title: 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 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 performs 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 on the reaction mechanisms of the two terminal enzymes in a distinct pathway employed by pseudomonads for the production of a potent antimicrobial agent.
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Carl R. Woese Institute for Genomic Biology, ∥Howard Hughes Medical Institute, and ⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Philip
authors.initials: P
authors.lastname: Olivares
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Carl R. Woese Institute for Genomic Biology, ∥Howard Hughes Medical Institute, and ⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Emily C
authors.initials: EC
authors.lastname: Ulrich
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Carl R. Woese Institute for Genomic Biology, ∥Howard Hughes Medical Institute, and ⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Chekan
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Carl R. Woese Institute for Genomic Biology, ∥Howard Hughes Medical Institute, and ⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Wilfred A
authors.identifier: 0000-0002-5467-7071
authors.initials: WA
authors.lastname: van der Donk
authors.affiliation: Department of Biochemistry, ‡Department of Chemistry, §Carl R. Woese Institute for Genomic Biology, ∥Howard Hughes Medical Institute, and ⊥Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
authors.forename: Satish K
authors.identifier: 0000-0003-1790-1334
authors.initials: SK
authors.lastname: Nair
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D005578:Fosfomycin / Q000096:biosynthesis*; D011487:Protein Conformation; D044224:Pseudomonas syringae / Q000201:enzymology / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D004798:Enzymes; D005578:Fosfomycin