Biochemical and spectroscopic studies of (S)-2-hydroxypropylphosphonic acid epoxidase in fosfomycin biosynthesis
Abstract
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-hydroxylpropanylphosphonic
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.
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