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1.  Structure of Staphylococcus aureus 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) in complex with acetoacetyl-CoA 
The structure of S. aureus MenB, an enzyme in the biosynthetic pathway to vitamin K2, has been determined and compared with the enzyme derived from another important pathogen, M. tuberculosis.
Vitamin K2, or menaquinone, is an essential cofactor for many organisms and the enzymes involved in its biosynthesis are potential antimicrobial drug targets. One of these enzymes, 1,4-dihydroxy-2-naphthoyl-CoA synthase (MenB) from the pathogen Staphylococcus aureus, has been obtained in recombinant form and its quaternary structure has been analyzed in solution. Cubic crystals of the enzyme allowed a low-resolution structure (2.9 Å) to be determined. The asymmetric unit consists of two subunits and a crystallographic threefold axis of symmetry generates a hexamer consistent with size-exclusion chromatography. Analytical ultracentrifugation indicates the presence of six states in solution, monomeric through to hexameric, with the dimer noted as being particularly stable. MenB displays the crotonase-family fold with distinct N- and C-terminal domains and a flexible segment of structure around the active site. The smaller C-terminal domain plays an important role in oligomerization and also in substrate binding. The presence of acetoacetyl-CoA in one of the two active sites present in the asymmetric unit indicates how part of the substrate binds and facilitates comparisons with the structure of Mycobacterium tuberculosis MenB.
doi:10.1107/S1744309107047720
PMCID: PMC2339762  PMID: 18007038
crotonase; synthase; vitamin biosynthesis; menaquinone; MenB
2.  A Structure-Based Approach to Ligand Discovery for 2C-Methyl-d-erythritol-2,4-cyclodiphosphate Synthase: A Target for Antimicrobial Therapy† 
Journal of Medicinal Chemistry  2009;52(8):2531-2542.
The nonmevalonate route to isoprenoid biosynthesis is essential in Gram-negative bacteria and apicomplexan parasites. The enzymes of this pathway are absent from mammals, contributing to their appeal as chemotherapeutic targets. One enzyme, 2C-methyl-d-erythritol-2,4-cyclodiphosphate synthase (IspF), has been validated as a target by genetic approaches in bacteria. Virtual screening against Escherichia coli IspF (EcIspF) was performed by combining a hierarchical filtering methodology with molecular docking. Docked compounds were inspected and 10 selected for experimental validation. A surface plasmon resonance assay was developed and two weak ligands identified. Crystal structures of EcIspF complexes were determined to support rational ligand development. Cytosine analogues and Zn2+-binding moieties were characterized. One of the putative Zn2+-binding compounds gave the lowest measured KD to date (1.92 ± 0.18 μM). These data provide a framework for the development of IspF inhibitors to generate lead compounds of therapeutic potential against microbial pathogens.
doi:10.1021/jm801475n
PMCID: PMC2669732  PMID: 19320487
3.  Characterization of the Mycobacterium tuberculosis 4-Diphosphocytidyl-2-C-Methyl-d-Erythritol Synthase: Potential for Drug Development▿  
Journal of Bacteriology  2007;189(24):8922-8927.
Mycobacterium tuberculosis utilizes the methylerythritol phosphate (MEP) pathway for biosynthesis of isopentenyl diphosphate and its isomer, dimethylallyl diphosphate, precursors of all isoprenoid compounds. This pathway is of interest as a source of new drug targets, as it is absent from humans and disruption of the responsible genes has shown a lethal phenotype for Escherichia coli. In the MEP pathway, 4-diphosphocytidyl-2-C-methyl-d-erythritol is formed from 2-C-methyl-d-erythritol 4-phosphate (MEP) and CTP in a reaction catalyzed by a 4-diphosphocytidyl-2-C-methyl-d-erythritol synthase (IspD). In the present work, we demonstrate that Rv3582c is essential for M. tuberculosis: Rv3582c has been cloned and expressed, and the encoded protein has been purified. The purified M. tuberculosis IspD protein was capable of catalyzing the formation of 4-diphosphocytidyl-2-C-methyl-d-erythritol in the presence of MEP and CTP. The enzyme was active over a broad pH range (pH 6.0 to 9.0), with peak activity at pH 8.0. The activity was absolutely dependent upon divalent cations, with 20 mM Mg2+ being optimal, and replacement of CTP with other nucleotide 5′-triphosphates did not support activity. Under the conditions tested, M. tuberculosis IspD had Km values of 58.5 μM for MEP and 53.2 μM for CTP. Calculated kcat and kcat/Km values were 0.72 min−1 and 12.3 mM−1 min−1 for MEP and 1.0 min−1 and 18.8 mM−1 min−1 for CTP, respectively.
doi:10.1128/JB.00925-07
PMCID: PMC2168624  PMID: 17921290
4.  The nucleotide-binding site of Aquifex aeolicus LpxC 
Acta Crystallographica Section F  2006;62(Pt 11):1082-1086.
The structure of UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP is reported. The complex allows for a description of how the enzyme recognizes and binds a nucleotide moiety and enables the construction of an LpxC-substrate model.
The structure of recombinant Aquifex aeolicus UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) in complex with UDP has been determined to a resolution of 2.2 Å. Previous studies have characterized the binding sites of the fatty-acid and sugar moieties of the substrate, UDP-(3-O-hydroxymyristoyl)-N-­acetylglucosamine, but not that of the nucleotide. The uracil-binding site is constructed from amino acids that are highly conserved across species. Hydrophobic associations with the Phe155 and Arg250 side chains in combination with hydrogen-bonding interactions with the main chain of Glu154 and the side chains of Tyr151 and Lys227 position the base. The phosphate and ribose groups are directed away from the active site and interact with Arg137, Lys156, Glu186 and Arg250. The orientation of the phosphate-ribose tail is not conducive to catalysis, perhaps owing to the position of an inhibitory Zn2+. However, based on the position of uracil revealed in this study and on the previously reported complex of LpxC with an inhibitor, a model is proposed for substrate binding.
doi:10.1107/S1744309106041893
PMCID: PMC2225228  PMID: 17077484
lipid A; Aquifex aeolicus; LpxC

Results 1-4 (4)