| Fosfomycin, produced by a few strains of Streptomyces, is a clinically useful antibiotic against both Gram-positive and Gram-negative bacteria. The unique structure of fosfomycin, characterized by a carbon-phosphorous bond and an epoxide ring, has attracted many attentions to its biosynthetic pathway. (S)-2-hydroxylpropanylphosphonic acid epoxidase (HppE) catalyzes the last step in the pathway, conversion of a secondary alcohol, (S)-2-hydroxypropanylphosphonic acid ((S)-HPP), to the final epoxide product. This is a completely new enzymatic reaction in contrast to many other biological epoxidation reactions that usually involve olefin oxidation. It is worth noting that the oxygen atom in the hydroxyl group of ( S)-HPP is retained during the ring closure.; The previous studies of HppE have led to the discovery of a novel biological epoxidation system, (S)-HPP + NADH + O2 + H + → fosfomycin + NAD+ + 2H2O. The further biochemical and EPR spectral analyses suggested that HppE is a new type of mononuclear non-heme iron-dependent enzyme carrying a 2-His-1-carboxylate triad iron-binding motif. In addition to three amino acid ligands, both phosphonate and hydroxyl groups of (S)-HPP also coordinate to the iron center in the enzyme-substrate complex. The bidentate substrate-binding mode is presumably responsible for the strict regiospecificity and stereospecificity in the HppE-catalyzed epoxidation.; A catecholate-to-FeIII charge transfer complex was identified in the HppE active site, by UV-visible absorption and resonance Raman spectroscopies. The catechol is derived from a hydroxylated amino acid residue, Tyr105, as a product of the self-catalytic hydroxylation. The oxidation of Try105 to DOPA is a side reaction that occurs only in the absence of substrate, and the presence of DOPA has no apparent effects on the epoxidase activity of HppE.; Mechanistic information of HppE has been continuously garnered by EPR spectral analysis. Using NO as a dioxygen analogue and a probe for ferrous center, the first step of the catalytic cycle has been revealed to be the reduction of substrate-bound iron center from its ferric state to ferrous state by FMN/NADH. Besides, the presence of a spin-coupled adduct of protein-centered radical and ferric center has been supported by experimental evidence. However, it is still not clear whether the "hidden" radical plays any roles in HppE catalysis. |
