Thiostrepton tryptophan methyltransferase (TsrM)

Methylation is a common modification that occurs on both nucleic and amino acids, and plays an important role in the function of biomolecules. The enzymes responsible for performing this methylation are called methyltransferases (MTase; E.C. 2.1) In many cases, MTases utilize the co-factor S-adenosylmethionine (SAM) as their source of methyl. In canonical methylation chemistry, the electrophilic methyl group reacts with a nucleophilic substrate to give the final product. These reactions typically occur with an inversion of stereochemistry (SN2), and are frequently seen on carbon, nitrogen or oxygen nucleophiles (1).

A growing class of enzymes, the radical SAM family, utilize SAM in a different manner. These enzymes are able to cleave SAM reductively and use the resultant 5'-deoxyadenosyl radical intermediate to perform a wide range of ususual chemical reactions, including cofactor biosynthesis, peptide modifications, and lipid metabolism (1, 2). Radical SAM enzymes are present in all domains of life, and share a conserved CxxxCxxC motif to coordinate an iron-sulfur cluster necessary to perform the radical chemistry (1). 

Radical SAM enzymes can be divided into 3 classes based on their sequence homology. However I'm going to focus on those in Class B, which contain both the conserved CxxxCxxC motif and a cobalamin-binding domain. There has been growing interest in the radical SAM community over the methyltransferase TsrM (a member of Class B) because of its unique mechanism of action. Recently, a report on TsrM by Pierre et al. appeared in Nature Chemical Biology (3).

TsrM is involved in the biosynthetic pathway of Thiostrepton A, a thiopeptide antibiotic that undergoes numerous post-translational modifications. TsrM contains a cobalamin co-factor and was hypothesized to methylate tryptophan to 2-methyltryptophan. This methylated tryptophan ultimately serves as a precursor to the quinaldic acid moiety in the antibiotic. In their study, Pierre et al. cloned and overexpressed TsrM in E. coli and characterized its activity in vitro.

Initial experiments confirmed the necessity of methylcobalamin during the reaction. Interestingly, while 2-methyltryptophan production is coupled to SAH, there was no observation 5'-deoxyadeonise (5'dA). The presence of 5'dA would suggest the formation of a 5'-deoxyadenosyl radical as an intermediate during the reaction. Because none of this product was observed, this suggests that SAM does not undergo homolytic cleavage, but is used only as a source of methyl (3).

Interestingly, when (methyl-d3)-SAM was used in labeling experiments, the product was exclusively (methyl-d3)-2-methyladenosine, even in the presence of excess unlabeled methylcob(III)lamin. This suggests that the enzyme binds free cobalamin, which is then methylated by SAM after it is bound. To further elucidate the mechanism, the authors performed UV-Vis spectroscopy to identify key cobalamin intermediate species. Spectroscopy was able to detect Co(II) and Co(III). Yet in contrast to other cobalamin-dependent enzymes, no formation of Co(I) was observed. However in the proposed mechanism, it was suggested that Co(II) is converted to Co(I) with help from the [4Fe-4S] cluster (3). 

Proposed Mechanism of TsrM (2,3):

While some questions remain, continued work on the mechanism of this enzyme, as well as other radical SAMs is expected to shed light on this unique methylation. As genomics efforts continue to identify novel gene clusters, it is likely that more fascinating radical SAM chemistry will soon be discovered.

References:
1. Zhang et al. "Radical-Mediated Enzymatic Methylation: A Tale of Two SAMs." Accounts Chem. Res. 2011, 45, 555-564.
2. Chan, et al. "The Mechanisms of Radical SAM/Cobalamin Methylations: An Evolving Working Hypothesis." ChemBioChem, 2013, doi: 10.1002/cbic.201200762
3. Pierre et al. "Thiostrepton tryptophan methyltransferase expands the chemistry of radical SAM enzymes." Nat. Chem. Biol., 2012, 8, 957-959. doi: 10.1038/nchembio.1091