Characterization of Two Late-Stage Enzymes Involved in Fosfomycin Biosynthesis in Pseudomonads

Abstract

The broad-spectrum phosphonate antibiotic fosfomycin is currently in use for clinical treatment of infections caused by both Gram-positive and Gram-negative uropathogens. The antibiotic is biosynthesized by various streptomycetes, as well as by pseudomonads. Notably, the biosynthetic strategies used by the two genera share only two steps: the first step in which the primary metabolite phosphoenolpyruvate (PEP) is converted to phosphonopyruvate (PnPy), and the terminal step in which 2-hydroxypropylphosphonate (2-HPP) is converted to fosfomycin. Otherwise, distinct enzymatic paths are employed. Here, we biochemically confirm the last two steps in the fosfomycin biosynthetic pathway of Pseudomonas syringae PB-5123, showing that Psf3 carries out the reduction of 2-oxopropylphosphonate (2-OPP) to (S)-2-HPP, followed by the Psf4-catalyzed epoxidation of (S)-2-HPP to fosfomycin. Psf4 can also accept (R)-2-HPP as a substrate, but instead performs an oxidation to make 2-OPP. We show that the combined activities of Psf3 and Psf4 can be used to convert racemic 2-HPP to fosfomycin in an enantioconvergent process. X-ray structures of each enzyme with bound substrates provide insights into the stereospecificity of each conversion. These studies shed light into the reaction mechanisms of the terminal two enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.

Stereospecific Radical-Mediated B12-Dependent Methyl Transfer by the Fosfomycin Biosynthesis Enzyme Fom3

“…11 In the last step, the epoxidase Fom4 converts exclusively the S enantiomer of 2-HPP to fosfomycin. 6,17,18 The stereospecificity of this reaction has been demonstrated both by feeding stereospecifically 2 H-labeled 2-HPP to fosfomycin-producing Streptomyces fradiae and by in vitro enzyme assays, 6,18–20 which have also shown that Fom4 converts ( R )-2-HPP to 2-oxopropylphosphonate (2-OPP). 18,20…”

Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase

“…10–13 In the final step of fosfomycin biosynthesis in Streptomyces, ( S )-HPP is converted to fosfomycin by the nonheme iron peroxidase Fom4, which is also referred to as HppE. 14–16…”

Enzymes, Reacting with Organophosphorus Compounds as Detoxifiers: Diversity and Functions

“…Interestingly, 2-hydroxypropylphosphonic acid epoxidase (HppE) implements partially similar biochemical mechanics as PhnZ. An enzyme from Streptomyces wedmorensis cells is the most investigated and located basally ( Figure 21 ) though other homologs, for example, evolutionally novel enzyme from Pseudomonas syringae (26% identity), are also issued [ 100 ]. HppE forms homotetramer and naturally participates in fosfomycin (i.e., natural antibiotic with phosphonate moiety) biosynthesis [ 86 ].…”