| Fosfomycin, produced by a few strains of Streptomyces , is a clinically useful antibiotic against both gram-positive and gram-negative bacteria. Fosfomycin blocks the assembly of the peptidoglycan layer of the bacterial cell wall by inhibiting the enzyme, UDP-GlcNAc-3-O-enolpyruvyltransferase. It has an unusual structure which is characterized by the presence of both a unique carbon-phosphorous bond and an epoxide. The proposed biosynthesis of fosfomycin involves four steps. Even though the first two steps have been well-studied and the mechanisms have been established, the mechanisms for the proposed last two steps remain elusive. The last two steps are a methylation reaction and an epoxidation reaction.;One unusual feature of the last step is that the starting material for the epoxidation is a saturated compound (a secondary alcohol) instead of an olefin as in other biological epoxidation reactions. Another unique feature is that the C-2 oxygen is retained during the formation of the epoxide ring. This makes the oxirane ring formation an overall dehydrogenation reaction. In order to study the mechanism of this unique reaction in detail, the corresponding enzymes, HPP epoxidase and HPP epoxidase reductase have been cloned, overexpressed. The utilizing of a sensitive bioassay led to the discovery a novel biological epoxidation system (HPP + NADH + O2 + H+ → fosfomycin + NAD+ + 2H2O). Further characterization of HPP epoxidase with UV-visible, Resonance Raman, EPR spectroscopies as well as x-ray crystallography revealed that HPP epoxidase is a new member of non-heme iron enzyme and the active site has iron-catecholate core structure. Also the detailed mechanism has been probed by the synthesis of HPP analogs and their subsequent biochemical evaluation. These studies indicated that HPP epoxidase is stereoselective and its substrate binding pocket is relatively flexible. These studies also indicated that HPP epoxidation is stepwise and the generation of carbon centered radical might be the initial step. |
