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

Methanogenesis marker protein 10 (Mmp10) from Methanosarcina acetivorans is a radical S-adenosylmethionine methylase that unexpectedly requires cobalamin

“…4, the addition of either MeCbl or OHCbl results in a dramatic increase in enzyme activity, as evidenced by the substantial time-dependent increase in SAH, 5Ј-dAH, and MeArg concentrations. The similar effects of MeCbl and OHCbl on the MaMmp10 reaction suggest that the enzyme readily converts OHCbl to MeCbl, an event that has been observed in other cobalamin-dependent RS enzymes (21,24,30).…”

“…As expected, the products of the reaction are 5Ј-dAH, methionine (Met) S-adenosylhomocysteine (SAH), and the methylated peptide. In the presence of the required low-potential reductant, Ti(III) citrate, MaMmp10 exhibits a k cat of 1.87 min Ϫ1 , which is comparable with that of other class B methylases that catalyze reactions via a 5Ј-dA ⅐ intermediate (24).…”

“…Ti(III) citrate was prepared as described previously (51). All other materials and reagents have been previously reported (21,24,30) or were of the highest available quality.…”

Structural basis for non-radical catalysis by TsrM, a radical SAM methylase

“…In this instance, both SAM and the substrate undergoing methylation are required to be bound simultaneously, because the 5’-dA• generated from the reductive cleavage of SAM would need to abstract a hydrogen atom directly from the substrate. This substrate radical would then attack the methyl moiety of MeCbl, yielding the methylated product and cob(II)alamin ( 25 ). In the TsrM structure, SAM binds near the FeS cluster, but does not ligate to it.…”

Crystallographic snapshots of a B12-dependent radical SAM methyltransferase

“…In addition, a hydrophobic pocket prevents water from converting the pentacoordinated MeCbl into its more stabilized hexacoordinated counterpart, which is a strategy conserved in other B 12 -dependent radical SAM enzymes. These enzymes thus appear to have evolved unique structures and mechanisms to alkylate sp 2 -and sp 3 -hybridized carbon atoms using the twin catalytic power of the cobalamin and SAM cofactors 8,13,14,[16][17][18][19]21,50,51 . In contrast to catalysis by known radical SAM enzymes, catalysis by Mmp10 requires active site reorganization and SAM flexibility within the active site.…”