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

Bo Wang, Anthony J. Blaszczyk, Hayley L. Knox, Shengbin Zhou, Elizabeth J. Blaesi, Carsten Krebs, Roy X. Wang, Squire J. Booker

Research output: Contribution to journalArticle

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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 (CH 3 ) rather than a methyl cation (CH 3 + ), directly forming cob(II)alamin in the process.

Original languageEnglish (US)
Pages (from-to)4972-4984
Number of pages13
JournalBiochemistry
Volume57
Issue number33
DOIs
StatePublished - Aug 21 2018

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Cytidine Monophosphate
S-Adenosylmethionine
Streptomyces
Vitamin B 12
Hydrogen
Methylation
Fosfomycin
Atoms
Stereoisomerism
Enantiomers
Biosynthesis
Electron Spin Resonance Spectroscopy
Substrates
Paramagnetic resonance
Cations
Spectrum Analysis
Carbon
Spectroscopy
Anti-Bacterial Agents
N-(4'-fluorobutyrophenone)-4-(4-chlorophenyl)pyridinium

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

@article{03b4cb9d928146e09a6f6675d8d12241,
title = "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 M{\"o}ssbauer 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 (CH 3 • ) rather than a methyl cation (CH 3 + ), directly forming cob(II)alamin in the process.",
author = "Bo Wang and Blaszczyk, {Anthony J.} and Knox, {Hayley L.} and Shengbin Zhou and Blaesi, {Elizabeth J.} and Carsten Krebs and Wang, {Roy X.} and Booker, {Squire J.}",
year = "2018",
month = "8",
day = "21",
doi = "10.1021/acs.biochem.8b00693",
language = "English (US)",
volume = "57",
pages = "4972--4984",
journal = "Biochemistry",
issn = "0006-2960",
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Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase. / Wang, Bo; Blaszczyk, Anthony J.; Knox, Hayley L.; Zhou, Shengbin; Blaesi, Elizabeth J.; Krebs, Carsten; Wang, Roy X.; Booker, Squire J.

In: Biochemistry, Vol. 57, No. 33, 21.08.2018, p. 4972-4984.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Stereochemical and Mechanistic Investigation of the Reaction Catalyzed by Fom3 from Streptomyces fradiae, a Cobalamin-Dependent Radical S-Adenosylmethionine Methylase

AU - Wang, Bo

AU - Blaszczyk, Anthony J.

AU - Knox, Hayley L.

AU - Zhou, Shengbin

AU - Blaesi, Elizabeth J.

AU - Krebs, Carsten

AU - Wang, Roy X.

AU - Booker, Squire J.

PY - 2018/8/21

Y1 - 2018/8/21

N2 - 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 (CH 3 • ) rather than a methyl cation (CH 3 + ), directly forming cob(II)alamin in the process.

AB - 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 (CH 3 • ) rather than a methyl cation (CH 3 + ), directly forming cob(II)alamin in the process.

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U2 - 10.1021/acs.biochem.8b00693

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JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 33

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