3 results for Pearce, FG

  • A tetrameric structure is not essential for activity in dihydrodipicolinate synthase (DHDPS) from Mycobacterium tuberculosis

    Evans, Genevieve; Schuldt, L; Griffin, MDW; Devenish, SRA; Pearce, FG; Perugini, MA; Dobson, RCJ; Jameson, GB; Weiss, MS; Gerrard, Juliet (2011-08-15)

    Journal article
    The University of Auckland Library

    Dihydrodipicolinate synthase (DHDPS) is a validated antibiotic target for which a new approach to inhibitor design has been proposed: disrupting native tetramer formation by targeting the dimer???dimer interface. In this study, rational design afforded a variant of Mycobacterium tuberculosis, Mtb-DHDPS-A204R, with disrupted quaternary structure. X-ray crystallography (at a resolution of 2.1 ??) revealed a dimeric protein with an identical fold and active-site structure to the tetrameric wild-type enzyme. Analytical ultracentrifugation confirmed the dimeric structure in solution, yet the dimeric mutant has similar activity to the wild-type enzyme. Although the affinity for both substrates was somewhat decreased, the high catalytic competency of the enzyme was surprising in the light of previous results showing that dimeric variants of the Escherichia coli and Bacillus anthracis DHDPS enzymes have dramatically reduced activity compared to their wild-type tetrameric counterparts. These results suggest that Mtb-DHDPS-A204R is similar to the natively dimeric enzyme from Staphylococcus aureus, and highlight our incomplete understanding of the role played by oligomerisation in relating protein structure and function.

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  • Inhibiting dihydrodipicolinate synthase across species: Towards specificity for pathogens?

    Mitsakos, V; Dobson, RCJ; Pearce, FG; Devenish, SR; Evans, Genevieve; Burgess, BR; Perugini, MA; Gerrard, Juliet; Hutton, CA (2008-01-15)

    Journal article
    The University of Auckland Library

    Dihydrodipicolinate synthase (DHDPS) is a key enzyme in lysine biosynthesis and an important antibiotic target. The specificity of a range of heterocyclic product analogues against DHDPS from three pathogenic species, Bacillus anthracis, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus, and the evolutionarily related N-acetylneuraminate lyase, has been determined. The results suggest that the development of species-specific inhibitors of DHDPS as potential antibacterials is achievable.

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  • Crystal structure and kinetic study of dihydrodipicolinate synthase from Mycobacterium tuberculosis

    Kefala, G; Evans, Genevieve; Griffin, MDW; Devenish, SRA; Pearce, FG; Perugini, MA; Gerrard, Juliet; Weiss, MS; Dobson, RCJ (2008-04)

    Journal article
    The University of Auckland Library

    The three-dimensional structure of the enzyme dihydrodipicolinate synthase (KEGG entry Rv2753c, EC 4.2.1.52) from Mycobacterium tuberculosis (Mtb-DHDPS) was determined and refined at 2.28 ?? (1 ??=0.1 nm) resolution. The asymmetric unit of the crystal contains two tetramers, each of which we propose to be the functional enzyme unit. This is supported by analytical ultracentrifugation studies, which show the enzyme to be tetrameric in solution. The structure of each subunit consists of an N-terminal (b/a)8-barrel followed by a C-terminal a-helical domain. The active site comprises residues from two adjacent subunits, across an interface, and is located at the C-terminal side of the (b/a)8-barrel domain. A comparison with the other known DHDPS structures shows that the overall architecture of the active site is largely conserved, albeit the proton relay motif comprising Tyr143, Thr54 and Tyr117 appears to be disrupted. The kinetic parameters of the enzyme are reported: KMASA=0.43??0.02 mM, KMpyruvate=0.17??0.01 mM and Vmax=4.42??0.08 mmol??s-1??mg-1. Interestingly, the Vmax of Mtb-DHDPS is 6-fold higher than the corresponding value for Escherichia coli DHDPS, and the enzyme is insensitive to feedback inhibition by (S)-lysine. This can be explained by the three-dimensional structure, which shows that the (S)-lysine-binding site is not conserved in Mtb-DHDPS, when compared with DHDPS enzymes that are known to be inhibited by (S)-lysine. A selection of metabolites from the aspartate family of amino acids do not inhibit this enzyme. A comprehensive understanding of the structure and function of this important enzyme from the (S)-lysine biosynthesis pathway may provide the key for the design of new antibiotics to combat tuberculosis.

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