strongly reduced, resulting in only small amounts of EtSH-derived volatiles accompanied by diethyl sulfide in P. gallaeciensis and O. indolifex, while D. shibae produced the related oxidation product diethyl sulfone. The formation of diethyl sulfide and diethyl sulfone requires the lysis pathway, which is not active for [2H6]DMSP. These observations can be explained by a shifted distribution between the two competing pathways due to a blocked dealkylation of ethylated substrates.

  3. Specific features of changes in levels of endogenous respiration substrates in Saccharomyces cerevisiae cells at low temperature.

    PubMed

    Aliverdieva, D A; Mamaev, D V; Lagutina, L S; Sholtz, K F

    2006-01-01

    The rate of endogenous respiration of Saccharomyces cerevisiae cells incubated at 0 degrees C under aerobic conditions in the absence of exogenous substrates decreased exponentially with a half-period of about 5 h when measured at 30 degrees C. This was associated with an indirectly shown decrease in the level of oxaloacetate in the mitochondria in situ. The initial concentration of oxaloacetate significantly decreased the activity of succinate dehydrogenase. The rate of cell respiration in the presence of acetate and other exogenous substrates producing acetyl-CoA in mitochondria also decreased, whereas the respiration rate on succinate increased. These changes were accompanied by an at least threefold increase in the L-malate concentration in the cells within 24 h. It is suggested that the increase in the L-malate level in the cells and the concurrent decrease in the oxaloacetate level in the mitochondria should be associated with a deceleration at 0 degrees C of the transport of endogenous respiration substrates from the cytosol into the mitochondria. This deceleration is likely to be caused by a high Arrhenius activation energy specific for transporters. The physiological significance of L-malate in regulation of the S. cerevisiae cell respiration is discussed.

  4. Substrate specificity, substrate channeling, and allostery in BphJ: an acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI.

    PubMed

    Baker, Perrin; Carere, Jason; Seah, Stephen Y K

    2012-06-01

    BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD(+) and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. PMID:22574886

  5. The SEEKING mind: primal neuro-affective substrates for appetitive incentive states and their pathological dynamics in addictions and depression.

    PubMed

    Alcaro, Antonio; Panksepp, Jaak

    2011-10-01

    Appetitive motivation and incentive states are essential functions sustained by a common emotional brain process, the SEEKING disposition, which drives explorative and approach behaviors, sustains goal-directed activity, promotes anticipatory cognitions, and evokes feelings of positive excitement which control reward-learning. All such functions are orchestrated by the same "archetypical" neural processes, activated in ancient subcortical areas and transported to the forebrain by the mesolimbic dopamine (ML-DA) system. In mammals, the neurophysiology of the SEEKING urge is expressed by DA-promoted high-frequency oscillations, in the form of transient and synchronized gamma waves (>30Hz) emerging in limbic forebrain and diffusing throughout basal ganglia-thalamocortical (BG-T-C) circuits. These patterns may be considered basic "SEEKING neurodynamic impulses" which represent the primary-process exploratory disposition getting integrated with information relative to the external and the internal environment. Abnormal manifestation of SEEKING and its neural substrates are evident in clinical depression and addiction. Specifically, depression is characterized by reduced recruitment of SEEKING, while addictions reflect re-organizations of the SEEKING disposition around ultra-specific appetitive memories and compulsive activities. PMID:21396397

  6. The SEEKING mind: primal neuro-affective substrates for appetitive incentive states and their pathological dynamics in addictions and depression.

    PubMed

    Alcaro, Antonio; Panksepp, Jaak

    2011-10-01

    Appetitive motivation and incentive states are essential functions sustained by a common emotional brain process, the SEEKING disposition, which drives explorative and approach behaviors, sustains goal-directed activity, promotes anticipatory cognitions, and evokes feelings of positive excitement which control reward-learning. All such functions are orchestrated by the same "archetypical" neural processes, activated in ancient subcortical areas and transported to the forebrain by the mesolimbic dopamine (ML-DA) system. In mammals, the neurophysiology of the SEEKING urge is expressed by DA-promoted high-frequency oscillations, in the form of transient and synchronized gamma waves (>30Hz) emerging in limbic forebrain and diffusing throughout basal ganglia-thalamocortical (BG-T-C) circuits. These patterns may be considered basic "SEEKING neurodynamic impulses" which represent the primary-process exploratory disposition getting integrated with information relative to the external and the internal environment. Abnormal manifestation of SEEKING and its neural substrates are evident in clinical depression and addiction. Specifically, depression is characterized by reduced recruitment of SEEKING, while addictions reflect re-organizations of the SEEKING disposition around ultra-specific appetitive memories and compulsive activities.

  7. Effects of temperature and substrate stoichiometry on microbial specific respiration rate, carbon use efficiency, and 13C fractionation

    NASA Astrophysics Data System (ADS)

    Min, K.; Lehmeier, C.; Sellers, M.; Chen, Y.; Ballantyne, F.; Billings, S. A.

    2013-12-01

    Microbial activity contributes up to 60% of soil respiration. However, uncertainty in microbial respiration with rising temperature has previously prevented better predictions of the amount and the source of carbon (C) respired from soil. Three key variables of microbial C economies are of particular interest for estimating microbially mediated C release with temperature: (1) specific respiration rate (SRR), which is microbial CO2 release per microbial biomass-C, (2) carbon use efficiency (CUE), which determines how much organic C consumed by microbes is transformed into biomass, and (3) changes in the δ13C of respired CO2 with temperature, which suggests the form of organic C mineralized and helps to partition soil respiration in plant- and microbe-derived CO2. However, it is difficult to obtain these variables in intact soils, due to confounding factors that influence the amount and δ13C of respired CO2. Here we present an experimental approach that allows us to grow an isolated microbial population on well-characterized organic substrates and directly measure SRR, CUE and δ13C of respired CO2. We explored the effect of temperature on those variables, and how it changes with C:N of the substrate provided. This is important given various substrates available for microbial decay, and the potential for changing microbial CUE with substrate C:N. This approach thus can help constrain potential microbial C loss with warming as soil organic substrates with varying C:N are decomposed. We hypothesized that (1) increased SRR and declined CUE with warming would be more evident at higher C:N, (2) apparent 13C fractionation between biomass and respired CO2 would decrease with temperature due to C limitation, and (3) this fractionation would be higher for high C:N. Pseudomonas fluorescens (a ubiquitous Gram-negative bacterium) was grown at 0.13 h-1 in a chemostat from 13 to 26.5°C. The concentration of cellobiose, the sole C source with constant δ13C, was adjusted to have

  8. Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A.

    PubMed

    Iturrioz, X; Vazeux, G; Célérier, J; Corvol, P; Llorens-Cortès, C

    2000-03-21

    Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound protease that contains the HEXXH consensus sequence found in the zinc metalloprotease family, the zincins. In addition to the catalytic zinc atom, APA contains a Ca2+ ion that increases its enzymatic activity. Aligning the sequences of the mouse APA, APN, and other monozinc aminopeptidases led to the identification of a conserved histidine (His 450 in mouse APA). Replacing this residue with a phenylalanine (Phe 450) by site-directed mutagenesis resulted in markedly lower levels of APA activity and in a change in the sensitivity of APA to Ca2+ (the EC50 for Ca2+ was 25 microM in the wild type and only 279 microM in the mutant). Kinetic studies, with a supramaximal Ca2+ concentration (4 mM), showed that the Km of the mutant enzyme for the substrate alpha-L-glutamyl-beta-naphthylamide was 25 times higher than that of the wild type, whereas the kcat value was much lower (factor of 22). Thus, overall, the wild-type enzyme had a cleavage efficiency that was 571 times higher than that of the mutant. The inhibitory potencies of two different classes of inhibitors, a glutamate thiol and a glutamate phosphonate compound, were significantly lower (factors of 19 and 22, respectively) for the mutated enzyme than for the wild-type enzyme. In contrast, inhibition by lysine thiol was unaffected. These data strongly suggest that His 450 is critical for catalytic activity and is involved in substrate binding via interaction with the P1 carboxylate side chain of the substrate. Furthermore, His 450, together with Ca2+, may contribute to the substrate specificity of APA for N-terminal acidic amino acid residues.

  9. Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations.

    PubMed

    Woodroof, Helen I; Pogson, Joe H; Begley, Mike; Cantley, Lewis C; Deak, Maria; Campbell, David G; van Aalten, Daan M F; Whitworth, Alexander J; Alessi, Dario R; Muqit, Miratul M K

    2011-11-01

    Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated-namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)-are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates. PMID:22645651

  10. Identification of ElpA, a Coxiella burnetii pathotype-specific Dot/Icm type IV secretion system substrate.

    PubMed

    Graham, Joseph G; Winchell, Caylin G; Sharma, Uma M; Voth, Daniel E

    2015-03-01

    Coxiella burnetii causes human Q fever, a zoonotic disease that presents with acute flu-like symptoms and can result in chronic life-threatening endocarditis. In human alveolar macrophages, C. burnetii uses a Dot/Icm type IV secretion system (T4SS) to generate a phagolysosome-like parasitophorous vacuole (PV) in which to replicate. The T4SS translocates effector proteins, or substrates, into the host cytosol, where they mediate critical cellular events, including interaction with autophagosomes, PV formation, and prevention of apoptosis. Over 100 C. burnetii Dot/Icm substrates have been identified, but the function of most remains undefined. Here, we identified a novel Dot/Icm substrate-encoding open reading frame (CbuD1884) present in all C. burnetii isolates except the Nine Mile reference isolate, where the gene is disrupted by a frameshift mutation, resulting in a pseudogene. The CbuD1884 protein contains two transmembrane helices (TMHs) and a coiled-coil domain predicted to mediate protein-protein interactions. The C-terminal region of the protein contains a predicted Dot/Icm translocation signal and was secreted by the T4SS, while the N-terminal portion of the protein was not secreted. When ectopically expressed in eukaryotic cells, the TMH-containing N-terminal region of the CbuD1884 protein trafficked to the endoplasmic reticulum (ER), with the C terminus dispersed nonspecifically in the host cytoplasm. This new Dot/Icm substrate is now termed ElpA (ER-localizing protein A). Full-length ElpA triggered substantial disruption of ER structure and host cell secretory transport. These results suggest that ElpA is a pathotype-specific T4SS effector that influences ER function during C. burnetii infection.

  11. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    PubMed

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

  12. Differential substrate specificity and kinetic behavior of Escherichia coli YfdW and Oxalobacter formigenes formyl coenzyme A transferase.

    PubMed

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

    2008-04-01

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

  13. Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

    PubMed Central

    Kaneko, Satoshi; Arimoto, Mitsue; Ohba, Misako; Kobayashi, Hideyuki; Ishii, Tadashi; Kusakabe, Isao

    1998-01-01

    α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan. PMID:9758835

  14. Purification, Characterization, and Substrate Specificities of Multiple Xylanases from Streptomyces sp. Strain B-12-2

    PubMed Central

    Elegir, Graziano; Szakács, George; Jeffries, Thomas W.

    1994-01-01

    The endoxylanase complex from Streptomyces sp. strain B-12-2 was purified and characterized. The organism forms five distinct xylanases in the absence of significant cellulase activity when grown on oat spelt xylan. This is the largest number of endoxylanases yet reported for a streptomycete. On the basis of their physiochemical characteristics, they can be divided into two groups: the first group (xyl 1a and xyl 1b) consists of low-molecular-mass (26.4 and 23.8 kDa, respectively) neutral- to high-pI (6.5 and 8.3, respectively) endoxylanases. Group 1 endoxylanases are unable to hydrolyze aryl-β-d-cellobioside, have low levels of activity against xylotetraose (X4) and limited activity against xylopentaose, produce little or no xylose, and form products having a higher degree of polymerization with complex substrates. These enzymes apparently carry out transglycosylation. The second group (xyl 2, xyl 3, and xyl 4) consists of high-molecular-mass (36.2, 36.2, and 40.5 kDa, respectively), low-pI (5.4, 5.0, and 4.8, respectively) xylanases. Group 2 endoxylanases are able to hydrolyze aryl-β-d-cellobioside, show higher levels of activity against X4, and hydrolyze xylopentaose completely with the formation of xylobiose and xylotriose plus limited amounts of X4 and xylose. The enzymes display intergroup synergism when acting on kraft pulp. Despite intragroup similarities, each enzyme exhibited a unique action pattern and physiochemical characteristic. xyl 2 was highly glycosylated, and xyl 1b (but no other enzyme) was completely inhibited by p-hydroxymercuribenzoate. Images PMID:16349337

  15. Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitor: a different substrate specificity and inhibitory regulation.

    PubMed

    Cho, Kyung-Hyun; An, Sojin; Lee, Woo-Song; Paik, Young-Ki; Kim, Young-Kook; Jeong, Tae-Sook

    2003-10-01

    Recently, acyl-CoA:cholesterol acyltransferase was found to be present as two isoforms, ACAT-1 and ACAT-2, in mammalian tissues with different metabolic functions and tissue-specific locations. In this study, the isoforms were mass-produced individually from insect cells to establish a more sensitive and reliable screening method for specific inhibitors against each isoform. The expressed hACAT-1 and hACAT-2 appeared as a 50 kDa- and a 46 kDa-band on SDS-PAGE, respectively, from Hi5 cells and they preferred to exist in oligomeric form, from dimer to tetramer, during the purification process. They also exhibited an approximate 3.4 to 3.7-fold increase in activities when compared to rat liver microsomal fractions at the same protein concentration. Known ACAT inhibitors, pyripyropene A, oleic acid anilide, and diethyl pyrocarbonate, were tested to evaluate the inhibitory specificity and sensitivity of the expressed enzymes. Interestingly, pyripyropene A inhibited only the hACAT-2 fraction with IC(50)=0.64 microM but not the hACAT-1 fraction; whereas the fatty acid anilide did not show a significant difference in inhibitory activity with either hACAT-1 or hACAT-2. Furthermore, cholesterol was more rapidly utilized by hACAT-1, but hACAT-2 esterified other cholic acid derivatives more efficiently. These results suggest that the specificity of each substrate and inhibitor was highly different, depending on each isoform from the viewpoint of the regulatory site and the substrate binding site location. PMID:13679053

  16. Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2.

    PubMed

    Chi, Yuling; Zhou, Bo; Wang, Wei-Qing; Chung, Sung-Kee; Kwon, Yong-Uk; Ahn, Young-Hoon; Chang, Young-Tae; Tsujishita, Yosuke; Hurley, James H; Zhang, Zhong-Yin

    2004-10-22

    Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective

  17. Quality of fresh organic matter affects priming of soil organic matter and substrate utilization patterns of microbes

    NASA Astrophysics Data System (ADS)

    Wang, Hui; Boutton, Thomas W.; Xu, Wenhua; Hu, Guoqing; Jiang, Ping; Bai, Edith

    2015-05-01

    Changes in biogeochemical cycles and the climate system due to human activities are expected to change the quantity and quality of plant litter inputs to soils. How changing quality of fresh organic matter (FOM) might influence the priming effect (PE) on soil organic matter (SOM) mineralization is still under debate. Here we determined the PE induced by two 13C-labeled FOMs with contrasting nutritional quality (leaf vs. stalk of Zea mays L.). Soils from two different forest types yielded consistent results: soils amended with leaf tissue switched faster from negative PE to positive PE due to greater microbial growth compared to soils amended with stalks. However, after 16 d of incubation, soils amended with stalks had a higher PE than those amended with leaf. Phospholipid fatty acid (PLFA) results suggested that microbial demand for carbon and other nutrients was one of the major determinants of the PE observed. Therefore, consideration of both microbial demands for nutrients and FOM supply simultaneously is essential to understand the underlying mechanisms of PE. Our study provided evidence that changes in FOM quality could affect microbial utilization of substrate and PE on SOM mineralization, which may exacerbate global warming problems under future climate change.

  18. Quality of fresh organic matter affects priming of soil organic matter and substrate utilization patterns of microbes.

    PubMed

    Wang, Hui; Boutton, Thomas W; Xu, Wenhua; Hu, Guoqing; Jiang, Ping; Bai, Edith

    2015-01-01

    Changes in biogeochemical cycles and the climate system due to human activities are expected to change the quantity and quality of plant litter inputs to soils. How changing quality of fresh organic matter (FOM) might influence the priming effect (PE) on soil organic matter (SOM) mineralization is still under debate. Here we determined the PE induced by two (13)C-labeled FOMs with contrasting nutritional quality (leaf vs. stalk of Zea mays L.). Soils from two different forest types yielded consistent results: soils amended with leaf tissue switched faster from negative PE to positive PE due to greater microbial growth compared to soils amended with stalks. However, after 16 d of incubation, soils amended with stalks had a higher PE than those amended with leaf. Phospholipid fatty acid (PLFA) results suggested that microbial demand for carbon and other nutrients was one of the major determinants of the PE observed. Therefore, consideration of both microbial demands for nutrients and FOM supply simultaneously is essential to understand the underlying mechanisms of PE. Our study provided evidence that changes in FOM quality could affect microbial utilization of substrate and PE on SOM mineralization, which may exacerbate global warming problems under future climate change. PMID:25960162

  19. Quality of fresh organic matter affects priming of soil organic matter and substrate utilization patterns of microbes.

    PubMed

    Wang, Hui; Boutton, Thomas W; Xu, Wenhua; Hu, Guoqing; Jiang, Ping; Bai, Edith

    2015-01-01

    Changes in biogeochemical cycles and the climate system due to human activities are expected to change the quantity and quality of plant litter inputs to soils. How changing quality of fresh organic matter (FOM) might influence the priming effect (PE) on soil organic matter (SOM) mineralization is still under debate. Here we determined the PE induced by two (13)C-labeled FOMs with contrasting nutritional quality (leaf vs. stalk of Zea mays L.). Soils from two different forest types yielded consistent results: soils amended with leaf tissue switched faster from negative PE to positive PE due to greater microbial growth compared to soils amended with stalks. However, after 16 d of incubation, soils amended with stalks had a higher PE than those amended with leaf. Phospholipid fatty acid (PLFA) results suggested that microbial demand for carbon and other nutrients was one of the major determinants of the PE observed. Therefore, consideration of both microbial demands for nutrients and FOM supply simultaneously is essential to understand the underlying mechanisms of PE. Our study provided evidence that changes in FOM quality could affect microbial utilization of substrate and PE on SOM mineralization, which may exacerbate global warming problems under future climate change.

  20. Substrate Specificity and Ligand Interactions of CYP26A1, the Human Liver Retinoic Acid Hydroxylase

    PubMed Central

    Thatcher, Jayne E.; Buttrick, Brian; Shaffer, Scott A.; Shimshoni, Jakob A.; Goodlett, David R.; Nelson, Wendel L.

    2011-01-01

    All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. atRA is also used as a drug, and synthetic atRA analogs and inhibitors of retinoic acid (RA) metabolism have been developed. The hepatic clearance of atRA is mediated primarily by CYP26A1, but design of CYP26A1 inhibitors is hindered by lack of information on CYP26A1 structure and structure-activity relationships of its ligands. The aim of this study was to identify the primary metabolites of atRA formed by CYP26A1 and to characterize the ligand selectivity and ligand interactions of CYP26A1. On the basis of high-resolution tandem mass spectrometry data, four metabolites formed from atRA by CYP26A1 were identified as 4-OH-RA, 4-oxo-RA, 16-OH-RA and 18-OH-RA. 9-cis-RA and 13-cis-RA were also substrates of CYP26A1. Forty-two compounds with diverse structural properties were tested for CYP26A1 inhibition using 9-cis-RA as a probe, and IC50 values for 10 inhibitors were determined. The imidazole- and triazole-containing inhibitors [S-(R*,R*)]-N-[4-[2-(dimethylamino)-1-(1H-imidazole-1-yl)propyl]-phenyl]2-benzothiazolamine (R116010) and (R)-N-[4-[2-ethyl-1-(1H-1,2,4-triazol-1-yl)butyl]phenyl]-2-benzothiazolamine (R115866) were the most potent inhibitors of CYP26A1 with IC50 values of 4.3 and 5.1 nM, respectively. Liarozole and ketoconazole were significantly less potent with IC50 values of 2100 and 550 nM, respectively. The retinoic acid receptor (RAR) γ agonist CD1530 was as potent an inhibitor of CYP26A1 as ketoconazole with an IC50 of 530 nM, whereas the RARα and RARβ agonists tested did not significantly inhibit CYP26A1. The pan-RAR agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid and the peroxisome proliferator-activated receptor ligands rosiglitazone and pioglitazone inhibited CYP26A1 with IC50 values of 3.7, 4.2, and 8.6 μM, respectively. These data demonstrate that CYP26A1 has high ligand selectivity but accepts structurally related nuclear

  1. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa.

  2. Substrate specificity of the Deazaflavin-Dependent Nitroreductase (Ddn) from Mycobacterium tuberculosis Responsible for the Bioreductive Activation of Bicyclic Nitroimidazoles

    PubMed Central

    Gurumurthy, Meera; Mukherjee, Tathagata; Dowd, Cynthia S.; Singh, Ramandeep; Niyomrattanakit, Pornwaratt; Tay, Jo Ann; Nayyar, Amit; Lee, Yong Sok; Cherian, Joseph; Boshoff, Helena I.; Dick, Thomas; Barry, Clifton E.; Manjunatha, Ujjini H.

    2012-01-01

    The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis (TB) and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F420) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions, cofactor and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. For nitroimidazo-oxazoles bearing only short alkyl substituents on the C-7 position of the oxazole, substrates were reduced by Ddn without stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereo-specificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turn-over independent assessment of affinity. Using this assay we found that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer likely has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F420. The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding. PMID:22023140

  3. Dihydroflavonol 4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in Fragaria species.

    PubMed

    Miosic, Silvija; Thill, Jana; Milosevic, Malvina; Gosch, Christian; Pober, Sabrina; Molitor, Christian; Ejaz, Shaghef; Rompel, Annette; Stich, Karl; Halbwirth, Heidi

    2014-01-01

    During fruit ripening, strawberries show distinct changes in the flavonoid classes that accumulate, switching from the formation of flavan 3-ols and flavonols in unripe fruits to the accumulation of anthocyanins in the ripe fruits. In the common garden strawberry (Fragaria×ananassa) this is accompanied by a distinct switch in the pattern of hydroxylation demonstrated by the almost exclusive accumulation of pelargonidin based pigments. In Fragaria vesca the proportion of anthocyanins showing one (pelargonidin) and two (cyanidin) hydroxyl groups within the B-ring is almost equal. We isolated two dihydroflavonol 4-reductase (DFR) cDNA clones from strawberry fruits, which show 82% sequence similarity. The encoded enzymes revealed a high variability in substrate specificity. One enzyme variant did not accept DHK (with one hydroxyl group present in the B-ring), whereas the other strongly preferred DHK as a substrate. This appears to be an uncharacterized DFR variant with novel substrate specificity. Both DFRs were expressed in the receptacle and the achenes of both Fragaria species and the DFR2 expression profile showed a pronounced dependence on fruit development, whereas DFR1 expression remained relatively stable. There were, however, significant differences in their relative rates of expression. The DFR1/DFR2 expression ratio was much higher in the Fragaria×ananassa and enzyme preparations from F.×ananassa receptacles showed higher capability to convert DHK than preparations from F. vesca. Anthocyanin concentrations in the F.×ananassa cultivar were more than twofold higher and the cyanidin:pelargonidin ratio was only 0.05 compared to 0.51 in the F. vesca cultivar. The differences in the fruit colour of the two Fragaria species can be explained by the higher expression of DFR1 in F.×ananassa as compared to F. vesca, a higher enzyme efficiency (Kcat/Km values) of DFR1 combined with the loss of F3'H activity late in fruit development of F.×ananassa. PMID:25393679

  4. A study of substrate specificity for a CTD phosphatase, SCP1, by proteomic screening of binding partners.

    PubMed

    Kim, Young Jun; Bahk, Young Yil

    2014-05-30

    RNA polymerase II carboxyl-terminal domain (RNAPII CTD) phosphatases are a newly emerging family of phosphatases. Recently a CTD-specific phosphatase, small CTD phosphatase 1 (SCP1), has shown to act as an evolutionarily conserved transcriptional corepressor for inhibiting neuronal gene transcription in non-neuronal cells. In this study, using the established NIH/3T3 and HEK293T cells, which are expressing human SCP1 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for SCP1. Although the present findings provide the possibility for new avenues to provide to a better understanding of cellular physiology of SCP1, now these proteomic and some immunological approaches for SCP1 interactome might not represent the accurate physiological relevance in vivo. In this presentation, we focus the substrate specificity to delineate an appearance of the dephosphorylation reaction catalyzed by SCP1 phosphatase. We compared the phosphorylated sequences of the immunologically confirmed binding partners with SCP1 searched in HPRD. We found the similar sequences from CdcA3 and validated the efficiency of enzymatic catalysis for synthetic phosphopeptides the recombinant SCP1. This approach led to the identification of several interacting partners with SCP1. We suggest that CdcA3 could be an enzymatic substrate for SCP1 and that SCP1 might have the relationship with cell cycle regulation through enzymatic activity against CdcA3. PMID:24769477

  5. EVIDENCE FOR BIDENTATE SUBSTRATE BINDING AS THE BASIS FOR THE K48 LINKAGE SPECIFICITY OF OTUBAIN 1

    PubMed Central

    Wang, Tao; Yin, Luming; Cooper, Eric M.; Lai, Ming-Yih; Dickey, Seth; Pickart, Cecile M.; Fushman, David; Wilkinson, Keith D.; Cohen, Robert E.; Wolberger, Cynthia

    2009-01-01

    Otubain 1 belongs to the ovarian tumor (OTU) domain class of cysteine protease deubiquitinating enzymes. We show here that human otubain 1 (hOtu1) is highly linkage-specific, cleaving Lys48 (K48)-linked polyubiquitin but not K63-, K29-, K6-, or K11-linked polyubiquitin, or linear α-linked polyubiquitin. Cleavage is not limited to either end of a polyubiquitin chain, and both free and substrate-linked polyubiquitin are disassembled. Intriguingly, cleavage of K48-diubiquitin by hOtu1 can be inhibited by diubiquitins of various linkage types, as well as by monoubiquitin. NMR studies and activity assays suggest that both the proximal and distal units of K48-diubiquitin bind to hOtu1. Reaction of Cys23 with ubiquitin-vinylsulfone identified a ubiquitin binding site that is distinct from the active site, which includes Cys91. Occupancy of the active site is needed to enable tight binding to the second site. We propose that distinct binding sites for the ubiquitins on either side of the scissile bond allow hOtu1 to discriminate among different isopeptide linkages in polyubiquitin substrates. Bidentate binding may be a general strategy used to achieve linkage-specific deubiquitination. PMID:19211026

  6. Restricted Location of PSEN2/γ-Secretase Determines Substrate Specificity and Generates an Intracellular Aβ Pool.

    PubMed

    Sannerud, Ragna; Esselens, Cary; Ejsmont, Paulina; Mattera, Rafael; Rochin, Leila; Tharkeshwar, Arun Kumar; De Baets, Greet; De Wever, Veerle; Habets, Roger; Baert, Veerle; Vermeire, Wendy; Michiels, Christine; Groot, Arjan J; Wouters, Rosanne; Dillen, Katleen; Vints, Katlijn; Baatsen, Pieter; Munck, Sebastian; Derua, Rita; Waelkens, Etienne; Basi, Guriqbal S; Mercken, Mark; Vooijs, Marc; Bollen, Mathieu; Schymkowitz, Joost; Rousseau, Frederic; Bonifacino, Juan S; Van Niel, Guillaume; De Strooper, Bart; Annaert, Wim

    2016-06-30

    γ-Secretases are a family of intramembrane-cleaving proteases involved in various signaling pathways and diseases, including Alzheimer's disease (AD). Cells co-express differing γ-secretase complexes, including two homologous presenilins (PSENs). We examined the significance of this heterogeneity and identified a unique motif in PSEN2 that directs this γ-secretase to late endosomes/lysosomes via a phosphorylation-dependent interaction with the AP-1 adaptor complex. Accordingly, PSEN2 selectively cleaves late endosomal/lysosomal localized substrates and generates the prominent pool of intracellular Aβ that contains longer Aβ; familial AD (FAD)-associated mutations in PSEN2 increased the levels of longer Aβ further. Moreover, a subset of FAD mutants in PSEN1, normally more broadly distributed in the cell, phenocopies PSEN2 and shifts its localization to late endosomes/lysosomes. Thus, localization of γ-secretases determines substrate specificity, while FAD-causing mutations strongly enhance accumulation of aggregation-prone Aβ42 in intracellular acidic compartments. The findings reveal potentially important roles for specific intracellular, localized reactions contributing to AD pathogenesis. PMID:27293189

  7. Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.

    PubMed

    Arndt, Joseph W; Chai, Qing; Christian, Todd; Stevens, Raymond C

    2006-03-14

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

  8. THE ACTIN BUNDLING PROTEIN PALLADIN IS AN AKT1-SPECIFIC SUBSTRATE THAT REGULATES BREAST CANCER CELL MIGRATION

    PubMed Central

    Chin, Y. Rebecca; Toker, Alex

    2010-01-01

    Summary The phosphoinositide 3-kinase (PI 3-K) signaling pathway is frequently deregulated in cancer. Downstream of PI 3-K, Akt1 and Akt2 have opposing roles in breast cancer invasive migration leading to metastatic dissemination. Here we identify palladin, an actin-associated protein, as an Akt1-specific substrate that modulates breast cancer cell invasive migration. Akt1, but not Akt2, phosphorylates palladin at S507 in a domain that is critical for F-actin bundling. Downregulation of palladin enhances migration and invasion of breast cancer cells and induces abnormal branching morphogenesis in 3D cultures. Palladin phosphorylation at S507 is required for Akt1-mediated inhibition of breast cancer cell migration and also for F-actin bundling leading to the maintenance of an organized actin cytoskeleton. These findings identify palladin as an Akt1-specific substrate that regulates cell motility and provide a molecular mechanism that accounts for the functional distinction between Akt isoforms in breast cancer cell signaling to cell migration. PMID:20471940

  9. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    PubMed

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing.

  10. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions

    PubMed Central

    Long, Yicheng; Jackman, Jane E.

    2015-01-01

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from A. castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  11. Enzymatic properties and subtle differences in the substrate specificity of phylogenetically distinct invertebrate N-glycan processing hexosaminidases

    PubMed Central

    Dragosits, Martin; Yan, Shi; Razzazi-Fazeli, Ebrahim; Wilson, Iain B H; Rendic, Dubravko

    2015-01-01

    Fused lobes (FDL) hexosaminidases are the most recently genetically defined glycosidases involved in the biosynthesis of N-glycans in invertebrates, and their narrow specificity is essential for the generation of paucimannosidic N-glycans in insects. In this study, we explored the potential of FDL hexosaminidases in the utilization of different artificial and natural substrates, both as purified, native compounds or generated in vitro using various relevant glycosyltransferases. In addition to the already-known FDL enzyme from Drosophila melanogaster, we now have identified and characterized the Apis mellifera FDL homolog. The enzymatic properties of the soluble forms of the affinity-purified insect FDL enzymes, expressed in both yeast and insect cells, were compared with those of the phylogenetically distinct recombinant Caenorhabditis elegans FDL-like enzymes and the N-acetylgalactosamine (GalNAc)-specific Caenorhabditis hexosaminidase HEX-4. In tests with a range of substrates, including natural N-glycans, we show that the invertebrate FDL(-like) enzymes are highly specific for N-acetylglucosamine attached to the α1,3-mannose, but under extreme conditions also remove other terminal GalNAc and N-acetylglucosamine residues. Recombinant FDL also proved useful in the analysis of complex mixtures of N-glycans originating from wild-type and mutant Caenorhabditis strains, thereby aiding isomeric definition of paucimannosidic and hybrid N-glycans in this organism. Furthermore, differences in activity and specificity were shown for two site-directed mutants of Drosophila FDL, compatible with the high structural similarity of chitinolytic and N-glycan degrading exohexosaminidases in insects. Our studies are another indication for the variety of structural and function aspects in the GH20 hexosaminidase family important for both catabolism and biosynthesis of glycoconjugates in eukaryotes. PMID:25488985

  12. Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

    PubMed

    Robinson, Ronald G; Smith, Sean M; Wolkenberg, Scott E; Kandebo, Monika; Yao, Lihang; Gibson, Christopher R; Harrison, Scott T; Polsky-Fisher, Stacey; Barrow, James C; Manley, Peter J; Mulhearn, James J; Nanda, Kausik K; Schubert, Jeffrey W; Trotter, B Wesley; Zhao, Zhijian; Sanders, John M; Smith, Robert F; McLoughlin, Debra; Sharma, Sujata; Hall, Dawn L; Walker, Tiffany L; Kershner, Jennifer L; Bhandari, Neetesh; Hutson, Pete H; Sachs, Nancy A

    2012-02-15

    Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity. PMID:22860182

  13. Excimer laser texturing of natural composite polymer surfaces for studying cell-to-substrate specific response

    NASA Astrophysics Data System (ADS)

    Dinca, V.; Alloncle, P.; Delaporte, P.; Ion, V.; Rusen, L.; Filipescu, M.; Mustaciosu, C.; Luculescu, C.; Dinescu, M.

    2015-10-01

    Surface modifications of biocompatible materials are among the main factors used for enhancing and promoting specific cellular activities (e.g. spreading, adhesion, migration, and differentiation) for various types of medical applications such as implants, microfluidic devices, or tissue engineering scaffolds. In this work an excimer laser at 193 nm was used to fabricate chitosan-collagen roughness gradients. The roughness gradients were obtained in one step by applying single laser pulses and sample tilting. Fourier transform infrared spectroscopy measurements, atomic force microscopy (AFM), scanning electron microscopy (SEM), and spectro-ellipsometry (SE) were used for sample characterization. The goal is to determine the optimal morpho-chemical characteristics of these structures for in vitro tailoring of protein adsorption and cell behavior. The response induced by the roughness gradient onto various cell lines (i.e. L 929 fibroblasts, HEP G2 hepatocytes, OLN 93 oligodendrocytes, M63 osteoblasts) and bovine serum albumin (BSA) protein absorption was investigated.

  14. Dissociating motivational direction and affective valence: specific emotions alter central motor processes.

    PubMed

    Coombes, Stephen A; Cauraugh, James H; Janelle, Christopher M

    2007-11-01

    We aimed to clarify the relation between affective valence and motivational direction by specifying how central and peripheral components of extension movements are altered according to specific unpleasant affective states. As predicted, premotor reaction time was quicker for extension movements initiated during exposure to attack than for extension movements initiated during exposure to all other valence categories (mutilation, erotic couples, opposite-sex nudes, neutral humans, household objects, blank). Exposure to erotic couples and mutilations yielded greater peak force than exposure to images of attack, neutral humans, and household objects. Finally, motor reaction time and peak electromyographic amplitude were not altered by valence. These findings indicate that unpleasant states do not unilaterally prime withdrawal movements, and that the quick execution of extension movements during exposure to threatening images is due to rapid premotor, rather than motor, reaction time. Collectively, our findings support the call for dissociating motivational direction and affective valence. PMID:17958705

  15. Purification, Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase from Aspergillus oryzae

    PubMed Central

    Riou, Christine; Salmon, Jean-Michel; Vallier, Marie-Jose; Günata, Ziya; Barre, Pierre

    1998-01-01

    Aspergillus oryzae was found to secrete two distinct β-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3′,4′,5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min−1 mg of protein−1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1.36 M) or glucono-δ-lactone (Ki, 12.5 mM), another powerful β-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter−1). Other flavor precursors (benzyl- and 2-phenylethyl-β-d-glucosides) and prunin (4′,5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel β-glucosidase is of great potential interest in wine and

  16. Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae.

    PubMed

    Riou, C; Salmon, J M; Vallier, M J; Günata, Z; Barre, P

    1998-10-01

    Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential

  17. Factors affecting allergen-specific IgE serum levels in cats

    PubMed Central

    Belova, S.; Wilhelm, S.; Linek, M.; Beco, L.; Fontaine, J.; Bergvall, K.; Favrot, C.

    2012-01-01

    Pruritic skin diseases are common in cats and demand rigorous diagnostic workup for finding an underlying etiology. Measurement of a serum allergen-specific IgE in a pruritic cat is often used to make or confirm the diagnosis of a skin hypersensitivity disease, although current evidence suggests that elevated allergen-specific IgE do not always correlate with a clinical disease and vice versa. The aim of the study was to to assess the possible influence of age, deworming status, lifestyle, flea treatment, and gender on allergen-specific IgE levels and to evaluate the reliability of IgE testing in predicting the final diagnosis of a pruritic cat. For this purpose sera of 179 cats with pruritus of different causes and 20 healthy cats were evaluated for allergen-specific IgE against environmental, food and flea allergens using the Fc-epsilon receptor based enzyme-linked immunosorbent assay (ELISA) test. The results of the study showed positive correlation between age, outdoor life style, absence of deworming, absence of flea control measures and levels of allergen-specific IgE. Gender and living area (urban versus rural) did not seem to affect the formation of allergen-specific IgE. According to these findings, evaluating allergen-specific IgE levels, is not a reliable test to diagnose hypersensitivity to food or environmental allergens in cats. On the contrary, this test can be successfully used for diagnosing feline flea bite hypersensitivity. PMID:22754094

  18. Substrate specificity and function of the pheromone receptor AinR in Vibrio fischeri ES114.

    PubMed

    Kimbrough, John H; Stabb, Eric V

    2013-11-01

    Two distinct but interrelated pheromone-signaling systems, LuxI/LuxR and AinS/AinR, positively control bioluminescence in Vibrio fischeri. Although each system generates an acyl-homoserine lactone (AHL) signal, the protein sequences of LuxI/LuxR and AinS/AinR are unrelated. AinS and LuxI generate the pheromones N-octanoyl-AHL (C8-AHL) and N-3-oxo-hexanoyl-AHL (3OC6-AHL), respectively. LuxR is a transcriptional activator that responds to 3OC6-AHL, and to a lesser extent to C8-AHL. AinR is hypothesized to respond to C8-AHL and, based on homology to Vibrio harveyi LuxN, to mediate the repression of a Qrr regulatory RNA. However, a ΔainR mutation decreased luminescence, which was not predicted based on V. harveyi LuxN, raising the possibility of a distinct regulatory mechanism for AinR. Here we show that ainR can complement a luxN mutant, suggesting functional similarity. Moreover, in V. fischeri, we observed ainR-dependent repression of a Pqrr-lacZ transcriptional reporter in the presence of C8-AHL, consistent with its hypothesized regulatory role. The system appears quite sensitive, with a half-maximal effect on a Pqrr reporter at 140 pM C8-AHL. Several other AHLs with substituted and unsubstituted acyl chains between 6 and 10 carbons also displayed an AinR-dependent effect on Pqrr-lacZ; however, AHLs with acyl chains of four carbons or 12 or more carbons lacked activity. Interestingly, 3OC6-AHL also affected expression from the qrr promoter, but this effect was largely luxR dependent, indicating a previously unknown connection between these systems. Finally, we propose a preliminary explanation for the unexpected luminescence phenotype of the ΔainR mutant.

  19. Substrate Specificity and Function of the Pheromone Receptor AinR in Vibrio fischeri ES114

    PubMed Central

    Kimbrough, John H.

    2013-01-01

    Two distinct but interrelated pheromone-signaling systems, LuxI/LuxR and AinS/AinR, positively control bioluminescence in Vibrio fischeri. Although each system generates an acyl-homoserine lactone (AHL) signal, the protein sequences of LuxI/LuxR and AinS/AinR are unrelated. AinS and LuxI generate the pheromones N-octanoyl-AHL (C8-AHL) and N-3-oxo-hexanoyl-AHL (3OC6-AHL), respectively. LuxR is a transcriptional activator that responds to 3OC6-AHL, and to a lesser extent to C8-AHL. AinR is hypothesized to respond to C8-AHL and, based on homology to Vibrio harveyi LuxN, to mediate the repression of a Qrr regulatory RNA. However, a ΔainR mutation decreased luminescence, which was not predicted based on V. harveyi LuxN, raising the possibility of a distinct regulatory mechanism for AinR. Here we show that ainR can complement a luxN mutant, suggesting functional similarity. Moreover, in V. fischeri, we observed ainR-dependent repression of a Pqrr-lacZ transcriptional reporter in the presence of C8-AHL, consistent with its hypothesized regulatory role. The system appears quite sensitive, with a half-maximal effect on a Pqrr reporter at 140 pM C8-AHL. Several other AHLs with substituted and unsubstituted acyl chains between 6 and 10 carbons also displayed an AinR-dependent effect on Pqrr-lacZ; however, AHLs with acyl chains of four carbons or 12 or more carbons lacked activity. Interestingly, 3OC6-AHL also affected expression from the qrr promoter, but this effect was largely luxR dependent, indicating a previously unknown connection between these systems. Finally, we propose a preliminary explanation for the unexpected luminescence phenotype of the ΔainR mutant. PMID:24056099

  20. Expression, purification and luminescence properties of coelenterazine-utilizing luciferases from Renilla, Oplophorus and Gaussia: comparison of substrate specificity for C2-modified coelenterazines.

    PubMed

    Inouye, Satoshi; Sahara-Miura, Yuiko; Sato, Jun-ichi; Iimori, Rie; Yoshida, Suguru; Hosoya, Takamitsu

    2013-03-01

    The cold-induced expression system in Escherichia coli is useful and we have applied this system to prepare the coelenterazine-utilizing luciferases including Renilla luciferase (RLase), a red-shifted variant of Renilla luciferase (RLase-547), the catalytic domain of Oplophorus luciferase (19kOLase) and Gaussia luciferase (GLase). The luminescence properties of the purified luciferases were characterized by using 10 kinds of C2-modified coelenterazine analogues as a substrate. The order of the maximal luminescence intensity for native coelenterazine was GLase (100%)>RLase (8.0%)>RLase-547 (0.73%)>19kOLase (0.09%) under our assay conditions. The substrate specificities of coelenterazine-utilizing luciferases for the C2-modified analogues showed significant differences, but the emission peaks catalyzed by coelenterazine-utilizing luciferases were not affected by the C2-substituted coelenterazine. These results suggest that the catalytic environment for the oxygenation process of coelenterazine and the excited species of coelenteramide might be different among coelenterazine-utilizing luciferases. PMID:23274053

  1. Neural substrates of defensive reactivity in two subtypes of specific phobia.

    PubMed

    Lueken, Ulrike; Hilbert, Kevin; Stolyar, Veronika; Maslowski, Nina I; Beesdo-Baum, Katja; Wittchen, Hans-Ulrich

    2014-11-01

    Depending on threat proximity, different defensive behaviours are mediated by a descending neural network involving forebrain (distal threat) vs midbrain areas (proximal threat). Compared to healthy subjects, it can be assumed that phobics are characterized by shortened defensive distances on a behavioural and neural level. This study aimed at characterizing defensive reactivity in two subtypes of specific phobia [snake (SP) and dental phobics (DP)]. Using functional magnetic resonance imaging (fMRI), n = 39 subjects (13 healthy controls, HC; 13 SP; 13 DP) underwent an event-related fMRI task employing an anticipation (5-10 s) and immediate perception phase (phobic pictures and matched neutral stimuli; 1250 ms) to modulate defensive distance. Although no differential brain activity in any comparisons was observed in DP, areas associated with defensive behaviours (e.g. amygdala, hippocampus, midbrain) were activated in SP. Decreasing defensive distance in SP was characterized by a shift to midbrain activity. Present findings substantiate differences between phobia types in their physiological and neural organization that can be expanded to early stages of defensive behaviours. Findings may contribute to a better understanding of the dynamic organization of defensive reactivity in different types of phobic fear.

  2. Neural substrates of defensive reactivity in two subtypes of specific phobia.

    PubMed

    Lueken, Ulrike; Hilbert, Kevin; Stolyar, Veronika; Maslowski, Nina I; Beesdo-Baum, Katja; Wittchen, Hans-Ulrich

    2014-11-01

    Depending on threat proximity, different defensive behaviours are mediated by a descending neural network involving forebrain (distal threat) vs midbrain areas (proximal threat). Compared to healthy subjects, it can be assumed that phobics are characterized by shortened defensive distances on a behavioural and neural level. This study aimed at characterizing defensive reactivity in two subtypes of specific phobia [snake (SP) and dental phobics (DP)]. Using functional magnetic resonance imaging (fMRI), n = 39 subjects (13 healthy controls, HC; 13 SP; 13 DP) underwent an event-related fMRI task employing an anticipation (5-10 s) and immediate perception phase (phobic pictures and matched neutral stimuli; 1250 ms) to modulate defensive distance. Although no differential brain activity in any comparisons was observed in DP, areas associated with defensive behaviours (e.g. amygdala, hippocampus, midbrain) were activated in SP. Decreasing defensive distance in SP was characterized by a shift to midbrain activity. Present findings substantiate differences between phobia types in their physiological and neural organization that can be expanded to early stages of defensive behaviours. Findings may contribute to a better understanding of the dynamic organization of defensive reactivity in different types of phobic fear. PMID:24174207

  3. Substrate Specificity of the Purified Primary Alcohol Dehydrogenases from Methanol-Oxidizing Bacteria

    PubMed Central

    Sperl, George T.; Forrest, Hugh S.; Gibson, David T.

    1974-01-01

    Hyphomicrobium strain WC, Pseudomonas strain TP-1, and Pseudomonas strain W1 are capable of growth on methanol as the sole source of carbon and energy. Methanol-grown cells of each organism contain a primary alcohol dehydrogenase that has been purified to homogeneity. Each enzyme has a molecular weight of 120,000 and shows an in vitro requirement for phenazine methosulfate and ammonium ions for enzymatic activity. Normal aliphatic alcohols are oxidized rapidly by each enzyme. The presence of a methyl group on the carbon atom adjacent to the primary alcohol group lowers the enzymatic activity. This effect is reduced as the methyl substituent is moved further away from the hydroxyl group. The effect of other substituents on enzymatic activity is reported. Methanol, formaldehyde, and to a limited extent acetaldehyde are oxidized by the primary alcohol dehydrogenases. Higher aldehydes are not oxidized. A possible explanation for this specificity, with regard to aldehydes, is presented in terms of degree of hydration of the aldehyde. Images PMID:4828309

  4. Substrate specificity and kinetic mechanism of purine nucleoside phosphorylase from Mycobacterium tuberculosis.

    PubMed

    Ducati, Rodrigo G; Santos, Diógenes S; Basso, Luiz A

    2009-06-15

    Purine nucleoside phosphorylase from Mycobacterium tuberculosis (MtPNP) is numbered among targets for persistence of the causative agent of tuberculosis. Here, it is shown that MtPNP is more specific to natural 6-oxopurine nucleosides and synthetic compounds, and does not catalyze the phosphorolysis of adenosine. Initial velocity, product inhibition and equilibrium binding data suggest that MtPNP catalyzes 2'-deoxyguanosine (2dGuo) phosphorolysis by a steady-state ordered bi bi kinetic mechanism, in which inorganic phosphate (P(i)) binds first followed by 2dGuo, and ribose 1-phosphate dissociates first followed by guanine. pH-rate profiles indicated a general acid as being essential for both catalysis and 2dGuo binding, and that deprotonation of a group abolishes P(i) binding. Proton inventory and solvent deuterium isotope effects indicate that a single solvent proton transfer makes a modest contribution to the rate-limiting step. Pre-steady-state kinetic data indicate that product release appears to contribute to the rate-limiting step for MtPNP-catalyzed reaction.

  5. Active transport, substrate specificity, and methylation of Hg(II) in anaerobic bacteria

    SciTech Connect

    Schasfer, Jeffra; Rocks, Sara; Zheng, Wang; Liang, Liyuan; Gu, Baohua; Morel, Francois M

    2011-01-01

    The formation of methylmercury (MeHg), which is biomagnified in aquatic food chains and poses a risk to human health, is effected by some iron- and sulfate-reducing bacteria (FeRB and SRB) in anaerobic environments. However, very little is known regarding the mechanism of uptake of inorganic Hg by these organisms, in part because of the inherent difficulty in measuring the intracellular Hg concentration. By using the FeRB Geobacter sulfurreducens and the SRB Desulfovibrio desulfuricans ND132 as model organisms, we demonstrate that Hg(II) uptake occurs by active transport. We also establish that Hg(II) uptake by G. sulfurreducens is highly dependent on the characteristics of the thiols that bind Hg(II) in the external medium, with some thiols promoting uptake and methylation and others inhibiting both. The Hg(II) uptake system of D. desulfuricans has a higher affinity than that of G. sulfurreducens and promotes Hg methylation in the presence of stronger complexing thiols. We observed a tight coupling between Hg methylation and MeHg export from the cell, suggesting that these two processes may serve to avoid the build up and toxicity of cellular Hg. Our results bring up the question of whether cellular Hg uptake is specific for Hg(II) or accidental, occurring via some essential metal importer. Our data also point at Hg(II) complexation by thiols as an important factor controlling Hg methylation in anaerobic environments.

  6. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

    PubMed

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

    2016-09-27

    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes. PMID:27580341

  7. Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation.

    PubMed

    Coitinho, Juliana B; Pereira, Mozart S; Costa, Débora M A; Guimarães, Samuel L; Araújo, Simara S; Hengge, Alvan C; Brandão, Tiago A S; Nagem, Ronaldo A P

    2016-09-27

    The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD(+) as a cofactor, the last reaction of the upper degradation pathway of naphthalene in Pseudomonas putida G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large kcat/Km values close to 10(6) M(-1) s(-1). The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes.

  8. Specific Neuron Placement on Gold and Silicon Nitride-Patterned Substrates through a Two-Step Functionalization Method.

    PubMed

    Mescola, Andrea; Canale, Claudio; Prato, Mirko; Diaspro, Alberto; Berdondini, Luca; Maccione, Alessandro; Dante, Silvia

    2016-06-28

    considered a good starting point to develop alternatives to the traditional chip coatings to provide selective and specific neuron-substrate adhesion. PMID:27268249

  9. Universal and culture-specific factors in the recognition and performance of musical affect expressions.

    PubMed

    Laukka, Petri; Eerola, Tuomas; Thingujam, Nutankumar S; Yamasaki, Teruo; Beller, Grégory

    2013-06-01

    We present a cross-cultural study on the performance and perception of affective expression in music. Professional bowed-string musicians from different musical traditions (Swedish folk music, Hindustani classical music, Japanese traditional music, and Western classical music) were instructed to perform short pieces of music to convey 11 emotions and related states to listeners. All musical stimuli were judged by Swedish, Indian, and Japanese participants in a balanced design, and a variety of acoustic and musical cues were extracted. Results first showed that the musicians' expressive intentions could be recognized with accuracy above chance both within and across musical cultures, but communication was, in general, more accurate for culturally familiar versus unfamiliar music, and for basic emotions versus nonbasic affective states. We further used a lens-model approach to describe the relations between the strategies that musicians use to convey various expressions and listeners' perceptions of the affective content of the music. Many acoustic and musical cues were similarly correlated with both the musicians' expressive intentions and the listeners' affective judgments across musical cultures, but the match between musicians' and listeners' uses of cues was better in within-cultural versus cross-cultural conditions. We conclude that affective expression in music may depend on a combination of universal and culture-specific factors.

  10. Emotional Stimuli and Context Moderate Effects of Nicotine on Specific, But Not Global Affects

    PubMed Central

    Gilbert, David G.; Riise, Hege; Dillon, Amber; Huber, Jamie; Rabinovich, Norka E.; Sugai, Chihiro

    2010-01-01

    We tested the hypothesis that the effects of nicotine on affect are moderated by the presence or absence of emotionally positive and negative stimuli and by attentional choice to avoid attending to emotionally negative stimuli. Thirty-two habitual smokers were assigned to tasks allowing attentional freedom to look back and forth at two simultaneously presented pictures, while another 32 habitual smokers viewed single pictures without attentional choice. Picture contents in both tasks were one of four combinations: emotionally negative + neutral, negative + positive, positive + neutral, or neutral + neutral. Participants wore a nicotine patch on one day and placebo patch on another day. Nicotine reduced anxiety most when negative pictures were presented in combination with neutral pictures, but had no effects on anxiety when negative pictures were presented in combination with positive pictures and when negative pictures were not presented. In contrast, nicotine only reduced depressive affect when the participant had attentional choice between positive and negative pictures. Nicotine also enhanced PANAS positive affect and reduced PANAS negative affect, but these effects were not moderated by task manipulations. Overall, the findings support the view that nicotine’s ability to reduce specific negative affects is moderated by emotional context and attentional freedom. Nicotine tended to enhance eye-gaze orientation to emotional pictures versus neutral pictures in women, but had no significant effect on eye-gaze in men. PMID:18266550

  11. Universal and culture-specific factors in the recognition and performance of musical affect expressions.

    PubMed

    Laukka, Petri; Eerola, Tuomas; Thingujam, Nutankumar S; Yamasaki, Teruo; Beller, Grégory

    2013-06-01

    We present a cross-cultural study on the performance and perception of affective expression in music. Professional bowed-string musicians from different musical traditions (Swedish folk music, Hindustani classical music, Japanese traditional music, and Western classical music) were instructed to perform short pieces of music to convey 11 emotions and related states to listeners. All musical stimuli were judged by Swedish, Indian, and Japanese participants in a balanced design, and a variety of acoustic and musical cues were extracted. Results first showed that the musicians' expressive intentions could be recognized with accuracy above chance both within and across musical cultures, but communication was, in general, more accurate for culturally familiar versus unfamiliar music, and for basic emotions versus nonbasic affective states. We further used a lens-model approach to describe the relations between the strategies that musicians use to convey various expressions and listeners' perceptions of the affective content of the music. Many acoustic and musical cues were similarly correlated with both the musicians' expressive intentions and the listeners' affective judgments across musical cultures, but the match between musicians' and listeners' uses of cues was better in within-cultural versus cross-cultural conditions. We conclude that affective expression in music may depend on a combination of universal and culture-specific factors. PMID:23398579

  12. Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5)

    PubMed Central

    2012-01-01

    Background The large Glycoside Hydrolase family 5 (GH5) groups together a wide range of enzymes acting on β-linked oligo- and polysaccharides, and glycoconjugates from a large spectrum of organisms. The long and complex evolution of this family of enzymes and its broad sequence diversity limits functional prediction. With the objective of improving the differentiation of enzyme specificities in a knowledge-based context, and to obtain new evolutionary insights, we present here a new, robust subfamily classification of family GH5. Results About 80% of the current sequences were assigned into 51 subfamilies in a global analysis of all publicly available GH5 sequences and associated biochemical data. Examination of subfamilies with catalytically-active members revealed that one third are monospecific (containing a single enzyme activity), although new functions may be discovered with biochemical characterization in the future. Furthermore, twenty subfamilies presently have no characterization whatsoever and many others have only limited structural and biochemical data. Mapping of functional knowledge onto the GH5 phylogenetic tree revealed that the sequence space of this historical and industrially important family is far from well dispersed, highlighting targets in need of further study. The analysis also uncovered a number of GH5 proteins which have lost their catalytic machinery, indicating evolution towards novel functions. Conclusion Overall, the subfamily division of GH5 provides an actively curated resource for large-scale protein sequence annotation for glycogenomics; the subfamily assignments are openly accessible via the Carbohydrate-Active Enzyme database at http://www.cazy.org/GH5.html. PMID:22992189

  13. Dictyostelium discoideum contains three inositol monophosphatase activities with different substrate specificities and sensitivities to lithium.

    PubMed Central

    Van Dijken, P; Bergsma, J C; Hiemstra, H S; De Vries, B; Van Der Kaay, J; Van Haastert, P J

    1996-01-01

    The small ion lithium, a very effective agent in the treatment of manic depressive patients, inhibits the mammalian enzyme inositol monophosphatase, which is proposed as the biological target for the effects of lithium. In this study we investigated Dictyostelium discoideum inositol monophosphatase activity. Partial purification of the proteins in the soluble cell fraction using anion-exchange chromatography revealed the presence of at least three enzyme activities capable of degrading inositol monophosphate isomers. The first activity was similar to the monophosphatase found in mammalian cells, as it degraded Ins(4)P, Ins(1)P and to a lesser extent Ins(3)P, was dependent on MgCl2 and inhibited by LiCl in a uncompetitive [corrected] manner. The second enzyme activity was specific for Ins(4)P; the enzyme activity was not dependent on MgCl2 and not inhibited by LiCl. The third monophosphatase activity degraded especially Ins(3)P, but also Ins(4)P and Ins(1)P; increasing concentrations of MgCl2 inhibited this enzyme activity, whereas LiCl had no effect. In vivo, LiCl induces a reduction of inositol levels by about 20%. In [3H]inositol-labelled cells LiCl causes a 6-fold increase in the radioactivity of [3H]Ins(1)P, a doubling of [3H]Ins(4)P and a slight decrease in the radioactivity in [3H]Ins(3)P. These data indicate that the biological effects of lithium in Dictyostelium are not due to depletion of the inositol pool by inhibition of inositol monophosphatase activity. PMID:8670062

  14. Possible participation of brain-specific proteins of the S100 group in the regulation of processes of phosphorylation-dephosphorylation of endogenous substrates of nerve tissue

    SciTech Connect

    Gruden, M.A.; Poletaev, A.B.

    1986-06-10

    The influence of brain-specific proteins of the S-100 group (BSP S100) and their endogenous cationic and anionic protein ligands on the phosphorylation of macromolecular substrates of the rat brain and liver was investigated. It was shown that BSP S100 has a substantial influence both on the phosphorylation and on the dephosphorylation of various substrates, primarily of nerve tissue; moreover, this action depends on endogenous protein ligands of BSP S100 and has characteristics of organ specificity.

  15. Nature of immobilization surface affects antibody specificity to placental alkaline phosphatase.

    PubMed

    Kumar, Mukesh; Khan, Imran; Sinha, Subrata

    2015-01-01

    Retention of native conformation of immobilized protein is essential for various applications including selection and detection of specific recombinant antibodies (scFvs). Placental alkaline phosphatase (PAP), an onco-fetal antigen expressed on the surface of several tumors, was immobilized on supermagnetic particles for selection of recombinant antibodies from a human phage display antibody library. The isolated antibodies were found to be cross-reactive to either of the isozymes of alkaline phosphatase, i.e., bone alkaline phosphatase (BAP) or intestinal alkaline phosphatase (IAP) and could not be used for tumor targeting. A specific anti-PAP monoclonal antibody H17E2 was tested for retention of specificity under these conditions. Binding of the antibody to magnetic beads conjugated IAP and BAP along with PAP and the ability of the two isozymes to inhibit its binding to PAP depicted the loss of isozyme specificity of the antibody. However, the antibody retained its specificity to PAP immobilized on polyvinyl chloride (PVC) surface. Enzyme activity was observed on both surfaces. This demonstrates that nature of immobilization may affect antigen-antibody binding in subtle ways, resulting in alteration of conformation of the epitopes. This may have consequences for determining the specificity of antibody binding for proteins that share a high degree of homology.

  16. The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

    PubMed

    Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

    2009-01-01

    Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling. PMID:19654415

  17. N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5.

    PubMed

    Zou, Shui; Toh, Joel D W; Wong, Kendra H Q; Gao, Yong-Gui; Hong, Wanjin; Woon, Esther C Y

    2016-01-01

    N(6)-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a 'conformational marker', which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins. PMID:27156733

  18. N6-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5

    PubMed Central

    Zou, Shui; Toh, Joel D. W.; Wong, Kendra H. Q.; Gao, Yong-Gui; Hong, Wanjin; Woon, Esther C. Y.

    2016-01-01

    N6-Methyladenosine (m6A) is currently one of the most intensively studied post-transcriptional modifications in RNA. Due to its critical role in epigenetics and physiological links to several human diseases, it is also of tremendous biological and medical interest. The m6A mark is dynamically reversed by human demethylases FTO and ALKBH5, however the mechanism by which these enzymes selectively recognise their target transcripts remains unclear. Here, we report combined biophysical and biochemical studies on the specificity determinants of m6A demethylases, which led to the identification of an m6A-mediated substrate discrimination mechanism. Our results reveal that m6A itself serves as a ‘conformational marker’, which induces different conformational outcomes in RNAs depending on sequence context. This critically impacts its interactions with several m6A-recognising proteins, including FTO and ALKBH5. Remarkably, through the RNA-remodelling effects of m6A, the demethylases were able to discriminate substrates with very similar nucleotide sequences. Our findings provide novel insights into the biological functions of m6A modifications. The mechanism identified in this work is likely of significance to other m6A-recognising proteins. PMID:27156733

  19. A Fungal α-Galactosidase from Tricholoma matsutake with Broad Substrate Specificity and Good Hydrolytic Activity on Raffinose Family Oligosaccharides.

    PubMed

    Geng, Xueran; Tian, Guoting; Zhao, Yongchang; Zhao, Liyan; Wang, Hexiang; Ng, Tzi Bun

    2015-07-24

    An acidic α-galactosidase designated as TMG was purified from the fruiting bodies The purification protocol entailed ion exchange chromatography on Q-Sepharose and of Tricholoma matsutake with 136-fold purification and a specific activity of 909 units/mg. Mono-Q and fast protein liquid chromatography on Superdex 75. TMG is a monomeric protein exhibiting a molecular mass of 47 kDa in SDS-PAGE and gel filtration. The purified enzyme was identified by LC-MS/MS and three inner amino acid sequences were obtained. The optimum pH and temperature for TMG with pNPGal as substrate were pH 4.5 and 55 °C, respectively. The α-galactosidase activity was strongly inhibited by K+, Ca2+, Cd2+, Hg2+, Ag+ and Zn2+ ions. The enzyme activity was inhibited by the chemical modification agent N-bromosuccinimide (NBS), indicating the importance of tryptophan residue(s) at or near the active site. Besides hydrolyzing pNPGal, TMG also efficaciously catalyzed the degradation of natural substrates such as stachyose, raffinose, and melibiose. Thus TMG can be exploited commercially for improving the nutritional value of soy milk by degradation of indigestible oligosaccharides.

  20. Rac1 changes the substrate specificity of gamma-secretase between amyloid precursor protein and Notch1.

    PubMed

    Boo, Jung Hyun; Sohn, Ji Hoon; Kim, Ji Eun; Song, Hyundong; Mook-Jung, Inhee

    2008-08-01

    Beta amyloid peptide is generated from amyloid precursor protein (APP) by proteolytic cleavage of beta- and gamma-secretases, and plays a critical role in the pathogenesis of Alzheimer's disease. Since gamma-secretase cleaves several proteins including APP and Notch in a number of cell types, it is important to understand the conditions determining gamma-secretase substrate specificity. In the present study, inhibition of Rac1 attenuated gamma-secretase activity for APP, resulting in decreased production of the APP intracellular domain but accumulated C-terminal fragments (APP-CTF). In contrast, Rac1 inhibitor, NSC23766 increased production of the Notch1 intracellular domain but slightly decreased the ectodomain-shed form of Notch1 (NotchDeltaE). To elucidate the mechanism underlying these observations, we performed co-immunoprecipitation experiments to analyze the interaction between Rac1 and presenilin1 (PS1), a component of the gamma-secretase complex. Inhibition of Rac1 enhanced its interaction with PS1. Under the same condition, PS1 interacted more strongly with NotchDeltaE than with APP-CTF. Our results suggested that PS1 determines the preferred substrate for gamma-secretase between APP and Notch1, depending on the activation status of Rac1.

  1. The Akt1-eNOS axis illustrates the specificity of kinase-substrate relationships in vivo.

    PubMed

    Schleicher, Michael; Yu, Jun; Murata, Takahisa; Derakhshan, Berhad; Atochin, Dimitriy; Qian, Li; Kashiwagi, Satoshi; Di Lorenzo, Annarita; Harrison, Kenneth D; Huang, Paul L; Sessa, William C

    2009-08-04

    Akt1 is critical for many in vivo functions; however, the cell-specific substrates responsible remain to be defined. Here, we examine the importance of endothelial nitric oxide synthase (eNOS) as an Akt1 substrate by generating Akt1-deficient mice (Akt1(-/-) mice) carrying knock-in mutations (serine to aspartate or serine to alanine substitutions) of the critical Akt1 phosphorylation site on eNOS (serine 1176) that render the enzyme "constitutively active" or "less active." The eNOS mutations did not influence several phenotypes in Akt1(-/-) mice; however, the defective postnatal angiogenesis characteristic of Akt1(-/-) mice was rescued by crossing the Akt1(-/-) mice with mice carrying the constitutively active form of eNOS, but not by crossing with mice carrying the less active eNOS mutant. This genetic rescue resulted in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) and increased production of HIF-1alpha-responsive genes in vivo and in vitro. Thus, Akt1 regulates angiogenesis largely through phosphorylation of eNOS and NO-dependent signaling.

  2. A family with a dystrophin gene mutation specifically affecting dystrophin expression in the heart

    SciTech Connect

    Muntoni, F.; Davies, K.; Dubowitz, V.

    1994-09-01

    We recently described a family with X-linked dilated cardiomyopathy where a large deletion in the muscle promoter region of the dystrophin gene was associated with a severe dilated cardiomyopathy in absence of clinical skeletal muscle involvement. The deletion removed the entire muscle promoter region, the first muscle exon and part of intron 1. The brain and Purkinje cell promoters were not affected by the deletion. Despite the lack of both the muscle promoter and the first muscle exon, dystrophin was detected immunocytochemically in relative high levels in the skeletal muscle of the affected males. We have now found that both the brain and Purkinje cell promoters were transcribed at high levels in the skeletal muscle of these individuals. This phenomenon, that does not occur in normal skeletal muscle, indicates that these two isoforms, physiologically expressed mainly in the central nervous system, can be transcribed and be functionally active in skeletal muscle under specific circumstances. Contrary to what is observed in skeletal muscle, dystrophin was not detected in the heart of one affected male using immunocytochemistry and an entire panel of anti-dystrophin antibodies. This was most likely the cause for the pronounced cardiac fibrosis observed and eventually responsible for the severe cardiac involvement invariably seen in seven affected males. In conclusion, the mutation of the muscle promoter, first muscle exon and part of intron 1 specifically affected expression of dystrophin in the heart. We believe that this deletion removes sequences involved in regulation of dystrophin expression in the heart and are at the moment characterizing other families with X-linked cardiomyopathy secondary to a dystrophinopathy.

  3. Sampling the Hudson River estuary for PCBs using multiplate artificial substrate samplers and congener-specific gas chromatography in 1991

    SciTech Connect

    Bush, B. . Wadsworth Center for Labs. and Research State Univ. o