Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase.
Biochemistry
; 57(33): 4972-4984, 2018 08 21.
Article
em En
| MEDLINE
| ID: mdl-30036047
ABSTRACT
Fom3, a cobalamin-dependent radical S-adenosylmethionine (SAM) methylase, has recently been shown to catalyze the methylation of carbon 2â³ 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'-dAâ¢) 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 C2â³- 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 C2â³ homolytic bond-dissociation energy. Our studies suggest a mechanism in which the substrate C2â³ radical, generated upon hydrogen atom abstraction by the 5'-dAâ¢, directly attacks MeCbl to transfer a methyl radical (CH3â¢) rather than a methyl cation (CH3+), directly forming cob(II)alamin in the process.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
S-Adenosilmetionina
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Streptomyces
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Proteínas de Bactérias
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Vitamina B 12
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Metiltransferases
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article