title: Evidence for radical-mediated catalysis by HppE: a study using cyclopropyl and methylenecyclopropyl substrate analogues.
abstract: (S)-2-Hydroxypropylphosphonic acid epoxidase (HppE) is an unusual mononuclear iron enzyme that catalyzes the oxidative epoxidation of (S)-2-hydroxypropylphosphonic acid ((S)-HPP) in the biosynthesis of the antibiotic fosfomycin. HppE also recognizes (R)-2-hydroxypropylphosphonic acid ((R)-HPP) as a substrate and converts it to 2-oxo-propylphosphonic acid. To probe the mechanisms of these HppE-catalyzed oxidations, cyclopropyl- and methylenecyclopropyl-containing compounds were synthesized and studied as radical clock substrate analogues. Enzymatic assays indicated that the (S)- and (R)-isomers of the cyclopropyl-containing analogues were efficiently converted to epoxide and ketone products by HppE, respectively. In contrast, the ultrafast methylenecyclopropyl-containing probe inactivated HppE, consistent with a rapid radical-triggered ring-opening process that leads to enzyme inactivation. Taken together, these findings provide, for the first time, experimental evidence for the involvement of a C2-centered radical intermediate with a lifetime on the order of nanoseconds in the HppE-catalyzed oxidation of (R)-HPP.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: Hui
authors.initials: H
authors.lastname: Huang
authors.forename: Wei-chen
authors.initials: WC
authors.lastname: Chang
authors.forename: Pei-Jing
authors.initials: PJ
authors.lastname: Pai
authors.forename: Anthony
authors.initials: A
authors.lastname: Romo
authors.forename: Steven O
authors.initials: SO
authors.lastname: Mansoorabadi
authors.forename: David H
authors.initials: DH
authors.lastname: Russell
authors.forename: Hung-wen
authors.initials: HW
authors.lastname: Liu
mesh_terms: D055162:Biocatalysis; D003521:Cyclopropanes / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D010757:Phosphorous Acids / Q000737:chemistry / Q000378:metabolism
chemical_list: D003521:Cyclopropanes; D005609:Free Radicals; D010757:Phosphorous Acids; C570063:phosphonic acid; C030797:cyclopropane; D010088:Oxidoreductases; C405402:2-hydroxypropylphosphonic acid epoxidase