Abstract
The oxirane (1R,2S)-1,2-epoxypropylphosphonic acid (fosfomycin) is a natural product antibiotic produced in Streptomyces wedmorensis by the metal-ion-dependent (S)-2-hydroxypropylphosphonic acid epoxidase. This epoxidase is highly unusual since it has no requirement for a haem prosthetic group. The gene encoding the enzyme, fom4, has been cloned and a highly efficient recombinant source of the enzyme established. Two different crystal forms, tetragonal and hexagonal, have been obtained. The hexagonal form displays symmetry consistent with space group P6(1/5)22 and unit-cell parameters a = 86.44, c = 221.56 A, gamma = 120 degrees. The Matthews coefficient, VM, of 2.7 A3 Da(-1) corresponds to two subunits, each of approximate weight 21.4 kDa, in the asymmetric unit with 55% solvent content. These crystals diffract to high resolution and experimental phases are being sought to determine the structure.
Highlights
Fosfomycin, (1R,2S)-1,2-epoxypropylphosphonic acid, is a broadspectrum antimicrobial produced by Streptomyces wedmorensis and Pseudomonas syringae (Hendlin et al, 1969; Shoji et al, 1986)
One interest in the enzymes involved in the production of this antimicrobial drug is the application of protein-engineering methods to assist in the development of new antimicrobials based upon the fosfomycin structure
The early steps of this pathway are well established; much less is understood about the remaining steps, especially the unusual mechanism of the oxiranyl ring formation (Liu et al, 2003).This final step in the pathway is the epoxidation of HPP to produce fosfomycin, a reaction that is catalysed by the enzyme HPP epoxidase (Zhao et al, 2002)
Summary
Fosfomycin, (1R,2S)-1,2-epoxypropylphosphonic acid, is a broadspectrum antimicrobial produced by Streptomyces wedmorensis and Pseudomonas syringae (Hendlin et al, 1969; Shoji et al, 1986). One interest in the enzymes involved in the production of this antimicrobial drug is the application of protein-engineering methods to assist in the development of new antimicrobials based upon the fosfomycin structure. The early steps of this pathway are well established; much less is understood about the remaining steps, especially the unusual mechanism of the oxiranyl ring formation (Liu et al, 2003).This final step in the pathway is the epoxidation of HPP to produce fosfomycin, a reaction that is catalysed by the enzyme HPP epoxidase (Zhao et al, 2002). We describe the cloning of the S. wedmorensis fom gene, which encodes HPP epoxidase, the construction of a highly efficient protein-expression system and purification of the recombinant enzyme along with crystallization and preliminary diffraction experiments
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