PEPTIDOGLYCAN BIOSYNTHESIS AND VANCOMYCIN RESISTANCE

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This proposal focuses on enzymes in bacterial cell wall assembly of the peptidoglycan (PG) component, a structure unique to bacteria and known to be the target of several clinically useful antibiotics. Experiments are proposed in two areas: (1) the first committed step in the PG biosynthesis, an unusual enolpyruvyl transfer from PEP to UDP-Nacetyl gluco-samine to produce UDPenolpyruvyl G1cNAc, the scaffolding element for peptide assembly and (2) the D-ala-D-ala termini of PG that form the high affinity site for the antibiotic vancomycin. We have recently cloned, sequenced and purified to homogeneity MurZ, the enolpyruvyl transferase, and propose to study its catalytic mechanism and the mechanism of time-dependent inactivation of this enzyme by the antibiotic fosfomycin, an epoxypropane phosphonate in clinical use in Europe. No molecular information is known about the specificity of fosfomycin for MurZ and structure/function studies on catalytic mechanism could lead to improved antibiotic design against this target. Vancomycin resistance arises in life-threatening gram positive bacterial infections (e.g., endocarditis) when Van resistance genes encode five new proteins, VanS, R, H, A, X. We have recently overproduced, purified and characterized VanH, a D-specific a-ketoacid reductase and VanA, a D-Ala- D-X ligase and shown that they act in concert to make D-Ala-D-Lactate (a depsipeptide) and allow replacement of the normal D-Ala-D-Ala PG terminus by D-Ala-D-Lactate and that no longer recognizes vancomycin. We propose to further study the molecular mechanism of vancomycin resistance by comparison of VanA with the chromosomal D-Ala-D-Ala ligases as well as to purify and characterize VanS and VanR, a proposed two component regulatory system for control of VanH, A, X transcription. Knowledge of the Van resistance proteins may permit design of drugs, such as phos- phinate dipeptidomimetics, to revert vancomycin resistant bacteria to sensitivity.