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

()-2-Hydroxypropylphosphonate [()-2-HPP, ] epoxidase (HppE) reduces HO at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 hydrogen of by its C2 oxygen, with inversion of configuration at C1, yields the -epoxide of the drug [(1,2)-epoxypropylphosphonic acid (-Fos, )]. Here we show that HppE achieves ∼95% selectivity for C1 inversion and -epoxide formation via steric guidance of a radical-coupling mechanism. Published structures of the HppE·Fe· and HppE·Zn· 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). of C1 configuration and -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 -epoxide by radicaloid C-O coupling.