This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The emergence of bacterial strains resistant to vancomycin group antibiotics has driven researchers to develop novel antibiotics to which resistant strains are susceptible. One approach is to pursue analogs of potent antibiotics that have even greater efficacy, as even slight changes to antibiotic structure can have dramatic effects on their activity. Chemical synthesis of such analogs is largely impractical, due to the technical difficulty and low yield. An alternative centers on the study of how these antibiotics are synthesized in vivo, with the ultimate goal of modifying the end product through the manipulation of the specificities or order of the enzymes involved in the biosynthesis. This proposal studies one aspect of this goal, the biosynthetic pathway of 3,5-dihydroxy-L-phenylglycine (Dpg), a non-proteinogenic amino acid used in vancomycin group antibiotics. Four enzymes are proposed to synthesize 3,5-dihydroxyphenylacetate (Dpa), a precursor to Dpg. This proposal focuses on the enzymatic mechanism of one enzyme in the pathway, DpgC. DpgC is a member of the crotonase superfamily of proteins characterized by the ability to process Coenzyme A (CoA) thioesters. DpgC posseses a remarkable dual function in catalysis, first catalyzing the two electron oxidation to Dpa-CoA, then hydrolyzing the CoA esters to form the observed acid product, Dpa. The oxidation step is particularly unique, in that the reaction apparently proceeds without the participation of soluble cofactors or bound metal ions. If the reaction is dependant only on molecular oxygen and the enzyme it would represent a novel mechanism in enzymology. Enzyme oxygenations, especially of unactivated methylene units, invariably use cofactors such as flavins and hemes and/or redux-active metals such as iron or copper. This proposal seeks to understand the mechanism of DpgC using X-ray crystallographic structural analysis. A detailed understanding of the mechanism of vancomycin biosynthesis can be applied toward the development on novel antibiotics through rational engineering or combinatorial biosynthesis.
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