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Comparative analysis of the Escherichia coli ketopantoate hydroxymethyltransferase crystal structure confirms that it is a member of the (betaalpha)8 phosphoenolpyruvate/pyruvate superfamily.

ABSTRACT: Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B(5)), the transfer of a hydroxymethyl group onto alpha-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within the phosphoenolpyruvate/pyruvate superfamily. Based on the analysis, we propose that in this superfamily there should be a subdivision into two groups. This paper completes our structural analysis of the E. coli enzymes in the pantothenate pathway.

SUBMITTER: Schmitzberger F

PROVIDER: S-EPMC164873 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Comparative analysis of the Escherichia coli ketopantoate hydroxymethyltransferase crystal structure confirms that it is a member of the (betaalpha)8 phosphoenolpyruvate/pyruvate superfamily.

Schmitzberger Florian F Smith Alison G AG Abell Chris C Blundell Tom L TL

Journal of bacteriology 20030701 14

Escherichia coli ketopantoate hydroxymethyltransferase (KPHMT) catalyzes the first step in the biosynthesis pathway of pantothenate (vitamin B(5)), the transfer of a hydroxymethyl group onto alpha-ketoisovalerate. Here we describe a detailed comparative analysis of the KPHMT crystal structure and the identification of structural homologues, some of which have remarkable similarities in their active sites, modes of binding to substrates, and mechanisms. We show that KPHMT forms a family within th ...[more]

PMID: 12837791

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Project description:Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.

Dataset Information

Comparative analysis of the Escherichia coli ketopantoate hydroxymethyltransferase crystal structure confirms that it is a member of the (betaalpha)8 phosphoenolpyruvate/pyruvate superfamily.

Publications

Comparative analysis of the Escherichia coli ketopantoate hydroxymethyltransferase crystal structure confirms that it is a member of the (betaalpha)8 phosphoenolpyruvate/pyruvate superfamily.

Similar Datasets

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