Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase

摘要

Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2 '' of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to form cytidylyl-2-hydroxypropylphosphonate (HPP-CMP) during the biosynthesis of fosfomycin, a broad-spectrum antibiotic. It has been hypothesized that a S'-deoxyadenosyl S'-radical (5'-dA(center dot)) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate for addition of a methyl group from methylcobalamin (MeCbl); however, the mechanistic details of this reaction remain elusive. Moreover, it has been reported that Fom3 catalyzes the methylation of HEP-CMP to give a mixture of the (S)-HPP and (R)-HPP stereoisomers, which is rare for an enzyme-catalyzed reaction. Herein, we describe a detailed biochemical investigation of a Fom3 that is purified with 1 equiv of its cobalamin cofactor bound, which is almost exclusively in the form of MeCbl. Electron paramagnetic resonance and Mossbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA(center dot) generated by Fom3 abstracts the C2 ''-pro-R hydrogen of HEP-CMP and that methyl addition takes place with inversion of configuration to yield solely (S)-HPP-CMP. Fom3 also sluggishly converts cytidylyl-ethylphosphonate to the corresponding methylated product but more readily acts on cytidyly1-2-fluoroethylphosphonate, which exhibits a lower C2 '' homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2 '' radical, generated upon hydrogen atom abstraction by the 5'-dA(center dot), directly attacks MeCbl to transfer a methyl radical (CH3 center dot) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.