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Sample records for promiscuous substrate recognition

  1. 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

  2. Promiscuous Substrate Recognition in Folding and Assembly Activities of the Trigger Factor Chaperone

    SciTech Connect

    Martinez-Hackert, E.; Hendrickson, W

    2009-01-01

    Trigger factor (TF) is a molecular chaperone that binds to bacterial ribosomes where it contacts emerging nascent chains, but TF is also abundant free in the cytosol where its activity is less well characterized. In vitro studies show that TF promotes protein refolding. We find here that ribosome-free TF stably associates with and rescues from misfolding a large repertoire of full-length proteins. We identify over 170 members of this cytosolic Escherichia coli TF substrate proteome, including ribosomal protein S7. We analyzed the biochemical properties of a TF:S7 complex from Thermotoga maritima and determined its crystal structure. Thereby, we obtained an atomic-level picture of a promiscuous chaperone in complex with a physiological substrate protein. The structure of the complex reveals the molecular basis of substrate recognition by TF, indicates how TF could accelerate protein folding, and suggests a role for TF in the biogenesis of protein complexes.

  3. NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism

    PubMed Central

    Kim, Youngchang; Cunningham, Mark A.; Mire, Joseph; Tesar, Christine; Sacchettini, James; Joachimiak, Andrzej

    2013-01-01

    The specter of a return to an era in which infectious disease looms as a significant threat to human health is not just hyperbole; there are serious concerns about the widespread overuse and misuse of antibiotics contributing to increased antibiotic resistance in pathogens. The recent discovery of a new enzyme, first identified in Klebsiella pneumoniae from a patient from New Delhi and denoted as NDM-1, represents an example of extreme promiscuity: It hydrolyzes and inactivates nearly all known β-lactam-based antibiotics with startling efficiency. NDM-1 can utilize different metal cofactors and seems to exploit an alternative mechanism based on the reaction conditions. Here we report the results of a combined experimental and theoretical study that examines the substrate, metal binding, and catalytic mechanism of the enzyme. We utilize structures obtained through X-ray crystallography, biochemical assays, and numerical simulation to construct a model of the enzyme catalytic pathway. The NDM-1 enzyme interacts with the substrate solely through zinc, or other metals, bound in the active site, explaining the observed lack of specificity against a broad range of β-lactam antibiotic agents. The zinc ions also serve to activate a water molecule that hydrolyzes the β-lactam ring through a proton shuttle.—Kim, Y., Cunningham, M. A.; Mire, J., Tesar, C., Sacchettini, J., Joachimiak, A. NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism. PMID:23363572

  4. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  5. 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

  6. Molecular mechanism underlying promiscuous polyamine recognition by spermidine acetyltransferase.

    PubMed

    Sugiyama, Shigeru; Ishikawa, Sae; Tomitori, Hideyuki; Niiyama, Mayumi; Hirose, Mika; Miyazaki, Yuma; Higashi, Kyohei; Murata, Michio; Adachi, Hiroaki; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Kashiwagi, Keiko; Igarashi, Kazuei; Matsumura, Hiroyoshi

    2016-07-01

    Spermidine acetyltransferase (SAT) from Escherichia coli, which catalyses the transfer of acetyl groups from acetyl-CoA to spermidine, is a key enzyme in controlling polyamine levels in prokaryotic cells. In this study, we determined the crystal structure of SAT in complex with spermidine (SPD) and CoA at 2.5Å resolution. SAT is a dodecamer organized as a hexamer of dimers. The secondary structural element and folding topology of the SAT dimer resemble those of spermidine/spermine N(1)-acetyltransferase (SSAT), suggesting an evolutionary link between SAT and SSAT. However, the polyamine specificity of SAT is distinct from that of SSAT and is promiscuous. The SPD molecule is also located at the inter-dimer interface. The distance between SPD and CoA molecules is 13Å. A deep, highly acidic, water-filled cavity encompasses the SPD and CoA binding sites. Structure-based mutagenesis and in-vitro assays identified SPD-bound residues, and the acidic residues lining the walls of the cavity are mostly essential for enzymatic activities. Based on mutagenesis and structural data, we propose an acetylation mechanism underlying promiscuous polyamine recognition for SAT. PMID:27163532

  7. Structural Basis for Substrate Promiscuity of dCK

    SciTech Connect

    Sabini, Elisabetti; Hazra, Saugata; Ort, Stephen; Konrad, Manfred; Lavie, Arnon

    2008-06-06

    Deoxycytidine kinase (dCK) is an essential nucleoside kinase critical for the production of nucleotide precursors for DNA synthesis. This enzyme catalyzes the initial conversion of the nucleosides deoxyadenosine (dA), deoxyguanosine (dG), and deoxycytidine (dC) into their monophosphate forms, with subsequent phosphorylation to the triphosphate forms performed by additional enzymes. Several nucleoside analog prodrugs are dependent on dCK for their pharmacological activation, and even nucleosides of the non-physiological L-chirality are phosphorylated by dCK. In addition to accepting dC and purine nucleosides (and their analogs) as phosphoryl acceptors, dCK can utilize either ATP or UTP as phosphoryl donors. To unravel the structural basis for substrate promiscuity of dCK at both the nucleoside acceptor and nucleotide donor sites, we solved the crystal structures of the enzyme as ternary complexes with the two enantiomeric forms of dA (D-dA, or L-dA), with either UDP or ADP bound to the donor site. The complexes with UDP revealed an open state of dCK in which the nucleoside, either D-dA or L-dA, is surprisingly bound in a manner not consistent with catalysis. In contrast, the complexes with ADP, with either D-dA or L-dA, adopted a closed and catalytically competent conformation. The differential states adopted by dCK in response to the nature of the nucleotide were also detected by tryptophan fluorescence experiments. Thus, we are in the unique position to observe differential effects at the acceptor site due to the nature of the nucleotide at the donor site, allowing us to rationalize the different kinetic properties observed with UTP to those with ATP.

  8. Multisite Promiscuity in the Processing of Endogenous Substrates By Human Carboxylesterase 1

    SciTech Connect

    Bencharit, S.; Edwards, C.C.; Morton, C.L.; Howard-Williams, E.L.; Kuhn, P.; Potter, P.M.; Redinbo, M.R.; /North Carolina U. /St. Jude Children's Hosp., Memphis /SLAC, SSRL

    2007-01-16

    Human carboxylesterase 1 (hCE1) is a drug and endobiotic-processing serine hydrolase that exhibits relatively broad substrate specificity. It has been implicated in a variety of endogenous cholesterol metabolism pathways including the following apparently disparate reactions: cholesterol ester hydrolysis (CEH), fatty acyl Coenzyme A hydrolysis (FACoAH), acyl-Coenzyme A:cholesterol acyltransfer (ACAT), and fatty acyl ethyl ester synthesis (FAEES). The structural basis for the ability of hCE1 to perform these catalytic actions involving large substrates and products has remained unclear. Here we present four crystal structures of the hCE1 glycoprotein in complexes with the following endogenous substrates or substrate analogues: Coenzyme A, the fatty acid palmitate, and the bile acids cholate and taurocholate. While the active site of hCE1 was known to be promiscuous and capable of interacting with a variety of chemically distinct ligands, these structures reveal that the enzyme contains two additional ligand-binding sites and that each site also exhibits relatively non-specific ligand-binding properties. Using this multisite promiscuity, hCE1 appears structurally capable of assembling several catalytic events depending, apparently, on the physiological state of the cellular environment. These results expand our understanding of enzyme promiscuity and indicate that, in the case of hCE1, multiple non-specific sites are employed to perform distinct catalytic actions.

  9. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes.

    PubMed

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-09-01

    Although one of an enzyme's hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. It is known that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. Here we report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination. PMID:26244568

  10. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    DOE PAGESBeta

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involvingmore » the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.« less

  11. Substrate-Assisted Catalysis in the Reaction Catalyzed by Salicylic Acid Binding Protein 2 (SABP2), a Potential Mechanism of Substrate Discrimination for Some Promiscuous Enzymes

    SciTech Connect

    Yao, Jianzhuang; Guo, Haobo; Chaiprasongsuk, Minta; Zhao, Nan; Chen, Feng; Yang, Xiaohan; Guo, Hong

    2015-08-05

    Although one of an enzyme’s hallmarks is the high specificity for their natural substrates, substrate promiscuity has been reported more frequently. We know that promiscuous enzymes generally show different catalytic efficiencies to different substrates, but our understanding of the origin of such differences is still lacking. We report the results of quantum mechanical/molecular mechanical simulations and an experimental study of salicylic acid binding protein 2 (SABP2). SABP2 has promiscuous esterase activity toward a series of substrates but shows a high activity toward its natural substrate, methyl salicylate (MeSA). Finally, our results demonstrate that this enzyme may use substrate-assisted catalysis involving the hydroxyl group from MeSA to enhance the activity and achieve substrate discrimination.

  12. Hyperstability and substrate promiscuity in laboratory resurrections of Precambrian β-lactamases.

    PubMed

    Risso, Valeria A; Gavira, Jose A; Mejia-Carmona, Diego F; Gaucher, Eric A; Sanchez-Ruiz, Jose M

    2013-02-27

    We report a sequence reconstruction analysis targeting several Precambrian nodes in the evolution of class-A β-lactamases and the preparation and experimental characterization of their encoded proteins. Despite extensive sequence differences with the modern enzymes (~100 amino acid differences), the proteins resurrected in the laboratory properly fold into the canonical lactamase structure. The encoded proteins from 2-3 billion years (Gyr)-old β-lactamase sequences undergo cooperative two-state thermal denaturation and display very large denaturation temperature enhancements (~35 °C) relative to modern β-lactamases. They degrade different antibiotics in vitro with catalytic efficiencies comparable to that of an average modern enzyme. This enhanced substrate promiscuity is not accompanied by significant changes in the active-site region as seen in static X-ray structures, suggesting a plausible role for dynamics in the evolution of function in these proteins. Laboratory resurrections of 2-3 Gyr-old β-lactamases also endowed modern microorganisms with significant levels of resistance toward a variety of antibiotics, opening up the possibility of performing laboratory replays of the molecular tape of lactamase evolution. Overall, these results support the notions that Precambrian life was thermophilic and that proteins can evolve from substrate-promiscuous generalists into specialists during the course of natural evolution. They also highlight the biotechnological potential of laboratory resurrection of Precambrian proteins, as both high stability and enhanced promiscuity (likely contributors to high evolvability) are advantageous features in protein scaffolds for molecular design and laboratory evolution. PMID:23394108

  13. Production of Sactipeptides in Escherichia coli: Probing the Substrate Promiscuity of Subtilosin A Biosynthesis.

    PubMed

    Himes, Paul M; Allen, Scott E; Hwang, Sungwon; Bowers, Albert A

    2016-06-17

    Sactipeptides are peptide-derived natural products that are processed by remarkable, radical-mediated cysteine sulfur to α-carbon coupling reactions. The resulting sactionine thioether linkages give rise to the unique defined structures and concomitant biological activities of sactipeptides. An E. coli heterologous expression system, based on the biosynthesis of one such sactipeptide, subtilosin A, is described and this expression system is exploited to probe the promiscuity of the subtilosin A sactionine bond-forming enzyme, AlbA. These efforts allowed the facile expression and isolation of a small library of mutant sactipeptides based on the subtilosin A precursor peptide, demonstrating broad substrate promiscuity where none was previously known. Importantly, we show that the positions of the sactionine linkages can be moved, giving rise to new, unnatural sactipeptide structures. E. coli heterologous expression also allowed incorporation of unnatural amino acids into sactipeptides by means of amber-suppression technology, potentially opening up new chemistry and new applications for unnatural sactipeptides. PMID:27019323

  14. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin.

    PubMed

    Eudes, Aymerick; Pereira, Jose H; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E K; Lee, Taek Soon; Adams, Paul D; Keasling, Jay D; Loqué, Dominique

    2016-03-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  15. Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase to Reduce Lignin

    PubMed Central

    Eudes, Aymerick; Pereira, Jose H.; Yogiswara, Sasha; Wang, George; Teixeira Benites, Veronica; Baidoo, Edward E.K.; Lee, Taek Soon; Adams, Paul D.; Keasling, Jay D.; Loqué, Dominique

    2016-01-01

    Lignin poses a major challenge in the processing of plant biomass for agro-industrial applications. For bioengineering purposes, there is a pressing interest in identifying and characterizing the enzymes responsible for the biosynthesis of lignin. Hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase (HCT; EC 2.3.1.133) is a key metabolic entry point for the synthesis of the most important lignin monomers: coniferyl and sinapyl alcohols. In this study, we investigated the substrate promiscuity of HCT from a bryophyte (Physcomitrella) and from five representatives of vascular plants (Arabidopsis, poplar, switchgrass, pine and Selaginella) using a yeast expression system. We demonstrate for these HCTs a conserved capacity to acylate with p-coumaroyl-CoA several phenolic compounds in addition to the canonical acceptor shikimate normally used during lignin biosynthesis. Using either recombinant HCT from switchgrass (PvHCT2a) or an Arabidopsis stem protein extract, we show evidence of the inhibitory effect of these phenolics on the synthesis of p-coumaroyl shikimate in vitro, which presumably occurs via a mechanism of competitive inhibition. A structural study of PvHCT2a confirmed the binding of a non-canonical acceptor in a similar manner to shikimate in the active site of the enzyme. Finally, we exploited in Arabidopsis the substrate flexibility of HCT to reduce lignin content and improve biomass saccharification by engineering transgenic lines that overproduce one of the HCT non-canonical acceptors. Our results demonstrate conservation of HCT substrate promiscuity and provide support for a new strategy for lignin reduction in the effort to improve the quality of plant biomass for forage and cellulosic biofuels. PMID:26858288

  16. Recognition sequences and substrate evolution in cyanobactin biosynthesis.

    PubMed

    Sardar, Debosmita; Pierce, Elizabeth; McIntosh, John A; Schmidt, Eric W

    2015-02-20

    Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit "substrate evolution", where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme-substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro. PMID:24625112

  17. Structural basis for promiscuous PAM recognition in type I-E Cascade from E. coli.

    PubMed

    Hayes, Robert P; Xiao, Yibei; Ding, Fran; van Erp, Paul B G; Rajashankar, Kanagalaghatta; Bailey, Scott; Wiedenheft, Blake; Ke, Ailong

    2016-02-25

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process. PMID:26863189

  18. A promiscuous recognition mechanism between GPR17 and SDF-1: Molecular insights.

    PubMed

    Parravicini, Chiara; Daniele, Simona; Palazzolo, Luca; Trincavelli, Maria Letizia; Martini, Claudia; Zaratin, Paola; Primi, Roberto; Coppolino, Giusy; Gianazza, Elisabetta; Abbracchio, Maria P; Eberini, Ivano

    2016-06-01

    Recent data and publications suggest a promiscuous behaviour for GPR17, a class-A GPCR operated by different classes of ligands, such as uracil nucleotides, cysteinyl-leukotrienes and oxysterols. This observation, together with the ability of several class-A GPCRs to form homo- and hetero-dimers, is likely to unveil new pathophysiological roles and novel emerging pharmacological properties for some of these GPCRs, including GPR17. This receptor shares structural, phylogenetic and functional properties with some chemokine receptors, CXCRs. Both GPR17 and CXCR2 are operated by oxysterols, and both GPR17 and CXCR ligands have been demonstrated to have a role in orchestrating inflammatory responses and oligodendrocyte precursor cell differentiation to myelinating cells in acute and chronic diseases of the central nervous system. Here, by combining in silico modelling data with in vitro validation in (i) a classical reference pharmacological assay for GPCR activity and (ii) a model of maturation of primary oligodendrocyte precursor cells, we demonstrate that GPR17 can be activated by SDF-1, a ligand of chemokine receptors CXCR4 and CXCR7, and investigate the underlying molecular recognition mechanism. We also demonstrate that cangrelor, a GPR17 orthosteric antagonist, can block the SDF-1-mediated activation of GPR17 in a concentration-dependent manner. The ability of GPR17 to respond to different classes of GPCR ligands suggests that this receptor modifies its function depending on the extracellular mileu changes occurring under specific pathophysiological conditions and advocates it as a strategic target for neurodegenerative diseases with an inflammatory/immune component. PMID:26971834

  19. Enzyme promiscuity in earthworm serine protease: substrate versatility and therapeutic potential.

    PubMed

    Verma, Mahendra Kumar; Pulicherla, K K

    2016-04-01

    Enzymes are the most versatile molecules in the biological world. These amazing molecules play an integral role in the regulation of various metabolic pathways and physiology subsequently. Promiscuity of an enzyme is the capacity to catalyze additional biochemical reactions besides their native one. Catalytic promiscuity has shown great impact in enzyme engineering for commercial enzyme and therapeutics with natural or engineered catalytic promiscuity. The earthworm serine protease (ESP) is a classic example of enzyme promiscuity and studied for its therapeutic potential over the last few decades. The ESP was reported for several therapeutic properties and fibrinolytic activity has been much explored. ESP, a complex enzyme exists as several isoforms of molecular weight ranging from 14 to 33 kDa. The fibrinolytic capacity of the enzyme has been studied in different species of earthworm and molecular mechanism is quite different from conventional thrombolytics. Cytotoxic and anti-tumor activities of ESP were evaluated using several cancer cell lines. Enzyme had shown tremendous scope in fighting against plant viruses and microbes. ESP is also reported for anti-inflammatory activity and anti-oxidant property. Apart from these, recently, ESP is reported for DNase activity. The daunting challenge for researchers is to understand the molecular mechanism for such diverse properties and possibility of enzyme promiscuity. This review emphasizes molecular mechanism of ESP governing various biochemical reactions. Further, the concept of enzyme promiscuity in ESP towards development of novel enzyme based drugs has been reviewed in this study. PMID:26739820

  20. Catalytic and substrate promiscuity: distinct multiple chemistries catalysed by the phosphatase domain of receptor protein tyrosine phosphatase.

    PubMed

    Srinivasan, Bharath; Marks, Hanna; Mitra, Sreyoshi; Smalley, David M; Skolnick, Jeffrey

    2016-07-15

    The presence of latent activities in enzymes is posited to underlie the natural evolution of new catalytic functions. However, the prevalence and extent of such substrate and catalytic ambiguity in evolved enzymes is difficult to address experimentally given the order-of-magnitude difference in the activities for native and, sometimes, promiscuous substrate/s. Further, such latent functions are of special interest when the activities concerned do not fall into the domain of substrate promiscuity. In the present study, we show a special case of such latent enzyme activity by demonstrating the presence of two mechanistically distinct reactions catalysed by the catalytic domain of receptor protein tyrosine phosphatase isoform δ (PTPRδ). The primary catalytic activity involves the hydrolysis of a phosphomonoester bond (C─O─P) with high catalytic efficiency, whereas the secondary activity is the hydrolysis of a glycosidic bond (C─O─C) with poorer catalytic efficiency. This enzyme also displays substrate promiscuity by hydrolysing diester bonds while being highly discriminative for its monoester substrates. To confirm these activities, we also demonstrated their presence on the catalytic domain of protein tyrosine phosphatase Ω (PTPRΩ), a homologue of PTPRδ. Studies on the rate, metal-ion dependence, pH dependence and inhibition of the respective activities showed that they are markedly different. This is the first study that demonstrates a novel sugar hydrolase and diesterase activity for the phosphatase domain (PD) of PTPRδ and PTPRΩ. This work has significant implications for both understanding the evolution of enzymatic activity and the possible physiological role of this new chemistry. Our findings suggest that the genome might harbour a wealth of such alternative latent enzyme activities in the same protein domain that renders our knowledge of metabolic networks incomplete. PMID:27208174

  1. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

    SciTech Connect

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl; Woolard, Frank; Ockey, Denise; McPhee, Derek; Renninger, Neil; Chang, Michelle; Baker, David; Keasling, Jay

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

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

    PubMed Central

    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. The substrate promiscuity of a phosphopantetheinyl transferase SchPPT for coenzyme A derivatives and acyl carrier proteins.

    PubMed

    Wang, Yue-Yue; Luo, Hong-Dou; Zhang, Xiao-Sheng; Lin, Tao; Jiang, Hui; Li, Yong-Quan

    2016-03-01

    Phosphopantetheinyl transferases (PPTases) catalyze the posttranslational modification of acyl carrier proteins (ACPs) in fatty acid synthases (FASs), ACPs in polyketide synthases, and peptidyl carrier proteins (PCPs) in nonribosomal peptide synthetases (NRPSs) in all organisms. Some bacterial PPTases have broad substrate specificities for ACPs/PCPs and/or coenzyme A (CoA)/CoA analogs, facilitating their application in metabolite production in hosts and/or labeling of ACPs/PCPs, respectively. Here, a group II PPTase SchPPT from Streptomyces chattanoogensis L10 was characterized to accept a heterologous ACP and acetyl-CoA. Thus, SchPPT is a promiscuous PPTase and may be used on polyketide production in heterologous bacterial host and labeling of ACPs. PMID:26748983

  4. Substrate Promiscuity: AglB, the Archaeal Oligosaccharyltransferase, Can Process a Variety of Lipid-Linked Glycans

    PubMed Central

    Cohen-Rosenzweig, Chen; Guan, Ziqiang; Shaanan, Boaz

    2014-01-01

    Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. PMID:24212570

  5. Substrate promiscuity: AglB, the archaeal oligosaccharyltransferase, can process a variety of lipid-linked glycans.

    PubMed

    Cohen-Rosenzweig, Chen; Guan, Ziqiang; Shaanan, Boaz; Eichler, Jerry

    2014-01-01

    Across evolution, N-glycosylation involves oligosaccharyltransferases that transfer lipid-linked glycans to selected Asn residues of target proteins. While these enzymes catalyze similar reactions in each domain, differences exist in terms of the chemical composition, length and degree of phosphorylation of the lipid glycan carrier, the sugar linking the glycan to the lipid carrier, and the composition and structure of the transferred glycan. To gain insight into how oligosaccharyltransferases cope with such substrate diversity, the present study analyzed the archaeal oligosaccharyltransferase AglB from four haloarchaeal species. Accordingly, it was shown that despite processing distinct lipid-linked glycans in their native hosts, AglB from Haloarcula marismortui, Halobacterium salinarum, and Haloferax mediterranei could readily replace their counterpart from Haloferax volcanii when introduced into Hfx. volcanii cells deleted of aglB. As the four enzymes show significant sequence and apparently structural homology, it appears that the functional similarity of the four AglB proteins reflects the relaxed substrate specificity of these enzymes. Such demonstration of AglB substrate promiscuity is important not only for better understanding of N-glycosylation in Archaea and elsewhere but also for efforts aimed at transforming Hfx. volcanii into a glycoengineering platform. PMID:24212570

  6. Self-recognition drives the preferential accumulation of promiscuous CD4+ T-cells in aged mice

    PubMed Central

    Deshpande, Neha R; Parrish, Heather L; Kuhns, Michael S

    2015-01-01

    T-cell recognition of self and foreign peptide antigens presented in major histocompatibility complex molecules (pMHC) is essential for life-long immunity. How the ability of the CD4+ T-cell compartment to bind self- and foreign-pMHC changes over the lifespan remains a fundamental aspect of T-cell biology that is largely unexplored. We report that, while old mice (18–22 months) contain fewer CD4+ T-cells compared with adults (8–12 weeks), those that remain have a higher intrinsic affinity for self-pMHC, as measured by CD5 expression. Old mice also have more cells that bind individual or multiple distinct foreign-pMHCs, and the fold increase in pMHC-binding populations is directly related to their CD5 levels. These data demonstrate that the CD4+ T-cell compartment preferentially accumulates promiscuous constituents with age as a consequence of higher affinity T-cell receptor interactions with self-pMHC. DOI: http://dx.doi.org/10.7554/eLife.05949.001 PMID:26173205

  7. Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis.

    PubMed

    Lairson, Luke L; Watts, Andrew G; Wakarchuk, Warren W; Withers, Stephen G

    2006-12-01

    Despite their unparalleled catalytic prowess and environmental compatibility, enzymes have yet to see widespread application in synthetic chemistry. This lack of application and the resulting underuse of their enormous potential stems not only from a wariness about aqueous biological catalysis on the part of the typical synthetic chemist but also from limitations on enzyme applicability that arise from the high degree of substrate specificity possessed by most enzymes. This latter perceived limitation is being successfully challenged through rational protein engineering and directed evolution efforts to alter substrate specificity. However, such programs require considerable effort to establish. Here we report an alternative strategy for expanding the substrate specificity, and therefore the synthetic utility, of a given enzyme through a process of "substrate engineering". The attachment of a readily removable functional group to an alternative glycosyltransferase substrate induces a productive binding mode, facilitating rational control of substrate specificity and regioselectivity using wild-type enzymes. PMID:17057723

  8. Promiscuous words

    PubMed Central

    2013-01-01

    Promiscuity is frequently used to describe animal mating behaviour, and especially to describe multiple mating by females. Yet this use of the term is incorrect, perhaps reflecting an erroneous adoption of common language to pique reader interest. We evaluated the patterns of use and misuse of the word ‘promiscuity’ in a representative journal of animal behaviour. This survey highlights how inappropriately the term is used, and how it can conceal critical features of animal mating strategies with intriguing evolutionary significance. Further analysis of the scientific impact of papers identified by the term promiscuous or polyandrous revealed that the former were cited less frequently. We argue that using promiscuity to describe animal mating strategies is anthropomorphic, inaccurate, and potentially misleading. Consistent with other biological disciplines, the word promiscuity should be used to describe indiscriminate mating behaviour only, and that polygyny and polyandry should be used to describe male and female mating frequency respectively. PMID:24209457

  9. Functional Characterization and Substrate Promiscuity of UGT71 Glycosyltransferases from Strawberry (Fragaria × ananassa).

    PubMed

    Song, Chuankui; Gu, Le; Liu, Jingyi; Zhao, Shuai; Hong, Xiaotong; Schulenburg, Katja; Schwab, Wilfried

    2015-12-01

    Glycosylation determines the complexity and diversity of plant natural products. To characterize fruit ripening-related UDP-dependent glycosyltransferases (UGTs) functionally in strawberry, we mined the publicly available Fragaria vesca genome sequence and found 199 putative UGT genes. Candidate UGTs whose expression levels were strongly up-regulated during fruit ripening were cloned from F.×ananassa and six were successfully expressed in Escherichia coli and biochemically characterized. UGT75T1 showed very strict substrate specificity and glucosylated only galangin out of 33 compounds. The other recombinant enzymes exhibited broad substrate tolerance, accepting numerous flavonoids, hydroxycoumarins, naphthols and the plant hormone, (+)-S-abscisic acid (ABA). UGT71W2 showed the highest activity towards 1-naphthol, while UGT71A33, UGT71A34a/b and UGT71A35 preferred 3-hydroxycoumarin and formed 3- and 7-O-glucosides as well as a diglucoside from flavonols. Screening of a strawberry physiological aglycone library identified kaempferol, quercetin, ABA and three unknown natural compounds as putative in planta substrates of UGT71A33, UGT71A34a and UGT71W2. Metabolite analyses of RNA interference (RNAi)-mediated silenced fruits demonstrated that UGT71W2 contributes to the glycosylation of flavonols, xenobiotics and, to a minor extent, of ABA, in planta. The study showed that both specialist and generalist UGTs were expressed during strawberry fruit ripening and the latter were probably not restricted to only one function in plants. PMID:26454881

  10. Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascena.

    PubMed

    Huang, Fong-Chin; Horváth, Györgyi; Molnár, Péter; Turcsi, Erika; Deli, József; Schrader, Jens; Sandmann, Gerhard; Schmidt, Holger; Schwab, Wilfried

    2009-03-01

    Several of the key flavor compounds in rose essential oil are C(13)-norisoprenoids, such as beta-damascenone, beta-damascone, and beta-ionone which are derived from carotenoid degradation. To search for genes putatively responsible for the cleavage of carotenoids, cloning of carotenoid cleavage (di-)oxygenase (CCD) genes from Rosa damascena was carried out by a degenerate primer approach and yielded a full-length cDNA (RdCCD1). The RdCCD1 gene was expressed in Escherichia coli and recombinant protein was assayed for its cleavage activity with a multitude of carotenoid substrates. The RdCCD1 protein was able to cleave a variety of carotenoids at the 9-10 and 9'-10' positions to produce a C(14) dialdehyde and two C(13) products, which vary depending on the carotenoid substrates. RdCCD1 could also cleave lycopene at the 5-6 and 5'-6' positions to produce 6-methyl-5-hepten-2-one. Expression of RdCCD1 was studied by real-time PCR in different tissues of rose. The RdCCD1 transcript was present predominantly in rose flower, where high levels of volatile C(13)-norisoprenoids are produced. Thus, the accumulation of C(13)-norisoprenoids in rose flower is correlated to the expression of RdCCD1. PMID:19264332

  11. The structural basis of substrate promiscuity in UDP-hexose 4-epimerase from the hyperthermophilic Eubacterium Thermotoga maritima.

    PubMed

    Shin, Sun-Mi; Choi, Jin Myung; di Luccio, Eric; Lee, Yong-Jik; Lee, Sang-Jae; Lee, Sang Jun; Lee, Sung Haeng; Lee, Dong-Woo

    2015-11-01

    UDP-galactose 4-epimerase (GalE) catalyzes the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal), which is a pivotal step in the Leloir pathway for d-galactose metabolism. Although GalE is widely distributed in prokaryotes and eukaryotes, little information is available regarding hyperthermophilic GalE. We overexpressed the TM0509 gene, encoding a putative GalE from Thermotoga maritima (TMGalE), in Escherichia coli and characterized the encoded protein. To further investigate the molecular basis of this enzyme's catalytic function, we determined the crystal structures of TMGalE and TMGalE bound to UDP-Glc at resolutions of 1.9 Å and 2.0 Å, respectively. The enzyme was determined to be a homodimer with a molecular mass of 70 kDa. The enzyme could reversibly catalyze the epimerization of UDP-GalNAc/UDP-GlcNAc as well as UDP-Gal/UDP-Glc at elevated temperatures, with an apparent optimal temperature and pH of 80 °C and 7.0, respectively. Our data showed that TM0509 is a UDP-galactosugar 4-epimerase involved in d-galactose metabolism; consequently, this study provides the first detailed characterization of a hyperthermophilic GalE. Moreover, the promiscuous substrate specificity of TMGalE, which is more similar to human GalE than E. coli GalE, supports the notion that TMGalE might exhibit the earliest form of sugar-epimerizing enzymes in the evolution of galactose metabolism. PMID:26344854

  12. Extreme Substrate Promiscuity of the Neisseria Oligosaccharyl Transferase Involved in Protein O-Glycosylation*S⃞

    PubMed Central

    Faridmoayer, Amirreza; Fentabil, Messele A.; Haurat, M. Florencia; Yi, Wen; Woodward, Robert; Wang, Peng George; Feldman, Mario F.

    2008-01-01

    Neisseria meningitidis PglL belongs to a novel family of bacterial oligosaccharyltransferases (OTases) responsible for O-glycosylation of type IV pilins. Although members of this family are widespread among pathogenic bacteria, there is little known about their mechanism. Understanding the O-glycosylation process may uncover potential targets for therapeutic intervention, and can open new avenues for the exploitation of these pathways for biotechnological purposes. In this work, we demonstrate that PglL is able to transfer virtually any glycan from the undecaprenyl pyrophosphate (UndPP) carrier to pilin in engineered Escherichia coli and Salmonella cells. Surprisingly, PglL was also able to interfere with the peptidoglycan biosynthetic machinery and transfer peptidoglycan subunits to pilin. This represents a previously unknown post-translational modification in bacteria. Given the wide range of glycans transferred by PglL, we reasoned that substrate specificity of PglL lies in the lipid carrier. To test this hypothesis we developed an in vitro glycosylation system that employed purified PglL, pilin, and the lipid farnesyl pyrophosphate (FarPP) carrying a pentasaccharide that had been synthesized by successive chemical and enzymatic steps. Although FarPP has different stereochemistry and a significantly shorter aliphatic chain than the natural lipid substrate, the pentasaccharide was still transferred to pilin in our system. We propose that the primary roles of the lipid carrier during O-glycosylation are the translocation of the glycan into the periplasm, and the positioning of the pyrophosphate linker and glycan adjacent to PglL. The unique characteristics of PglL make this enzyme a promising tool for glycoengineering novel glycan-based vaccines and therapeutics. PMID:18930921

  13. ERAD substrate recognition in budding yeast.

    PubMed

    Xie, Wei; Ng, Davis T W

    2010-07-01

    During protein synthesis, the orderly progression of folding, modification, and assembly is paramount to function and vis-à-vis cellular viability. Accordingly, sophisticated quality control mechanisms have evolved to monitor protein maturation throughout the cell. Proteins failing at any step are segregated and degraded as a preventative measure against potential toxicity. Although protein quality control is generally poorly understood, recent research advances in endoplasmic reticulum-associated degradation (ERAD) pathways have provided the most detailed view so far. The discovery of distinct substrate processing sites established a biochemical basis for genetic profiles of model misfolded proteins. Detailed mechanisms for substrate recognition were recently uncovered. For some proteins, sequential glycan trimming steps set a time window for folding. Proteins still unfolded at the final stage expose a specific degradation signal recognized by the ERAD machinery. Through this mechanism, the system does not in fact know that a molecule is "misfolded". Instead, it goes by the premise that proteins past due have veered off their normal folding pathways and therefore aberrant. PMID:20178855

  14. Donor substrate promiscuity of bacterial β1-3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1-4-galactosyltransferases.

    PubMed

    Li, Yanhong; Xue, Mengyang; Sheng, Xue; Yu, Hai; Zeng, Jie; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Wang, Peng G; Chen, Xi

    2016-04-15

    β1-3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1-4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1-3Galβ1-4GlcβProN3 and Galβ1-3Galβ1-4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents. PMID:26968649

  15. The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for Archaeal isopentenyl phosphate kinase

    PubMed Central

    Mabanglo, Mark F.; Serohijos, Adrian W. R.; Poulter, C. Dale

    2011-01-01

    Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate (IPP) in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK, and H58 and K18 in S. wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase superfamily. We measured the fosfomycin kinase activity of THA IPK, Km = 15.1 ± 1.0 mM and kcat = (4.0 ± 0.1) × 10−2 s−1, resulting in a catalytic efficiency, kcat/Km = 2.6 M−1s−1, that is five orders of magnitude less than the native reaction. Fosfomycin is a competitive inhibitor of IPK, Ki = 3.6 ± 0.2 mM. Molecular dynamics simulation of the IPK•fosfomycin•MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK•IP•ATP complex that it engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin producing organisms. PMID:22148590

  16. Donor substrate promiscuity of the N-acetylglucosaminyltransferase activities of Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA.

    PubMed

    Li, Yanhong; Yu, Hai; Thon, Vireak; Chen, Yi; Muthana, Musleh M; Qu, Jingyao; Hie, Liana; Chen, Xi

    2014-02-01

    The biological activities of heparan sulfate (HS) and heparin (HP) are closely related to their molecular structures. Both Pasteurella multocida heparosan synthase 2 (PmHS2) and Escherichia coli K5 KfiA have been used for enzymatic and chemoenzymatic synthesis of HS and HP oligosaccharides and their derivatives. We show here that cloning using the pET15b vector and expressing PmHS2 as an N-His6-tagged fusion protein improve its expression level in E. coli. Investigation of the donor substrate specificity of the N-acetylglucosaminyltransferase activities of P. multocida heparosan synthase 2 (PmHS2) and E. coli K5 KfiA indicates the substrate promiscuities of PmHS2 and KfiA. Overall, both PmHS2 and KfiA can use uridine 5'-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and some of its C2'- and C6'-derivatives as donor substrates for their α1-4-GlcNAcT activities. Nevertheless, PmHS2 has a broader tolerance towards substrate modifications. Other than the UDP-sugars that can be used by KfiA, additional C6'-derivatives of UDP-GlcNAc, UDP-glucose, and UDP-N-acetylgalactosamine (UDP-GalNAc) are tolerable substrates for the α1-4-GlcNAcT activity of PmHS2. The substrate promiscuities of PmHS2 and KfiA will allow efficient chemoenzymatic synthesis of diverse HS and HP oligosaccharide derivatives which may have improved or altered activities compared to their natural counterparts. PMID:23661084

  17. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant. PMID:17539607

  18. Dynamics connect substrate recognition to catalysis in protein kinase A

    PubMed Central

    Masterson, Larry R.; Cheng, Cecilia; Yu, Tao; Tonelli, Marco; Kornev, Alexandr; Taylor, Susan S.; Veglia, Gianluigi

    2012-01-01

    Atomic resolution studies of protein kinases have traditionally been carried out in the inhibitory state, limiting our current knowledge on the mechanisms of substrate recognition and catalysis. Using NMR, x-ray crystallography, and thermodynamic measurements we analyzed the substrate recognition process of cAMP-dependent protein kinase (PKA), finding that entropy and protein dynamics play a prominent role. The nucleotide acts as a dynamic and allosteric activator by coupling the two lobes of apo PKA, enhancing the enzyme dynamics synchronously, and priming it for catalysis. The formation of the ternary complex is entropically driven and NMR spin relaxation data reveal that both substrate and PKA are dynamic in the closed state. Our results show that the enzyme toggles between open and closed states, which indicate that a population shift/conformational selection rather than an induced-fit mechanism governs substrate recognition. PMID:20890288

  19. U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs.

    PubMed

    Jenkins, Jermaine L; Agrawal, Anant A; Gupta, Ankit; Green, Michael R; Kielkopf, Clara L

    2013-04-01

    Degenerate splice site sequences mark the intron boundaries of pre-mRNA transcripts in multicellular eukaryotes. The essential pre-mRNA splicing factor U2AF(65) is faced with the paradoxical tasks of accurately targeting polypyrimidine (Py) tracts preceding 3' splice sites while adapting to both cytidine and uridine nucleotides with nearly equivalent frequencies. To understand how U2AF(65) recognizes degenerate Py tracts, we determined six crystal structures of human U2AF(65) bound to cytidine-containing Py tracts. As deoxy-ribose backbones were required for co-crystallization with these Py tracts, we also determined two baseline structures of U2AF(65) bound to the deoxy-uridine counterparts and compared the original, RNA-bound structure. Local structural changes suggest that the N-terminal RNA recognition motif 1 (RRM1) is more promiscuous for cytosine-containing Py tracts than the C-terminal RRM2. These structural differences between the RRMs were reinforced by the specificities of wild-type and site-directed mutant U2AF(65) for region-dependent cytosine- and uracil-containing RNA sites. Small-angle X-ray scattering analyses further demonstrated that Py tract variations select distinct inter-RRM spacings from a pre-existing ensemble of U2AF(65) conformations. Our results highlight both local and global conformational selection as a means for universal 3' splice site recognition by U2AF(65). PMID:23376934

  20. Structural basis for ligand and substrate recognition by torovirus hemagglutinin esterases

    PubMed Central

    Langereis, Martijn A.; Zeng, Qinghong; Gerwig, Gerrit J.; Frey, Barbara; von Itzstein, Mark; Kamerling, Johannis P.; de Groot, Raoul J.; Huizinga, Eric G.

    2009-01-01

    Hemagglutinin esterases (HEs), closely related envelope glycoproteins in influenza C and corona- and toroviruses, mediate reversible attachment to O-acetylated sialic acids (Sias). They do so by acting both as lectins and as receptor-destroying enzymes, functions exerted by separate protein domains. HE divergence was accompanied by changes in quaternary structure and in receptor and substrate specificity. The selective forces underlying HE diversity and the molecular basis for Sia specificity are poorly understood. Here we present crystal structures of porcine and bovine torovirus HEs in complex with receptor analogs. Torovirus HEs form homodimers with sialate-O-acetylesterase domains almost identical to corresponding domains in orthomyxo- and coronavirus HEs, but with unique lectin sites. Structure-guided biochemical analysis of the esterase domains revealed that a functionally, but not structurally conserved arginine–Sia carboxylate interaction is critical for the binding and positioning of glycosidically bound Sias in the catalytic pocket. Although essential for efficient de-O-acetylation of Sias, this interaction is not required for catalysis nor does it affect substrate specificity. In fact, the distinct preference of the porcine torovirus enzyme for 9-mono- over 7,9-di-O-acetylated Sias can be explained from a single-residue difference with HEs of more promiscuous specificity. Apparently, esterase and lectin pockets coevolved; also the porcine torovirus HE receptor-binding site seems to have been designed to use 9-mono- and exclude di-O-acetylated Sias, possibly as an adaptation to replication in swine. Our findings shed light on HE evolution and provide fundamental insight into mechanisms of substrate binding, substrate recognition, and receptor selection in this important class of virion proteins. PMID:19721004

  1. The neuronal substrates of human olfactory based kin recognition.

    PubMed

    Lundström, Johan N; Boyle, Julie A; Zatorre, Robert J; Jones-Gotman, Marilyn

    2009-08-01

    Kin recognition, an evolutionary phenomenon ubiquitous among phyla, is thought to promote an individual's genes by facilitating nepotism and avoidance of inbreeding. Whereas isolating and studying kin recognition mechanisms in humans using auditory and visual stimuli is problematic because of the high degree of conscious recognition of the individual involved, kin recognition based on body odors is done predominantly without conscious recognition. Using this, we mapped the neural substrates of human kin recognition by acquiring measures of regional cerebral blood flow from women smelling the body odors of either their sister or their same-sex friend. The initial behavioral experiment demonstrated that accurate identification of kin is performed with a low conscious recognition. The subsequent neuroimaging experiment demonstrated that olfactory based kin recognition in women recruited the frontal-temporal junction, the insula, and the dorsomedial prefrontal cortex; the latter area is implicated in the coding of self-referent processing and kin recognition. We further show that the neuronal response is seemingly independent of conscious identification of the individual source, demonstrating that humans have an odor based kin detection system akin to what has been shown for other mammals. PMID:19067327

  2. Local and spatial factors determining HIV-1 protease substrate recognition.

    PubMed Central

    Hazebrouck, S; Machtelinckx-Delmas, V; Kupiec, J J; Sonigo, P

    2001-01-01

    Insertional mutagenesis of the Escherichia coli thymidylate synthase (TS) was used to address substrate recognition of HIV-1 protease in a well characterized structural context. By modifying the TS conformation while maintaining its enzymic activity, we investigated the influence of protein folding on protease-substrate recognition. A slight destabilization of the TS structure permitted the cleavage of a target site, which was resistant in the native TS. This result supports a dynamic interpretation of HIV-1 protease specificity. Exposure time of the potential cleavage site, which depends on the stability of the global conformation, must be compatible with the cleavage kinetics, which are determined by the local sequence. Cleavage specificity has been described as the consequence of cumulative interactions, globally favourable, between at least six amino acids around the cleavage site. To investigate influence of local sequence, we introduced insertions of variable lengths in two exposed loops of the TS. In both environments, insertion of only two amino acids could determine specific cleavage. We then inserted libraries of dipeptides naturally cleaved by the HIV-1 protease in order to assess the limitations of established classifications of substrates in different conformational contexts. PMID:11513751

  3. Characterization of cyclo-Acetoacetyl-L-Tryptophan Dimethylallyltransferase in Cyclopiazonic Acid Biosynthesis: Substrate Promiscuity and Site Directed Mutagenesis Studies

    PubMed Central

    Liu, Xinyu; Walsh, Christopher T.

    2009-01-01

    The fungal neurotoxin α-cyclopiazonic acid (CPA), a nanomolar inhibitor of Ca2+-ATPase with a unique pentacyclic indole tetramic acid scaffold is assembled by a three enzyme pathway CpaS, CpaD and CpaO in Aspergillus sp. We recently characterized the first pathway-specific enzyme CpaS, a hybrid two module polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) that generates cyclo-acetoacetyl-L-tryptophan (cAATrp). Here we report the characterization of the second pathway-specific enzyme CpaD that regiospecifically dimethylallylates cAATrp to form β-cyclopiazonic acid. By exploring the tryptophan and tetramate moieties of cAATrp, we demonstrate that CpaD discriminates against free Trp but accepts tryptophan-containing thiohydantoins, diketopiperazines and linear peptides as substrates for C4-prenylation and also acts as regiospecific O-dimethylallyltransferase (DMAT) on a tyrosine-derived tetramic acid. Comparative evaluation of CpaDs from A. oryzae RIB40 and A. flavus NRRL3357 indicated the importance of the N-terminal region for its activity. Sequence alignment of CpaD with eleven homologous fungal Trp-DMATs revealed five regions of conservation suggesting the presense of critical motifs that could be diagonostic for discovering additional Trp-DMATs. Subsequent site-directed mutagenesis studies identified five polar/charged residues and five tyrosine residues within these motifs that are critical for CpaD activity. This motif characerization will enable a gene probe-based approach to discover additional biosynthetic Trp-DMATs. PMID:19877600

  4. Clostridial neurotoxins: mode of substrate recognition and novel therapy development.

    PubMed

    Chen, Sheng

    2014-01-01

    The clostridial neurotoxins (CNTs) are among the most potent protein toxins known to humans. CNTs include seven serotypes (A~G) of botulinum toxins (BoNTs), which cause botulism, a flaccid paralysis, and tetanus toxin (TeNT), which causes spastic paralysis. BoNTs are classified as category A agent and may be used as potential bioterrorism weapons. On the other hand, the ability of an extremely low dosage of BoNTs (less than 1 ng) to cause reversible partial paralysis upon injection into muscle has turned BoNTs, in particular serotypes A and B, from deadly agents to novel therapeutic agents for treatment of a wide range of clinical conditions associated with involuntary muscle spasm and contractions. In addition to clinical use, they may also be used in cosmetics. Further indications for BoNTs will continue to be developed, although current BoNT therapies have encountered some limitations due to the pharmacological properties of BoNTs, such as their ability to elicit immunoresistance in patients upon periodical injections. This review summarizes the present knowledge of the mechanisms of action of CNTs, with particular focus on the mode of substrate recognition by CNT catalytic domains and knowledge regarding substrate recognition can be utilized to develop novel BoNT products to extend its usefulness in therapeutic interventions and overcome the immunoresistance problems. PMID:24106961

  5. Substrate recognition by ADAR1 and ADAR2.

    PubMed Central

    Wong, S K; Sato, S; Lazinski, D W

    2001-01-01

    RNA editing catalyzed by ADAR1 and ADAR2 involves the site-specific conversion of adenosine to inosine within imperfectly duplexed RNA. ADAR1- and ADAR2-mediated editing occurs within transcripts of glutamate receptors (GluR) in the brain and in hepatitis delta virus (HDV) RNA in the liver. Although the Q/R site within the GluR-B premessage is edited more efficiently by ADAR2 than it is by ADAR1, the converse is true for the +60 site within this same transcript. ADAR1 and ADAR2 are homologs having two common functional regions, an N-terminal double-stranded RNA-binding domain and a C-terminal deaminase domain. It is neither understood why only certain adenosines within a substrate molecule serve as targets for ADARs, nor is it known which domain of an ADAR confers its specificity for particular editing sites. To assess the importance of several aspects of RNA sequence and structure on editing, we evaluated 20 different mutated substrates, derived from four editing sites, for their ability to be edited by either ADAR1 or ADAR2. We found that when these derivatives contained an A:C mismatch at the editing site, editing by both ADARs was enhanced compared to when A:A or A:G mismatches or A:U base pairs occurred at the same site. Hence substrate recognition and/or catalysis by ADARs could involve the base that opposes the edited adenosine. In addition, by using protein chimeras in which the deaminase domains were exchanged between ADAR1 and ADAR2, we found that this domain played a dominant role in defining the substrate specificity of the resulting enzyme. PMID:11421361

  6. Structural characterization of human heparanase reveals insights into substrate recognition.

    PubMed

    Wu, Liang; Viola, Cristina M; Brzozowski, Andrzej M; Davies, Gideon J

    2015-12-01

    Heparan sulfate (HS) is a glycosaminoglycan that forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-β-glucuronidase of the glycoside hydrolase 79 (GH79) family. Overexpression of HPSE results in breakdown of extracellular HS and release of stored growth factors and hence is strongly linked to cancer metastasis. Here we present crystal structures of human HPSE at 1.6-Å to 1.9-Å resolution that reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. We used oligosaccharide complexes to map the substrate-binding and sulfate-recognition motifs. These data shed light on the structure and interactions of a key enzyme involved in ECM maintenance and provide a starting point for the design of HPSE inhibitors for use as biochemical tools and anticancer therapeutics. PMID:26575439

  7. Structural characterization of human heparanase reveals insights into substrate recognition

    PubMed Central

    Wu, Liang; Viola, Cristina M.; Brzozowski, Andrzej M.; Davies, Gideon J.

    2016-01-01

    Heparan Sulfate (HS) is a glycosaminoglycan (GAG) which forms a key component of the extracellular matrix (ECM). Breakdown of HS is carried out by heparanase (HPSE), an endo-β-glucuronidase of the glycoside hydrolase (GH)79 family. Overexpression of HPSE is strongly linked to cancer metastases - reflecting breakdown of extracellular HS and release of stored growth factors. Here we present crystal structures of human HPSE at 1.6-1.9 Å resolution reveal how an endo-acting binding cleft is exposed by proteolytic activation of latent proHPSE. Oligosaccharide complexes map the substrate-binding and sulfate recognition motifs. These data shed light on the structure and interactions for a key enzyme involved in ECM maintenance, and provide a starting point for design of HPSE inhibitors as biochemical tools and anti-cancer therapeutics. PMID:26575439

  8. Fibrinogen Substrate Recognition by Staphylocoagulase·(Pro)thrombin Complexes*

    PubMed Central

    Panizzi, Peter; Friedrich, Rainer; Fuentes-Prior, Pablo; Richter, Klaus; Bock, Paul E.; Bode, Wolfram

    2008-01-01

    Thrombin generation and fibrinogen (Fbg) clotting are the ultimate proteolytic reactions in the blood coagulation pathway. Staphylocoagulase (SC), a protein secreted by the human pathogen Staphylococcus aureus, activates prothrombin (ProT) without proteolysis. The SC·(pro)thrombin complex recognizes Fbg as a specific substrate, converting it directly into fibrin. The crystal structure of a fully active SC fragment containing residues 1–325 (SC-(1–325)) bound to human prethrombin 2 showed previously that SC inserts its Ile1-Val2 N terminus into the Ile16 pocket of prethrombin 2, inducing a functional active site in the cognate zymogen conformationally. Exosite I of α-thrombin, the Fbg recognition site, and proexosite I on ProT are blocked by domain 2 of SC-(1–325). In the present studies, active site-labeled fluorescent ProT analogs were used to quantitate Fbg binding to the SC-(1–325)·ProT complex. Fbg binding and cleavage are mediated by expression of a new Fbg-binding exosite on the SC-(1–325)·ProT complex, resulting in formation of an (SC-(1–325)·ProT)2·Fbg pentameric complex with a dissociation constant of 8–34 nM. In both crystal structures, the SC-(1–325)·(pre)thrombin complexes form dimers, with both pro-teinases/zymogens facing each other over a large U-shaped cleft, through which the Fbg substrate could thread. On this basis, a molecular model of the pentameric (SC-(1–325)·thrombin)2·Fbg encounter complex was generated, which explains the coagulant properties and efficient Fbg conversion. The results provide new insight into the mechanism that mediates high affinity Fbg binding and cleavage as a substrate of SC·(pro)thrombin complexes, a process that is central to the molecular pathology of S. aureus endocarditis. PMID:16230339

  9. Substrate Recognition and Modification by the Nosiheptide Resistance Methyltransferase

    PubMed Central

    Chen, Dongrong; Murchie, Alastair I. H.

    2015-01-01

    Background The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2ʹO-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear. Methodology/Principal Findings We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5ʹ) to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation. Conclusion/Significance Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2’ hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an

  10. Neural Substrates of Auditory Emotion Recognition Deficits in Schizophrenia

    PubMed Central

    Hoptman, Matthew J.; Leitman, David I.; Moreno-Ortega, Marta; Lehrfeld, Jonathan M.; Dias, Elisa; Sehatpour, Pejman; Laukka, Petri; Silipo, Gail

    2015-01-01

    Deficits in auditory emotion recognition (AER) are a core feature of schizophrenia and a key component of social cognitive impairment. AER deficits are tied behaviorally to impaired ability to interpret tonal (“prosodic”) features of speech that normally convey emotion, such as modulations in base pitch (F0M) and pitch variability (F0SD). These modulations can be recreated using synthetic frequency modulated (FM) tones that mimic the prosodic contours of specific emotional stimuli. The present study investigates neural mechanisms underlying impaired AER using a combined event-related potential/resting-state functional connectivity (rsfMRI) approach in 84 schizophrenia/schizoaffective disorder patients and 66 healthy comparison subjects. Mismatch negativity (MMN) to FM tones was assessed in 43 patients/36 controls. rsfMRI between auditory cortex and medial temporal (insula) regions was assessed in 55 patients/51 controls. The relationship between AER, MMN to FM tones, and rsfMRI was assessed in the subset who performed all assessments (14 patients, 21 controls). As predicted, patients showed robust reductions in MMN across FM stimulus type (p = 0.005), particularly to modulations in F0M, along with impairments in AER and FM tone discrimination. MMN source analysis indicated dipoles in both auditory cortex and anterior insula, whereas rsfMRI analyses showed reduced auditory-insula connectivity. MMN to FM tones and functional connectivity together accounted for ∼50% of the variance in AER performance across individuals. These findings demonstrate that impaired preattentive processing of tonal information and reduced auditory-insula connectivity are critical determinants of social cognitive dysfunction in schizophrenia, and thus represent key targets for future research and clinical intervention. SIGNIFICANCE STATEMENT Schizophrenia patients show deficits in the ability to infer emotion based upon tone of voice [auditory emotion recognition (AER)] that drive

  11. Promiscuity and electrostatic flexibility in the alkaline phosphatase superfamily.

    PubMed

    Pabis, Anna; Kamerlin, Shina Caroline Lynn

    2016-04-01

    Catalytic promiscuity, that is, the ability of single enzymes to facilitate the turnover of multiple, chemically distinct substrates, is a widespread phenomenon that plays an important role in the evolution of enzyme function. Additionally, such pre-existing multifunctionality can be harnessed in artificial enzyme design. The members of the alkaline phosphatase superfamily have served extensively as both experimental and computational model systems for enhancing our understanding of catalytic promiscuity. In this Opinion, we present key recent computational studies into the catalytic activity of these highly promiscuous enzymes, highlighting the valuable insight they have provided into both the molecular basis for catalytic promiscuity in general, and its implications for the evolution of phosphatase activity. PMID:26716576

  12. Substrate Recognition by β-Ketoacyl-ACP Synthases†

    PubMed Central

    Borgaro, Janine G.; Chang, Andrew; Machutta, Carl A.; Zhang, Xujie; Tonge, Peter J.

    2011-01-01

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14 residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the kcat/Km for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  13. Substrate recognition by β-ketoacyl-ACP synthases.

    PubMed

    Borgaro, Janine G; Chang, Andrew; Machutta, Carl A; Zhang, Xujie; Tonge, Peter J

    2011-12-13

    β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway. PMID:22017312

  14. Sequence-specific intramembrane proteolysis: identification of a recognition motif in rhomboid substrates.

    PubMed

    Strisovsky, Kvido; Sharpe, Hayley J; Freeman, Matthew

    2009-12-25

    Members of the widespread rhomboid family of intramembrane proteases cleave transmembrane domain (TMD) proteins to regulate processes as diverse as EGF receptor signaling, mitochondrial dynamics, and invasion by apicomplexan parasites. However, lack of information about their substrates means that the biological role of most rhomboids remains obscure. Knowledge of how rhomboids recognize their substrates would illuminate their mechanism and might also allow substrate prediction. Previous work has suggested that rhomboid substrates are specified by helical instability in their TMD. Here we demonstrate that rhomboids instead primarily recognize a specific sequence surrounding the cleavage site. This recognition motif is necessary for substrate cleavage, it determines the cleavage site, and it is more strictly required than TM helix-destabilizing residues. Our work demonstrates that intramembrane proteases can be sequence specific and that genome-wide substrate prediction based on their recognition motifs is feasible. PMID:20064469

  15. Fractionating the Neural Substrates of Incidental Recognition Memory

    ERIC Educational Resources Information Center

    Greene, Ciara M.; Vidaki, Kleio; Soto, David

    2015-01-01

    Familiar stimuli are typically accompanied by decreases in neural response relative to the presentation of novel items, but these studies often include explicit instructions to discriminate old and new items; this creates difficulties in partialling out the contribution of top-down intentional orientation to the items based on recognition goals.…

  16. Neural Substrates for Episodic Encoding and Recognition of Unfamiliar Faces

    ERIC Educational Resources Information Center

    Hofer, Alex; Siedentopf, Christian M.; Ischebeck, Anja; Rettenbacher, Maria A.; Verius, Michael; Golaszewski, Stefan M.; Felber, Stephan; Fleischhacker, W. Wolfgang

    2007-01-01

    Functional MRI was used to investigate brain activation in healthy volunteers during encoding of unfamiliar faces as well as during correct recognition of newly learned faces (CR) compared to correct identification of distractor faces (CF), missed alarms (not recognizing previously presented faces, MA), and false alarms (incorrectly recognizing…

  17. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    SciTech Connect

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  18. Substrate Recognition of Histone H2B by DUBm

    NASA Astrophysics Data System (ADS)

    Henderson, Elizabeth; Berndsen, Christopher; Wolberger, Cynthia

    2011-03-01

    The SAGA complex is a transcriptional coactivator that regulates gene expression in eukaryotes via histone acetylation and deubiquitination, which are crucial for transcription. Our lab is investigating the SAGA-dependent deubiquitination of histone H2B. The deubiquitinating module (DUBm) of SAGA is comprised of a ubiquitin-specific protease, Ubp8, and three other proteins. It is known that Ubp8 cleaves ubiquitin from histone H2B, however, the specific way in which the enzyme binds to the substrate remains elusive. In order to unravel this mechanism, we attempted to determine the crystal structure of the substrate binding complex. We obtained this substrate by exploiting the techniques of intein chemistry to artificially ubiquitinate a histone H2B peptide, which we then co-crystallized with DUBm. Additionally, we synthesized Ub-K63R-linked chains and Ub-K48-linked chains and co-crystallized them with DUBm.

  19. Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer.

    PubMed

    Pabis, Anna; Duarte, Fernanda; Kamerlin, Shina C L

    2016-06-01

    The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

  20. Promiscuity in the Enzymatic Catalysis of Phosphate and Sulfate Transfer

    PubMed Central

    2016-01-01

    The enzymes that facilitate phosphate and sulfate hydrolysis are among the most proficient natural catalysts known to date. Interestingly, a large number of these enzymes are promiscuous catalysts that exhibit both phosphatase and sulfatase activities in the same active site and, on top of that, have also been demonstrated to efficiently catalyze the hydrolysis of other additional substrates with varying degrees of efficiency. Understanding the factors that underlie such multifunctionality is crucial both for understanding functional evolution in enzyme superfamilies and for the development of artificial enzymes. In this Current Topic, we have primarily focused on the structural and mechanistic basis for catalytic promiscuity among enzymes that facilitate both phosphoryl and sulfuryl transfer in the same active site, while comparing this to how catalytic promiscuity manifests in other promiscuous phosphatases. We have also drawn on the large number of experimental and computational studies of selected model systems in the literature to explore the different features driving the catalytic promiscuity of such enzymes. Finally, on the basis of this comparative analysis, we probe the plausible origins and determinants of catalytic promiscuity in enzymes that catalyze phosphoryl and sulfuryl transfer. PMID:27187273

  1. Basis for substrate recognition and distinction by matrix metalloproteinases

    PubMed Central

    Ratnikov, Boris I.; Cieplak, Piotr; Gramatikoff, Kosi; Pierce, James; Eroshkin, Alexey; Igarashi, Yoshinobu; Kazanov, Marat; Sun, Qing; Godzik, Adam; Osterman, Andrei; Stec, Boguslaw; Strongin, Alex; Smith, Jeffrey W.

    2014-01-01

    Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure–function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221–227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for function-related predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure–function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k(obs)] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50–57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs. PMID:25246591

  2. Basis for substrate recognition and distinction by matrix metalloproteinases.

    PubMed

    Ratnikov, Boris I; Cieplak, Piotr; Gramatikoff, Kosi; Pierce, James; Eroshkin, Alexey; Igarashi, Yoshinobu; Kazanov, Marat; Sun, Qing; Godzik, Adam; Osterman, Andrei; Stec, Boguslaw; Strongin, Alex; Smith, Jeffrey W

    2014-10-01

    Genomic sequencing and structural genomics produced a vast amount of sequence and structural data, creating an opportunity for structure-function analysis in silico [Radivojac P, et al. (2013) Nat Methods 10(3):221-227]. Unfortunately, only a few large experimental datasets exist to serve as benchmarks for function-related predictions. Furthermore, currently there are no reliable means to predict the extent of functional similarity among proteins. Here, we quantify structure-function relationships among three phylogenetic branches of the matrix metalloproteinase (MMP) family by comparing their cleavage efficiencies toward an extended set of phage peptide substrates that were selected from ∼ 64 million peptide sequences (i.e., a large unbiased representation of substrate space). The observed second-order rate constants [k(obs)] across the substrate space provide a distance measure of functional similarity among the MMPs. These functional distances directly correlate with MMP phylogenetic distance. There is also a remarkable and near-perfect correlation between the MMP substrate preference and sequence identity of 50-57 discontinuous residues surrounding the catalytic groove. We conclude that these residues represent the specificity-determining positions (SDPs) that allowed for the expansion of MMP proteolytic function during evolution. A transmutation of only a few selected SDPs proximal to the bound substrate peptide, and contributing the most to selectivity among the MMPs, is sufficient to enact a global change in the substrate preference of one MMP to that of another, indicating the potential for the rational and focused redesign of cleavage specificity in MMPs. PMID:25246591

  3. Cation–π Interactions Contribute to Substrate Recognition in γ‐Butyrobetaine Hydroxylase Catalysis

    PubMed Central

    Kamps, Jos J. A. G.; Khan, Amjad; Choi, Hwanho; Lesniak, Robert K.; Brem, Jürgen; Rydzik, Anna M.; McDonough, Michael A.; Schofield, Christopher J.; Claridge, Timothy D. W.

    2015-01-01

    Abstract γ‐Butyrobetaine hydroxylase (BBOX) is a non‐heme FeII‐ and 2‐oxoglutarate‐dependent oxygenase that catalyzes the stereoselective hydroxylation of an unactivated C−H bond of γ‐butyrobetaine (γBB) in the final step of carnitine biosynthesis. BBOX contains an aromatic cage for the recognition of the positively charged trimethylammonium group of the γBB substrate. Enzyme binding and kinetic analyses on substrate analogues with P and As substituting for N in the trimethylammonium group show that the analogues are good BBOX substrates, which follow the efficiency trend N+>P+>As+. The results reveal that an uncharged carbon analogue of γBB is not a BBOX substrate, thus highlighting the importance of the energetically favorable cation–π interactions in productive substrate recognition. PMID:26660433

  4. Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

    PubMed

    Chen, Sheng; Wan, Hoi Ying

    2011-01-15

    BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions. PMID:21029044

  5. Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins.

    PubMed

    Sikorra, Stefan; Henke, Tina; Galli, Thierry; Binz, Thomas

    2008-07-25

    Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond. PMID:18511418

  6. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

    PubMed

    Joachimiak, Lukasz A; Walzthoeni, Thomas; Liu, Corey W; Aebersold, Ruedi; Frydman, Judith

    2014-11-20

    The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical, and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate-binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to TRiC's unique ability to fold obligate substrates. PMID:25416944

  7. The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT

    PubMed Central

    Joachimiak, Lukasz A.; Walzthoeni, Thomas; Liu, Corey; Aebersold, Ruedi; Frydman, Judith

    2014-01-01

    Summary The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion. Here, we integrate structural, biophysical and modeling approaches to identify the hitherto unknown substrate-binding site in TRiC and uncover the basis of substrate recognition. NMR and modeling provided a structural model of a chaperonin-substrate complex. Mutagenesis and crosslinking-mass spectrometry validated the identified substrate binding interface and demonstrate that TRiC contacts full-length substrates combinatorially in a subunit-specific manner. The binding site of each subunit has a distinct, evolutionarily conserved, pattern of polar and hydrophobic residues specifying recognition of discrete substrate motifs. The combinatorial recognition of polypeptides broadens the specificity of TRiC and may direct the topology of bound polypeptides along a productive folding trajectory, contributing to its unique ability to fold obligate substrates. PMID:25416944

  8. Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5.

    PubMed

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-01-01

    Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L(54)-E(55), of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20-65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2', P1', P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development. PMID:26794648

  9. Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5

    PubMed Central

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-01-01

    Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L54-E55, of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20–65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2′, P1′, P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development. PMID:26794648

  10. Substrate recognition by the cell surface palmitoyl transferase DHHC5

    PubMed Central

    Howie, Jacqueline; Reilly, Louise; Fraser, Niall J.; Vlachaki Walker, Julia M.; Wypijewski, Krzysztof J.; Ashford, Michael L. J.; Calaghan, Sarah C.; McClafferty, Heather; Tian, Lijun; Shipston, Michael J.; Boguslavskyi, Andrii; Shattock, Michael J.; Fuller, William

    2014-01-01

    The cardiac phosphoprotein phospholemman (PLM) regulates the cardiac sodium pump, activating the pump when phosphorylated and inhibiting it when palmitoylated. Protein palmitoylation, the reversible attachment of a 16 carbon fatty acid to a cysteine thiol, is catalyzed by the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases. The cell surface palmitoyl acyltransferase DHHC5 regulates a growing number of cellular processes, but relatively few DHHC5 substrates have been identified to date. We examined the expression of DHHC isoforms in ventricular muscle and report that DHHC5 is among the most abundantly expressed DHHCs in the heart and localizes to caveolin-enriched cell surface microdomains. DHHC5 coimmunoprecipitates with PLM in ventricular myocytes and transiently transfected cells. Overexpression and silencing experiments indicate that DHHC5 palmitoylates PLM at two juxtamembrane cysteines, C40 and C42, although C40 is the principal palmitoylation site. PLM interaction with and palmitoylation by DHHC5 is independent of the DHHC5 PSD-95/Discs-large/ZO-1 homology (PDZ) binding motif, but requires a ∼120 amino acid region of the DHHC5 intracellular C-tail immediately after the fourth transmembrane domain. PLM C42A but not PLM C40A inhibits the Na pump, indicating PLM palmitoylation at C40 but not C42 is required for PLM-mediated inhibition of pump activity. In conclusion, we demonstrate an enzyme–substrate relationship for DHHC5 and PLM and describe a means of substrate recruitment not hitherto described for this acyltransferase. We propose that PLM palmitoylation by DHHC5 promotes phospholipid interactions that inhibit the Na pump. PMID:25422474

  11. Extensive HLA class I allele promiscuity among viral CTL epitopes

    PubMed Central

    Frahm, Nicole; Yusim, Karina; Suscovich, Todd J.; Adams, Sharon; Sidney, John; Hraber, Peter; Hewitt, Hannah S.; Linde, Caitlyn H.; Kavanagh, Daniel G.; Woodberry, Tonia; Henry, Leah M.; Faircloth, Kellie; Listgarten, Jennifer; Kadie, Carl; Jojic, Nebojsa; Sango, Kaori; Brown, Nancy V.; Pae, Eunice; Zaman, M. Tauheed; Bihl, Florian; Khatri, Ashok; John, Mina; Mallal, Simon; Marincola, Francesco M.; Walker, Bruce D.; Sette, Alessandro; Heckerman, David; Korber, Bette T.; Brander, Christian

    2008-01-01

    Summary Promiscuous binding of T helper epitopes to MHC class II molecules has been well established, but few examples of promiscuous class I restricted epitopes exist. To address the extent of promiscuity of HLA class I peptides, responses to 242 well-defined viral epitopes were tested in 100 subjects regardless of the individuals’ HLA type. Surprisingly, half of all detected responses were seen in the absence of the originally reported restricting HLA class I allele, and only 3% of epitopes were recognized exclusively in the presence of their original allele. Functional assays confirmed the frequent recognition of HLA class I-restricted T cell epitopes on several alternative alleles across HLA class I supertypes and encoded on different class I loci. These data have significant implications for the understanding of MHC class I restricted antigen presentation and vaccine development. PMID:17705138

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

    SciTech Connect

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-12-08

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

  13. Recognition of nucleoside monophosphate substrates by Haemophilus influenzae class C acid phosphatase.

    PubMed

    Singh, Harkewal; Schuermann, Jonathan P; Reilly, Thomas J; Calcutt, Michael J; Tanner, John J

    2010-12-10

    The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD(+) utilization pathway by dephosphorylating nicotinamide mononucleotide to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases (CCAPs), which are nonspecific 5',3'-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with nicotinamide mononucleotide, 5'-AMP, 3'-AMP, and 2'-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and the hydrogen-bonding edge of the base. The span between the hydrophobic box and the phosphoryl site is optimal for recognizing nucleoside monophosphates, explaining the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, consistent with an observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5'-nucleotides and 3'-nucleotides. These pockets minimize the enzyme's direct interactions with the ribose and provide sufficient space to accommodate 5' substrates in an anti conformation and 3' substrates in a syn conformation. Finally, the structures suggest that class B acid phosphatases and CCAPs share a common strategy for nucleotide recognition. PMID:20934434

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

    PubMed Central

    Singh, Harkewal; Schuermann, Jonathan P.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2010-01-01

    Summary The e (P4) phosphatase from Haemophilus influenzae functions in a vestigial NAD+ utilization pathway by dephosphorylating NMN to nicotinamide riboside. P4 is also the prototype of class C acid phosphatases, which are nonspecific 5′-, 3′-nucleotidases localized to the bacterial outer membrane. To understand substrate recognition by P4 and other class C phosphatases, we have determined the crystal structures of a substrate-trapping mutant P4 enzyme complexed with NMN, 5′-AMP, 3′-AMP, and 2′-AMP. The structures reveal an anchor-shaped substrate-binding cavity comprising a conserved hydrophobic box that clamps the nucleotide base, a buried phosphoryl binding site, and three solvent-filled pockets that contact the ribose and hydrogen-bonding edge of the base. The span between the hydrophobic box and phosphoryl site is optimal for recognizing nucleoside monophosphates, which explains the general preference for this class of substrate. The base makes no hydrogen bonds with the enzyme, which is consistent with observed lack of base specificity. Two solvent-filled pockets flanking the ribose are key to the dual recognition of 5′- and 3′-nucleotides. These pockets minimize the enzyme’s direct interactions with the ribose and provide sufficient space to accommodate 5′ substrates in an anti conformation and 3′ substrates in a syn conformation. Finally, the structures suggest that class B and C acid phosphatases share a common strategy for nucleotide recognition. PMID:20934434

  15. Conformational selection in the recognition of phosphorylated substrates by the catalytic domain of human Pin1.

    PubMed

    Velazquez, Hector A; Hamelberg, Donald

    2011-11-01

    Post-translational phosphorylation and the related conformational changes in signaling proteins are responsible for regulating a wide range of subcellular processes. Human Pin1 is central to many of these cell signaling pathways in normal and aberrant subcellular processes, catalyzing cis-trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. Pin1 has therefore been identified as a possible drug target in many diseases, including cancer and Alzheimer's. The effects of phosphorylation on Pin1 substrates, and the atomistic basis for Pin1 recognition and catalysis, are not well understood. Here, we determine the conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 using all-atom molecular dynamics simulations. We show that phosphorylation induces backbone conformational changes on the peptide substrate analogues. We also show that Pin1 recognizes specific conformations of its substrate by conformational selection. Furthermore, dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme-substrate complex when the substrate is in the transition state configuration, suggesting that these motions play significant roles during catalytic turnover. These results provide a detailed atomistic picture of the mechanism of Pin1 recognition that can be exploited for drug design purposes and further our understanding of the synergistic complexities of post-translational phosphorylation and cis-trans isomerization. PMID:21967280

  16. Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin.

    PubMed

    Cooke, Heather A; Guenther, Elizabeth L; Luo, Yinggang; Shen, Ben; Bruner, Steven D

    2009-10-13

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization. PMID:19702337

  17. Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

    SciTech Connect

    Cooke, H.; Guenther, E; Luo, Y; Shen, B; Bruner, S

    2009-01-01

    The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

  18. Conserved Sequence Preferences Contribute to Substrate Recognition by the Proteasome*

    PubMed Central

    Yu, Houqing; Singh Gautam, Amit K.; Wilmington, Shameika R.; Wylie, Dennis; Martinez-Fonts, Kirby; Kago, Grace; Warburton, Marie; Chavali, Sreenivas; Inobe, Tomonao; Finkelstein, Ilya J.; Babu, M. Madan

    2016-01-01

    The proteasome has pronounced preferences for the amino acid sequence of its substrates at the site where it initiates degradation. Here, we report that modulating these sequences can tune the steady-state abundance of proteins over 2 orders of magnitude in cells. This is the same dynamic range as seen for inducing ubiquitination through a classic N-end rule degron. The stability and abundance of His3 constructs dictated by the initiation site affect survival of yeast cells and show that variation in proteasomal initiation can affect fitness. The proteasome's sequence preferences are linked directly to the affinity of the initiation sites to their receptor on the proteasome and are conserved between Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human cells. These findings establish that the sequence composition of unstructured initiation sites influences protein abundance in vivo in an evolutionarily conserved manner and can affect phenotype and fitness. PMID:27226608

  19. Steroid promiscuity: Diversity of enzyme action. Preface.

    PubMed

    Lathe, Richard; Kotelevtsev, Yuri; Mason, J Ian

    2015-07-01

    This Special Issue on the topic of Steroid and Sterol Signaling: Promiscuity and Diversity, dwells on the growing realization that the 'one ligand, one binding site' and 'one enzyme, one reaction' concepts are out of date. Focusing on cytochromes P450 (CYP), hydroxysteroid dehydrogenases (HSDs), and related enzymes, the Special Issue highlights that a single enzyme can bind to diverse substrates, and in different conformations, and can catalyze multiple different conversions (and in different directions), thereby, generating an unexpectedly wide spectrum of ligands that can have subtly different biological actions. This article is part of a Special Issue entitled 'Steroid/Sterol Signaling' . PMID:25596328

  20. Mechanism and Substrate Recognition of 2-Hydroxyethylphosphonate Dioxygenase

    SciTech Connect

    Peck, Spencer C.; Cooke, Heather A.; Cicchillo, Robert M.; Malova, Petra; Hammerschmidt, Friedrich; Nair, Satish K.; van der Donk, Wilfred A.

    2011-09-20

    HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased k{sub cat}/K{sub m,2-HEP}. Furthermore, the latter mutants could not be saturated in O{sub 2}. These results suggest that proper binding of 2-HEP is important for O{sub 2} activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O{sub 2}. The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.

  1. The CouPSTU and TarPQM Transporters in Rhodopseudomonas palustris: Redundant, Promiscuous Uptake Systems for Lignin-Derived Aromatic Substrates

    PubMed Central

    Salmon, Robert C.; Cliff, Matthew J.; Rafferty, John B.; Kelly, David J.

    2013-01-01

    The biodegradation of lignin, one of the most abundant carbon compounds on Earth, has important biotechnological applications in the derivation of useful products from lignocellulosic wastes. The purple photosynthetic bacterium Rhodopseudomonas palustris is able to grow photoheterotrophically under anaerobic conditions on a range of phenylpropeneoid lignin monomers, including coumarate, ferulate, caffeate, and cinnamate. RPA1789 (CouP) is the periplasmic binding-protein component of an ABC system (CouPSTU; RPA1789, RPA1791–1793), which has previously been implicated in the active transport of this class of aromatic substrate. Here, we show using both intrinsic tryptophan fluorescence and isothermal titration calorimetry that CouP binds a range of phenylpropeneoid ligands with Kd values in the nanomolar range. The crystal structure of CouP with ferulate as the bound ligand shows H-bond interactions between the 4-OH group of the aromatic ring with His309 and Gln305. H-bonds are also made between the carboxyl group on the ferulate side chain and Arg197, Ser222, and Thr102. An additional transport system (TarPQM; RPA1782–1784), a member of the tripartite ATP-independent periplasmic (TRAP) transporter family, is encoded at the same locus as rpa1789 and several other genes involved in coumarate metabolism. We show that the periplasmic binding-protein of this system (TarP; RPA1782) also binds coumarate, ferulate, caffeate, and cinnamate with nanomolar Kd values. Thus, we conclude that R. palustris uses two redundant but energetically distinct primary and secondary transporters that both employ high-affinity periplasmic binding-proteins to maximise the uptake of lignin-derived aromatic substrates from the environment. Our data provide a detailed thermodynamic and structural basis for understanding the interaction of lignin-derived aromatic substrates with proteins and will be of use in the further exploitation of the flexible metabolism of R. palustris for anaerobic

  2. The Substrate Recognition Domains of the N-end Rule Pathway*

    PubMed Central

    Tasaki, Takafumi; Zakrzewska, Adriana; Dudgeon, Drew D.; Jiang, Yonghua; Lazo, John S.; Kwon, Yong Tae

    2009-01-01

    The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway. PMID:19008229

  3. Atomic substitution reveals the structural basis for substrate adenine recognition and removal by adenine DNA glycosylase

    SciTech Connect

    Lee, Seongmin; Verdine, Gregory L.

    2010-01-14

    Adenine DNA glycosylase catalyzes the glycolytic removal of adenine from the promutagenic A {center_dot} oxoG base pair in DNA. The general features of DNA recognition by an adenine DNA glycosylase, Bacillus stearothermophilus MutY, have previously been revealed via the X-ray structure of a catalytically inactive mutant protein bound to an A:oxoG-containing DNA duplex. Although the structure revealed the substrate adenine to be, as expected, extruded from the DNA helix and inserted into an extrahelical active site pocket on the enzyme, the substrate adenine engaged in no direct contacts with active site residues. This feature was paradoxical, because other glycosylases have been observed to engage their substrates primarily through direct contacts. The lack of direct contacts in the case of MutY suggested that either MutY uses a distinctive logic for substrate recognition or that the X-ray structure had captured a noncatalytically competent state in lesion recognition. To gain further insight into this issue, we crystallized wild-type MutY bound to DNA containing a catalytically inactive analog of 2'-deoxyadenosine in which a single 2'-H atom was replaced by fluorine. The structure of this fluorinated lesion-recognition complex (FLRC) reveals the substrate adenine buried more deeply into the active site pocket than in the prior structure and now engaged in multiple direct hydrogen bonding and hydrophobic interactions. This structure appears to capture the catalytically competent state of adenine DNA glycosylases, and it suggests a catalytic mechanism for this class of enzymes, one in which general acid-catalyzed protonation of the nucleobase promotes glycosidic bond cleavage.

  4. Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

    SciTech Connect

    Agarwal, R.; Schmidt, J; Stafford, R; Swaminathan, S

    2009-01-01

    Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

  5. Native Serotonin Membrane Receptors Recognize 5-Hydroxytryptophan-Functionalized Substrates: Enabling Small-Molecule Recognition

    PubMed Central

    2010-01-01

    Recognition of small diffusible molecules by large biomolecules is ubiquitous in biology. To investigate these interactions, it is important to be able to immobilize small ligands on substrates; however, preserving recognition by biomolecule-binding partners under these circumstances is challenging. We have developed methods to modify substrates with serotonin, a small-molecule neurotransmitter important in brain function and psychiatric disorders. To mimic soluble serotonin, we attached its amino acid precursor, 5-hydroxytryptophan, via the ancillary carboxyl group to oligo(ethylene glycol)-terminated alkanethiols self-assembled on gold. Anti-5-hydroxytryptophan antibodies recognize these substrates, demonstrating bioavailability. Interestingly, 5-hydroxytryptophan-functionalized surfaces capture membrane-associated serotonin receptors enantiospecifically. By contrast, surfaces functionalized with serotonin itself fail to bind serotonin receptors. We infer that recognition by biomolecules evolved to distinguish small-molecule ligands in solution requires tethering of the latter via ectopic moieties. Membrane proteins, which are notoriously difficult to isolate, or other binding partners can be captured for identification, mapping, expression, and other purposes using this generalizable approach. PMID:22778841

  6. A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

    PubMed Central

    Bouchard, Patricia; Legault, Pascale

    2014-01-01

    Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem–loop I (SLI) substrate and stem–loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem–loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8–3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7–8 kcal/mol than predicted for a comparable duplex containing three Watson–Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6–7 Watson–Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2–3 Watson–Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme. PMID:25051972

  7. An allosteric inhibitor of substrate recognition by the SCF[superscript Cdc4] ubiquitin ligase

    SciTech Connect

    Orlicky, Stephen; Tang, Xiaojing; Neduva, Victor; Elowe, Nadine; Brown, Eric D.; Sicheri, Frank; Tyers, Mike

    2010-09-17

    The specificity of SCF ubiquitin ligase-mediated protein degradation is determined by F-box proteins. We identified a biplanar dicarboxylic acid compound, called SCF-I2, as an inhibitor of substrate recognition by the yeast F-box protein Cdc4 using a fluorescence polarization screen to monitor the displacement of a fluorescein-labeled phosphodegron peptide. SCF-I2 inhibits the binding and ubiquitination of full-length phosphorylated substrates by SCF{sup Cdc4}. A co-crystal structure reveals that SCF-I2 inserts itself between the {beta}-strands of blades 5 and 6 of the WD40 propeller domain of Cdc4 at a site that is 25 {angstrom} away from the substrate binding site. Long-range transmission of SCF-I2 interactions distorts the substrate binding pocket and impedes recognition of key determinants in the Cdc4 phosphodegron. Mutation of the SCF-I2 binding site abrogates its inhibitory effect and explains specificity in the allosteric inhibition mechanism. Mammalian WD40 domain proteins may exhibit similar allosteric responsiveness and hence represent an extensive class of druggable target.

  8. Development of conformation independent computational models for the early recognition of breast cancer resistance protein substrates.

    PubMed

    Gantner, Melisa Edith; Di Ianni, Mauricio Emiliano; Ruiz, María Esperanza; Talevi, Alan; Bruno-Blanch, Luis E

    2013-01-01

    ABC efflux transporters are polyspecific members of the ABC superfamily that, acting as drug and metabolite carriers, provide a biochemical barrier against drug penetration and contribute to detoxification. Their overexpression is linked to multidrug resistance issues in a diversity of diseases. Breast cancer resistance protein (BCRP) is the most expressed ABC efflux transporter throughout the intestine and the blood-brain barrier, limiting oral absorption and brain bioavailability of its substrates. Early recognition of BCRP substrates is thus essential to optimize oral drug absorption, design of novel therapeutics for central nervous system conditions, and overcome BCRP-mediated cross-resistance issues. We present the development of an ensemble of ligand-based machine learning algorithms for the early recognition of BCRP substrates, from a database of 262 substrates and nonsubstrates compiled from the literature. Such dataset was rationally partitioned into training and test sets by application of a 2-step clustering procedure. The models were developed through application of linear discriminant analysis to random subsamples of Dragon molecular descriptors. Simple data fusion and statistical comparison of partial areas under the curve of ROC curves were applied to obtain the best 2-model combination, which presented 82% and 74.5% of overall accuracy in the training and test set, respectively. PMID:23984415

  9. Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme

    PubMed Central

    Bouchard, Patricia; Lacroix-Labonté, Julie; Desjardins, Geneviève; Lampron, Philipe; Lisi, Véronique; Lemieux, Sébastien; Major, François; Legault, Pascale

    2008-01-01

    Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual 31P and 15N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface. PMID:18314503

  10. 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

  11. Differential substrate recognition by isozymes of plant protein-only Ribonuclease P.

    PubMed

    Howard, Michael J; Karasik, Agnes; Klemm, Bradley P; Mei, Christine; Shanmuganathan, Aranganathan; Fierke, Carol A; Koutmos, Markos

    2016-05-01

    Ribonuclease P (RNase P) catalyzes the cleavage of leader sequences from precursor tRNA (pre-tRNA). Typically, these enzymes are ribonucleic protein complexes that are found in all domains of life. However, a new class of RNase P has been discovered that is composed entirely of protein, termed protein-only RNase P (PRORP). To investigate the molecular determinants of PRORP substrate recognition, we measured the binding affinities and cleavage kinetics ofArabidopsisPRORP1 for varied pre-tRNA substrates. This analysis revealed that PRORP1 does not make significant contacts within the trailer or beyond N-1of the leader, indicating that this enzyme recognizes primarily the tRNA body. To determine the extent to which sequence variation within the tRNA body modulates substrate selectivity and to provide insight into the evolution and function of PRORP enzymes, we measured the reactivity of the threeArabidopsisPRORP isozymes (PRORP1-3) with four pre-tRNA substrates. A 13-fold range in catalytic efficiencies (10(4)-10(5)M(-1)s(-1)) was observed, demonstrating moderate selectivity for pre-tRNA substrates. Although PRORPs bind the different pre-tRNA species with affinities varying by as much as 100-fold, the three isozymes have similar affinities for a given pre-tRNA, suggesting similar binding modes. However, PRORP isozymes have varying degrees of cleavage fidelity, which is dependent on the pre-tRNA species and the presence of a 3'-discriminator base. This work defines molecular determinants of PRORP substrate recognition that provides insight into this new class of RNA processing enzymes. PMID:26966150

  12. Crystallographic and mutational analyses of substrate recognition of endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum.

    PubMed

    Suzuki, Ryuichiro; Katayama, Takane; Kitaoka, Motomitsu; Kumagai, Hidehiko; Wakagi, Takayoshi; Shoun, Hirofumi; Ashida, Hisashi; Yamamoto, Kenji; Fushinobu, Shinya

    2009-09-01

    Endo-alpha-N-acetylgalactosaminidase (endo-alpha-GalNAc-ase), a member of the glycoside hydrolase (GH) family 101, hydrolyses the O-glycosidic bonds in mucin-type O-glycan between alpha-GalNAc and Ser/Thr. Endo-alpha-GalNAc-ase from Bifidobacterium longum JCM1217 (EngBF) is highly specific for the core 1-type O-glycan to release the disaccharide Galbeta1-3GalNAc (GNB), whereas endo-alpha-GalNAc-ase from Clostridium perfringens (EngCP) exhibits broader substrate specificity. We determined the crystal structure of EngBF at 2.0 A resolution and performed automated docking analysis to investigate possible binding modes of GNB. Mutational analysis revealed important residues for substrate binding, and two Trp residues (Trp748 and Trp750) appeared to form stacking interactions with the beta-faces of sugar rings of GNB by substrate-induced fit. The difference in substrate specificities between EngBF and EngCP is attributed to the variations in amino acid sequences in the regions forming the substrate-binding pocket. Our results provide a structural basis for substrate recognition by GH101 endo-alpha-GalNAc-ases and will help structure-based engineering of these enzymes to produce various kinds of neo-glycoconjugates. PMID:19502354

  13. 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

  14. Molecular Basis of Symbiotic Promiscuity

    PubMed Central

    Perret, Xavier; Staehelin, Christian; Broughton, William J.

    2000-01-01

    Eukaryotes often form symbioses with microorganisms. Among these, associations between plants and nitrogen-fixing bacteria are responsible for the nitrogen input into various ecological niches. Plants of many different families have evolved the capacity to develop root or stem nodules with diverse genera of soil bacteria. Of these, symbioses between legumes and rhizobia (Azorhizobium, Bradyrhizobium, Mesorhizobium, and Rhizobium) are the most important from an agricultural perspective. Nitrogen-fixing nodules arise when symbiotic rhizobia penetrate their hosts in a strictly controlled and coordinated manner. Molecular codes are exchanged between the symbionts in the rhizosphere to select compatible rhizobia from pathogens. Entry into the plant is restricted to bacteria that have the “keys” to a succession of legume “doors”. Some symbionts intimately associate with many different partners (and are thus promiscuous), while others are more selective and have a narrow host range. For historical reasons, narrow host range has been more intensively investigated than promiscuity. In our view, this has given a false impression of specificity in legume-Rhizobium associations. Rather, we suggest that restricted host ranges are limited to specific niches and represent specialization of widespread and more ancestral promiscuous symbioses. Here we analyze the molecular mechanisms governing symbiotic promiscuity in rhizobia and show that it is controlled by a number of molecular keys. PMID:10704479

  15. Promiscuous Feminisms for Troubling Times

    ERIC Educational Resources Information Center

    Voithofer, Rick

    2013-01-01

    Looking across the six articles in this issue, this paper argues that promiscuous uses of feminist methodologies offer a unique constellation of conceptual, pragmatic, material, and ethical strategies with which to understand and engage some of the social and cultural tensions that are occurring within and outside schools. It presents a…

  16. Computational and Experimental Insights into the Mechanism of Substrate Recognition and Feedback Inhibition of Protoporphyrinogen Oxidase

    PubMed Central

    Yang, Sheng-Gang; Wang, Zhi-Fang; Zhan, Chang-Guo; Xi, Zhen; Yang, Guang-Fu

    2013-01-01

    Protoporphyrinogen IX oxidase (PPO; EC 1.3.3.4) is an essential enzyme catalyzing the last common step in the pathway leading to heme and chlorophyll biosynthesis. Great interest in PPO inhibitors arises from both its significance to agriculture and medicine. However, the discovery of PPO inhibitors with ultrahigh potency and selectivity is hampered due to lack of structural and mechanistic understanding about the substrate recognition, which remains a longstanding question central in porphyrin biology. To understand the mechanism, a novel binding model of protogen (protoporphyrinogen IX, the substrate) was developed through extensive computational simulations. Subsequently, amino acid residues that are critical for protogen binding identified by computational simulations were substituted by mutagenesis. Kinetic analyses of these mutants indicated that these residues were critical for protogen binding. In addition, the calculated free energies of protogen binding with these mutants correlated well with the experimental data, indicating the reasonability of the binding model. On the basis of this novel model, the fundamental mechanism of substrate recognition was investigated by performing potential of mean force (PMF) calculations, which provided an atomic level description of conformational changes and pathway intermediates. The free energy profile revealed a feedback inhibition mechanism of proto (protoporphyrin IX, the product), which was also in agreement with experimental evidence. The novel mechanistic insights obtained from this study present a new starting point for future rational design of more efficient PPO inhibitors based on the product-bound PPO structure. PMID:23935953

  17. Substrate recognition of PLCγ1 via a specific docking surface on Itk.

    PubMed

    Xie, Qian; Joseph, Raji E; Fulton, D Bruce; Andreotti, Amy H

    2013-02-22

    Itk (interleukin-2 inducible T cell kinase) is a non-receptor protein tyrosine kinase expressed primarily in T cells. Itk catalyzes phosphorylation on tyrosine residues within a number of its natural substrates, including the well-characterized Y783 of PLCγ1. However, the molecular mechanisms Itk exploits to recognize its substrates are not completely understood. We have previously identified a specific docking interaction between the kinase domain of Itk and the C-terminal Src homology 2 (SH2C) domain of PLCγ1 that promotes substrate specificity for this enzyme/substrate pair. In the current study, we identify and map the interaction surface on the Itk kinase domain as an acidic patch centered on the G helix. Mutation of the residues on and adjacent to the G helix within the Itk kinase domain impairs the catalytic efficacy of PLCγ1 substrate phosphorylation by specifically altering the protein-protein interaction interface and not the inherent catalytic activity of Itk. NMR titration experiments using a Btk (Bruton's tyrosine kinase) kinase domain as a surrogate for the Itk kinase domain provide further support for an Itk/PLCγ1 SH2C interaction surrounding the G helix of the kinase domain. The work presented here provides structural insight into how the Itk kinase uses the G helix to single out Y783 of PLCγ1 for specific phosphorylation. Comparing these results to other well-characterized kinase/substrate systems suggests that the G helix is a general structural feature used by kinases for substrate recognition during signaling. PMID:23219468

  18. Comparative characterization of botulinum neurotoxin subtypes F1 and F7 featuring differential substrate recognition and cleavage mechanisms.

    PubMed

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-03-01

    BoNT/F7, one of the seven subtypes of botulinum neurotoxin type F (F1 to F7), is the second-most divergent subtype of this group. Despite sharing >60% identity with BoNT/F1 at both holotoxin and enzymatic domain levels, it requires an N-terminal extended peptide substrate for efficient substrate cleavage, suggesting its unique substrate recognition and specificity mechanism. Substrate mapping and saturation mutagenesis analysis revealed that VAMP2 (20-65) was likely a minimally effective substrate for LC/F7 (light chain of BoNT/F7), and in addition, LC/F7 recognized VAMP2 in a unique way, which differed significantly from that of LC/F1, although both of them share similar substrate binding and hydrolysis mode. LC/F7 utilizes distinct pockets for specific substrate binding and recognition in particular for the B1, B2 and S2 sites recognitions. Our findings provide insights into the distinct substrate recognition features of BoNT subtypes and useful information for therapy development for BoNT/F. PMID:26748154

  19. Simulation studies of substrate recognition by the exocellulase CelF from Clostridium cellulolyticum.

    PubMed

    Chen, Mo; Himmel, Michael E; Wilson, David B; Brady, John W

    2016-07-01

    Molecular dynamics (MD) simulations were used to study substrate recognition by the family 48 exocellulase CelF from Clostridium cellulolyticum. It was hypothesized that residues around the entrance of the active site tunnel of this enzyme might serve to recognize and bind the substrate through an affinity for the cellulose monomer repeat unit, β-d-glucopyranose. Simulations were conducted of the catalytic domain of this enzyme surrounded by a concentrated solution of β-d-glucopyranose, and the full three-dimensional probability distribution for finding sugar molecules adjacent to the enzyme was calculated from the trajectory. A significant probability of finding the sugar stacked against the planar faces of Trp 310 and Trp 312 at the entrance of the active site tunnel was observed. Biotechnol. Bioeng. 2016;113: 1433-1440. © 2015 Wiley Periodicals, Inc. PMID:26693961

  20. Vitamin K-dependent carboxylase: possible role of the substrate "propeptide" as an intracellular recognition site.

    PubMed Central

    Suttie, J W; Hoskins, J A; Engelke, J; Hopfgartner, A; Ehrlich, H; Bang, N U; Belagaje, R M; Schoner, B; Long, G L

    1987-01-01

    The liver microsomal vitamin K-dependent carboxylase catalyzes the posttranslational conversion of specific glutamate residues to gamma-carboxyglutamate residues in a limited number of proteins. A number of these proteins have been shown to contain a homologous basic amino acid-rich "propeptide" between the leader sequence and the amino terminus of the mature protein. Plasmids encoding protein C, a vitamin K-dependent protein, containing or lacking a propeptide region were constructed and the protein was expressed in Escherichia coli. The protein products were assayed as substrates in an in vitro vitamin K-dependent carboxylase system. Only proteins containing a propeptide region were substrates for the enzyme. These data support the hypothesis that this sequence of the primary gene product is an important recognition site for this processing enzyme. PMID:3543932

  1. Substrate Recognition and Activity Regulation of the Escherichia coli mRNA Endonuclease MazF.

    PubMed

    Zorzini, Valentina; Mernik, Andrej; Lah, Jurij; Sterckx, Yann G J; De Jonge, Natalie; Garcia-Pino, Abel; De Greve, Henri; Versées, Wim; Loris, Remy

    2016-05-20

    Escherichia coli MazF (EcMazF) is the archetype of a large family of ribonucleases involved in bacterial stress response. The crystal structure of EcMazF in complex with a 7-nucleotide substrate mimic explains the relaxed substrate specificity of the E. coli enzyme relative to its Bacillus subtilis counterpart and provides a framework for rationalizing specificity in this enzyme family. In contrast to a conserved mode of substrate recognition and a conserved active site, regulation of enzymatic activity by the antitoxin EcMazE diverges from its B. subtilis homolog. Central in this regulation is an EcMazE-induced double conformational change as follows: a rearrangement of a crucial active site loop and a relative rotation of the two monomers in the EcMazF dimer. Both are induced by the C-terminal residues Asp-78-Trp-82 of EcMazE, which are also responsible for strong negative cooperativity in EcMazE-EcMazF binding. This situation shows unexpected parallels to the regulation of the F-plasmid CcdB activity by CcdA and further supports a common ancestor despite the different activities of the MazF and CcdB toxins. In addition, we pinpoint the origin of the lack of activity of the E24A point mutant of EcMazF in its inability to support the substrate binding-competent conformation of EcMazF. PMID:27026704

  2. Protein-Protein Interactions, Not Substrate Recognition, Dominate the Turnover of Chimeric Assembly Line Polyketide Synthases.

    PubMed

    Klaus, Maja; Ostrowski, Matthew P; Austerjost, Jonas; Robbins, Thomas; Lowry, Brian; Cane, David E; Khosla, Chaitan

    2016-07-29

    The potential for recombining intact polyketide synthase (PKS) modules has been extensively explored. Both enzyme-substrate and protein-protein interactions influence chimeric PKS activity, but their relative contributions are unclear. We now address this issue by studying a library of 11 bimodular and 8 trimodular chimeric PKSs harboring modules from the erythromycin, rifamycin, and rapamycin synthases. Although many chimeras yielded detectable products, nearly all had specific activities below 10% of the reference natural PKSs. Analysis of selected bimodular chimeras, each with the same upstream module, revealed that turnover correlated with the efficiency of intermodular chain translocation. Mutation of the acyl carrier protein (ACP) domain of the upstream module in one chimera at a residue predicted to influence ketosynthase-ACP recognition led to improved turnover. In contrast, replacement of the ketoreductase domain of the upstream module by a paralog that produced the enantiomeric ACP-bound diketide caused no changes in processing rates for each of six heterologous downstream modules compared with those of the native diketide. Taken together, these results demonstrate that protein-protein interactions play a larger role than enzyme-substrate recognition in the evolution or design of catalytically efficient chimeric PKSs. PMID:27246853

  3. Molecular Understanding of USP7 Substrate Recognition and C-Terminal Activation.

    PubMed

    Rougé, Lionel; Bainbridge, Travis W; Kwok, Michael; Tong, Raymond; Di Lello, Paola; Wertz, Ingrid E; Maurer, Till; Ernst, James A; Murray, Jeremy

    2016-08-01

    The deubiquitinating enzyme USP7 has a pivotal role in regulating the stability of proteins involved in fundamental cellular processes of normal biology and disease. Despite the importance of USP7, the mechanisms underlying substrate recognition and catalytic activation are poorly understood. Here we present structural, biochemical, and biophysical analyses elucidating the molecular mechanism by which the C-terminal 19 amino acids of USP7 (residues 1084-1102) enhance the ubiquitin cleavage activity of the deubiquitinase (DUB) domain. Our data demonstrate that the C-terminal peptide binds the activation cleft in the catalytic domain and stabilizes the catalytically competent conformation of USP7. Additional structures of longer fragments of USP7, as well as solution studies, provide insight into full-length USP7, the role of the UBL domains, and demonstrate that both substrate recognition and deubiquitinase activity are highly regulated by the catalytic and noncatalytic domains of USP7, a feature that could be essential for the proper function of multi-domain DUBs. PMID:27452404

  4. The structural basis for substrate recognition by mammalian polynucleotide kinase 3’ phosphatase

    PubMed Central

    Garces, Fernando; Pearl, Laurence H.; Oliver, Antony W.

    2016-01-01

    Mammalian polynucleotide kinase 3’ phosphatase (PNK) plays a key role in the repair of DNA damage, functioning as part of both the non-homologous end-joining (NHEJ) and base-excision repair (BER) pathways. Through its two catalytic activities, PNK ensures that DNA termini are compatible with extension and ligation by either removing 3’-phosphates from, or by phosphorylating 5’-hydroxyl groups on, the ribose sugar of the DNA backbone. We have now determined crystal structures of murine PNK with DNA molecules bound to both of its active sites. The structure of ssDNA engaged with the 3’-phosphatase domain suggests a mechanism of substrate interaction that assists DNA end-seeking. The structure of dsDNA bound to the 5’-kinase domain reveals a mechanism of DNA bending that facilitates recognition of DNA-ends in the context of single-strand and double-strand breaks, and suggests a close functional cooperation in substrate recognition between the kinase and phosphatase active sites. PMID:22055185

  5. Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.

    PubMed

    Feng, Chong; Liu, Yang; Wang, Guoqiang; Deng, Zengqin; Zhang, Qi; Wu, Wei; Tong, Yufeng; Cheng, Changmei; Chen, Zhongzhou

    2014-04-25

    N(6)-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct "lid" region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a "flipping" motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members. PMID:24616105

  6. Crystal Structures of the Human RNA Demethylase Alkbh5 Reveal Basis for Substrate Recognition*

    PubMed Central

    Feng, Chong; Liu, Yang; Wang, Guoqiang; Deng, Zengqin; Zhang, Qi; Wu, Wei; Tong, Yufeng; Cheng, Changmei; Chen, Zhongzhou

    2014-01-01

    N6-Methylation of adenosine is the most ubiquitous and abundant modification of nucleoside in eukaryotic mRNA and long non-coding RNA. This modification plays an essential role in the regulation of mRNA translation and RNA metabolism. Recently, human AlkB homolog 5 (Alkbh5) and fat mass- and obesity-associated protein (FTO) were shown to erase this methyl modification on mRNA. Here, we report five high resolution crystal structures of the catalytic core of Alkbh5 in complex with different ligands. Compared with other AlkB proteins, Alkbh5 displays several unique structural features on top of the conserved double-stranded β-helix fold typical of this protein family. Among the unique features, a distinct “lid” region of Alkbh5 plays a vital role in substrate recognition and catalysis. An unexpected disulfide bond between Cys-230 and Cys-267 is crucial for the selective binding of Alkbh5 to single-stranded RNA/DNA by bringing a “flipping” motif toward the central β-helix fold. We generated a substrate binding model of Alkbh5 based on a demethylation activity assay of several structure-guided site-directed mutants. Crystallographic and biochemical studies using various analogs of α-ketoglutarate revealed that the active site cavity of Alkbh5 is much smaller than that of FTO and preferentially binds small molecule inhibitors. Taken together, our findings provide a structural basis for understanding the substrate recognition specificity of Alkbh5 and offer a foundation for selective drug design against AlkB members. PMID:24616105

  7. Analysis on substrate specificity of Escherichia coli ribonuclease P using shape variants of pre-tRNA: proposal of subsites model for substrate shape recognition.

    PubMed

    Suwa, Satoshi; Nagai, Yasuhiro; Fujimoto, Akihiro; Kikuchi, Yo; Tanaka, Terumichi

    2009-02-01

    We prepared a series of shape variants of a pre-tRNA and examined substrate shape recognition by bacterial RNase P ribozyme and holoenzyme. Cleavage site analysis revealed two new subsites for accepting the T-arm and the bottom half of pre-tRNA in the substrate-binding site of the enzyme. These two subsites take part in cleavage site selection of substrate by the enzyme: the cleavage site is not always selected according to the relative position of the 3'-CCA sequence of the substrate. Kinetic studies indicated that the substrate shape is recognized mainly in the transition state of the reaction, and neither the shape nor position of either the T-arm or the bottom half of the substrate affected the Michaelis complex formation. These results strongly suggest that the 5' and 3' termini of a substrate are trapped by the enzyme first, then the position and the shape of the T-arm and the bottom half are examined by the cognate subsites. From these facts, we propose a new substrate recognition model that can explain many experimental facts that have been seen as enigmatic. PMID:19008262

  8. Evolutionary bases of carbohydrate recognition and substrate discrimination in the ROK protein family.

    PubMed

    Conejo, Maria S; Thompson, Steven M; Miller, Brian G

    2010-06-01

    The ROK (repressor, open reading frame, kinase) protein family (Pfam 00480) is a large collection of bacterial polypeptides that includes sugar kinases, carbohydrate responsive transcriptional repressors, and many functionally uncharacterized gene products. ROK family sugar kinases phosphorylate a range of structurally distinct hexoses including the key carbon source D: -glucose, various glucose epimers, and several acetylated hexosamines. The primary sequence elements responsible for carbohydrate recognition within different functional categories of ROK polypeptides are largely unknown due to a limited structural characterization of this protein family. In order to identify the structural bases for substrate discrimination in individual ROK proteins, and to better understand the evolutionary processes that led to the divergent evolution of function in this family, we constructed an inclusive alignment of 227 representative ROK polypeptides. Phylogenetic analyses and ancestral sequence reconstructions of the resulting tree reveal a discrete collection of active site residues that dictate substrate specificity. The results also suggest a series of mutational events within the carbohydrate-binding sites of ROK proteins that facilitated the expansion of substrate specificity within this family. This study provides new insight into the evolutionary relationship of ROK glucokinases and non-ROK glucokinases (Pfam 02685), revealing the primary sequence elements shared between these two protein families, which diverged from a common ancestor in ancient times. PMID:20512568

  9. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme

    PubMed Central

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-01-01

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg2+ ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg2+ ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process. PMID:27440438

  10. Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases

    PubMed Central

    Srivastav, Rajpal; Kumar, Dilip; Grover, Amit; Singh, Ajit; Manjasetty, Babu A.; Sharma, Rakesh; Taneja, Bhupesh

    2014-01-01

    DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3′-5′ exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress. PMID:24878921

  11. Multibody cofactor and substrate molecular recognition in the myo-inositol monophosphatase enzyme.

    PubMed

    Ferruz, Noelia; Tresadern, Gary; Pineda-Lucena, Antonio; De Fabritiis, Gianni

    2016-01-01

    Molecular recognition is rarely a two-body protein-ligand problem, as it often involves the dynamic interplay of multiple molecules that together control the binding process. Myo-inositol monophosphatase (IMPase), a drug target for bipolar disorder, depends on 3 Mg(2+) ions as cofactor for its catalytic activity. Although the crystallographic pose of the pre-catalytic complex is well characterized, the binding process by which substrate, cofactor and protein cooperate is essentially unknown. Here, we have characterized cofactor and substrate cooperative binding by means of large-scale molecular dynamics. Our study showed the first and second Mg(2+) ions identify the binding pocket with fast kinetics whereas the third ion presents a much higher energy barrier. Substrate binding can occur in cooperation with cofactor, or alone to a binary or ternary cofactor-IMPase complex, although the last scenario occurs several orders of magnitude faster. Our atomic description of the three-body mechanism offers a particularly challenging example of pathway reconstruction, and may prove particularly useful in realistic contexts where water, ions, cofactors or other entities cooperate and modulate the binding process. PMID:27440438

  12. Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition.

    PubMed

    Wang, Hui; Klein, Michael G; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching

    2016-07-01

    Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction. PMID:27220631

  13. Substrate recognition and catalysis by LytB, a pneumococcal peptidoglycan hydrolase involved in virulence

    PubMed Central

    Rico-Lastres, Palma; Díez-Martínez, Roberto; Iglesias-Bexiga, Manuel; Bustamante, Noemí; Aldridge, Christine; Hesek, Dusan; Lee, Mijoon; Mobashery, Shahriar; Gray, Joe; Vollmer, Waldemar; García, Pedro; Menéndez, Margarita

    2015-01-01

    Streptococcus pneumoniae is a major cause of life-threatening diseases worldwide. Here we provide an in-depth functional characterization of LytB, the peptidoglycan hydrolase responsible for physical separation of daughter cells. Identified herein as an N-acetylglucosaminidase, LytB is involved also in colonization and invasion of the nasopharynx, biofilm formation and evasion of host immunity as previously demonstrated. We have shown that LytB cleaves the GlcNAc-β-(1,4)-MurNAc glycosidic bond of peptidoglycan building units. The hydrolysis occurs at sites with fully acetylated GlcNAc moieties, with preference for uncross-linked muropeptides. The necessity of GlcN acetylation and the presence of a single acidic moiety (Glu585) essential for catalysis strongly suggest a substrate-assisted mechanism with anchimeric assistance of the acetamido group of GlcNAc moieties. Additionally, modelling of the catalytic region bound to a hexasaccharide tripentapeptide provided insights into substrate-binding subsites and peptidoglycan recognition. Besides, cell-wall digestion products and solubilisation rates might indicate a tight control of LytB activity to prevent unrestrained breakdown of the cell wall. Choline-independent localization at the poles of the cell, mediated by the choline-binding domain, peptidoglycan modification, and choline-mediated (lipo)teichoic-acid attachment contribute to the high selectivity of LytB. Moreover, so far unknown chitin hydrolase and glycosyltransferase activities were detected using GlcNAc oligomers as substrate. PMID:26537571

  14. The MPN domain of Caenorhabditis elegans UfSP modulates both substrate recognition and deufmylation activity.

    PubMed

    Ha, Byung Hak; Kim, Kyung Hee; Yoo, Hee Min; Lee, Weontae; EunKyeong Kim, Eunice

    2016-08-01

    Ubiquitin-fold modifier 1 (Ufm1) specific protease (UfSP) is a novel cysteine protease that activates Ufm1 from its precursor by processing the C-terminus to expose the conserved Gly necessary for substrate conjugation and de-conjugates Ufm1 from the substrate. There are two forms: UfSP1 and UfSP2, the later with an additional domain at the N-terminus. Ufm1 and both the conjugating and deconjugating enzymes are highly conserved. However, in Caenorhabditis elegans there is one UfSP which has extra 136 residues at the N terminus compared to UfSP2. The crystal structure of cUfSP reveals that these additional residues display a MPN fold while the rest of the structure mimics that of UfSP2. The MPN domain does not have the metalloprotease activity found in some MPN-domain containing protein, rather it is required for the recognition and deufmylation of the substrate of cUfSP, UfBP1. In addition, the MPN domain is also required for localization to the endoplasmic reticulum. PMID:27240952

  15. A cell-signaling network temporally resolves specific versus promiscuous phosphorylation.

    PubMed

    Kanshin, Evgeny; Bergeron-Sandoval, Louis-Philippe; Isik, S Sinan; Thibault, Pierre; Michnick, Stephen W

    2015-02-24

    If specific and functional kinase- or phosphatase-substrate interactions are optimized for binding compared to promiscuous interactions, then changes in phosphorylation should occur faster on functional versus promiscuous substrates. To test this hypothesis, we designed a high temporal resolution global phosphoproteomics protocol to study the high-osmolarity glycerol (HOG) response in the budding yeast Saccharomyces cerevisiae. The method provides accurate, stimulus-specific measurement of phosphoproteome changes, quantitative analysis of phosphodynamics at sub-minute temporal resolution, and detection of more phosphosites. Rates of evolution of dynamic phosphosites were comparable to those of known functional phosphosites and significantly lower than static or longer-time-frame dynamic phosphosites. Kinetic profile analyses indicated that putatively functional kinase- or phosphatase-substrate interactions occur more rapidly, within 60 s, than promiscuous interactions. Finally, we report many changes in phosphorylation of proteins implicated in cytoskeletal and mitotic spindle dynamics that may underlie regulation of cell cycle and morphogenesis. PMID:25704821

  16. Multiple sequence signals direct recognition and degradation of protein substrates by the AAA+ protease HslUV.

    PubMed

    Sundar, Shankar; McGinness, Kathleen E; Baker, Tania A; Sauer, Robert T

    2010-10-29

    Proteolysis is important for protein quality control and for the proper regulation of many intracellular processes in prokaryotes and eukaryotes. Discerning substrates from other cellular proteins is a key aspect of proteolytic function. The Escherichia coli HslUV protease is a member of a major family of ATP-dependent AAA+ degradation machines. HslU hexamers recognize and unfold native protein substrates and then translocate the polypeptide into the degradation chamber of the HslV peptidase. Although a wealth of structural information is available for this system, relatively little is known about mechanisms of substrate recognition. Here, we demonstrate that mutations in the unstructured N-terminal and C-terminal sequences of two model substrates alter HslUV recognition and degradation kinetics, including changes in V(max). By introducing N- or C-terminal sequences that serve as recognition sites for specific peptide-binding proteins, we show that blocking either terminus of the substrate interferes with HslUV degradation, with synergistic effects when both termini are obstructed. These results support a model in which one terminus of the substrate is tethered to the protease and the other terminus is engaged by the translocation/unfolding machinery in the HslU pore. Thus, degradation appears to consist of discrete steps, which involve the interaction of different terminal sequence signals in the substrate with different receptor sites in the HslUV protease. PMID:20837023

  17. Crystal Structures of the Histidine Acid Phosphatase from Francisella tularensis Provide Insight into Substrate Recognition

    SciTech Connect

    Singh, Harkewal; Felts, Richard L.; Schuermann, Jonathan P.; Reilly, Thomas J.; Tanner, John J.

    2009-12-01

    Histidine acid phosphatases catalyze the transfer of a phosphoryl group from phosphomonoesters to water at acidic pH using an active-site histidine. The histidine acid phosphatase from the category A pathogen Francisella tularensis (FtHAP) has been implicated in intramacrophage survival and virulence, motivating interest in understanding the structure and mechanism of this enzyme. Here, we report a structure-based study of ligand recognition by FtHAP. The 1.70-{angstrom}-resolution structure of FtHAP complexed with the competitive inhibitor L(+)-tartrate was solved using single-wavelength anomalous diffraction phasing. Structures of the ligand-free enzyme and the complex with inorganic phosphate were determined at resolutions of 1.85 and 1.70 {angstrom}, respectively. The structure of the Asp261Ala mutant enzyme complexed with the substrate 3'-AMP was determined at 1.50 {angstrom} resolution to gain insight into substrate recognition. FtHAP exhibits a two-domain fold similar to that of human prostatic acid phosphatase, consisting of an {alpha}/{beta} core domain and a smaller domain that caps the core domain. The structures show that the core domain supplies the phosphoryl binding site, catalytic histidine (His17), and an aspartic acid residue (Asp261) that protonates the leaving group, while the cap domain contributes residues that enforce substrate preference. FtHAP and human prostatic acid phosphatase differ in the orientation of the crucial first helix of the cap domain, implying differences in the substrate preferences of the two enzymes. 3'-AMP binds in one end of a 15-{angstrom}-long tunnel, with the adenine clamped between Phe23 and Tyr135, and the ribose 2'-hydroxyl interacting with Gln132. The importance of the clamp is confirmed with site-directed mutagenesis; mutation of Phe23 and Tyr135 individually to Ala increases K{sub m} by factors of 7 and 10, respectively. The structural data are consistent with a role for FtHAP in scavenging phosphate from small

  18. Biological messiness vs. biological genius: Mechanistic aspects and roles of protein promiscuity.

    PubMed

    Atkins, William M

    2015-07-01

    In contrast to the traditional biological paradigms focused on 'specificity', recent research and theoretical efforts have focused on functional 'promiscuity' exhibited by proteins and enzymes in many biological settings, including enzymatic detoxication, steroid biochemistry, signal transduction and immune responses. In addition, divergent evolutionary processes are apparently facilitated by random mutations that yield promiscuous enzyme intermediates. The intermediates, in turn, provide opportunities for further evolution to optimize new functions from existing protein scaffolds. In some cases, promiscuity may simply represent the inherent plasticity of proteins resulting from their polymeric nature with distributed conformational ensembles. Enzymes or proteins that bind or metabolize noncognate substrates create 'messiness' or noise in the systems they contribute to. With our increasing awareness of the frequency of these promiscuous behaviors it becomes interesting and important to understand the molecular bases for promiscuous behavior and to distinguish between evolutionarily selected promiscuity and evolutionarily tolerated messiness. This review provides an overview of current understanding of these aspects of protein biochemistry and enzymology. PMID:25218442

  19. Level of Education, Sexual Promiscuity, and AIDS.

    ERIC Educational Resources Information Center

    Krull, Catherine D.

    1994-01-01

    Responses of 595 unmarried persons on the 1991 General Social Survey suggest that educational attainment indirectly increases promiscuous sexual behavior by liberalizing sexual attitudes. In a second study, the incidence of AIDS was partly explained by educational attainment, liberal sexual attitudes, and promiscuous sexual practices. (KS)

  20. Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

    PubMed Central

    Kuznetsov, Nikita A.; Koval, Vladimir V.; Zharkov, Dmitry O.; Nevinsky, Georgy A.; Douglas, Kenneth T.; Fedorova, Olga S.

    2005-01-01

    Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG. PMID:16024742

  1. Regulation of Structural Dynamics within a Signal Recognition Particle Promotes Binding of Protein Targeting Substrates*

    PubMed Central

    Gao, Feng; Kight, Alicia D.; Henderson, Rory; Jayanthi, Srinivas; Patel, Parth; Murchison, Marissa; Sharma, Priyanka; Goforth, Robyn L.; Kumar, Thallapuranam Krishnaswamy Suresh; Henry, Ralph L.; Heyes, Colin D.

    2015-01-01

    Protein targeting is critical in all living organisms and involves a signal recognition particle (SRP), an SRP receptor, and a translocase. In co-translational targeting, interactions among these proteins are mediated by the ribosome. In chloroplasts, the light-harvesting chlorophyll-binding protein (LHCP) in the thylakoid membrane is targeted post-translationally without a ribosome. A multidomain chloroplast-specific subunit of the SRP, cpSRP43, is proposed to take on the role of coordinating the sequence of targeting events. Here, we demonstrate that cpSRP43 exhibits significant interdomain dynamics that are reduced upon binding its SRP binding partner, cpSRP54. We showed that the affinity of cpSRP43 for the binding motif of LHCP (L18) increases when cpSRP43 is complexed to the binding motif of cpSRP54 (cpSRP54pep). These results support the conclusion that substrate binding to the chloroplast SRP is modulated by protein structural dynamics in which a major role of cpSRP54 is to improve substrate binding efficiency to the cpSRP. PMID:25918165

  2. The exosome subunit Rrp44 plays a direct role in RNA substrate recognition.

    PubMed

    Schneider, Claudia; Anderson, James T; Tollervey, David

    2007-07-20

    The exosome plays key roles in RNA maturation and surveillance, but it is unclear how target RNAs are identified. We report the functional characterization of the yeast exosome component Rrp44, a member of the RNase II family. Recombinant Rrp44 and the purified TRAMP polyadenylation complex each specifically recognized tRNA(i)(Met) lacking a single m(1)A(58) modification, even in the presence of a large excess of total tRNA. This tRNA is otherwise mature and functional in translation in vivo but is presumably subtly misfolded. Complete degradation of the hypomodified tRNA required both Rrp44 and the poly(A) polymerase activity of TRAMP. The intact exosome lacking only the catalytic activity of Rrp44 failed to degrade tRNA(i)(Met), showing this to be a specific Rrp44 substrate. Recognition of hypomodified tRNA(i)(Met) by Rrp44 is genetically separable from its catalytic activity on other substrates, with the mutations mapping to distinct regions of the protein. PMID:17643380

  3. RNA structure-dependent uncoupling of substrate recognition and cleavage by Escherichia coli ribonuclease III

    PubMed Central

    Calin-Jageman, Irina; Nicholson, Allen W.

    2003-01-01

    Members of the ribonuclease III superfamily of double-strand-specific endoribonucleases participate in diverse RNA maturation and decay pathways. Ribonuclease III of the gram-negative bacterium Escherichia coli processes rRNA and mRNA precursors, and its catalytic action can regulate gene expression by controlling mRNA translation and stability. It has been proposed that E.coli RNase III can function in a non-catalytic manner, by binding RNA without cleaving phosphodiesters. However, there has been no direct evidence for this mode of action. We describe here an RNA, derived from the T7 phage R1.1 RNase III substrate, that is resistant to cleavage in vitro by E.coli RNase III but retains comparable binding affinity. R1.1[CL3B] RNA is recognized by RNase III in the same manner as R1.1 RNA, as revealed by the similar inhibitory effects of a specific mutation in both substrates. Structure-probing assays and Mfold analysis indicate that R1.1[CL3B] RNA possesses a bulge– helix–bulge motif in place of the R1.1 asymmetric internal loop. The presence of both bulges is required for uncoupling. The bulge–helix–bulge motif acts as a ‘catalytic’ antideterminant, which is distinct from recognition antideterminants, which inhibit RNase III binding. PMID:12711683

  4. Nosology, ontology and promiscuous realism.

    PubMed

    Binney, Nicholas

    2015-06-01

    Medics may consider worrying about their metaphysics and ontology to be a waste of time. I will argue here that this is not the case. Promiscuous realism is a metaphysical position which holds that multiple, equally valid, classification schemes should be applied to objects (such as patients) to capture different aspects of their complex and heterogeneous nature. As medics at the bedside may need to capture different aspects of their patients' problems, they may need to use multiple classification schemes (multiple nosologies), and thus consider adopting a different metaphysics to the one commonly in use. PMID:25389077

  5. Structural Basis of Substrate Recognition by Aldehyde Dehydrogenase 7A1.

    PubMed

    Luo, Min; Tanner, John J

    2015-09-01

    Aldehyde dehydrogenase 7A1 (ALDH7A1) is part of lysine catabolism and catalyzes the NAD(+)-dependent oxidation of α-aminoadipate semialdehyde to α-aminoadipate. Herein, we describe a structural study of human ALDH7A1 focused on substrate recognition. Five crystal structures and small-angle X-ray scattering data are reported, including the first crystal structure of any ALDH7 family member complexed with α-aminoadipate. The product binds with the ε-carboxylate in the oxyanion hole, the aliphatic chain packed into an aromatic box, and the distal end of the product anchored by electrostatic interactions with five conserved residues. This binding mode resembles that of glutamate bound to the proline catabolic enzyme ALDH4A1. Analysis of ALDH7A1 and ALDH4A1 structures suggests key interactions that underlie substrate discrimination. Structures of apo ALDH7A1 reveal dramatic conformational differences from the product complex. Product binding is associated with a 16 Å movement of the C-terminus into the active site, which stabilizes the active conformation of the aldehyde substrate anchor loop. The fact that the C-terminus is part of the active site was hitherto unknown. Interestingly, the C-terminus and aldehyde anchor loop are disordered in a new tetragonal crystal form of the apoenzyme, implying that these parts of the enzyme are highly flexible. Our results suggest that the active site of ALDH7A1 is disassembled when the aldehyde site is vacant, and the C-terminus is a mobile element that forms quaternary structural interactions that aid aldehyde binding. These results are relevant to the c.1512delG genetic deletion associated with pyridoxine-dependent epilepsy, which alters the C-terminus of ALDH7A1. PMID:26260980

  6. Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)

    SciTech Connect

    Critton, D.; Tortajada, A; Stetson, G; Peti, W; Page, R

    2008-01-01

    Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPs as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.

  7. Modeling catalytic promiscuity in the alkaline phosphatase superfamily

    PubMed Central

    Duarte, Fernanda; Amrein, Beat Anton

    2013-01-01

    In recent years, it has become increasingly clear that promiscuity plays a key role in the evolution of new enzyme function. This finding has helped to elucidate fundamental aspects of molecular evolution. While there has been extensive experimental work on enzyme promiscuity, computational modeling of the chemical details of such promiscuity has traditionally fallen behind the advances in experimental studies, not least due to the nearly prohibitive computational cost involved in examining multiple substrates with multiple potential mechanisms and binding modes in atomic detail with a reasonable degree of accuracy. However, recent advances in both computational methodologies and power have allowed us to reach a stage in the field where we can start to overcome this problem, and molecular simulations can now provide accurate and efficient descriptions of complex biological systems with substantially less computational cost. This has led to significant advances in our understanding of enzyme function and evolution in a broader sense. Here, we will discuss currently available computational approaches that can allow us to probe the underlying molecular basis for enzyme specificity and selectivity, discussing the inherent strengths and weaknesses of each approach. As a case study, we will discuss recent computational work on different members of the alkaline phosphatase superfamily (AP) using a range of different approaches, showing the complementary insights they have provided. We have selected this particular superfamily, as it poses a number of significant challenges for theory, ranging from the complexity of the actual reaction mechanisms involved to the reliable modeling of the catalytic metal centers, as well as the very large system sizes. We will demonstrate that, through current advances in methodologies, computational tools can provide significant insight into the molecular basis for catalytic promiscuity, and, therefore, in turn, the mechanisms of protein

  8. Substrate recognition by the zinc metalloprotease effector NleC from enteropathogenic Escherichia coli.

    PubMed

    Giogha, Cristina; Lung, Tania Wong Fok; Mühlen, Sabrina; Pearson, Jaclyn S; Hartland, Elizabeth L

    2015-12-01

    Upon infection of epithelial cells, enteropathogenic Escherichia coli suppresses host cell inflammatory signalling in a type III secretion system (T3SS) dependent manner. Two key T3SS effector proteins involved in this response are NleE and NleC. NleC is a zinc metalloprotease effector that degrades the p65 subunit of NF-κB. Although the site of p65 cleavage by NleC is now well described, other areas of interaction have not been precisely defined. Here we constructed overlapping truncations of p65 to identify regions required for NleC cleavage. We determined that NleC cleaved both p65 and p50 within the Rel homology domain (RHD) and that two motifs, E22IIE25 and P177VLS180 , within the RHD of p65 were important for recognition and binding by NleC. Alanine substitution of one or both of these motifs protected p65 from binding and degradation by NleC. The E22IIE25 and P177VLS180 motifs were located within the structurally distinct N-terminal subdomain of the RHD involved in DNA binding by p65 on adjacent, parallel strands. Although these motifs have not been recognized previously, both were needed for the correct localization and function of p65. In summary, this work has identified two regions of p65 within the RHD needed for binding and cleavage by NleC and provides further insight into the molecular basis of substrate recognition by a T3SS effector. PMID:26096513

  9. Conformational Changes and Substrate Recognition in Pseudomonas aeruginosa d-Arginine Dehydrogenase

    SciTech Connect

    Fu, Guoxing; Yuan, Hongling; Li, Congran; Lu, Chung-Dar; Gadda, Giovanni; Weber, Irene T.

    2010-11-15

    DADH catalyzes the flavin-dependent oxidative deamination of D-amino acids to the corresponding {alpha}-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 {angstrom} resolution and its complexes with iminoarginine (DADH{sub red}/iminoarginine) and iminohistidine (DADH{sub red}/iminohistidine) at 1.30 {angstrom} resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADH{sub red}/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an 'active site lid', similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in k{sub cat}/K{sub m} values for D-arginine and D-histidine. Comparison of the kinetic data for the activity of DADH on different D-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic D-amino acids bound within a flask-like cavity.

  10. N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

    PubMed Central

    Hassan, Yousef I.; Moriyama, Hideaki; Olsen, Lars J.; Bi, Xin; Zempleni, Janos

    2009-01-01

    Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS. PMID:19157941

  11. Promiscuity and diversity in 3-ketosteroid reductases

    PubMed Central

    Penning, Trevor M.; Chen, Mo; Jin, Yi

    2014-01-01

    Many steroid hormones contain a Δ4-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1–AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1–AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled ‘Steroid/Sterol signaling’. PMID:25500069

  12. Promiscuity and diversity in 3-ketosteroid reductases.

    PubMed

    Penning, Trevor M; Chen, Mo; Jin, Yi

    2015-07-01

    Many steroid hormones contain a Δ(4)-3-ketosteroid functionality that undergoes sequential reduction by 5α- or 5β- steroid reductases to produce 5α- or 5β-dihydrosteroids; and a subsequent 3-keto-reduction to produce a series of isomeric tetrahydrosteroids. Apart from steroid 5α-reductase all the remaining enzymes involved in the two step reduction process in humans belong to the aldo-keto reductase (AKR) superfamily. The enzymes involved in 3-ketosteroid reduction are AKR1C1-AKR1C4. These enzymes are promiscuous and also catalyze 20-keto- and 17-keto-steroid reduction. Interest in these reactions exist since they regulate steroid hormone metabolism in the liver, and in steroid target tissues, they may regulate steroid hormone receptor occupancy. In addition many of the dihydrosteroids are not biologically inert. The same enzymes are also involved in the metabolism of synthetic steroids e.g., hormone replacement therapeutics, contraceptive agents and inhaled glucocorticoids, and may regulate drug efficacy at their cognate receptors. This article reviews these reactions and the structural basis for substrate diversity in AKR1C1-AKR1C4, ketosteroid reductases. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25500069

  13. Sequence correlations shape protein promiscuity

    NASA Astrophysics Data System (ADS)

    Lukatsky, David B.; Afek, Ariel; Shakhnovich, Eugene I.

    2011-08-01

    We predict analytically that diagonal correlations of amino acid positions within protein sequences statistically enhance protein propensity for nonspecific binding. We use the term "promiscuity" to describe such nonspecific binding. Diagonal correlations represent statistically significant repeats of sequence patterns where amino acids of the same type are clustered together. The predicted effect is qualitatively robust with respect to the form of the microscopic interaction potentials and the average amino acid composition. Our analytical results provide an explanation for the enhanced diagonal correlations observed in hubs of eukaryotic organismal proteomes [J. Mol. Biol. 409, 439 (2011)], 10.1016/j.jmb.2011.03.056. We suggest experiments that will allow direct testing of the predicted effect.

  14. PubChem promiscuity: a web resource for gathering compound promiscuity data from PubChem

    PubMed Central

    Canny, Stephanie A.; Cruz, Yasel; Southern, Mark R.; Griffin, Patrick R.

    2012-01-01

    Summary: Promiscuity counts allow for a better understanding of a compound's assay activity profile and drug potential. Although PubChem contains a vast amount of compound and assay data, it currently does not have a convenient or efficient method to obtain in-depth promiscuity counts for compounds. PubChem promiscuity fills this gap. It is a Java servlet that uses NCBI Entrez (eUtils) web services to interact with PubChem and provide promiscuity counts in a variety of categories along with compound descriptors, including PAINS-based functional group detection. Availability: http://chemutils.florida.scripps.edu/pcpromiscuity Contact: southern@scripps.edu PMID:22084255

  15. Neutral genetic drift can alter promiscuous protein functions, potentially aiding functional evolution

    PubMed Central

    Bloom, Jesse D; Romero, Philip A; Lu, Zhongyi; Arnold, Frances H

    2007-01-01

    Background Many of the mutations accumulated by naturally evolving proteins are neutral in the sense that they do not significantly alter a protein's ability to perform its primary biological function. However, new protein functions evolve when selection begins to favor other, "promiscuous" functions that are incidental to a protein's original biological role. If mutations that are neutral with respect to a protein's primary biological function cause substantial changes in promiscuous functions, these mutations could enable future functional evolution. Results Here we investigate this possibility experimentally by examining how cytochrome P450 enzymes that have evolved neutrally with respect to activity on a single substrate have changed in their abilities to catalyze reactions on five other substrates. We find that the enzymes have sometimes changed as much as four-fold in the promiscuous activities. The changes in promiscuous activities tend to increase with the number of mutations, and can be largely rationalized in terms of the chemical structures of the substrates. The activities on chemically similar substrates tend to change in a coordinated fashion, potentially providing a route for systematically predicting the change in one activity based on the measurement of several others. Conclusion Our work suggests that initially neutral genetic drift can lead to substantial changes in protein functions that are not currently under selection, in effect poising the proteins to more readily undergo functional evolution should selection favor new functions in the future. Reviewers This article was reviewed by Martijn Huynen, Fyodor Kondrashov, and Dan Tawfik (nominated by Christoph Adami). PMID:17598905

  16. Functional Promiscuity of the COG0720 Family

    PubMed Central

    Phillips, Gabriela; Grochowski, Laura L.; Bonnett, Shilah; Xu, Huimin; Bailly, Marc; Haas-Blaby, Crysten; El Yacoubi, Basma; Iwata-Reuyl, Dirk; White, Robert H.; de Crécy-Lagard, Valérie

    2011-01-01

    The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH4), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G+) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins include the 6-pyruvoyl-tetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologs, all members of the COG0720 family, that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H2NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH4 pathway. PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway. PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, and phylogenetic co-distribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate, or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis, and resulted in the identification PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families, extensive comparative genomic analysis beyond homology is required to correctly predict function. PMID:21999246

  17. Investigation of the intermolecular recognition mechanism between the E3 ubiquitin ligase Keap1 and substrate based on multiple substrates analysis.

    PubMed

    Jiang, Zheng-Yu; Xu, Li-Li; Lu, Meng-Chen; Pan, Yang; Huang, Hao-Ze; Zhang, Xiao-Jin; Sun, Hao-Peng; You, Qi-Dong

    2014-12-01

    E3 ubiquitin ligases are attractive drug targets due to their specificity to the ubiquitin machinery. However, the development of E3 ligase inhibitors has proven challenging for the fact that they must disrupt protein-protein interactions (PPIs). The E3 ligase involved in interactome provide new hope for the discovery of the E3 ligase inhibitors. These currently known natural binding partners of the E3 ligase can benefit the discovery of other unknown substrates and also the E3 ligase inhibitors. Herein, we present a novel strategy that using multiple substrates to elucidate the molecular recognition mechanism of E3 ubiquitin ligase. Molecular dynamics simulation, molecular mechanics-generalized born surface area (MM-GBSA) binding energy calculation and energy decomposition scheme were incorporated to evaluate the quantitative contributions of sub-pocket and per-residue to binding. In this case, Kelch-like ECH-associated protein-1 (Keap1), a substrate adaptor component of the Cullin-RING ubiquitin ligases complex, is applied for the investigation of how it recognize its substrates, especially Nrf2, a master regulator of the antioxidant response. By analyzing multiple substrates binding determinants, we found that both the polar sub-pockets (P1 and P2) and the nonpolar sub-pockets (P4 and P5) of Keap1 can make remarkable contributions to intermolecular interactions. This finding stresses the requirement for substrates to interact with the polar and nonpolar sub-pockets simultaneously. The results discussed in this paper not only show the binding determinants of the Keap1 substrates but also provide valuable implications for both Keap1 substrate discovery and PPI inhibitor design. PMID:25301376

  18. A Single-Stranded Junction Modulates Nanosecond Motional Ordering of the Substrate Recognition Duplex of a Group I Ribozyme

    PubMed Central

    Nguyen, Phuong; Shi, Xuesong; Sigurdsson, Snorri Th.; Herschlag, Daniel

    2013-01-01

    Rigid spinning: Site-directed spin-labeling studies using a rigid nitroxide spin label (Ç) reveal that both length and sequence of a single-stranded junction (J1/2) modulate nanosecond motional ordering of the substrate-recognition duplex (P1) of the 120 kD group I ribozyme. The studies demonstrate an approach for experimental measurements of nanosecond dynamics in high-molecular-weight RNA complexes. PMID:23900919

  19. Substrate recognition of holocytochrome c synthase: N-terminal region and CXXCH motif of mitochondrial cytochrome c

    PubMed Central

    Zhang, Yulin; Stevens, Julie M.; Ferguson, Stuart J.

    2014-01-01

    Holocytochrome c synthase (HCCS) attaches heme covalently to mitochondrial respiratory cytochromes c. Little is known about the reaction of heme attachment to apocytochromes c by HCCS, although recently it has been established that the CXXCH motif and the N-terminus of the apocytochrome polypeptide are important protein–protein recognition motifs. Here, we explore further the important features of the N-terminal sequence and investigate what variations in the CXXCH residues are productively recognised by HCCS in its substrate. PMID:25084480

  20. Structural Basis of Substrate Recognition in Human Nicotinamide N-Methyltransferase

    SciTech Connect

    Peng, Yi; Sartini, Davide; Pozzi, Valentina; Wilk, Dennis; Emanuelli, Monica; Yee, Vivien C.

    2012-05-02

    Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of nicotinamide, pyridines, and other analogues using S-adenosyl-L-methionine as donor. NNMT plays a significant role in the regulation of metabolic pathways and is expressed at markedly high levels in several kinds of cancers, presenting it as a potential molecular target for cancer therapy. We have determined the crystal structure of human NNMT as a ternary complex bound to both the demethylated donor S-adenosyl-L-homocysteine and the acceptor substrate nicotinamide, to 2.7 {angstrom} resolution. These studies reveal the structural basis for nicotinamide binding and highlight several residues in the active site which may play roles in nicotinamide recognition and NNMT catalysis. The functional importance of these residues was probed by mutagenesis. Of three residues near the nicotinamide's amide group, substitution of S201 and S213 had no effect on enzyme activity while replacement of D197 dramatically decreased activity. Substitutions of Y20, whose side chain hydroxyl interacts with both the nicotinamide aromatic ring and AdoHcy carboxylate, also compromised activity. Enzyme kinetics analysis revealed k{sub cat}/K{sub m} decreases of 2-3 orders of magnitude for the D197A and Y20A mutants, confirming the functional importance of these active site residues. The mutants exhibited substantially increased K{sub m} for both NCA and AdoMet and modestly decreased k{sub cat}. MD simulations revealed long-range conformational effects which provide an explanation for the large increase in K{sub m}(AdoMet) for the D197A mutant, which interacts directly only with nicotinamide in the ternary complex crystal structure.

  1. Beyond perceptual expertise: revisiting the neural substrates of expert object recognition.

    PubMed

    Harel, Assaf; Kravitz, Dwight; Baker, Chris I

    2013-01-01

    Real-world expertise provides a valuable opportunity to understand how experience shapes human behavior and neural function. In the visual domain, the study of expert object recognition, such as in car enthusiasts or bird watchers, has produced a large, growing, and often-controversial literature. Here, we synthesize this literature, focusing primarily on results from functional brain imaging, and propose an interactive framework that incorporates the impact of high-level factors, such as attention and conceptual knowledge, in supporting expertise. This framework contrasts with the perceptual view of object expertise that has concentrated largely on stimulus-driven processing in visual cortex. One prominent version of this perceptual account has almost exclusively focused on the relation of expertise to face processing and, in terms of the neural substrates, has centered on face-selective cortical regions such as the Fusiform Face Area (FFA). We discuss the limitations of this face-centric approach as well as the more general perceptual view, and highlight that expert related activity is: (i) found throughout visual cortex, not just FFA, with a strong relationship between neural response and behavioral expertise even in the earliest stages of visual processing, (ii) found outside visual cortex in areas such as parietal and prefrontal cortices, and (iii) modulated by the attentional engagement of the observer suggesting that it is neither automatic nor driven solely by stimulus properties. These findings strongly support a framework in which object expertise emerges from extensive interactions within and between the visual system and other cognitive systems, resulting in widespread, distributed patterns of expertise-related activity across the entire cortex. PMID:24409134

  2. Beyond perceptual expertise: revisiting the neural substrates of expert object recognition

    PubMed Central

    Harel, Assaf; Kravitz, Dwight; Baker, Chris I.

    2013-01-01

    Real-world expertise provides a valuable opportunity to understand how experience shapes human behavior and neural function. In the visual domain, the study of expert object recognition, such as in car enthusiasts or bird watchers, has produced a large, growing, and often-controversial literature. Here, we synthesize this literature, focusing primarily on results from functional brain imaging, and propose an interactive framework that incorporates the impact of high-level factors, such as attention and conceptual knowledge, in supporting expertise. This framework contrasts with the perceptual view of object expertise that has concentrated largely on stimulus-driven processing in visual cortex. One prominent version of this perceptual account has almost exclusively focused on the relation of expertise to face processing and, in terms of the neural substrates, has centered on face-selective cortical regions such as the Fusiform Face Area (FFA). We discuss the limitations of this face-centric approach as well as the more general perceptual view, and highlight that expert related activity is: (i) found throughout visual cortex, not just FFA, with a strong relationship between neural response and behavioral expertise even in the earliest stages of visual processing, (ii) found outside visual cortex in areas such as parietal and prefrontal cortices, and (iii) modulated by the attentional engagement of the observer suggesting that it is neither automatic nor driven solely by stimulus properties. These findings strongly support a framework in which object expertise emerges from extensive interactions within and between the visual system and other cognitive systems, resulting in widespread, distributed patterns of expertise-related activity across the entire cortex. PMID:24409134

  3. Substrate recognition of N,N'-diacetylchitobiose deacetylase from Pyrococcus horikoshii.

    PubMed

    Nakamura, Tsutomu; Yonezawa, Yasushige; Tsuchiya, Yuko; Niiyama, Mayumi; Ida, Kurumi; Oshima, Maki; Morita, Junji; Uegaki, Koichi

    2016-09-01

    Enzymes of carbohydrate esterase (CE) family 14 catalyze hydrolysis of N-acetyl groups at the non-reducing end of the N-acetylglucosamine (GlcNAc) residue of chitooligosaccharides or related compounds. N,N'-diacetylchitobiose deacetylase (Dac) belongs to the CE-14 family and plays a role in the chitinolytic pathway in archaea by deacetylating N,N'-diacetylchitobiose (GlcNAc2), which is the end product of chitinase. In this study, we revealed the structural basis of reaction specificity in CE-14 deacetylases by solving a crystal structure of Dac from Pyrococcus horikoshii (Ph-Dac) in complex with a novel reaction intermediate analog. We developed 2-deoxy-2-methylphosphoramido-d-glucose (MPG) as the analog of the tetrahedral oxyanion intermediate of the monosaccharide substrate GlcNAc. The crystal structure of Ph-Dac in complex with MPG demonstrated that Arg92, Asp115, and His152 side chains interact with hydroxyl groups of the glucose moiety of the non-reducing-end GlcNAc residue. The amino acid residues responsible for recognition of the MPG glucose moiety are spatially conserved in other CE-14 deacetylases. Molecular dynamics simulation of the structure of the Ph-Dac-GlcNAc2 complex indicated that the reducing GlcNAc residue is placed in a large intermolecular cleft and is not involved with specific interactions with the enzyme. This observation was consistent with results indicating that Ph-Dac displayed similar kinetic parameters for both GlcNAc and GlcNAc2. This study provides the structural basis of reaction-site specificity of Dac and related CE-14 enzymes. PMID:27456364

  4. Mechanism of Dis3l2 substrate recognition in the Lin28-let-7 pathway.

    PubMed

    Faehnle, Christopher R; Walleshauser, Jack; Joshua-Tor, Leemor

    2014-10-01

    The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development, glucose metabolism and tissue regeneration. Overexpression of Lin28 is correlated with the onset of numerous cancers, whereas let-7, a tumour suppressor, silences several human oncogenes. Lin28 binds to precursor let-7 (pre-let-7) hairpins, triggering the 3' oligo-uridylation activity of TUT4 and TUT7 (refs 10-12). The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3l2 (refs 13, 14), a homologue of the catalytic subunit of the RNA exosome. The molecular basis of Lin28-mediated recruitment of TUT4 and TUT7 to pre-let-7 and its subsequent degradation by Dis3l2 is largely unknown. To examine the mechanism of Dis3l2 substrate recognition we determined the structure of mouse Dis3l2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA-binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from that of its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first 12 nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3l2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28-let-7 pathway. PMID:25119025

  5. Mechanism of Dis3L2 substrate recognition in the Lin28/let-7 pathway

    PubMed Central

    Joshua-Tor, Leemor

    2014-01-01

    Summary paragraph The pluripotency factor Lin28 inhibits the biogenesis of the let-7 family of mammalian microRNAs1–4. Lin28 is highly expressed in embryonic stem cells and has a fundamental role in regulation of development5, glucose metabolism6 and tissue regeneration7. Alternatively, Lin28 overexpression is correlated with the onset of numerous cancers8, while let-7, a tumor suppressor, silences several human oncogenes5. Lin28 binds to precursor let-7 (pre-let-7) hairpins9, triggering the 3' oligo-uridylation activity of TUT4/710–12. The oligoU tail added to pre-let-7 serves as a decay signal, as it is rapidly degraded by Dis3L213,14, a homolog of the catalytic subunit of the RNA exosome. The molecular basis of Lin28 mediated recruitment of TUT4/7 to pre-let-7 and its subsequent degradation by Dis3L2 is largely unknown. To examine the mechanism of Dis3L2 substrate recognition we determined the structure of mouse Dis3L2 in complex with an oligoU RNA to mimic the uridylated tail of pre-let-7. Three RNA binding domains form an open funnel on one face of the catalytic domain that allows RNA to navigate a path to the active site different from its exosome counterpart. The resulting path reveals an extensive network of uracil-specific interactions spanning the first twelve nucleotides of an oligoU-tailed RNA. We identify three U-specificity zones that explain how Dis3L2 recognizes, binds and processes uridylated pre-let-7 in the final step of the Lin28/let-7 pathway. PMID:25119025

  6. Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin.

    PubMed

    Chen, Sheng; Hall, Cherisse; Barbieri, Joseph T

    2008-07-25

    Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates. PMID:18511417

  7. Acyl-ACP Substrate Recognition in Burkholderia mallei BmaI1 Acyl-Homoserine Lactone Synthase

    PubMed Central

    2015-01-01

    The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme. PMID:25215658

  8. Promiscuous Recognition of a Trypanosoma cruzi CD8+ T Cell Epitope among HLA-A2, HLA-A24 and HLA-A1 Supertypes in Chagasic Patients

    PubMed Central

    Guzmán, Fanny; Rosas, Fernando; Thomas, M. Carmen; López, Manuel Carlos; González, John Mario; Cuéllar, Adriana; Puerta, Concepción J.

    2016-01-01

    Background TcTLE is a nonamer peptide from Trypanosoma cruzi KMP-11 protein that is conserved among different parasite strains and that is presented by different HLA-A molecules from the A2 supertype. Because peptides presented by several major histocompatibility complex (MHC) supertypes are potential targets for immunotherapy, the aim of this study was to determine whether MHC molecules other than the A2 supertype present the TcTLE peptide. Methodology/Principal Findings From 36 HLA-A2-negative chagasic patients, the HLA-A genotypes of twenty-eight patients with CD8+ T cells that recognized the TcTLE peptide using tetramer (twenty) or functional (eight) assays, were determined. SSP-PCR was used to identify the A locus and the allelic variants. Flow cytometry was used to analyze the frequency of TcTLE-specific CD8+ T cells, and their functional activity (IFN-γ, TNFα, IL-2, perforin, granzyme and CD107a/b production) was induced by exposure to the TcTLE peptide. All patients tested had TcTLE-specific CD8+ T cells with frequencies ranging from 0.07–0.37%. Interestingly, seven of the twenty-eight patients had HLA-A homozygous alleles: A*24 (5 patients), A*23 (1 patient) and A*01 (1 patient), which belong to the A24 and A1 supertypes. In the remaining 21 patients with HLA-A heterozygous alleles, the most prominent alleles were A24 and A68. The most common allele sub-type was A*2402 (sixteen patients), which belongs to the A24 supertype, followed by A*6802 (six patients) from the A2 supertype. Additionally, the A*3002/A*3201 alleles from the A1 supertype were detected in one patient. All patients presented CD8+ T cells producing at least one cytokine after TcTLE peptide stimulation. Conclusion/Significance These results show that TcTLE is a promiscuous peptide that is presented by the A24 and A1 supertypes, in addition to the A2 supertype, suggesting its potential as a target for immunotherapy. PMID:26974162

  9. Reprogramming acyl carrier protein interactions of an acyl-CoA promiscuous trans-acyltransferase

    PubMed Central

    Ye, Zhixia; Musiol, Ewa M; Weber, Tilmann; Williams, Gavin J

    2014-01-01

    SUMMARY Protein interactions between acyl carrier proteins (ACP’s) and trans-acting acyltransferase domains (trans-AT’s) are critical for regioselective extender unit installation by many polyketide synthases. Yet, little is known regarding the specificity of these interactions, particularly for trans-AT’s with unusual extender unit specificities. Currently, the best-studied trans-AT with non-malonyl specificity is KirCII from kirromycin biosynthesis. Here, we developed a new assay to probe ACP interactions based on leveraging the extender unit promiscuity of KirCII. The assay allows us to identify residues on the ACP surface that contribute to specific recognition by KirCII. This information proved sufficient to modify a non-cognate ACP from a different biosynthetic system to be a substrate for KirCII. The findings form a foundation for further understanding the specificity of trans-AT:ACP protein interactions, and for engineering modular polyketide synthases to produce analogues. PMID:24726832

  10. Molecular Recognition of Fluorine Impacts Substrate Selectivity in the Fluoroacetyl-CoA Thioesterase FlK

    PubMed Central

    2015-01-01

    The fluoroacetate-producing bacterium Streptomyces cattleya has evolved a fluoroacetyl-CoA thioesterase (FlK) that exhibits a remarkably high level of discrimination for its cognate substrate compared to the cellularly abundant analogue acetyl-CoA, which differs only by the absence of the fluorine substitution. A major determinant of FlK specificity derives from its ability to take advantage of the unique properties of fluorine to enhance the reaction rate, allowing fluorine discrimination under physiological conditions where both substrates are likely to be present at saturating concentrations. Using a combination of pH–rate profiles, pre-steady-state kinetic experiments, and Taft analysis of wild-type and mutant FlKs with a set of substrate analogues, we explore the role of fluorine in controlling the enzyme acylation and deacylation steps. Further analysis of chiral (R)- and (S)-[2H1]fluoroacetyl-CoA substrates demonstrates that a kinetic isotope effect (1.7 ± 0.2) is observed for only the (R)-2H1 isomer, indicating that deacylation requires recognition of the prochiral fluoromethyl group to position the α-carbon for proton abstraction. Taken together, the selectivity for the fluoroacetyl-CoA substrate appears to rely not only on the enhanced polarization provided by the electronegative fluorine substitution but also on molecular recognition of fluorine in both formation and breakdown of the acyl-enzyme intermediate to control active site reactivity. These studies provide insights into the basis of fluorine selectivity in a naturally occurring enzyme–substrate pair, with implications for drug design and the development of fluorine-selective biocatalysts. PMID:24635371

  11. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  12. Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2'-O-methyltransferase.

    PubMed

    Zhou, Mi; Long, Tao; Fang, Zhi-Peng; Zhou, Xiao-Long; Liu, Ru-Juan; Wang, En-Duo

    2015-01-01

    Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNA(Leu)CAA and tRNA(Leu)UAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2'-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i(6)) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2'-O-methylation relies on prior N(6)-isopentenyladenosine modification at position 37. PMID:26106808

  13. Motions of the Substrate Recognition Duplex in a Group I Intron Assessed by Site-Directed Spin Labeling

    SciTech Connect

    Grant, Gian Paola G; Boyd, Nathan; Herschlag, Daniel; Qin, Peter Z

    2009-03-11

    The Tetrahymena group I intron recognizes its oligonucleotide substrate in a two-step process. First, a substrate recognition duplex, called the P1 duplex, is formed. The P1 duplex then docks into the prefolded ribozyme core by forming tertiary contacts. P1 docking controls both the rate and the fidelity of substrate cleavage and has been extensively studied as a model for the formation of RNA tertiary structure. However, previous work has been limited to studying millisecond or slower motions. Here we investigated nanosecond P1 motions in the context of the ribozyme using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A nitroxide spin label (R5a) was covalently attached to a specific site of the substrate oligonucleotide, the labeled substrate was bound to a prefolded ribozyme to form the P1 duplex, and X-band EPR spectroscopy was used to monitor nitroxide motions in the 0.1-50 ns regime. Using substrates that favor the docked or the undocked states, it was established that R5a was capable of reporting P1 duplex motions. Using R5a-labeled substrates it was found that the J1/2 junction connecting P1 to the ribozyme core controls nanosecond P1 mobility in the undocked state. This may account for previous observations that J1/2 mutations weaken substrate binding and give rise to cryptic cleavage. This study establishes the use of SDSL to probe nanosecond dynamic behaviors of individual helices within large RNA and RNA/protein complexes. This approach may help in understanding the relationship between RNA structure, dynamics, and function.

  14. Determining the Degree of Promiscuity of Extensively Assayed Compounds

    PubMed Central

    Jasial, Swarit; Hu, Ye; Bajorath, Jürgen

    2016-01-01

    In the context of polypharmacology, an emerging concept in drug discovery, promiscuity is rationalized as the ability of compounds to specifically interact with multiple targets. Promiscuity of drugs and bioactive compounds has thus far been analyzed computationally on the basis of activity annotations, without taking assay frequencies or inactivity records into account. Most recent estimates have indicated that bioactive compounds interact on average with only one to two targets, whereas drugs interact with six or more. In this study, we have further extended promiscuity analysis by identifying the most extensively assayed public domain compounds and systematically determining their promiscuity. These compounds were tested in hundreds of assays against hundreds of targets. In our analysis, assay promiscuity was distinguished from target promiscuity and separately analyzed for primary and confirmatory assays. Differences between the degree of assay and target promiscuity were surprisingly small and average and median degrees of target promiscuity of 2.6 to 3.4 and 2.0 were determined, respectively. Thus, target promiscuity remained at a low level even for most extensively tested active compounds. These findings provide further evidence that bioactive compounds are less promiscuous than drugs and have implications for pharmaceutical research. In addition to a possible explanation that drugs are more extensively tested for additional targets, the results would also support a “promiscuity enrichment model” according to which promiscuous compounds might be preferentially selected for therapeutic efficacy during clinical evaluation to ultimately become drugs. PMID:27082988

  15. An ordered water channel in Staphylococcus aureus FabI: unraveling the mechanism of substrate recognition and reduction.

    PubMed

    Schiebel, Johannes; Chang, Andrew; Merget, Benjamin; Bommineni, Gopal R; Yu, Weixuan; Spagnuolo, Lauren A; Baxter, Michael V; Tareilus, Mona; Tonge, Peter J; Kisker, Caroline; Sotriffer, Christoph A

    2015-03-17

    One third of all drugs in clinical use owe their pharmacological activity to the functional inhibition of enzymes, highlighting the importance of enzymatic targets for drug development. Because of the close relationship between inhibition and catalysis, understanding the recognition and turnover of enzymatic substrates is essential for rational drug design. Although the Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI) involved in bacterial fatty acid biosynthesis constitutes a very promising target for the development of novel, urgently needed anti-staphylococcal agents, the substrate binding mode and catalytic mechanism remained unclear for this enzyme. Using a combined crystallographic, kinetic, and computational approach, we have explored the chemical properties of the saFabI binding cavity, obtaining a consistent mechanistic model for substrate binding and turnover. We identified a water-molecule network linking the active site with a water basin inside the homo-tetrameric protein, which seems to be crucial for the closure of the flexible substrate binding loop as well as for an effective hydride and proton transfer during catalysis. On the basis of our results, we also derive a new model for the FabI-ACP complex that reveals how the ACP-bound acyl-substrate is injected into the FabI binding crevice. These findings support the future development of novel FabI inhibitors that target the FabI-ACP interface leading to the disruption of the interaction between these two proteins. PMID:25706582

  16. An Ordered Water Channel in Staphylococcus aureus FabI: Unraveling the Mechanism of Substrate Recognition and Reduction

    PubMed Central

    Schiebel, Johannes; Chang, Andrew; Merget, Benjamin; Bommineni, Gopal R.; Yu, Weixuan; Spagnuolo, Lauren A.; Baxter, Michael V.; Tareilus, Mona; Tonge, Peter J.; Kisker, Caroline; Sotriffer, Christoph A.

    2015-01-01

    One third of all drugs in clinical use owe their pharmacological activity to the functional inhibition of enzymes, highlighting the importance of enzymatic targets for drug development. Because of the close relationship between inhibition and catalysis, understanding the recognition and turnover of enzymatic substrates is essential for rational drug design. Although the Staphylococcus aureus enoyl-acyl carrier protein reductase (saFabI) involved in bacterial fatty acid biosynthesis constitutes a very promising target for the development of novel, urgently needed anti-staphylococcal agents, the substrate binding mode and catalytic mechanism remained unclear for this enzyme. Using a combined crystallographic, kinetic and computational approach, we have explored the chemical properties of the saFabI binding cavity, obtaining a consistent mechanistic model for substrate binding and turnover. We identified a water-molecule network linking the active site with a water basin inside the homo-tetrameric protein, which seems to be crucial for the closure of the flexible substrate binding loop as well as for an effective hydride and proton transfer during catalysis. Based on our results, we also derive a new model for the FabI-ACP complex that reveals how the ACP-bound acyl-substrate is injected into the FabI binding crevice. These findings support the future development of novel FabI inhibitors that target the FabI-ACP interface leading to the disruption of the interaction between these two proteins. PMID:25706582

  17. Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea

    PubMed Central

    Hiromoto, Takeshi; Honjo, Eijiro; Noda, Naonobu; Tamada, Taro; Kazuma, Kohei; Suzuki, Masahiko; Blaber, Michael; Kuroki, Ryota

    2015-01-01

    UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3′- and 5′-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1–O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments. PMID:25556637

  18. Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-O-glucosyltransferase from Clitoria ternatea.

    PubMed

    Hiromoto, Takeshi; Honjo, Eijiro; Noda, Naonobu; Tamada, Taro; Kazuma, Kohei; Suzuki, Masahiko; Blaber, Michael; Kuroki, Ryota

    2015-03-01

    UDP-glucose: anthocyanidin 3-O-glucosyltransferase (UGT78K6) from Clitoria ternatea catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. After the acylation of the 3-O-glucosyl residue, the 3'- and 5'-hydroxyl groups of the product are further glucosylated by a glucosyltransferase in the biosynthesis of ternatins, which are anthocyanin pigments. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 Å, 2.55 Å, 2.70 Å, and 1.75 Å, respectively. The enzyme recognition of unstable anthocyanidin aglycones was initially observed in this structural determination. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The 3-hydroxyl groups of the acceptor substrates were located at hydrogen-bonding distances to the Nε2 atom of the His17 catalytic residue, supporting a role for glucosyl transfer to the 3-hydroxyl groups of anthocyanidins and flavonols. However, the molecular orientations of these three acceptors are different from those of the known flavonoid glycosyltransferases, VvGT1 and UGT78G1. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180° rotation about the O1-O3 axis of the flavonoid backbones observed in VvGT1 and UGT78G1; consequently, the 5- and 7-hydroxyl groups are protected from glucosylation. These substrate recognition schemes are useful to understand the unique reaction mechanism of UGT78K6 for the ternatin biosynthesis, and suggest the potential for controlled synthesis of natural pigments. PMID:25556637

  19. Substrate Recognition Mechanism and Substrate-Dependent Conformational Changes of an ROK Family Glucokinase from Streptomyces griseus

    PubMed Central

    Miyazono, Ken-ichi; Tabei, Nobumitsu; Morita, Sho; Ohnishi, Yasuo; Horinouchi, Sueharu

    2012-01-01

    Carbon catabolite repression (CCR) is a widespread phenomenon in many bacteria that is defined as the repression of catabolic enzyme activities for an unfavorable carbon source by the presence of a preferable carbon source. In Streptomyces, secondary metabolite production often is negatively affected by the carbon source, indicating the involvement of CCR in secondary metabolism. Although the CCR mechanism in Streptomyces still is unclear, glucokinase is presumably a central player in CCR. SgGlkA, a glucokinase from S. griseus, belongs to the ROK family glucokinases, which have two consensus sequence motifs (1 and 2). Here, we report the crystal structures of apo-SgGlkA, SgGlkA in complex with glucose, and SgGlkA in complex with glucose and adenylyl imidodiphosphate (AMPPNP), which are the first structures of an ROK family glucokinase. SgGlkA is divided into a small α/β domain and a large α+β domain, and it forms a dimer-of-dimer tetrameric configuration. SgGlkA binds a β-anomer of glucose between the two domains, and His157 in consensus sequence 1 plays an important role in the glucose-binding mechanism and anomer specificity of SgGlkA. In the structures of SgGlkA, His157 forms an HC3-type zinc finger motif with three cysteine residues in consensus sequence 2 to bind a zinc ion, and it forms two hydrogen bonds with the C1 and C2 hydroxyls of glucose. When the three structures are compared, the structure of SgGlkA is found to be modified by the binding of substrates. The substrate-dependent conformational changes of SgGlkA may be related to the CCR mechanism in Streptomyces. PMID:22101842

  20. Substrate Recognition of Anthrax Lethal Factor Examined by Combinatorial and Pre-steady-state Kinetic Approaches*

    PubMed Central

    Zakharova, Maria Yu.; Kuznetsov, Nikita A.; Dubiley, Svetlana A.; Kozyr, Arina V.; Fedorova, Olga S.; Chudakov, Dmitry M.; Knorre, Dmitry G.; Shemyakin, Igor G.; Gabibov, Alexander G.; Kolesnikov, Alexander V.

    2009-01-01

    Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca2+ and Mn2+. Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors. PMID:19359249

  1. Crystal structure of the substrate-recognition domain of the Shigella E3 ligase IpaH9.8.

    PubMed

    Takagi, Kenji; Kim, Minsoo; Sasakawa, Chihiro; Mizushima, Tsunehiro

    2016-04-01

    Infectious diseases caused by bacteria have significant impacts on global public health. During infection, pathogenic bacteria deliver a variety of virulence factors, called effectors, into host cells. The Shigella effector IpaH9.8 functions as an ubiquitin ligase, ubiquitinating the NF-κB essential modulator (NEMO)/IKK-γ to inhibit host inflammatory responses. IpaH9.8 contains leucine-rich repeats (LRRs) involved in substrate recognition and an E3 ligase domain. To elucidate the structural basis of the function of IpaH9.8, the crystal structure of the LRR domain of Shigella IpaH9.8 was determined and this structure was compared with the known structures of other IpaH family members. This model provides insights into the structural features involved in substrate specificity. PMID:27050259

  2. Molecular cloning of matrix Gla protein: implications for substrate recognition by the vitamin K-dependent gamma-carboxylase.

    PubMed Central

    Price, P A; Fraser, J D; Metz-Virca, G

    1987-01-01

    Matrix Gla protein (MGP), a low molecular weight protein found in bone, dentin, and cartilage, contains 5 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid (Gla). We have used antibodies raised against MGP and oligonucleotide probes to screen a lambda gt11 cDNA library constructed from the rat osteosarcoma cells (line ROS 17/2) that had been pretreated with 1 alpha,25-dihydroxyvitamin D3. By sequencing several cloned cDNAs, we established a 523-base-pair sequence that predicts an 84-residue mature MGP and a 19-residue hydrophobic signal peptide. The 84-residue mature rat MGP predicted from the cDNA sequence has an additional 5 residues at its C terminus (-Arg-Arg-Gly-Ala-Lys) not seen in the sequence of MGP isolated from bovine bone. The structure of rat MGP provides insight into the mechanisms by which the vitamin K-dependent gamma-carboxylase recognizes substrate. The present studies show that MGP, unlike other vitamin K-dependent proteins, lacks a propeptide. The absence of an MGP propeptide demonstrates that gamma-carboxylation and secretion of vitamin K-dependent proteins need not be linked to the presence of a propeptide or to its proteolytic removal. The propeptides of other vitamin K-dependent proteins are structurally homologous, and there is evidence that this homologous propeptide domain is important to substrate recognition by the gamma-carboxylase. Mature MGP has a sequence segment (residues 15-30) that is homologous to the propeptide of other vitamin K-dependent proteins and probably serves the same role in gamma-carboxylase recognition. Rat MGP also has a second sequence that has recently been identified in all known vitamin K-dependent vertebrate proteins, the invariant unit Glu-Xaa-Xaa-Xaa-Glu-Xaa-Cys (EXXXEXC). Since the glutamic residues in this unit are sites of gamma-carboxylation, it has been suggested that the EXXXEXC unit could allow the gamma-carboxylase to discriminate between substrate and product. The

  3. Structural basis for the dimerization and substrate recognition specificity of porcine epidemic diarrhea virus 3C-like protease.

    PubMed

    Ye, Gang; Deng, Feng; Shen, Zhou; Luo, Rui; Zhao, Ling; Xiao, Shaobo; Fu, Zhen F; Peng, Guiqing

    2016-07-01

    Porcine epidemic diarrhea virus (PEDV), a member of the genus Alphacoronavirus, has caused significant damage to the Asian and American pork industries. Coronavirus 3C-like protease (3CL(pro)), which is involved in the processing of viral polyproteins for viral replication, is an appealing antiviral drug target. Here, we present the crystal structures of PEDV 3CL(pro) and a molecular complex between an inactive PEDV 3CL(pro) variant C144A bound to a peptide substrate. Structural characterization, mutagenesis and biochemical analysis reveal the substrate-binding pockets and the residues that comprise the active site of PEDV 3CL(pro). The dimerization of PEDV 3CL(pro) is similar to that of other Alphacoronavirus 3CL(pro)s but has several differences from that of SARS-CoV 3CL(pro) from the genus Betacoronavirus. Furthermore, the non-conserved motifs in the pockets cause different cleavage of substrate between PEDV and SARS-CoV 3CL(pro)s, which may provide new insights into the recognition of substrates by 3CL(pro)s in various coronavirus genera. PMID:27128350

  4. Topolins and Hydroxylated Thidiazuron Derivatives Are Substrates of Cytokinin O-Glucosyltransferase with Position Specificity Related to Receptor Recognition1

    PubMed Central

    Mok, Machteld C.; Martin, Ruth C.; Dobrev, Petre I.; Vanková, Radomira; Ho, P. Shing; Yonekura-Sakakibara, Keiko; Sakakibara, Hitoshi; Mok, David W.S.

    2005-01-01

    Glucosides of trans-zeatin occur widely in plant tissues, formed either by O-glucosylation of the hydroxylated side chain or N-glucosylation of the purine ring structure. O-Glucosylation is stereo-specific: the O-glucosyltransferase encoded by the Phaseolus lunatus ZOG1 gene has high affinity for trans-zeatin as the substrate, whereas the enzyme encoded by the maize (Zea mays) cisZOG1 gene prefers cis-zeatin. Here we show that hydroxylated derivatives of benzyladenine (topolins) are also substrates of ZOG1 and cisZOG1. The m-OH and o-OH derivatives are the preferred substrate of ZOG1 and cisZOG1, respectively. Among the hydroxylated derivatives of thidiazuron tested, the only enzyme/substrate combination resulting in conversion was cisZOG1/(o-OH) thidiazuron. The abilities of these cytokinins to serve as substrates to the glucosyltransferases were in a large part correlated with their biological activities in the P. lunatus callus bioassay, indicating that there may be similarities between cytokinin-binding sites on the enzymes and cytokinin receptors. Further support for this interpretation is provided by cytokinin recognition studies involving the Arabidopsis (Arabidopsis thaliana) CRE1/WOL/AHK4 and maize ZmHK1 receptors. The AHK4 receptor responded to trans-zeatin and m-topolin, while the ZmHK1 receptor responded also to cis-zeatin and o-topolin. Three-dimensional molecular models of the substrates were applied to explain the results. PMID:15728338

  5. Molecular bases for the recognition of short peptide substrates and cysteine-directed modifications of human insulin-degrading enzyme

    PubMed Central

    Malito, Enrico; Ralat, Luis A.; Manolopoulou, Marika; Tsay, Julie L.; Wadlington, Natasha L.; Tang, Wei-Jen

    2009-01-01

    Insulin degrading enzyme (IDE) utilizes a large catalytic chamber to selectively bind and degrade peptide substrates such as insulin and amyloid β (Aβ). Tight interactions with substrates occur at an exosite located ~30Å away from the catalytic center that anchors the N-terminus of substrates to facilitate binding and subsequent cleavages at the catalytic site. However, IDE also degrades peptide substrates that are too short to occupy both the catalytic site and the exosite simultaneously. Here, we use kinins as a model system to address the kinetics and regulation of human IDE with short peptides. IDE specifically degrades bradykinin and kallidin at the Pro/Phe site. A 1.9Å crystal structure of bradykinin-bound IDE reveals the binding of bradykinin to the exosite, and not to the catalytic site. In agreement with observed high Km values, this suggests low affinity of bradykinin for IDE. This structure also provides the molecular basis on how the binding of short peptides at the exosite could regulate substrate recognition. We also found that human IDE is potently inhibited by physiologically relevant concentrations of S-nitrosylation and oxidation agents. Cysteine-directed modifications play a key role, since an IDE mutant devoid of all thirteen cysteines is insensitive to the inhibition by S-nitroso-glutathione, hydrogen peroxide, or N-ethylmaleimide. Specifically, cysteine 819 of human IDE is located inside the catalytic chamber pointing towards an extended hydrophobic pocket and is critical for the inactivation. Thiol-directed modification of this residue likely causes local structural perturbation to reduce substrate binding and catalysis. PMID:18986166

  6. 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. PMID:27032039

  7. Differential Active Site Loop Conformations Mediate Promiscuous Activities in the Lactonase SsoPox

    PubMed Central

    Elias, Mikael; Chabriere, Eric

    2013-01-01

    Enzymes are proficient catalysts that enable fast rates of Michaelis-complex formation, the chemical step and products release. These different steps may require different conformational states of the active site that have distinct binding properties. Moreover, the conformational flexibility of the active site mediates alternative, promiscuous functions. Here we focused on the lactonase SsoPox from Sulfolobus solfataricus. SsoPox is a native lactonase endowed with promiscuous phosphotriesterase activity. We identified a position in the active site loop (W263) that governs its flexibility, and thereby affects the substrate specificity of the enzyme. We isolated two different sets of substitutions at position 263 that induce two distinct conformational sampling of the active loop and characterized the structural and kinetic effects of these substitutions. These sets of mutations selectively and distinctly mediate the improvement of the promiscuous phosphotriesterase and oxo-lactonase activities of SsoPox by increasing active-site loop flexibility. These observations corroborate the idea that conformational diversity governs enzymatic promiscuity and is a key feature of protein evolvability. PMID:24086491

  8. Structural basis of substrate recognition by a bacterial deubiquitinase important for dynamics of phagosome ubiquitination

    PubMed Central

    Sheedlo, Michael J.; Qiu, Jiazhang; Tan, Yunhao; Paul, Lake N.; Luo, Zhao-Qing; Das, Chittaranjan

    2015-01-01

    Manipulation of the host’s ubiquitin network is emerging as an important strategy for counteracting and repurposing the posttranslational modification machineries of the host by pathogens. Ubiquitin E3 ligases encoded by infectious agents are well known, as are a variety of viral deubiquitinases (DUBs). Bacterial DUBs have been discovered, but little is known about the structure and mechanism underlying their ubiquitin recognition. In this report, we found that members of the Legionella pneumophila SidE effector family harbor a DUB module important for ubiquitin dynamics on the bacterial phagosome. Structural analysis of this domain alone and in complex with ubiquitin vinyl methyl ester (Ub-VME) reveals unique molecular contacts used in ubiquitin recognition. Instead of relying on the Ile44 patch of ubiquitin, as commonly used in eukaryotic counterparts, the SdeADub module engages Gln40 of ubiquitin. The architecture of the active-site cleft presents an open arrangement with conformational plasticity, permitting deubiquitination of three of the most abundant polyubiquitin chains, with a distinct preference for Lys63 linkages. We have shown that this preference enables efficient removal of Lys63 linkages from the phagosomal surface. Remarkably, the structure reveals by far the most parsimonious use of molecular contacts to achieve deubiquitination, with less than 1,000 Å2 of accessible surface area buried upon complex formation with ubiquitin. This type of molecular recognition appears to enable dual specificity toward ubiquitin and the ubiquitin-like modifier NEDD8. PMID:26598703

  9. Structural Recognition of an Optimized Substrate for the Ephrin family of Receptor Tyrosine Kinases

    PubMed Central

    Davis, Tara L.; Walker, John R.; Allali-Hassani, Abdellah; Parker, Sirlester A.; Turk, Benjamin E.; Dhe-Paganon, Sirano

    2010-01-01

    Summary Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor- expressing cells. Consequently the ephrins and ephrin RTKs often mediate processes involving cell:cell contact, including cellular adhesion or repulsion, developmental remodeling, and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and that is suitable for cocrystallization studies. Complex structures show multiple contacts between kinase and substrates, and in particular two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes. PMID:19678838

  10. β-Amyrin Biosynthesis: Promiscuity for Steric Bulk at Position 23 in the Oxidosqualene Substrate and the Significance of Hydrophobic Interaction between the Methyl Group at Position 30 and the Binding Site.

    PubMed

    Kaneko, Ikki; Hoshino, Tsutomu

    2016-08-01

    To examine how the sterics at the 23 position of (3S)-2,3-oxidosqualene 1 influence the polycyclization cascade in β-amyrin biosynthesis, substrate analogues substituted with an ethyl group (10, 11), a hydrogen atom (12, 13), or a propyl residue (14) at the 23 position were incubated with β-amyrin synthase. The bulkier ethyl group was accepted as a substrate, leading to formation of the β-amyrin skeleton (42, 43) without truncation of the multiple cyclization reactions. Analogue 13, possessing a hydrogen atom and an ethyl group at the 23E and 23Z positions, respectively, was also converted into the β-amyrin skeleton 45. However, the analogue lacking an ethyl group at the 23Z position (12) underwent almost no conversion, strongly indicating that an alkyl group must exist at the Z position. The cyclization of the analogue with a propyl substituent at the Z position (14) was poor. Analogue 15 possessing CH2OH at the 23E position afforded a new compound 47 in a high yield as a result of trapping of the final oleanyl cation. Conversely, 16 with 23Z-CH2OH afforded novel compounds 48-50 in low yields, which resulted from the intermediary dammarenyl and baccharenyl cations. Therefore, the hydrophobic interaction between the 23Z-alkyl group and its binding site (possibly via CH/π interaction) is critical for adopting the correct chair-chair-chair-boat-boat conformation and for the full cyclization cascade. PMID:27419810

  11. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

    PubMed

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-03-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  12. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  13. Structural Insights into Separase Architecture and Substrate Recognition through Computational Modelling of Caspase-Like and Death Domains

    PubMed Central

    Winter, Anja; Schmid, Ralf; Bayliss, Richard

    2015-01-01

    Separases are large proteins that mediate sister chromatid disjunction in all eukaryotes. They belong to clan CD of cysteine peptidases and contain a well-conserved C-terminal catalytic protease domain similar to caspases and gingipains. However, unlike other well-characterized groups of clan CD peptidases, there are no high-resolution structures of separases and the details of their regulation and substrate recognition are poorly understood. Here we undertook an in-depth bioinformatical analysis of separases from different species with respect to their similarity in amino acid sequence and protein fold in comparison to caspases, MALT-1 proteins (mucosa-associated lymphoidtissue lymphoma translocation protein 1) and gingipain-R. A comparative model of the single C-terminal caspase-like domain in separase from C. elegans suggests similar binding modes of substrate peptides between these protein subfamilies, and enables differences in substrate specificity of separase proteins to be rationalised. We also modelled a newly identified putative death domain, located N-terminal to the caspase-like domain. The surface features of this domain identify potential sites of protein-protein interactions. Notably, we identified a novel conserved region with the consensus sequence WWxxRxxLD predicted to be exposed on the surface of the death domain, which we termed the WR motif. We envisage that findings from our study will guide structural and functional studies of this important protein family. PMID:26513470

  14. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    SciTech Connect

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A.

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  15. Detection of mercury ions using silver telluride nanoparticles as a substrate and recognition element through surface-enhanced Raman scattering

    PubMed Central

    Wang, Chia-Wei; Lin, Zong-Hong; Roy, Prathik; Chang, Huan-Tsung

    2013-01-01

    In this paper we unveil a new sensing strategy for sensitive and selective detection of Hg2+ through surface-enhanced Raman scattering (SERS) using Ag2Te nanoparticles (NPs) as a substrate and recognition element and rhodamine 6G (R6G) as a reporter. Ag2Te NPs prepared from tellurium dioxide and silver nitrate and hydrazine in aqueous solution containing sodium dodecyl sulfate at 90°C with an average size of 26.8 ± 4.1 nm (100 counts) have strong SERS activity. The Ag2Te substrate provides strong SERS signals of R6G with an enhancement factor of 3.6 × 105 at 1360 cm−1, which is comparable to Ag NPs. After interaction of Ag2Te NPs with Hg2+, some HgTe NPs are formed, leading to decreases in the SERS signal of R6G, mainly because HgTe NPs relative to Ag2Te NPs have weaker SERS activity. Under optimum conditions, this SERS approach using Ag2Te as substrates is selective for the detection of Hg2+, with a limit of detection of 3 nM and linearity over 10–150 nM. The practicality of this approach has been validated for the determination of the concentrations of spiked Hg2+ in a pond water sample. PMID:24790948

  16. Dissecting the substrate recognition of 3-O-sulfotransferase for the biosynthesis of anticoagulant heparin

    SciTech Connect

    Moon, Andrea F.; Xu, Yongmei; Woody, Susan M.; Krahn, Joseph M.; Linhardt, Robert J.; Liu, Jian; Pedersen, Lars C.

    2012-05-29

    Heparin is a polysaccharide-based natural product that is used clinically as an anticoagulant drug. Heparan sulfate 3-O-sulfotransferase (3-OST) is an enzyme that transfers a sulfo group to the 3-OH position of a glucosamine unit. 3-OST is present in multiple isoforms, and the polysaccharides modified by these different isoforms perform distinct biological functions. 3-OST isoform 1 (3-OST-1) is the key enzyme for the biosynthesis of anticoagulant heparin. Here, we report the crystal structure of the ternary complex of 3-OST-1, 3'-phosphoadenosine 5'-phosphate, and a heptasaccharide substrate. Comparisons to previously determined structures of 3-OST-3 reveal unique binding modes used by the different isoforms of 3-OST for distinguishing the fine structures of saccharide substrates. Our data demonstrate that the saccharide substrates display distinct conformations when interacting with the different 3-OST isoforms. Site-directed mutagenesis data suggest that several key amino residues, including Lys259, Thr256, and Trp283 in 3-OST-3 and Arg268 in 3-OST-1, play important roles in substrate binding and specificity between isoforms. These results deepen our understanding of the biosynthetic mechanism of heparan sulfate and provide structural information for engineering enzymes for an enhanced biosynthetic approach to heparin production.

  17. Structural basis for substrate recognition and hydrolysis by mouse carnosinase CN2.

    PubMed

    Unno, Hideaki; Yamashita, Tetsuo; Ujita, Sayuri; Okumura, Nobuaki; Otani, Hiroto; Okumura, Akiko; Nagai, Katsuya; Kusunoki, Masami

    2008-10-01

    L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes. PMID:18550540

  18. Structures of Human Pumilio with Noncognate RNAs Reveal Molecular Mechanisms for Binding Promiscuity

    SciTech Connect

    Gupta,Y.; Nair, D.; Wharton, R.; Aggarwal, A.

    2008-01-01

    Pumilio is a founder member of the evolutionarily conserved Puf family of RNA-binding proteins that control a number of physiological processes in eukaryotes. A structure of human Pumilio (hPum) Puf domain bound to a Drosophila regulatory sequence showed that each Puf repeat recognizes a single nucleotide. Puf domains in general bind promiscuously to a large set of degenerate sequences, but the structural basis for this promiscuity has been unclear. Here, we describe the structures of hPum Puf domain complexed to two noncognate RNAs, CycBreverse and Puf5. In each complex, one of the nucleotides is ejected from the binding surface, in effect, acting as a 'spacer.' The complexes also reveal the plasticity of several Puf repeats, which recognize noncanonical nucleotides. Together, these complexes provide a molecular basis for recognition of degenerate binding sites, which significantly increases the number of mRNAs targeted for regulation by Puf proteins in vivo.

  19. Substrate ectodomain is critical for substrate preference and inhibition of γ-secretase

    PubMed Central

    Funamoto, Satoru; Sasaki, Toru; Ishihara, Seiko; Nobuhara, Mika; Nakano, Masaki; Watanabe-Takahashi, Miho; Saito, Takashi; Kakuda, Nobuto; Miyasaka, Tomohiro; Nishikawa, Kiyotaka; Saido, Takaomi C.; Ihara, Yasuo

    2013-01-01

    Understanding the substrate recognition mechanism of γ-secretase is a key step for establishing substrate-specific inhibition of amyloid β-protein (Aβ) production. However, it is widely believed that γ-secretase is a promiscuous protease and that its substrate-specific inhibition is elusive. Here we show that γ-secretase distinguishes the ectodomain length of substrates and preferentially captures and cleaves substrates containing a short ectodomain. We also show that a subset of peptides containing the CDCYCxxxxCxCxSC motif binds to the amino terminus of C99 and inhibits Aβ production in a substrate-specific manner. Interestingly, these peptides suppress β-secretase-dependent cleavage of APP, but not that of sialyltransferase 1. Most importantly, intraperitoneal administration of peptides into mice results in a significant reduction in cerebral Aβ levels. This report provides direct evidence of the substrate preference of γ-secretase and its mechanism. Our results demonstrate that the ectodomain of C99 is a potent target for substrate-specific anti-Aβ therapeutics to combat Alzheimer’s disease. PMID:24108142

  20. No genome barriers to promiscuous DNA

    NASA Astrophysics Data System (ADS)

    Lewin, R.

    1984-06-01

    Farrelly and Butow (1983) used the term 'promiscuous DNA' in their report of the apparent natural transfer of yeast mitochondrial DNA sequences into the nuclear genome. Ellis (1982) applied the same term in an editorial comment. It is pointed out since that time the subject of DNA's promiscuity has exploded with a series of reports. According to a report by Stern (1984), movement of DNA sequences between chloroplasts and mitochondria is not just a rare event but is a rampant process. It was recently concluded that 'the widespread presence of ctDNA sequences in plant mtDNA is best regarded as a dramatic demonstration of the dynamo nature of interactions between the chloroplast and the mitochondrion, similar to the ongoing process of interorganellar DNA transfer already documented between mitochondrion and nucleus and between chloroplast and nucleus'.

  1. Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

    PubMed Central

    Kretz, Colin A.; Dai, Manhong; Soylemez, Onuralp; Yee, Andrew; Desch, Karl C.; Siemieniak, David; Tomberg, Kärt; Kondrashov, Fyodor A.; Meng, Fan; Ginsburg, David

    2015-01-01

    Proteases play important roles in many biologic processes and are key mediators of cancer, inflammation, and thrombosis. However, comprehensive and quantitative techniques to define the substrate specificity profile of proteases are lacking. The metalloprotease ADAMTS13 regulates blood coagulation by cleaving von Willebrand factor (VWF), reducing its procoagulant activity. A mutagenized substrate phage display library based on a 73-amino acid fragment of VWF was constructed, and the ADAMTS13-dependent change in library complexity was evaluated over reaction time points, using high-throughput sequencing. Reaction rate constants (kcat/KM) were calculated for nearly every possible single amino acid substitution within this fragment. This massively parallel enzyme kinetics analysis detailed the specificity of ADAMTS13 and demonstrated the critical importance of the P1-P1′ substrate residues while defining exosite binding domains. These data provided empirical evidence for the propensity for epistasis within VWF and showed strong correlation to conservation across orthologs, highlighting evolutionary selective pressures for VWF. PMID:26170332

  2. [Proximity, intimacy and promiscuity in care].

    PubMed

    Flicourt, Nadia

    2015-04-01

    Lying at the heart of the intimacy of the other person, the nature of care supposes that the caregiver identifies the components resulting from the proximity and the invasion of the patient's personal space, where perceptions and representations give rise to reactive emotions and behaviour. Between modesty and nudity, proximity and promiscuity, caregivers have to adjust their approach of proper care, limiting the risks of intrusion. PMID:26043630

  3. Fluctuations of an Exposed π-Helix Involved in Lipoxygenase Substrate Recognition

    PubMed Central

    2014-01-01

    The second helix in lipoxygenases adapts to permit substrate access to the active site, but details of this process are varied and poorly understood. We therefore examined the dynamics of helix 2 in solutions of spin-labeled soybean lipoxygenase-1 and spin relaxation at 60 K of the spin-labels by catalytic iron. Helix 2 in soybean lipoxygenase structures is surface-exposed and contains one turn of π-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the π-helical segment became unusually mobile, on a nanosecond time scale, under conditions favoring substrate binding (pH 9 and lipid addition), while segments before and after had relatively unchanged dynamics. Backbone dynamics of residues in the π-helical segment appeared to be correlated, at pH 9. Samples also were frozen to examine the polarity and proticity of the local environments, the effect of the local environment on intrinsic relaxation, and dipolar relaxation by two symmetries of catalytic iron. The average hyperfine tensor component, Azz, of four π-helix residues decreased by 1.75 G, with an increase in pH from 7 to 9, while it remained unaffected for nearby buried residues. Power saturation data suggested the change in polarity specific to the π-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a π-helix in the second helix of plant lipoxygenases is highly dynamic and is the site where lipid chains penetrate to inner helices that outline the substrate pocket. PMID:25036469

  4. Molecular Insights into Substrate Recognition and Catalysis by Tryptophan 2,3-dioxygenase

    SciTech Connect

    Forouhar,F.; Ross Anderson, J.; Mowat, C.; Vorobiev, S.; Hussain, A.; Abashidze, M.; Bruckmann, C.; Thackray, S.; Seetharaman, J.; et al.

    2007-01-01

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-{angstrom} resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

  5. AGXT2: a promiscuous aminotransferase

    PubMed Central

    Rodionov, Roman N.; Jarzebska, Natalia; Weiss, Norbert; Lentz, Steven R.

    2014-01-01

    Alanine-glyoxylate aminotransferase 2 (AGXT2) is a multifunctional mitochondrial aminotransferase that was first identified in 1978. The physiological importance of AGXT2 was largely overlooked for three decades because AGXT2 is less active in glyoxylate metabolism than AGXT1, the enzyme that is deficient in primary hyperoxaluria type I. Recently, several novel functions of AGXT2 have been “rediscovered” in the setting of modern genomic and metabolomic studies. It is now apparent that AGXT2 has multiple substrates and products and that altered AGXT2 activity may contribute to the pathogenesis of cardiovascular, renal, neurological and hematological diseases. This article reviews the biochemical properties and physiological functions of AGXT2, its unique role at the intersection of key mitochondrial pathways, and its potential as a drug target. PMID:25294000

  6. Drug resistance against HCV NS3/4A inhibitors is defined by the balance of substrate recognition versus inhibitor binding

    PubMed Central

    Romano, Keith P.; Ali, Akbar; Royer, William E.; Schiffer, Celia A.

    2010-01-01

    Hepatitis C virus infects an estimated 180 million people worldwide, prompting enormous efforts to develop inhibitors targeting the essential NS3/4A protease. Resistance against the most promising protease inhibitors, telaprevir, boceprevir, and ITMN-191, has emerged in clinical trials. In this study, crystal structures of the NS3/4A protease domain reveal that viral substrates bind to the protease active site in a conserved manner defining a consensus volume, or substrate envelope. Mutations that confer the most severe resistance in the clinic occur where the inhibitors protrude from the substrate envelope, as these changes selectively weaken inhibitor binding without compromising the binding of substrates. These findings suggest a general model for predicting the susceptibility of protease inhibitors to resistance: drugs designed to fit within the substrate envelope will be less susceptible to resistance, as mutations affecting inhibitor binding would simultaneously interfere with the recognition of viral substrates. PMID:21084633

  7. Ultrahigh-throughput discovery of promiscuous enzymes by picodroplet functional metagenomics

    PubMed Central

    Colin, Pierre-Yves; Kintses, Balint; Gielen, Fabrice; Miton, Charlotte M.; Fischer, Gerhard; Mohamed, Mark F.; Hyvönen, Marko; Morgavi, Diego P.; Janssen, Dick B; Hollfelder, Florian

    2015-01-01

    Unculturable bacterial communities provide a rich source of biocatalysts, but their experimental discovery by functional metagenomics is difficult, because the odds are stacked against the experimentor. Here we demonstrate functional screening of a million-membered metagenomic library in microfluidic picolitre droplet compartments. Using bait substrates, new hydrolases for sulfate monoesters and phosphotriesters were identified, mostly based on promiscuous activities presumed not to be under selection pressure. Spanning three protein superfamilies, these break new ground in sequence space: promiscuity now connects enzymes with only distantly related sequences. Most hits could not have been predicted by sequence analysis, because the desired activities have never been ascribed to similar sequences, showing how this approach complements bioinformatic harvesting of metagenomic sequencing data. Functional screening of a library of unprecedented size with excellent assay sensitivity has been instrumental in identifying rare genes constituting catalytically versatile hubs in sequence space as potential starting points for the acquisition of new functions. PMID:26639611

  8. RNase P RNA from Prochlorococcus marinus: contribution of substrate domains to recognition by a cyanobacterial ribozyme.

    PubMed

    Hess, W R; Fingerhut, C; Schön, A

    1998-07-17

    The molecular organisation of the Prochlorococcus marinus rnpB gene and the catalytic activity of the encoded RNA were characterised. Kinetic parameters for several pre-tRNA substrates were comparable to those from other eubacterial RNase P RNAs, although unusually high cation concentrations were required. The CCA-end of pre-tRNAs is essential for efficient turnover despite the lack of the canonical binding motif in P. marinus RNase P RNA. A trnR gene is located only 38 nt upstream the rnpB 5' end on the complementary strand. This arrangement resembles those in the plastids of Cyanophora and Porphyra but not in any other bacterium. PMID:9708890

  9. High-throughput functional screening of steroid substrates with wild-type and chimeric P450 enzymes.

    PubMed

    Urban, Philippe; Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  10. High-Throughput Functional Screening of Steroid Substrates with Wild-Type and Chimeric P450 Enzymes

    PubMed Central

    Truan, Gilles; Pompon, Denis

    2014-01-01

    The promiscuity of a collection of enzymes consisting of 31 wild-type and synthetic variants of CYP1A enzymes was evaluated using a series of 14 steroids and 2 steroid-like chemicals, namely, nootkatone, a terpenoid, and mifepristone, a drug. For each enzyme-substrate couple, the initial steady-state velocity of metabolite formation was determined at a substrate saturating concentration. For that, a high-throughput approach was designed involving automatized incubations in 96-well microplate with sixteen 6-point kinetics per microplate and data acquisition using LC/MS system accepting 96-well microplate for injections. The resulting dataset was used for multivariate statistics aimed at sorting out the correlations existing between tested enzyme variants and ability to metabolize steroid substrates. Functional classifications of both CYP1A enzyme variants and steroid substrate structures were obtained allowing the delineation of global structural features for both substrate recognition and regioselectivity of oxidation. PMID:25243177

  11. Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium.

    PubMed

    Ishida, Takuya; Yaoi, Katsuro; Hiyoshi, Ayako; Igarashi, Kiyohiko; Samejima, Masahiro

    2007-11-01

    The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate-binding module, in cellulose-grown culture. The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other beta-1,4-glucans examined were poor substrates for the enzyme. The existence of the carbohydrate-binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme. HPLC and MALDI-TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases. However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases. Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16-18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7-9. In addition, the ratio of oligosaccharides of DP 7-9 versus those of DP 16-18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16-18 is affected by the ionic environment at the active site. PMID:17922847

  12. Mechanism of Substrate Recognition And PLP-Induced Conformational Changes in II-Diaminopimelate Aminotransferase From Arabidopsis Thaliana

    SciTech Connect

    Watanabe, N.; Clay, M.D.; Belkum, M.J.van; Cherney, M.M.; Vederas, J.C.; James, M.N.G.

    2009-05-26

    LL-Diaminopimelate aminotransferase (LL-DAP-AT), a pyridoxal phosphate (PLP)-dependent enzyme in the lysine biosynthetic pathways of plants and Chlamydia, is a potential target for the development of herbicides or antibiotics. This homodimeric enzyme converts L-tetrahydrodipicolinic acid (THDP) directly to LL-DAP using L-glutamate as the source of the amino group. Earlier, we described the 3D structures of native and malate-bound LL-DAP-AT from Arabidopsis thaliana (AtDAP-AT). Seven additional crystal structures of AtDAP-AT and its variants are reported here as part of an investigation into the mechanism of substrate recognition and catalysis. Two structures are of AtDAP-AT with reduced external aldimine analogues: N-(5'-phosphopyridoxyl)-L-glutamate (PLP-Glu) and N-(5'-phosphopyridoxyl)- LL-Diaminopimelate (PLP-DAP) bound in the active site. Surprisingly, they reveal that both L-glutamate and LL-DAP are recognized in a very similar fashion by the same sets of amino acid residues; both molecules adopt twisted V-shaped conformations. With both substrates, the {alpha}-carboxylates are bound in a salt bridge with Arg404, whereas the distal carboxylates are recognized via hydrogen bonds to the well-conserved side chains of Tyr37, Tyr125 and Lys129. The distal C{sup {var_epsilon}} amino group of LL-DAP is specifically recognized by several non-covalent interactions with residues from the other subunit (Asn309*, Tyr94*, Gly95*, and Glu97* (Amino acid designators followed by an asterisk (*) indicate that the residues originate in the other subunit of the dimer)) and by three bound water molecules. Two catalytically inactive variants of AtDAP-AT were created via site-directed mutagenesis of the active site lysine (K270N and K270Q). The structures of these variants permitted the observation of the unreduced external aldimines of PLP with L-glutamate and with LL-DAP in the active site, and revealed differences in the torsion angle about the PLP-substrate bond. Lastly, an apo

  13. Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis

    PubMed Central

    Miao, Yi; Tenor, Jennifer L.; Toffaletti, Dena L.; Washington, Erica J.; Liu, Jiuyu; Shadrick, William R.; Schumacher, Maria A.; Lee, Richard E.; Perfect, John R.; Brennan, Richard G.

    2016-01-01

    Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD–BeF3–trehalose and Tps2PD(D24N)–T6P complex structures reveal a “closed” conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate–protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD–BeF3–trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an “open” conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102–103 and 184–188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens. PMID:27307435

  14. The Role of Flexibility and Conformational Selection in the Binding Promiscuity of PDZ Domains

    PubMed Central

    Münz, Márton; Hein, Jotun; Biggin, Philip C.

    2012-01-01

    In molecular recognition, it is often the case that ligand binding is coupled to conformational change in one or both of the binding partners. Two hypotheses describe the limiting cases involved; the first is the induced fit and the second is the conformational selection model. The conformational selection model requires that the protein adopts conformations that are similar to the ligand-bound conformation in the absence of ligand, whilst the induced-fit model predicts that the ligand-bound conformation of the protein is only accessible when the ligand is actually bound. The flexibility of the apo protein clearly plays a major role in these interpretations. For many proteins involved in signaling pathways there is the added complication that they are often promiscuous in that they are capable of binding to different ligand partners. The relationship between protein flexibility and promiscuity is an area of active research and is perhaps best exemplified by the PDZ domain family of proteins. In this study we use molecular dynamics simulations to examine the relationship between flexibility and promiscuity in five PDZ domains: the human Dvl2 (Dishevelled-2) PDZ domain, the human Erbin PDZ domain, the PDZ1 domain of InaD (inactivation no after-potential D protein) from fruit fly, the PDZ7 domain of GRIP1 (glutamate receptor interacting protein 1) from rat and the PDZ2 domain of PTP-BL (protein tyrosine phosphatase) from mouse. We show that despite their high structural similarity, the PDZ binding sites have significantly different dynamics. Importantly, the degree of binding pocket flexibility was found to be closely related to the various characteristics of peptide binding specificity and promiscuity of the five PDZ domains. Our findings suggest that the intrinsic motions of the apo structures play a key role in distinguishing functional properties of different PDZ domains and allow us to make predictions that can be experimentally tested. PMID:23133356

  15. Statistically enhanced promiscuity of structurally correlated patterns

    NASA Astrophysics Data System (ADS)

    Lukatsky, D. B.; Shakhnovich, E. I.

    2008-02-01

    We predict that patterns with correlated surface density of atoms have statistically higher promiscuity (ability to bind stronger to an arbitrary pattern) as compared with noncorrelated patterns with the same average surface density. We suggest that this constitutes a generic design principle for highly connected proteins (hubs) in protein interaction networks. We develop an analytical theory for this effect. We show that our key predictions are generic and independent, qualitatively, on the specific form of the interatomic interaction potential, provided it has a finite range.

  16. Mutations, kataegis, and translocations in B lymphocytes: towards a mechanistic understanding of AID promiscuous activity

    PubMed Central

    Casellas, Rafael; Basu, Uttiya; Yewdell, William T.; Chaudhuri, Jayanta; Robbiani, Davide F.; Di Noia, Javier M.

    2016-01-01

    As B cells engage in the immune response they express the deaminase AID to initiate the hypermutation and recombination of immunoglobulin genes, which are crucial processes for the efficient recognition and disposal of pathogens, However, AID must be tightly controlled in B cells to minimize off-targeting mutations, which can drive chromosomal translocations and the development of B cell malignancies, such as lymphomas. Recent genomic and biochemical analyses have begun to unravel the crucial question of how AID-mediated deamination is targeted outside immunoglobulin genes. Here, we discuss the transcriptional and topological features that are emerging as key drivers of AID promiscuous activity. PMID:26898111

  17. Improved QM/MM Linear-Interaction Energy Model for Substrate Recognition in Zinc-Containing Metalloenzymes.

    PubMed

    Miranda, Williams E; Ngo, Van A; Valiente, Pedro A; Noskov, Sergei Yu

    2016-08-18

    One of the essential challenges in the description of receptor-drug interactions in the presence of various polyvalent cations (such as zinc, magnesium, or iron) is the accurate assessment of the electronic effects due to cofactor binding. The effects can range from partial electronic polarization of the proximal atoms in a receptor and bound substrate to long-range effects related to partial charge transfer and electronic delocalization effects between the cofactor and the drug. Here, we examine the role of the explicit account for electronic effects for a panel of small-molecule inhibitors binding to the zinc-aminopeptidase PfA-M1, an essential target for antimalarial drug development. Our study on PfA-M1:inhibitor interactions at the QM level reveals that the partial charge and proton transfer due to bound zinc ion are important mechanisms in the inhibitors' recognition and catalysis. The combination of classical MD simulations with a posteriori QM/MM corrections with novel DFTB parameters for the zinc cation and the linear-interaction energy (LIE) approach offers by far the most accurate estimates for the PfA-M1:inhibitor binding affinities, opening the door for future inhibitor design. PMID:27448039

  18. Roles of the N domain of the AAA+ Lon protease in substrate recognition, allosteric regulation and chaperone activity.

    PubMed

    Wohlever, Matthew L; Baker, Tania A; Sauer, Robert T

    2014-01-01

    Degron binding regulates the activities of the AAA+ Lon protease in addition to targeting proteins for degradation. The sul20 degron from the cell-division inhibitor SulA is shown here to bind to the N domain of Escherichia coli Lon, and the recognition site is identified by cross-linking and scanning for mutations that prevent sul20-peptide binding. These N-domain mutations limit the rates of proteolysis of model sul20-tagged substrates and ATP hydrolysis by an allosteric mechanism. Lon inactivation of SulA in vivo requires binding to the N domain and robust ATP hydrolysis but does not require degradation or translocation into the proteolytic chamber. Lon-mediated relief of proteotoxic stress and protein aggregation in vivo can also occur without degradation but is not dependent on robust ATP hydrolysis. In combination, these results demonstrate that Lon can function as a protease or a chaperone and reveal that some of its ATP-dependent biological activities do not require translocation. PMID:24205897

  19. Targeting bacterial membranes: NMR spectroscopy characterization of substrate recognition and binding requirements of D-arabinose-5-phosphate isomerase.

    PubMed

    Airoldi, Cristina; Sommaruga, Silvia; Merlo, Silvia; Sperandeo, Paola; Cipolla, Laura; Polissi, Alessandra; Nicotra, Francesco

    2010-02-01

    Lipopolysaccharide (LPS) is an essential component of the outer membrane of gram-negative bacteria and consists of three elements: lipid A, the core oligosaccharide, and the O-antigen. The inner-core region is highly conserved and contains at least one residue of 3-deoxy-D-manno-octulosonate (Kdo). Arabinose-5-phosphate isomerase (API) is an aldo-keto isomerase catalyzing the reversible isomerization of D-ribulose-5-phosphate (Ru5P) to D-arabinose-5-phosphate (A5P), the first step of Kdo biosynthesis. By exploiting saturation transfer difference (STD) NMR spectroscopy, the structural requirements necessary for API substrate recognition and binding were identified, with the aim of designing new API inhibitors. In addition, simple experimental conditions for the STD experiments to perform a fast, robust, and efficient screening of small libraries of potential API inhibitors, allowing the identification of new potential leads, were set up. Due to the essential role of API enzymes in LPS biosynthesis and gram-negative bacteria survival, by exploiting these data, a new generation of potent antibacterial drugs could be developed. PMID:20039350

  20. Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P.

    PubMed

    Brillante, Nadia; Gößringer, Markus; Lindenhofer, Dominik; Toth, Ursula; Rossmanith, Walter; Hartmann, Roland K

    2016-03-18

    RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship. PMID:26896801

  1. Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P

    PubMed Central

    Brillante, Nadia; Gößringer, Markus; Lindenhofer, Dominik; Toth, Ursula; Rossmanith, Walter; Hartmann, Roland K.

    2016-01-01

    RNase P is the enzyme that removes 5′ extensions from tRNA precursors. With its diversity of enzyme forms—either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins—the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5′ or 3′ extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship. PMID:26896801

  2. Molecular Insight into Substrate Recognition and Catalysis of Baeyer-Villiger Monooxygenase MtmOIV, the Key Frame-Modifying Enzyme in the Biosynthesis of Anticancer Agent Mithramycin

    SciTech Connect

    Bosserman, Mary A.; Downey, Theresa; Noinaj, Nicholas; Buchanan, Susan K.; Rohr, Jürgen

    2014-02-14

    Baeyer–Villiger monooxygenases (BVMOs) have been shown to play key roles for the biosynthesis of important natural products. MtmOIV, a homodimeric FAD- and NADPH-dependent BVMO, catalyzes the key frame-modifying steps of the mithramycin biosynthetic pathway, including an oxidative C–C bond cleavage, by converting its natural substrate premithramycin B into mithramycin DK, the immediate precursor of mithramycin. The drastically improved protein structure of MtmOIV along with the high-resolution structure of MtmOIV in complex with its natural substrate premithramycin B are reported here, revealing previously undetected key residues that are important for substrate recognition and catalysis. Kinetic analyses of selected mutants allowed us to probe the substrate binding pocket of MtmOIV and also to discover the putative NADPH binding site. This is the first substrate-bound structure of MtmOIV providing new insights into substrate recognition and catalysis, which paves the way for the future design of a tailored enzyme for the chemo-enzymatic preparation of novel mithramycin analogues.

  3. Structural Determinants of Substrate Recognition in the HAD Superfamily Member D-glycero-D-manno-Heptose-1,7-bisphosphate Phosphatase (GmhB)

    SciTech Connect

    Nguyen, H.; Wang, L; Huang, H; Peisach, E; Dunaway-Mariano, D; Allen, K

    2010-01-01

    The haloalkanoic acid dehalogenase (HAD) enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases, a cap domain functions in substrate recognition. However, for the HAD phosphatases that lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily, was selected for structure-function analysis. Herein, the X-ray crystallographic structures of Escherichia coli GmhB in the apo form (1.6 {angstrom} resolution), in a complex with Mg{sup 2+} and orthophosphate (1.8 {angstrom} resolution), and in a complex with Mg{sup 2+} and D-glycero-D-manno-heptose 1{beta},7-bisphosphate (2.2 {angstrom} resolution) were determined, in addition to the structure of Bordetella bronchiseptica GmhB bound to Mg{sup 2+} and orthophosphate (1.7 {angstrom} resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was conducted in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histidinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate recognition loops inserted into the cap domain yet, through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function.

  4. Structural Determinants of Substrate Recognition in the HAD Superfamily Member D-Glycero-D-manno-Heptose 1,7-bisphosphate Phosphatase, GmhB†

    PubMed Central

    Nguyen, Henry; Wang, Liangbing; Huang, Hua; Peisach, Ezra; Dunaway-Mariano, Debra; Allen, Karen N.

    2010-01-01

    The Haloalkanoic Acid Dehalogenase (HAD)1 enzyme superfamily is the largest family of phosphohydrolases. In HAD members, the structural elements that provide the binding interactions that support substrate specificity are separated from those that orchestrate catalysis. For most HAD phosphatases a cap domain functions in substrate recognition. However, for the HAD phosphatases which lack a cap domain, an alternate strategy for substrate selection must be operative. One such HAD phosphatase, GmhB of the HisB subfamily was selected for structure-function analysis. Herein, the X-ray crystallographic structures of E. coli GmhB in the apo form (1.6 Å resolution), complexed with Mg2+ and orthophosphate (1.8 Å resolution), and with Mg2+ and Dglycero-D-manno-heptose-1β,7-bisphosphate (2.2 Å resolution) were determined, in addition to the structure of B. bronchiseptica GmhB bound to Mg2+ and orthophosphate (1.7 Å resolution). The structures show that in place of a cap domain, the GmhB catalytic site is elaborated by three peptide inserts or loops that pack to form a concave, semicircular surface around the substrate leaving group. Structure-guided kinetic analysis of site-directed mutants was carried out in parallel with a bioinformatics study of sequence diversification within the HisB subfamily to identify loop residues that serve as substrate recognition elements and that distinguish GmhB from its subfamily counterpart, the histdinol-phosphate phosphatase domain of HisB. We show that GmhB and the histidinol-phosphate phosphatase domain use the same design of three substrate-recognition loops inserted into the cap domain, yet through selective residue usage on the loops, have achieved unique substrate specificity and thus novel biochemical function. PMID:20050614

  5. The Promiscuous Nature of Stars in Clusters

    NASA Astrophysics Data System (ADS)

    Hurley, Jarrod R.; Shara, Michael M.

    2002-05-01

    The recent availability of special-purpose computers designed for calculating gravitational interactions of N bodies at extremely high speed has provided the means to model globular clusters on a star-by-star basis for the first time. By endeavoring to make the N-body codes that operate on these machines as realistic as possible, the addition of stellar evolution being one example, we are learning much about the interaction between the star cluster itself and the stars it contains. A fascinating aspect of this research is the ability to follow the orbits of individual stars in detail and to document the formation of observed exotic systems. This has revealed that many stars within a star cluster lead wildly promiscuous lives, interacting often intimately and in rapid succession with a variety of neighbors.

  6. Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases.

    PubMed

    Steffen-Munsberg, Fabian; Matzel, Philipp; Sowa, Miriam A; Berglund, Per; Bornscheuer, Uwe T; Höhne, Matthias

    2016-05-01

    Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme's function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved 'flipping' arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases. PMID:26795966

  7. Insight into determinants of substrate binding and transport in a multidrug efflux protein

    PubMed Central

    Alegre, Kamela O.; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J.

    2016-01-01

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that ‘aromatic clamps’ formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such ‘clamps’ may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins. PMID:26961153

  8. Insight into determinants of substrate binding and transport in a multidrug efflux protein.

    PubMed

    Alegre, Kamela O; Paul, Stephanie; Labarbuta, Paola; Law, Christopher J

    2016-01-01

    Multidrug resistance arising from the activity of integral membrane transporter proteins presents a global public health threat. In bacteria such as Escherichia coli, transporter proteins belonging to the major facilitator superfamily make a considerable contribution to multidrug resistance by catalysing efflux of myriad structurally and chemically different antimicrobial compounds. Despite their clinical relevance, questions pertaining to mechanistic details of how these promiscuous proteins function remain outstanding, and the role(s) played by individual amino acid residues in recognition, binding and subsequent transport of different antimicrobial substrates by multidrug efflux members of the major facilitator superfamily requires illumination. Using in silico homology modelling, molecular docking and mutagenesis studies in combination with substrate binding and transport assays, we identified several amino acid residues that play important roles in antimicrobial substrate recognition, binding and transport by Escherichia coli MdtM, a representative multidrug efflux protein of the major facilitator superfamily. Furthermore, our studies suggested that 'aromatic clamps' formed by tyrosine and phenylalanine residues located within the substrate binding pocket of MdtM may be important for antimicrobial substrate recognition and transport by the protein. Such 'clamps' may be a structurally and functionally important feature of all major facilitator multidrug efflux proteins. PMID:26961153

  9. Sexual promiscuity: knowledge of dangers in institutions of higher learning.

    PubMed

    Ebong, R D

    1994-06-01

    Knowledge of dangers of sexual promiscuity was assessed in 2 institutions of higher learning. The objectives were to find out the knowledge of medical and social consequences as well as the factors responsible for sexual promiscuity among Nigerian youths. The study also assessed the discrepancies in societal concept of sex norms for males and females. The result was used as an index to determine the need for sex education for Nigerian youths. A total of 200 students (100 from each school) was assessed by random selection and use of a questionnaire. The result showed that students had a fair knowledge of sexual promiscuity, although in terms of medical consequences the knowledge was low for both groups. On social consequences, the knowledge was fair for both groups. Students agreed that lack of financial support and of supervision from parents and teachers were among the causes of sexual promiscuity. Recommendations were made for Health Education in these areas in institutions of higher learning. Also, recommendations were made for parental education on how to bring up, and care for, their adolescents to reduce the problems of sexual promiscuity. It was also recommended that a compulsory course on sexual promiscuity should be included in the syllabus in institutions of higher learning. PMID:7932483

  10. Molecular Evolution of the CYP2D Subfamily in Primates: Purifying Selection on Substrate Recognition Sites without the Frequent or Long-Tract Gene Conversion

    PubMed Central

    Yasukochi, Yoshiki; Satta, Yoko

    2015-01-01

    The human cytochrome P450 (CYP) 2D6 gene is a member of the CYP2D gene subfamily, along with the CYP2D7P and CYP2D8P pseudogenes. Although the CYP2D6 enzyme has been studied extensively because of its clinical importance, the evolution of the CYP2D subfamily has not yet been fully understood. Therefore, the goal of this study was to reveal the evolutionary process of the human drug metabolic system. Here, we investigate molecular evolution of the CYP2D subfamily in primates by comparing 14 CYP2D sequences from humans to New World monkey genomes. Window analysis and statistical tests revealed that entire genomic sequences of paralogous genes were extensively homogenized by gene conversion during molecular evolution of CYP2D genes in primates. A neighbor-joining tree based on genomic sequences at the nonsubstrate recognition sites showed that CYP2D6 and CYP2D8 genes were clustered together due to gene conversion. In contrast, a phylogenetic tree using amino acid sequences at substrate recognition sites did not cluster the CYP2D6 and CYP2D8 genes, suggesting that the functional constraint on substrate specificity is one of the causes for purifying selection at the substrate recognition sites. Our results suggest that the CYP2D gene subfamily in primates has evolved to maintain the regioselectivity for a substrate hydroxylation activity between individual enzymes, even though extensive gene conversion has occurred across CYP2D coding sequences. PMID:25808902

  11. Identification of determinants for tRNA substrate recognition by Escherichia coli C/U34 2′-O-methyltransferase

    PubMed Central

    Zhou, Mi; Long, Tao; Fang, Zhi-Peng; Zhou, Xiao-Long; Liu, Ru-Juan; Wang, En-Duo

    2015-01-01

    Post-transcriptional modifications bring chemical diversity to tRNAs, especially at positions 34 and 37 of the anticodon stem-loop (ASL). TrmL is the prokaryotic methyltransferase that catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the wobble base of tRNALeuCAA and tRNALeuUAA isoacceptors. This Cm34/Um34 modification affects codon-anticodon interactions and is essential for translational fidelity. TrmL-catalyzed 2′-O-methylation requires its homodimerization; however, understanding of the tRNA recognition mechanism by TrmL remains elusive. In the current study, by measuring tRNA methylation by TrmL and performing kinetic analysis of tRNA mutants, we found that TrmL exhibits a fine-tuned tRNA substrate recognition mechanism. Anticodon stem-loop minihelices with an extension of 2 base pairs are the minimal substrate for EcTrmL methylation. A35 is a key residue for TrmL recognition, while A36-A37-A38 are important either via direct interaction with TrmL or due to the necessity for prior isopentenylation (i6) at A37. In addition, TrmL only methylates pyrimidines but not purine residues at the wobble position, and the 2′-O-methylation relies on prior N6-isopentenyladenosine modification at position 37. PMID:26106808

  12. Dual roles of a conserved pair, Arg23 and Ser20, in recognition of multiple substrates in {alpha}-aminoadipate aminotransferase from Thermus thermophilus

    SciTech Connect

    Ouchi, Takuya; Tomita, Takeo; Miyagawa, Tomoharu; Kuzuyama, Tomohisa; Nishiyama, Makoto

    2009-10-09

    To clarify the mechanism for substrate recognition of {alpha}-aminoadipate aminotransferase (AAA-AT) from Thermus thermophilus, the crystal structure of AAA-AT complexed with N-(5'-phosphopyridoxyl)-L-glutamate (PPE) was determined at 1.67 A resolution. The crystal structure revealed that PPE is recognized by amino acid residues the same as those seen in N-(5'-phosphopyridoxyl)-L-{alpha}-aminoadipate (PPA) recognition; however, to bind the {gamma}-carboxyl group of Glu at a fixed position, the C{alpha} atom of the Glu moiety moves 0.80 A toward the {gamma}-carboxyl group in the PPE complex. Markedly decreased activity for Asp can be explained by the shortness of the aspartyl side chain to be recognized by Arg23 and further dislocation of the C{alpha} atom of bound Asp. Site-directed mutagenesis revealed that Arg23 has dual functions for reaction, (i) recognition of {gamma} ({delta})-carboxyl group of Glu (AAA) and (ii) rearrangement of {alpha}2 helix by changing the interacting partners to place the hydrophobic substrate at the suitable position.

  13. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  14. Finding promiscuous old drugs for new uses.

    PubMed

    Ekins, Sean; Williams, Antony J

    2011-08-01

    From research published in the last six years we have identified 34 studies that have screened libraries of FDA-approved drugs against various whole cell or target assays. These studies have each identified one or more compounds with a suggested new bioactivity that had not been described previously. We now show that 13 of these drugs were active against more than one additional disease, thereby suggesting a degree of promiscuity. We also show that following compilation of all the studies, 109 molecules were identified by screening in vitro. These molecules appear to be statistically more hydrophobic with a higher molecular weight and AlogP than orphan-designated products with at least one marketing approval for a common disease indication or one marketing approval for a rare disease from the FDA's rare disease research database. Capturing these in vitro data on old drugs for new uses will be important for potential reuse and analysis by others to repurpose or reposition these or other existing drugs. We have created databases which can be searched by the public and envisage that these can be updated as more studies are published. PMID:21607776

  15. Galactokinase promiscuity: a question of flexibility?

    PubMed

    McAuley, Megan; Kristiansson, Helena; Huang, Meilan; Pey, Angel L; Timson, David J

    2016-02-01

    Galactokinase catalyses the first committed step of the Leloir pathway, i.e. the ATP-dependent phosphorylation of α-D-galactose at C1-OH. Reduced galactokinase activity results in the inherited metabolic disease type II galactosaemia. However, inhibition of galactokinase is considered a viable approach to treating more severe forms of galactosaemia (types I and III). Considerable progress has been made in the identification of high affinity, selective inhibitors. Although the structure of galactokinase from a variety of species is known, its catalytic mechanism remains uncertain. Although the bulk of evidence suggests that the reaction proceeds via an active site base mechanism, some experimental and theoretical studies contradict this. The enzyme has potential as a biocatalyst in the production of sugar 1-phosphates. This potential is limited by its high specificity. A variety of approaches have been taken to identify galactokinase variants which are more promiscuous. These have broadened galactokinase's specificity to include a wide range of D- and L-sugars. Initial studies suggest that some of these alterations result in increased flexibility at the active site. It is suggested that modulation of protein flexibility is at least as important as structural modifications in determining the success or failure of enzyme engineering. PMID:26862196

  16. VARIATION IN RELATIVE SUBSTRATE SPECIFICTY OF BIFUNCTIONAL BETA-D-XYLOSIDASE/ALPHA-L-ARABINOFURANOSIDASE BY SINGLE-SITE MUTATIONS: ROLES OF SUBSTRATE DISTORTION AND RECOGNITION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To probe differential control of substrate specificities for 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-D-xylopyranoside (4NPX), residues of the glycone binding pocket (subsite -1) of the bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminanti...

  17. Structural and Thermodymamic Basis for Enhanced DNA Binding by a Promiscuous Mutant EcoRI Endonuclease

    SciTech Connect

    Sapienza,P.; Rosenberg, J.; Jen-Jacobson, L.

    2007-01-01

    Promiscuous mutant EcoRI endonucleases bind to the canonical site GAATTC more tightly than does the wild-type endonuclease, yet cleave variant (EcoRI*) sites more rapidly than does wild-type. The crystal structure of the A138T promiscuous mutant homodimer in complex with a GAATTC site is nearly identical to that of the wild-type complex, except that the Thr138 side chains make packing interactions with bases in the 5'-flanking regions outside the recognition hexanucleotide while excluding two bound water molecules seen in the wild-type complex. Molecular dynamics simulations confirm exclusion of these waters. The structure and simulations suggest possible reasons why binding of the A138T protein to the GAATTC site has S more favorable and H less favorable than for wild-type endonuclease binding. The interactions of Thr138 with flanking bases may permit A138T, unlike wild-type enzyme, to form complexes with EcoRI* sites that structurally resemble the specific wild-type complex with GAATTC.

  18. New structural insights into phosphorylation-free mechanism for full cyclin-dependent kinase (CDK)-cyclin activity and substrate recognition.

    PubMed

    Zheng, Fei; Quiocho, Florante A

    2013-10-18

    Pho85 is a versatile cyclin-dependent kinase (CDK) found in budding yeast that regulates a myriad of eukaryotic cellular functions in concert with 10 cyclins (called Pcls). Unlike cell cycle CDKs that require phosphorylation of a serine/threonine residue by a CDK-activating kinase (CAK) for full activation, Pho85 requires no phosphorylation despite the presence of an equivalent residue. The Pho85-Pcl10 complex is a key regulator of glycogen metabolism by phosphorylating the substrate Gsy2, the predominant, nutritionally regulated form of glycogen synthase. Here we report the crystal structures of Pho85-Pcl10 and its complex with the ATP analog, ATPγS. The structure solidified the mechanism for bypassing CDK phosphorylation to achieve full catalytic activity. An aspartate residue, invariant in all Pcls, acts as a surrogate for the phosphoryl adduct of the phosphorylated, fully activated CDK2, the prototypic cell cycle CDK, complexed with cyclin A. Unlike the canonical recognition motif, SPX(K/R), of phosphorylation sites of substrates of several cell cycle CDKs, the motif in the Gys2 substrate of Pho85-Pcl10 is SPXX. CDK5, an important signal transducer in neural development and the closest known functional homolog of Pho85, does not require phosphorylation either, and we found that in its crystal structure complexed with p25 cyclin a water/hydroxide molecule remarkably plays a similar role to the phosphoryl or aspartate group. Comparison between Pho85-Pcl10, phosphorylated CDK2-cyclin A, and CDK5-p25 complexes reveals the convergent structural characteristics necessary for full kinase activity and the variations in the substrate recognition mechanism. PMID:24022486

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

    PubMed

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

    2016-03-01

    In Ruminococcus albus, 4-O-β-d-mannosyl-d-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-d-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. PMID:26913570

  20. Site-directed mutagenesis maps interactions that enhance cognate and limit promiscuous catalysis by an alkaline phosphatase superfamily phosphodiesterase.

    PubMed

    Wiersma-Koch, Helen; Sunden, Fanny; Herschlag, Daniel

    2013-12-23

    Catalytic promiscuity, an evolutionary concept, also provides a powerful tool for gaining mechanistic insights into enzymatic reactions. Members of the alkaline phosphatase (AP) superfamily are highly amenable to such investigation, with several members having been shown to exhibit promiscuous activity for the cognate reactions of other superfamily members. Previous work has shown that nucleotide pyrophosphatase/phosphodiesterase (NPP) exhibits a >10⁶-fold preference for the hydrolysis of phosphate diesters over phosphate monoesters, and that the reaction specificity is reduced 10³-fold when the size of the substituent on the transferred phosphoryl group of phosphate diester substrates is reduced to a methyl group. Here we show additional specificity contributions from the binding pocket for this substituent (herein termed the R' substituent) that account for an additional ~250-fold differential specificity with the minimal methyl substituent. Removal of four hydrophobic side chains suggested on the basis of structural inspection to interact favorably with R' substituents decreases phosphate diester reactivity 10⁴-fold with an optimal diester substrate (R' = 5'-deoxythymidine) and 50-fold with a minimal diester substrate (R' = CH₃). These mutations also enhance the enzyme's promiscuous phosphate monoesterase activity by nearly an order of magnitude, an effect that is traced by mutation to the reduction of unfavorable interactions with the two residues closest to the nonbridging phosphoryl oxygen atoms. The quadruple R' pocket mutant exhibits the same activity toward phosphate diester and phosphate monoester substrates that have identical leaving groups, with substantial rate enhancements of ~10¹¹-fold. This observation suggests that the Zn²⁺ bimetallo core of AP superfamily enzymes, which is equipotent in phosphate monoester and diester catalysis, has the potential to become specialized for the hydrolysis of each class of phosphate esters via addition

  1. Structural Insights Into Substrate Recognition by the Neurospora Varkud Satellite Ribozyme: Importance of U-Turns at the Kissing-Loop Junction

    PubMed Central

    2013-01-01

    Substrate recognition by the Neurospora Varkud satellite ribozyme depends on the formation of a magnesium-dependent kissing-loop interaction between the stem-loop I (SLI) substrate and stem-loop V (SLV) of the catalytic domain. From mutagenesis studies, it has been established that this I/V kissing-loop interaction involves three Watson–Crick base pairs and is associated with a structural rearrangement of the SLI substrate that facilitates catalysis. Here, we report the NMR structural characterization of this I/V kissing-loop using isolated stem-loops. NMR studies were performed on different SLI/SLV complexes containing a common SLV and shiftable, preshifted, or double-stranded SLI variants. These studies confirm the presence of three Watson–Crick base pairs at the kissing-loop junction and provide evidence for the structural rearrangement of shiftable SLI variants upon SLV binding. NMR structure determination of an SLI/SLV complex demonstrates that both the SLI and SLV loops adopt U-turn structures, which facilitates intermolecular Watson–Crick base pairing. Several other interactions at the I/V interface, including base triples and base stacking, help create a continuously stacked structure. These NMR studies provide a structural basis to understand the stability of the I/V kissing-loop interaction and lead us to propose a kinetic model for substrate activation in the VS ribozyme. PMID:24325625

  2. Evolving new protein-protein interaction specificity through promiscuous intermediates.

    PubMed

    Aakre, Christopher D; Herrou, Julien; Phung, Tuyen N; Perchuk, Barrett S; Crosson, Sean; Laub, Michael T

    2015-10-22

    Interacting proteins typically coevolve, and the identification of coevolving amino acids can pinpoint residues required for interaction specificity. This approach often assumes that an interface-disrupting mutation in one protein drives selection of a compensatory mutation in its partner during evolution. However, this model requires a non-functional intermediate state prior to the compensatory change. Alternatively, a mutation in one protein could first broaden its specificity, allowing changes in its partner, followed by a specificity-restricting mutation. Using bacterial toxin-antitoxin systems, we demonstrate the plausibility of this second, promiscuity-based model. By screening large libraries of interface mutants, we show that toxins and antitoxins with high specificity are frequently connected in sequence space to more promiscuous variants that can serve as intermediates during a reprogramming of interaction specificity. We propose that the abundance of promiscuous variants promotes the expansion and diversification of toxin-antitoxin systems and other paralogous protein families during evolution. PMID:26478181

  3. Promiscuity and the evolution of sexual transmitted diseases

    NASA Astrophysics Data System (ADS)

    Gonçalves, Sebastián; Kuperman, Marcelo; Ferreira da Costa Gomes, Marcelo

    2003-09-01

    We study the relation between different social behaviors and the onset of epidemics in a model for the dynamics of sexual transmitted diseases. The model considers the society as a system of individual sexuated agents that can be organized in couples and interact with each other. The different social behaviors are incorporated assigning what we call a promiscuity value to each individual agent. The individual promiscuity is taken from a distribution and represents the daily probability of going out to look for a sexual partner, abandoning its eventual mate. In terms of this parameter we find a threshold for the epidemic which is much lower than the classical SIR model prediction, i.e., R0 (basic reproductive number)=1. Different forms for the distribution of the population promiscuity are considered showing that the threshold is weakly sensitive to them. We study the homosexual and the heterosexual case as well.

  4. Why Polyphenols have Promiscuous Actions? An Investigation by Chemical Bioinformatics.

    PubMed

    Tang, Guang-Yan

    2016-05-01

    Despite their diverse pharmacological effects, polyphenols are poor for use as drugs, which have been traditionally ascribed to their low bioavailability. However, Baell and co-workers recently proposed that the redox potential of polyphenols also plays an important role in this, because redox reactions bring promiscuous actions on various protein targets and thus produce non-specific pharmacological effects. To investigate whether the redox reactivity behaves as a critical factor in polyphenol promiscuity, we performed a chemical bioinformatics analysis on the structure-activity relationships of twenty polyphenols. It was found that the gene expression profiles of human cell lines induced by polyphenols were not correlated with the presence or not of redox moieties in the polyphenols, but significantly correlated with their molecular structures. Therefore, it is concluded that the promiscuous actions of polyphenols are likely to result from their inherent structural features rather than their redox potential. PMID:27319142

  5. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily.

    PubMed

    Baas, Bert-Jan; Zandvoort, Ellen; Geertsema, Edzard M; Poelarends, Gerrit J

    2013-05-27

    Catalytic promiscuity and evolution: Many enzymes exhibit catalytic promiscuity--the ability to catalyze reactions other than their biologically relevant one. These reactions can serve as starting points for both natural and laboratory evolution of new enzymatic functions. Recent advances in the study of enzyme promiscuity in the tautomerase superfamily are discussed. PMID:23649962

  6. Promiscuity and selectivity of bitter molecules and their receptors.

    PubMed

    Di Pizio, Antonella; Niv, Masha Y

    2015-07-15

    Bitter taste is essential for survival, as it protects against consuming poisonous compounds, which are often bitter. Bitter taste perception is mediated by bitter taste receptors (TAS2Rs), a subfamily of G-protein coupled receptors (GPCRs). The number of TAS2R subtypes is species-dependent, and varies from 3 in chicken to 50 in frog. TAS2Rs present an intriguing case for studying promiscuity: some of the receptors are still orphan, or have few known agonists, while others can be activated by numerous, structurally dissimilar compounds. The ligands also vary in the repertoire of TAS2Rs that they activate: some bitter compounds are selective toward a single TAS2R, while others activate multiple TAS2Rs. Selectivity/promiscuity profile of bitter taste receptors and their compounds was explored by a chemoinformatic approach. TAS2R-promiscuous and TAS2R-selective bitter molecules were found to differ in chemical features, such as AlogP, E-state, total charge, number of rings, globularity, and heavy atom count. This allowed the prediction of bitter ligand selectivity toward TAS2Rs. Interestingly, while promiscuous TAS2Rs are activated by both TAS2R-promiscuous and TAS2R-selective compounds, almost all selective TAS2Rs in human are activated by promiscuous compounds, which are recognized by other TAS2Rs anyway. Thus, unique ligands, that may have been the evolutionary driving force for development of selective TAS2Rs, still need to be unraveled. PMID:25934224

  7. Harnessing natural product assembly lines: structure, promiscuity, and engineering.

    PubMed

    Ladner, Christopher C; Williams, Gavin J

    2016-03-01

    Many therapeutically relevant natural products are biosynthesized by the action of giant mega-enzyme assembly lines. By leveraging the specificity, promiscuity, and modularity of assembly lines, a variety of strategies has been developed that enables the biosynthesis of modified natural products. This review briefly summarizes recent structural advances related to natural product assembly lines, discusses chemical approaches to probing assembly line structures in the absence of traditional biophysical data, and surveys efforts that harness the inherent or engineered promiscuity of assembly lines for the synthesis of non-natural polyketides and non-ribosomal peptide analogues. PMID:26527577

  8. Fidelity and Promiscuity in an Ant-Plant Mutualism: A Case Study of Triplaris and Pseudomyrmex

    PubMed Central

    Sanchez, Adriana

    2015-01-01

    The association between the myrmecophyte Triplaris and ants of the genus Pseudomyrmex is an often-reported example of mutualism but no molecular studies have examined this association to date. In this study, the interspecific relationships of Triplaris were reconstructed using five molecular markers (two chloroplast and three nuclear), and the relationships of the associated Pseudomyrmex using two molecular regions (one mitochondrial and one nuclear). A data set including all known collections of plant hosts and resident ants was also compiled. The pattern of distribution of both organisms reveals that there are varying degrees of host specificity; most ants show broader host usage (promiscuous) but one species (P. dendroicus) is faithful to a single species of Triplaris. In most ant-plant interactions, host usage is not specific at the species level and preferences may result from geographical or ecological sorting. The specificity of P. dendroicus could be based on chemical recognition of the host they were raised on. PMID:26630384

  9. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    PubMed

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. PMID:26682704

  10. Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

    PubMed

    Lacroix-Labonté, Julie; Girard, Nicolas; Dagenais, Pierre; Legault, Pascale

    2016-08-19

    The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates. PMID:27166370

  11. Structural analysis of a function-associated loop mutant of the substrate-recognition domain of Fbs1 ubiquitin ligase.

    PubMed

    Nishio, Kazuya; Yoshida, Yukiko; Tanaka, Keiji; Mizushima, Tsunehiro

    2016-08-01

    The SCF ubiquitin ligase comprises four components: Skp1, Cul1, Rbx1 and a variable-subunit F-box protein. The F-box protein Fbs1, which recognizes the N-linked glycoproteins, is involved in the endoplasmic reticulum-associated degradation pathway. Although FBG3, another F-box protein, shares 51% sequence identity with Fbs1, FBG3 does not bind glycoproteins. To investigate the sequence-structure relationship of the substrate-binding pocket, the crystal structure of a mutant substrate-binding domain of Fbs1 in which the six nonconserved regions (β1, β2-β3, β3-β4, β5-β6, β7-β8 and β9-β10) of Fbs1 were substituted with those of FBG3 was determined. The substrate-binding pocket of this model exhibits structural features that differ from those of Fsb1. PMID:27487926

  12. Much More than Power: The Pedagogy of Promiscuous Black Feminism

    ERIC Educational Resources Information Center

    Huckaby, M. Francyne

    2013-01-01

    This paper explores promiscuous black feminism by juxtaposing black feminism, Foucualt's poststructuralism, and my grandmother. The tensions created by these juxtapositions illuminate the ways black feminism and poststructuralism are resources and challenges to each other, and how both offer understandings of the relations at play that shape…

  13. Promiscuity in mice is associated with increased vaginal bacterial diversity

    NASA Astrophysics Data System (ADS)

    Macmanes, Matthew David

    2011-11-01

    Differences in the number of sexual partners (i.e., mating system) have the potential to exert a strong influence on the bacterial communities present in reproductive structures like the vagina. Because this structure serves as a conduit for gametes, bacteria present there may have a pronounced, direct effect on host reproductive success. As a first step towards the identification of the relationship between sexual behavior and potentially pathogenic bacterial communities inhabiting vital reproductive structures, as well as their potential effects on fitness, I sought to quantify differences in bacterial diversity in a promiscuous and monogamous mammal species. To accomplish this, I used two sympatric species of Peromyscus rodents— Peromyscus californicus and Peromyscus maniculatus that differ with regard to the number of sexual partners per individual to test the hypothesis that bacterial diversity should be greater in the promiscuous P. maniculatus relative to the monogamous P. californicus. As predicted, phylogenetically controlled and operational taxonomic unit-based indices of bacterial diversity indicated that diversity is greater in the promiscuous species. These results provide important new insights into the effects of mating system on bacterial diversity in free-living vertebrates, and may suggest a potential cost of promiscuity.

  14. Studies of the Maltose Transport System Reveal a Mechanism for Coupling ATP Hydrolysis to Substrate Translocation without Direct Recognition of Substrate

    SciTech Connect

    Gould, Alister D.; Shilton, Brian H.

    2010-10-11

    The ATPase activity of the maltose transporter (MalFGK{sub 2}) is dependent on interactions with the maltose-binding protein (MBP). To determine whether direct interactions between the translocated sugar and MalFGK{sub 2} are important for the regulation of ATP hydrolysis, we used an MBP mutant (sMBP) that is able to bind either maltose or sucrose. We observed that maltose- and sucrose-bound sMBP stimulate equal levels of MalFGK{sub 2} ATPase activity. Therefore, the ATPase activity of MalFGK{sub 2} is coupled to translocation of maltose solely by interactions between MalFGK{sub 2} and MBP. For both maltose and sucrose, the ability of sMBP to stimulate the MalFGK{sub 2} ATPase was greatly reduced compared with wild-type MBP, indicating that the mutations in sMBP have interfered with important interactions between MBP and MalFGK{sub 2}. High resolution crystal structure analysis of sMBP shows that in the closed conformation with bound sucrose, three of four mutations are buried, and the fourth causes only a minor change in the accessible surface. In contrast, in the open form of sMBP, all of the mutations are accessible, and the main chain of Tyr{sup 62}-Gly{sup 69} is destabilized and occupies an alternative conformation due to the W62Y mutation. On this basis, the compromised ability of sMBP to stimulate ATP hydrolysis by MalFGK{sub 2} is most likely due to a disruption of interactions between MalFGK{sub 2} and the open, rather than the closed, conformation of sMBP. Modeling the open sMBP structure bound to MalFGK{sub 2} in the transition state for ATP hydrolysis points to an important site of interaction and suggests a mechanism for coupling ATP hydrolysis to substrate translocation that is independent of the exact structure of the substrate.

  15. Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis.

    PubMed

    Mafu, Sibongile; Jia, Meirong; Zi, Jiachen; Morrone, Dana; Wu, Yisheng; Xu, Meimei; Hillwig, Matthew L; Peters, Reuben J

    2016-03-01

    The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis. PMID:26884192

  16. A New Family of HEAT-Like Repeat Proteins Lacking a Critical Substrate Recognition Motif Present in Related DNA Glycosylases.

    PubMed

    Mullins, Elwood A; Shi, Rongxin; Kotsch, Lyle A; Eichman, Brandt F

    2015-01-01

    DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases. PMID:25978435

  17. Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies

    PubMed Central

    Kaufmann, Kristian W.; Dawson, Eric S.; Henry, L. Keith; Field, Julie R.; Blakely, Randy D.; Meiler, Jens

    2009-01-01

    To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuTAa) structure reported by Yamashita et al. (Nature 2005;437:215–223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuTAa is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to cHitically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuTAa structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 Å of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

  18. Structural determinants of species-selective substrate recognition in human and Drosophila serotonin transporters revealed through computational docking studies.

    PubMed

    Kaufmann, Kristian W; Dawson, Eric S; Henry, L Keith; Field, Julie R; Blakely, Randy D; Meiler, Jens

    2009-02-15

    To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected. PMID:18704946

  19. The Structural Basis of Substrate Recognition in an exo-beta-d-Glucosaminidase Involved in Chitosan Hydrolysis

    SciTech Connect

    Lammerts van Bueren, A.; Ghinet, M; Gregg, K; Fleury, A; Brzezinski, R; Boraston, A

    2009-01-01

    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with beta-galactosidase activity (Escherichia coli LacZ), beta-glucuronidase activity (Homo sapiens GusB), and beta-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-beta-D-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural beta-1,4-D-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-beta-D-glucosaminide synthetic substrate provide insight into interactions in the +1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

  20. The Structural Basis of Substrate Recognition in an exo-b-d-glucosaminidase Involved in Chitosan Hydrolysis

    SciTech Connect

    Van Bueren, A.; Ghinet, M; Gregg, K; Fleury, A; Brzezinski, R; Boraston, A

    2009-01-01

    Family 2 of the glycoside hydrolase classification is one of the largest families. Structurally characterized members of this family include enzymes with ?-galactosidase activity (Escherichia coli LacZ), ?-glucuronidase activity (Homo sapiens GusB), and ?-mannosidase activity (Bacteroides thetaiotaomicron BtMan2A). Here, we describe the structure of a family 2 glycoside hydrolase, CsxA, from Amycolatopsis orientalis that has exo-?-d-glucosaminidase (exo-chitosanase) activity. Analysis of a product complex (1.85 A resolution) reveals a unique negatively charged pocket that specifically accommodates the nitrogen of nonreducing end glucosamine residues, allowing this enzyme to discriminate between glucose and glucosamine. This also provides structural evidence for the role of E541 as the catalytic nucleophile and D469 as the catalytic acid/base. The structures of an E541A mutant in complex with a natural ?-1,4-d-glucosamine tetrasaccharide substrate and both E541A and D469A mutants in complex with a pNP-?-d-glucosaminide synthetic substrate provide insight into interactions in the + 1 subsite of this enzyme. Overall, a comparison with the active sites of other GH2 enzymes highlights the unique architecture of the CsxA active site, which imparts specificity for its cationic substrate.

  1. Understanding the structural basis of substrate recognition by Plasmodium falciparum plasmepsin V to aid in the design of potent inhibitors

    PubMed Central

    Bedi, Rajiv K.; Patel, Chandan; Mishra, Vandana; Xiao, Huogen; Yada, Rickey Y.; Bhaumik, Prasenjit

    2016-01-01

    Plasmodium falciparum plasmepsin V (PfPMV) is an essential aspartic protease required for parasite survival, thus, considered as a potential drug target. This study reports the first detailed structural analysis and molecular dynamics simulation of PfPMV as an apoenzyme and its complexes with the substrate PEXEL as well as with the inhibitor saquinavir. The presence of pro-peptide in PfPMV may not structurally hinder the formation of a functionally competent catalytic active site. The structure of PfPMV-PEXEL complex shows that the unique positions of Glu179 and Gln222 are responsible for providing the specificity of PEXEL substrate with arginine at P3 position. The structural analysis also reveals that the S4 binding pocket in PfPMV is occupied by Ile94, Ala98, Phe370 and Tyr472, and therefore, does not allow binding of pepstatin, a potent inhibitor of most pepsin-like aspartic proteases. Among the screened inhibitors, the HIV-1 protease inhibitors and KNI compounds have higher binding affinities for PfPMV with saquinavir having the highest value. The presence of a flexible group at P2 and a bulky hydrophobic group at P3 position of the inhibitor is preferred in the PfPMV substrate binding pocket. Results from the present study will aid in the design of potent inhibitors of PMV. PMID:27531685

  2. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone. PMID:23828603

  3. Understanding the structural basis of substrate recognition by Plasmodium falciparum plasmepsin V to aid in the design of potent inhibitors.

    PubMed

    Bedi, Rajiv K; Patel, Chandan; Mishra, Vandana; Xiao, Huogen; Yada, Rickey Y; Bhaumik, Prasenjit

    2016-01-01

    Plasmodium falciparum plasmepsin V (PfPMV) is an essential aspartic protease required for parasite survival, thus, considered as a potential drug target. This study reports the first detailed structural analysis and molecular dynamics simulation of PfPMV as an apoenzyme and its complexes with the substrate PEXEL as well as with the inhibitor saquinavir. The presence of pro-peptide in PfPMV may not structurally hinder the formation of a functionally competent catalytic active site. The structure of PfPMV-PEXEL complex shows that the unique positions of Glu179 and Gln222 are responsible for providing the specificity of PEXEL substrate with arginine at P3 position. The structural analysis also reveals that the S4 binding pocket in PfPMV is occupied by Ile94, Ala98, Phe370 and Tyr472, and therefore, does not allow binding of pepstatin, a potent inhibitor of most pepsin-like aspartic proteases. Among the screened inhibitors, the HIV-1 protease inhibitors and KNI compounds have higher binding affinities for PfPMV with saquinavir having the highest value. The presence of a flexible group at P2 and a bulky hydrophobic group at P3 position of the inhibitor is preferred in the PfPMV substrate binding pocket. Results from the present study will aid in the design of potent inhibitors of PMV. PMID:27531685

  4. Unraveling the substrate recognition mechanism and specificity of the unusual glycosyl hydrolase family 29 BT2192 from Bacteroides thetaiotaomicron.

    PubMed

    Guillotin, Laure; Lafite, Pierre; Daniellou, Richard

    2014-03-11

    Glycosyl hydrolase (GH) family 29 (CAZy database) consists of retaining α-l-fucosidases. We have identified BT2192, a protein from Bacteroides thetaiotaomicron, as the first GH29 representative exhibiting both weak α-l-fucosidase and β-d-galactosidase activities. Determination and analysis of X-ray structures of BT2192 in complex with β-d-galactoside competitive inhibitors showed a new binding mode different from that of known GH29 enzymes. Three point mutations, specific to BT2192, prevent the canonical GH29 substrate α-l-fucose from binding efficiently to the fucosidase-like active site relative to other GH29 enzymes. β-d-Galactoside analogues bind and interact in a second pocket, which is not visible in other reported GH29 structures. Molecular simulations helped in the assessment of the flexibility of both substrates in their respective pocket. Hydrolysis of the fucosyl moiety from the putative natural substrates like 3-fucosyllactose or Lewis(X) antigen would be mainly due to the efficient interactions with the galactosyl moiety, in the second binding site, located more than 6-7 Å apart. PMID:24527659

  5. Complete abolition of reading and writing ability with a third ventricle colloid cyst: implications for surgical intervention and proposed neural substrates of visual recognition and visual imaging ability.

    PubMed

    Barker, Lynne Ann; Morton, Nicholas; Romanowski, Charles A J; Gosden, Kevin

    2013-01-01

    We report a rare case of a patient unable to read (alexic) and write (agraphic) after a mild head injury. He had preserved speech and comprehension, could spell aloud, identify words spelt aloud and copy letter features. He was unable to visualise letters but showed no problems with digits. Neuropsychological testing revealed general visual memory, processing speed and imaging deficits. Imaging data revealed an 8 mm colloid cyst of the third ventricle that splayed the fornix. Little is known about functions mediated by fornical connectivity, but this region is thought to contribute to memory recall. Other regions thought to mediate letter recognition and letter imagery, visual word form area and visual pathways were intact. We remediated reading and writing by multimodal letter retraining. The study raises issues about the neural substrates of reading, role of fornical tracts to selective memory in the absence of other pathology, and effective remediation strategies for selective functional deficits. PMID:24158300

  6. Complete abolition of reading and writing ability with a third ventricle colloid cyst: implications for surgical intervention and proposed neural substrates of visual recognition and visual imaging ability

    PubMed Central

    Barker, Lynne Ann; Morton, Nicholas; Romanowski, Charles A J; Gosden, Kevin

    2013-01-01

    We report a rare case of a patient unable to read (alexic) and write (agraphic) after a mild head injury. He had preserved speech and comprehension, could spell aloud, identify words spelt aloud and copy letter features. He was unable to visualise letters but showed no problems with digits. Neuropsychological testing revealed general visual memory, processing speed and imaging deficits. Imaging data revealed an 8 mm colloid cyst of the third ventricle that splayed the fornix. Little is known about functions mediated by fornical connectivity, but this region is thought to contribute to memory recall. Other regions thought to mediate letter recognition and letter imagery, visual word form area and visual pathways were intact. We remediated reading and writing by multimodal letter retraining. The study raises issues about the neural substrates of reading, role of fornical tracts to selective memory in the absence of other pathology, and effective remediation strategies for selective functional deficits. PMID:24158300

  7. Critical determinants for substrate recognition and catalysis in the M. tuberculosis class II AP-endonuclease/3'-5' exonuclease III.

    PubMed

    Khanam, Taran; Shukla, Ankita; Rai, Niyati; Ramachandran, Ravishankar

    2015-05-01

    The Mycobacterium tuberculosis AP-endonuclease/3'-5' exodeoxyribonuclease (MtbXthA) is an important player in DNA base excision repair (BER). We demonstrate that the enzyme has robust apurinic/apyrimidinic (AP) endonuclease activity, 3'-5' exonuclease, phosphatase, and phosphodiesterase activities. The enzyme functions as an AP-endonuclease at high ionic environments, while the 3'-5'-exonuclease activity is predominant at low ionic environments. Our molecular modelling and mutational experiments show that E57 and D251 are critical for catalysis. Although nicked DNA and gapped DNA are fair substrates of MtbXthA, the gap-size did not affect the excision activity and furthermore, a substrate with a recessed 3'-end is preferred. To understand the determinants of abasic-site recognition, we examined the possible roles of (i) the base opposite the abasic site, (ii) the abasic ribose ring itself, (iii) local distortions in the AP-site, and (iv) conserved residues located near the active site. Our experiments demonstrate that the first three determinants do not play a role in MtbXthA, and in fact the enzyme exhibits robust endonucleolytic activity against single-stranded AP DNA also. Regarding the fourth determinant, it is known that the catalytic-site of AP endonucleases is surrounded by conserved aromatic residues and intriguingly, the exact residues that are directly involved in abasic site recognition vary with the individual proteins. We therefore, used a combination of mutational analysis, kinetic assays, and structure-based modelling, to identify that Y237, supported by Y137, mediates the formation of the MtbXthA-AP-DNA complex and AP-site incision. PMID:25748880

  8. Evolution of conformational dynamics determines the conversion of a promiscuous generalist into a specialist enzyme.

    PubMed

    Zou, Taisong; Risso, Valeria A; Gavira, Jose A; Sanchez-Ruiz, Jose M; Ozkan, S Banu

    2015-01-01

    β-Lactamases are produced by many modern bacteria as a mechanism of resistance toward β-lactam antibiotics, the most common antibiotics in use. β-Lactamases, however, are ancient enzymes that originated billions of years ago. Recently, proteins corresponding to 2- to 3-Gy-old Precambrian nodes in the evolution of Class A β-lactamases have been prepared and shown to be moderately efficient promiscuous catalysts, able to degrade a variety of antibiotics with catalytic efficiency levels similar to those of an average modern enzyme. Remarkably, there are few structural differences (in particular at the active-site regions) between the resurrected enzymes and a penicillin-specialist modern β-lactamase. Here, we propose that the ancestral promiscuity originates from conformational dynamics. We investigate the differences in conformational dynamics of the ancient and extant β-lactamases through MD simulations and quantify the contribution of each position to functionally related dynamics through Dynamic Flexibility Index. The modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate (penicillin), whereas enhanced deformability at the active-site neighborhood in the ancestral resurrected proteins likely accounts for the binding and subsequent degradation of antibiotic molecules of different size and shape. Clustering of the conformational dynamics on the basis of Principal Component Analysis is in agreement with the functional divergence, as the ancient β-lactamases cluster together, separated from their modern descendant. Finally, our analysis leads to testable predictions, as sites of potential relevance for the evolution of dynamics are identified and mutations at those sites are expected to alter substrate-specificity. PMID:25312912

  9. A Polyketide Synthase Acyltransferase Domain Structure Suggests a Recognition Mechanism for Its Hydroxymalonyl-Acyl Carrier Protein Substrate

    PubMed Central

    Park, Hyunjun; Kevany, Brian M.; Dyer, David H.; Thomas, Michael G.; Forest, Katrina T.

    2014-01-01

    We have previously shown that the acyl transferase domain of ZmaA (ZmaA-AT) is involved in the biosynthesis of the aminopolyol polyketide/nonribosomal peptide hybrid molecule zwittermicin A from cereus UW85, and that it specifically recognizes the precursor hydroxymalonyl-acyl carrier protein (ACP) and transfers the hydroxymalonyl extender unit to a downstream second ACP via a transacylated AT domain intermediate. We now present the X-ray crystal structure of ZmaA-AT at a resolution of 1.7 Å. The structure shows a patch of solvent-exposed hydrophobic residues in the area where the AT is proposed to interact with the precursor ACP. We addressed the significance of the AT/ACP interaction in precursor specificity of the AT by testing whether malonyl- or methylmalonyl-ACP can be recognized by ZmaA-AT. We found that the ACP itself biases extender unit selection. Until now, structural information for ATs has been limited to ATs specific for the CoA-linked precursors malonyl-CoA and (2S)-methylmalonyl-CoA. This work contributes to polyketide synthase engineering efforts by expanding our knowledge of AT/substrate interactions with the structure of an AT domain that recognizes an ACP-linked substrate, the rare hydroxymalonate. Our structure suggests a model in which ACP interaction with a hydrophobic motif promotes secondary structure formation at the binding site, and opening of the adjacent substrate pocket lid to allow extender unit binding in the AT active site. PMID:25340352

  10. The Crystal Structure of Burkholderia cenocepacia DfsA Provides Insights into Substrate Recognition and Quorum Sensing Fatty Acid Biosynthesis.

    PubMed

    Spadaro, Francesca; Scoffone, Viola C; Chiarelli, Laurent R; Fumagalli, Marco; Buroni, Silvia; Riccardi, Giovanna; Forneris, Federico

    2016-06-14

    Burkholderia cenocepacia is a major concern among respiratory tract infections in cystic fibrosis patients. This pathogen is particularly difficult to treat because of its high level of resistance to the clinically relevant antimicrobial agents. In B. cenocepacia, the quorum sensing cell-cell communication system is involved in different processes that are important for bacterial virulence, such as biofilm formation and protease and siderophore production. Targeting the enzymes involved in this process represents a promising therapeutic approach. With the aim of finding effective quorum sensing inhibitors, we have determined the three-dimensional structure of B. cenocepacia diffusible factor synthase A, DfsA. This bifunctional crotonase (dehydratase/thioesterase) produces the characteristic quorum sensing molecule of B. cenocepacia, cis-2-dodecenoic acid or BDSF, starting from 3-hydroxydodecanoyl-acyl carrier protein. Unexpectedly, the crystal structure revealed the presence of a lipid molecule in the catalytic site of the enzyme, which was identified as dodecanoic acid. Our biochemical characterization shows that DfsA is able to use dodecanoyl-acyl carrier protein as a substrate, demonstrating that dodecanoic acid, the product of this reaction, is released very slowly from the DfsA active site, therefore acting as a DfsA inhibitor. This molecule shows an unprecedented conformational arrangement inside the DfsA active site. In contrast with previous hypotheses, our data illustrate how DfsA and closely related homologous enzymes can recognize long hydrophobic substrates without large conformational changes or assistance by additional regulator molecules. The elucidation of the substrate binding mode in DfsA provides the starting point for structure-based drug discovery studies targeting B. cenocepacia quorum sensing-assisted virulence. PMID:27198181