Mechanism of S-Adenosyl-l-methionine C-Methylation by Cobalamin-dependent Radical S-Adenosyl-l-methionine Methylase in 1-Amino-2-methylcyclopropanecarboxylic Acid Biosynthesis

Abstract

The radical S-adenosyl-L-methionine (SAM) methylase Orf29 catalyzes the C-methylation of SAM in the biosynthesis of 1-amino-2methylcyclopropanecarboxylic acid. Here, we determined that the methylation product is (4″R)-4″-methyl-SAM. Furthermore, we found that the 5′deoxyadenosyl radical generated by Orf29 abstracts the pro-R hydrogen atom from the C-4″ position of SAM to generate the radical intermediate, which reacts with methylcobalamin to give (4″R)-4″-methyl-SAM. Consequently, the Orf29-catalyzed C-methylation was confirmed to proceed with retention of configuration.

Changing Fates of the Substrate Radicals Generated in the Active Sites of the B12-Dependent Radical SAM Enzymes OxsB and AlsB

“…Methyl transfer proceeds with inversion of configuration consistent with the relative positioning of the radical SAM and cobalamin components within the OxsB active site . Similar arrangements have also been observed in two other structurally characterized B 12 -dependent radical SAM methylases (i.e., Mmp10 and TokK). , However, exceptions are known where methylation catalyzed by cobalamin-dependent radical SAM enzymes occurs with the retention of configuration, such as GenK involved in the biosynthesis of gentamicin, , a glutamine C -methyltransferase during the maturation of coenzyme M reductase, and a SAM methylase Orf29 during the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid . The inversion of configuration during the OxsB and AlsB catalyzed C2′-methylations places the hydrogen atom that was originally in the 2′- pro-S position of the substrate 3 now in position for H atom abstraction in a subsequent round of catalysis.…”

“…21,27 However, exceptions are known where methylation catalyzed by cobalamin-dependent radical SAM enzymes occurs with the retention of configuration, such as GenK involved in the biosynthesis of gentamicin, 47,55 a glutamine C-methyltransferase during the maturation of coenzyme M reductase, 22 and a SAM methylase Orf29 during the biosynthesis of 1-amino-2-methylcyclopropanecarboxylic acid. 56 The inversion of configuration during the OxsB and AlsB catalyzed C2′-methylations places the hydrogen atom that was originally in the 2′-pro-S position of the substrate 3 now in position for H atom abstraction in a subsequent round of catalysis. This resembles the repeated hydrogen atom abstraction seen for CysS, where the same carbon presents the H atom for abstraction during tert-butyl group formation, 24,26 as well as ThnK, TokK, and TmoD, where the added methyl group instead serves as the subsequent H atom donor.…”

S-Adenosylmethionine: more than just a methyl donor

“…241 The B 12dependent radical SAM methylase Orf29 encoded by a gene fragment upstream of Orf30 was later demonstrated to catalyze methylation of SAM yielding (4 ′′ R)-4 ′′ -methyl-SAM (171). 244 This SAM derivative ( 171) can be efficiently cyclized to give MeACC (170) under the action of Orf30 (Fig. 33).…”