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Active-site Cleft (active-site + cleft)
Selected AbstractsCrystal structure of the Streptococcus pneumoniae mevalonate kinase in complex with diphosphomevalonatePROTEIN SCIENCE, Issue 5 2007John L. Andreassi II Abstract Streptococcuspneumoniae, a ubiquitous gram-positive pathogen with an alarming, steadily evolving resistance to frontline antimicrobials, poses a severe global health threat both in the community and in the clinic. The recent discovery that diphosphomevalonate (DPM), an essential intermediate in the isoprenoid biosynthetic pathway, potently and allosterically inhibits S. pneumoniae mevalonate kinase (SpMK) without affecting the human isozyme established a new target and lead compound for antimicrobial design. Here we present the crystal structure of the first S. pneumoniae mevalonate kinase, at a resolution of 2.5 Å and in complex with DPM·Mg2+ in the active-site cleft. Structural comparison of SpMK with other members of the GHMP kinase family reveals that DPM functions as a partial bisubstrate analog (mevalonate linked to the pyrophosphoryl moiety of ATP) in that it elicits a ternary-complexlike form of the enzyme, except for localized disordering in a region that would otherwise interact with the missing portion of the nucleotide. Features of the SpMK-binding pockets are discussed in the context of established mechanistic findings and inherited human diseases linked to MK deficiency. [source] The apo structure of sucrose hydrolase from Xanthomonas campestris pv. campestris shows an open active-site grooveACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Elise Champion Glycoside hydrolase family 13 (GH-13) mainly contains starch-degrading or starch-modifying enzymes. Sucrose hydrolases utilize sucrose instead of amylose as the primary glucosyl donor. Here, the catalytic properties and X-ray structure of sucrose hydrolase from Xanthomonas campestris pv. campestris are reported. Sucrose hydrolysis catalyzed by the enzyme follows Michaelis,Menten kinetics, with a Km of 60.7,mM and a kcat of 21.7,s,1. The structure of the enzyme was solved at a resolution of 1.9,Å in the resting state with an empty active site. This represents the first apo structure from subfamily 4 of GH-13. Comparisons with structures of the highly similar sucrose hydrolase from X. axonopodis pv. glycines most notably showed that residues Arg516 and Asp138, which form a salt bridge in the X. axonopodis sucrose complex and define part of the subsite ,1 glucosyl-binding determinants, are not engaged in salt-bridge formation in the resting X. campestris enzyme. In the absence of the salt bridge an opening is created which gives access to subsite ,1 from the `nonreducing' end. Binding of the glucosyl moiety in subsite ,1 is therefore likely to induce changes in the conformation of the active-site cleft of the X. campestris enzyme. These changes lead to salt-bridge formation that shortens the groove. Additionally, this finding has implications for understanding the molecular mechanism of the closely related subfamily 4 glucosyl transferase amylosucrase, as it indicates that sucrose could enter the active site from the `nonreducing' end during the glucan-elongation cycle. [source] Structure of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with CDP- d -xyloseACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2005Nicole M. Koropatkin Tyvelose is a unique 3,6-dideoxyhexose found in the O antigens of some pathogenic species of Yersinia and Salmonella. It is produced via a complex biochemical pathway that employs CDP- d -glucose as the starting ligand. CDP- d -glucose 4,6-dehydratase catalyzes the first irreversible step in the synthesis of this 3,6-dideoxysugar by converting CDP- d -glucose to CDP-4-keto-6-deoxyglucose via an NAD+ -dependent intramolecular oxidation,reduction reaction. Here, the cloning, protein purification and X-ray crystallographic analysis of CDP- d -glucose 4,6-dehydratase from Salmonella typhi complexed with the substrate analog CDP- d -xylose are described. Each subunit of the tetrameric enzyme folds into two domains. The N-terminal region contains a Rossmann fold and provides the platform for NAD(H) binding. The C-terminal motif is primarily composed of ,-helices and houses the binding pocket for the CDP portion of the CDP- d -xylose ligand. The xylose moiety extends into the active-site cleft that is located between the two domains. Key residues involved in anchoring the sugar group to the protein include Ser134, Tyr159, Asn197 and Arg208. Strikingly, Ser134,O, and Tyr159,O, sit within 2.9,Å of the 4,-hydroxyl group of xylose. Additionally, the side chains of Asp135 and Lys136 are located at 3.5 and 3.2,Å, respectively, from C-5 of xylose. In the structurally related dTDP- d -glucose 4,6-dehydratase, the Asp/Lys pair is replaced with an Asp/Glu couple. On the basis of this investigation, it can be speculated that Tyr159 serves as the catalytic base to abstract the 4,-hydroxyl proton from the sugar and that Asp135 and Lys136 play critical roles in the subsequent dehydration step that leads to the final product. [source] Lysosomal cysteine proteases (cathepsins): promising drug targetsACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003an Turk Papain-like lysosomal cysteine proteases are processive and digestive enzymes expressed in organisms from bacteria to humans. Their ubiquity alone makes them potential drug targets, with the assumption that appropriate specificities may be achieved. These enzymes have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1,) and additionally have comparatively broad binding areas (S4, S3, S2,, S3,). This geometry distinguishes them from other protease classes, such as serine and aspartic proteases, with six and eight substrate-binding sites, respectively. Exopeptidases (cathepsins B, C, H and X), in contrast to endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate binding of N- and C-terminal groups of substrates in the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from specific exclusions of substrate type. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments can produce the desired result. The small number of papain-like lysosomal cysteine proteases (11 human enzymes are known) and the small number of substrate-binding sites calls for a innovative and empirical approach. [source] Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinaseACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2002Patricia A. Elkins The X-ray crystal structure of the proform of human matrix metalloproteinase MMP9 has been solved to 2.5,Å resolution. The construct includes the prodomain, the catalytic domain and three FnII (fibronectin type II) domains. The prodomain is inserted into the active-site cleft, blocking access to the catalytic zinc. Comparison with the crystal structure of the most closely related MMP, MMP2, indicates that the conformations of residues in the active-site cleft and in the cysteine-switch peptide of the prodomain are highly conserved and that design of MMP9-specific inhibitors will be challenging. In common with MMP2, the MMP9 S1, inhibitor-binding pocket is large compared with that of other MMPs. One small point of difference in the S1, binding pockets of MMP9 and MMP2 may provide an opportunity to explore the design of specific inhibitors. The side chain of Arg424 in MMP9 is angled slightly away from the S1, pocket when compared with the corresponding residue in MMP2, Thr424. The secondary structure of the FnII domains is conserved between the two closely related MMPs, although the second FnII domain makes no contact with the catalytic domain in MMP9, while the same domain in MMP2 has a substantial area of interaction with the catalytic domain. [source] Structure of d -lactate dehydrogenase from Aquifex aeolicus complexed with NAD+ and lactic acid (or pyruvate)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 12 2009Svetlana V. Antonyuk The crystal structure of d -lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12,Å resolution in space group P212121, with unit-cell parameters a = 90.94, b = 94.43, c = 188.85,Å. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticusd -lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a `closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft. [source] Structure of a d -tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritimaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Haruhiko Sakuraba The crystal structure of a d -tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2,Å. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of d -tagatose 3-epimerase from Pseudomonas cichorii and d -psicose 3-epimerase from Agrobacterium tumefaciens; however, TM0416p exhibited a unique solvent-accessible substrate-binding pocket that reflected the absence of an ,-helix that covers the active-site cleft in the two aforementioned ketohexose 3-epimerases. In addition, the residues responsible for creating a hydrophobic environment around the substrate in TM0416p differ entirely from those in the other two enzymes. Collectively, these findings suggest that the substrate specificity of TM0416p is likely to differ substantially from those of other d -tagatose 3-epimerase family enzymes. [source] Structure of HsaD, a steroid-degrading hydrolase, from Mycobacterium tuberculosisACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 1 2008Nathan Lack Tuberculosis is a major cause of death worldwide. Understanding of the pathogenicity of Mycobacterium tuberculosis has been advanced by gene analysis and has led to the identification of genes that are important for intracellular survival in macrophages. One of these genes encodes HsaD, a meta -cleavage product (MCP) hydrolase that catalyzes the hydrolytic cleavage of a carbon,carbon bond in cholesterol metabolism. This paper describes the production of HsaD as a recombinant protein and, following crystallization, the determination of its three-dimensional structure to 2.35,Å resolution by X-ray crystallography at the Diamond Light Source in Oxfordshire, England. To the authors' knowledge, this study constitutes the first report of a structure determined at the new synchrotron facility. The volume of the active-site cleft of the HsaD enzyme is more than double the corresponding active-site volumes of related MCP hydrolases involved in the catabolism of aromatic compounds, consistent with the specificity of HsaD for steroids such as cholesterol. Knowledge of the structure of the enzyme facilitates the design of inhibitors. [source] A new crystal form of bovine pancreatic RNase A in complex with 2,-deoxyguanosine-5,-monophosphateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2007Steven B. Larson The structure of bovine pancreatic RNase A has been determined in complex with 2,-deoxyguanosine-5,-monophosphate (dGMP) at 1.33,Å resolution at room temperature in a previously unreported unit cell belonging to space group P31. There are two molecules of nucleotide per enzyme molecule, one of which lies in the active-site cleft in the productive binding mode, whilst the guanine base of the other dGMP occupies the pyrimidine-specific binding site in a nonproductive mode such that it forms hydrogen bonds to the phosphate group of the first dGMP. This is the first RNase A structure containing a guanine base in the B2 binding site. Each dGMP molecule is involved in intermolecular interactions with adjacent RNase A molecules in the lattice and the two nucleotides appear to direct the formation of the crystal lattice. Because GMP may be produced during degradation of RNA, this association could represent an inhibitor complex and thereby affect the observed enzyme kinetics. [source] Lysosomal cysteine proteases (cathepsins): promising drug targetsACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2003an Turk Papain-like lysosomal cysteine proteases are processive and digestive enzymes expressed in organisms from bacteria to humans. Their ubiquity alone makes them potential drug targets, with the assumption that appropriate specificities may be achieved. These enzymes have rather short active-site clefts, comprising three well defined substrate-binding subsites (S2, S1 and S1,) and additionally have comparatively broad binding areas (S4, S3, S2,, S3,). This geometry distinguishes them from other protease classes, such as serine and aspartic proteases, with six and eight substrate-binding sites, respectively. Exopeptidases (cathepsins B, C, H and X), in contrast to endopeptidases (such as cathepsins L, S, V and F), possess structural features that facilitate binding of N- and C-terminal groups of substrates in the active-site cleft. Other than a clear preference for free chain termini in the case of exopeptidases, the substrate-binding sites exhibit no strict specificities. Instead, their subsite preferences arise more from specific exclusions of substrate type. This presents a challenge for the design of inhibitors to target a specific cathepsin: only the cumulative effect of an assembly of inhibitor fragments can produce the desired result. The small number of papain-like lysosomal cysteine proteases (11 human enzymes are known) and the small number of substrate-binding sites calls for a innovative and empirical approach. [source] | ||||||||||