Structural analysis of hydroxypropylphosphonic acid epoxidase : a fosfomycin biosynthetic enzyme

Abstract

An X-ray crystallographic study of the fosfomycin biosynthetic enzyme hydroxypropylphosphonic acid epoxidase (HppE) from Streptomyces wedmorensis is presented. Structural analysis of this cupin mononuclear iron enzyme in complex with its in vivo substrate, and comparison with apo- and holo-enzyme structures, provides insight into the mechanism of fosfomycin biosynthesis. In vivo, the enzyme catalyzes the biosynthesis of the epoxide fosfomycin from its substrate, S-2-hydroxypropylphosphonic acid (S-HPP). In vitro, HppE is able to catalyze the conversion of the substrate enantiomer, R-2-hydroxypropylphosphonic acid (R-HPP), to a single ketone product in a stereospecific manner. X-ray crystal structures of HppE in complex with the R-HPP substrate suggest a mechanism for the regiospecific reactions catalyzed by the enzyme. This regiospecificity of HppE catalysis is also observed when substrates R- and S-2-phenylethlylphosphonic acid (HPEP) are used, where a methyl group has been substituted by a larger phenyl substituent. X-ray crystal structures in complex with S-HPEP suggest a conserved mechanism of regioselectivity and a mechanism of ligand rearrangement upon co-substrate (i.e. dioxygen) binding. Finally, HppE is compared to enzymes and proteins within the cupin superfamily, defining a new structural subfamily of cupin mononuclear iron enzymes.