Steric Enforcement of cis-Epoxide Formation in the Radical C–O-Coupling Reaction by Which (S)‑2-Hydroxypropylphosphonate Epoxidase (HppE) Produces Fosfomycin

• Published in: Journal of the American Chemical Society Vol. 141; no. 51; pp. 20397 - 20406
• Main Authors: Zhou, Shengbin, Pan, Juan, Davis, Katherine M, Schaperdoth, Irene, Wang, Bo, Boal, Amie K, Krebs, Carsten, Bollinger, J. Martin
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26-12-2019
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.9b10974

(S)-2-Hydroxypropylphosphonate [(S)-2-HPP, 1] epoxidase (HppE) reduces H2O2 at its nonheme-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.