Steric enforcement of cis-epoxide formation in the radical C–O-coupling reaction by which (S)-2-hydroxypropylphosphonate epoxidase (HppE) produces Fosfomycin

• Type of Material: Article
• Authors: Shengbin Zhou, Pennsylvania State University; Juan Pan, Pennsylvania State University; Katherine M. Davis, Emory University; Irene Schaperdoth, Pennsylvania State University; Bo Wang, Pennsylvania State University; Amie K. Boal, Pennsylvania State University; Carsten Krebs, Pennsylvania State University; J. Martin Bollinger Jr., Pennsylvania State University
• Language: English
• Date: 2019-12-11
• Publisher: American Chemical Society
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2019 American Chemical Society
• Final Published Version (URL): https://doi.org/10.1021/jacs.9b10974
• Title of Journal or Parent Work: Journal of the American Chemical Society
• Volume: 141
• Issue: 51
• Start Page: 20397
• End Page: 20406
• Grant/Funding Agency: National Institutes of Health
• Grant/Funding Information: This work was supported by the National Institutes of Health (GM113106 to J.M.B. and C.K., GM127079 to C.K., GM119707 to A.K.B., and 1K99GM129460-01 to K.M.D.)
• Keywords: peroxidase; stereochemistry; iron; oxirane; antibacterial
• Subject - Topics: Chemical biology; Bioinorganic chemistry; Enzymology

Abstract

(S)-2-hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its non-heme-iron cofactor to install the oxirane “warhead” of the antibiotic fosfomycin. The net replacement of the C1 pro-R hydrogen of 1 by its C2 oxygen, with inversion of configuration at C1, yields the cis epoxide of the drug [(1R,2S)-epoxypropylphosphonic acid (cis-Fos, 2)]. Here we show that HppE achieves ~ 95% selectivity for C1 inversion and cis-epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE•FeII•1 and HppE•ZnII•2 complexes reveal distinct pockets for C3 of the substrate and product and identify four hydrophobic residues – Leu120, Leu144, Phe182, and Leu193 – close to C3 in one of the complexes. Replacement of Leu193 in the substrate C3 pocket with the bulkier Phe enhances stereoselectivity (cis:trans ~ 99:1), whereas the Leu120Phe substitution in the product C3 pocket diminishes it (~ 82:18). Retention of C1 configuration and trans-epoxide formation become predominant with the bulk-reducing Phe182Ala substitution in the substrate C3 pocket (~ 13:87), trifluorination of C3 (~ 23:77), or both (~ 1:99). The effect of C3 trifluorination is counteracted by the more constrained substrate C3 pockets in the Leu193Phe (~ 56:44) and Leu144Phe/Leu193Phe (~ 90:10) variants. The ability of HppE to epoxidize substrate analogues bearing halogens at C3, C1, or both is inconsistent with a published hypothesis of polar cyclization via a C1 carbocation. Rather, specific enzyme-substrate contacts drive inversion of the C1 radical – as proposed in a recent computational study – to direct formation of the more potently antibacterial cis epoxide by radicaloid C–O coupling.