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


ABSTRACT: (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.

SUBMITTER: Zhou S 

PROVIDER: S-EPMC6933072 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Zhou Shengbin S   Pan Juan J   Davis Katherine M KM   Schaperdoth Irene I   Wang Bo B   Boal Amie K AK   Krebs Carsten C   Bollinger J Martin JM  

Journal of the American Chemical Society 20191211 51


(<i>S</i>)-2-Hydroxypropylphosphonate [(<i>S</i>)-2-HPP, <b>1</b>] epoxidase (HppE) reduces H<sub>2</sub>O<sub>2</sub> at its nonheme-iron cofactor to install the oxirane "warhead" of the antibiotic fosfomycin. The net replacement of the C1 <i>pro-R</i> hydrogen of <b>1</b> by its C2 oxygen, with inversion of configuration at C1, yields the <i>cis</i>-epoxide of the drug [(1<i>R</i>,2<i>S</i>)-epoxypropylphosphonic acid (<i>cis</i>-Fos, <b>2</b>)]. Here we show that HppE achieves ∼95% selectivit  ...[more]

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