title: Mechanistic consequences of chiral radical clock probes: analysis of the mononuclear non-heme iron enzyme HppE with 2-hydroxy-3-methylenecyclopropyl radical clock substrates.
abstract: (S)-2-Hydroxypropylphosphonic acid [(S)-HPP] epoxidase (HppE) is a mononuclear iron enzyme that catalyzes the last step in the biosynthesis of the antibiotic fosfomycin. HppE also processes the (R)-enantiomer of HPP but converts it to 2-oxo-propylphosphonic acid. In this study, all four stereoisomers of 3-methylenecyclopropyl-containing substrate analogues, (2R, 3R)-8, (2R, 3S)-8, (2S, 3R)-8, and (2S, 3S)-8, were synthesized and used as radical probes to investigate the mechanism of the HppE-catalyzed reaction. Upon treatment with HppE, (2S, 3R)-8 and (2S, 3S)-8 were converted via a C1 radical intermediate to the corresponding epoxide products, as anticipated. In contrast, incubation of HppE with (2R, 3R)-8 led to enzyme inactivation, and incubation of HppE with (2R, 3S)-8 yielded the 2-keto product. The former finding is consistent with the formation of a C2 radical intermediate, where the inactivation is likely triggered by radical-induced ring cleavage of the methylenecyclopropyl group. Reaction with (2R, 3S)-8 is predicted to also proceed via a C2 radical intermediate, but no enzyme inactivation and no ring-opened product were detected. These results strongly suggest that an internal electron transfer to the iron center subsequent to C-H homolysis competes with ring-opening in the processing of the C2 radical intermediate. The different outcomes of the reactions with (2R, 3R)-8 and (2R, 3S)-8 demonstrate the need to carefully consider the chirality of substituted cyclopropyl groups as radical reporting groups in studies of enzymatic mechanisms.
authors: Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry, University of Texas at Austin , Austin, Texas 78712, United States.
authors: Hui
authors: H
authors: Huang
authors: Wei-Chen
authors: WC
authors: Chang
authors: Geng-Min
authors: GM
authors: Lin
authors: Anthony
authors: A
authors: Romo
authors: Pei-Jing
authors: PJ
authors: Pai
authors: William K
authors: WK
authors: Russell
authors: David H
authors: DH
authors: Russell
authors: Hung-Wen
authors: HW
authors: Liu
mesh_terms: D005578:Fosfomycin / Q000096:biosynthesis*; D063065:Organophosphonates / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D013237:Stereoisomerism
chemical_list: C531865:2-hydroxypropylphosphonic acid; D063065:Organophosphonates; D005578:Fosfomycin; D010088:Oxidoreductases; C023111:epoxidase