The thiostrepton A tryptophan methyltransferase TsrM catalyses a cob(II)alamin-dependent methyl transfer reaction.

Alhosna Benjdia,Olivier Berteau,Patricia Amara,Manon Carmona,Ruma Banerjee,Carmen Gherasim,Alain Guillot,Stéphane Pierre

doi:10.1038/ncomms9377

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a novel class of natural products including several antibiotics and bacterial toxins. In countless RiPP biosynthetic pathways, cobalamin-dependent radical SAM (B12/rSAM) enzymes play a pivotal role. In the biosynthetic pathway of the antibiotic and anti-cancer agent thiostrepton A, TsrM, a B12/rSAM enzyme, catalyses the transfer of a methyl group to an electrophilic carbon atom of tryptophan. Here we show that methylcob(III)alamin is the probable physiological enzyme cofactor, and cob(II)alamin rather than cob(I)alamin is a key reaction intermediate. Furthermore, we establish that TsrM and a triple-alanine mutant alkylate cob(II)alamin efficiently leading to the synthesis of MeCbl. Exploiting TsrM substrate ambiguity, we demonstrate that TsrM does not catalyse substrate H-atom abstraction like most radical SAM enzymes. Based on these data, we propose an unprecedented radical-based C-methylation mechanism, which further expands the chemical versatility of rSAM enzymes.

Highlights

Synthesized and post-translationally modified peptides (RiPPs) are a novel class of natural products including several antibiotics and bacterial toxins
We recently identified TsrM as the B12/rSAM enzyme responsible for this unconventional methyl transfer reaction and demonstrated that its activity is dependent on S-adenosyl-L-methionine (SAM) and the presence of a cobalamin derivative[1]
We propose an unprecedented mechanism for enzymatic C-methylation that explains how TsrM, and possibly other B12/rSAM enzymes, catalyses efficient methyl transfer to an unsaturated electrophilic carbon atom in Ribosomally synthesized and post-translationally modified peptides (RiPPs) biosynthesis

Summary

Introduction

Synthesized and post-translationally modified peptides (RiPPs) are a novel class of natural products including several antibiotics and bacterial toxins. More recently a novel B12/rSAM enzyme, GenK, a key enzyme in the gentamicin biosynthetic pathway, was shown to catalyse methyl transfer to an unactivated carbon with the concomitant production of 50-deoxyadenosine (50-dA)[7] This latter result demonstrated that GenK belongs to the superfamily of radical SAM enzymes and suggested that GenK, like most other rSAM enzymes investigated so far, produces a 50-deoxyadenosyl radical (50-dA) for the radical activation of its substrate[11]. TsrM exhibits high turnover and a broad substrate ambiguity unlike other radical SAM enzymes investigated to date These data are consistent with TsrM making unique use of MeCbl and cob(II)alamin being a central intermediates during catalysis. We propose an unprecedented mechanism for enzymatic C-methylation that explains how TsrM, and possibly other B12/rSAM enzymes, catalyses efficient methyl transfer to an unsaturated electrophilic carbon atom in RiPPs biosynthesis

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Conclusion