Unknown

Dataset Information

0

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


ABSTRACT: 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 5'-deoxyadenosyl 5'-radical (5'-dA•) 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 Mössbauer spectroscopies confirm that Fom3 contains one [4Fe-4S] cluster. Using deuterated enantiomers of HEP-CMP, we demonstrate that the 5'-dA• 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 cytidylyl-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•, directly attacks MeCbl to transfer a methyl radical (CH3•) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.

SUBMITTER: Wang B 

PROVIDER: S-EPMC6554712 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Wang Bo B   Blaszczyk Anthony J AJ   Knox Hayley L HL   Zhou Shengbin S   Blaesi Elizabeth J EJ   Krebs Carsten C   Wang Roy X RX   Booker Squire J SJ  

Biochemistry 20180809 33


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 5'-deoxyadenosyl 5'-radical (5'-dA<sup>•</sup>) generated from the reductive cleavage of SAM abstracts a hydrogen atom from HEP-CMP to prime the substrate fo  ...[more]

Similar Datasets

| S-EPMC6934041 | biostudies-literature
| S-EPMC7061316 | biostudies-literature
| S-EPMC6682749 | biostudies-literature
| S-EPMC5602403 | biostudies-literature
| S-EPMC3162382 | biostudies-literature
| S-EPMC4326810 | biostudies-literature
| S-EPMC4135684 | biostudies-literature
| S-EPMC9204698 | biostudies-literature
| S-EPMC5546040 | biostudies-literature
| S-EPMC10127265 | biostudies-literature