Evolution of Methods for the Study of Cobalamin-Dependent Radical SAM Enzymes

Abstract

While bioinformatic evidence of cobalamin-dependent radical S-adenosylmethionine (SAM) enzymes has existed since the naming of the radical SAM superfamily in 2001, none were biochemically characterized until 2011. In the past decade, the field has flourished as methodological advances have facilitated study of the subfamily. Because of the ingenuity and perseverance of researchers in this field, we now have functional, mechanistic, and structural insight into how this class of enzymes harnesses the power of both the cobalamin and radical SAM cofactors to achieve catalysis. All of the early characterized enzymes in this subfamily were methylases, but the activity of these enzymes has recently been expanded beyond methylation. We anticipate that the characterized functions of these enzymes will become both better understood and increasingly diverse with continued study.

Stereochemical course of cobalamin-dependent radical SAM methylation by TokK and ThnK

“…The substrate radical is methylated by the homolytic cleavage of methylcobalamin (MeCbl), which is formed by the nucleophilic attack of cob(I)alamin on a second equivalent of SAM. [6][7][8] A similar mechanistic strategy is employed by the related sequential methylase CysS, which, unlike TokK and ThnK, acts on a carrier protein-bound substrate. 9 The first methylation by both TokK and ThnK yields Me-PCPM (2) with the R configuration at C6 1,2 (Scheme 1).…”

Biomimetic S-Adenosylmethionine Regeneration Starting from Different Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes

“…Ti 3+ has been shown to reduce the [4Fe4S] 2+ -cluster into the required [4Fe4S] 1+ oxidation state as well as the cob(II)alamin into the cob(I)alamin state, and is therefore a well-suited reductant for class B radical SAM MTs. [52,78] Adding cobalamin to the assay mixture was not necessary as the enzyme was reconstituted with hydroxocobalamin after purification, which remains tightly bound to the enzyme. [52] A 24-mer peptide fragment of methyl-coenzyme M reductase was used as the substrate as described previously.…”

“…Also, 1 mM Ti(III)citrate was added to the assays as a reducing agent as described before. [56,78] Adding cobalamin to the assay mixture was not necessary as the enzyme was reconstituted with hydroxocobalamin after purification, which remains tightly bound to the enzyme. [56] A 24-mer peptide fragment of methyl-coenzyme M reductase was used as the substrate as described previously.…”

Consecutive Methylation Catalyzed by TsrM, an Atypical Class B Radical SAM Methylase

“…[ 28‐30 ] Class B RSMs require a cobalamin factor to transfer the methyl group from SAM to the substrate. [ 31‐34 ] Class C RSMs are HemN‐like enzymes that bind two SAM molecules simultaneously in the active site. These enzymes convert the first SAM to a dAdo radical, which abstracts the hydrogen atom from the methyl group of the second SAM, generating a SAM‐based methylene radical to initiate the subsequent radical‐based methylation reaction.…”