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

Abstract

TsrM methylates C2 of the indole ring of L-tryptophan (Trp) during the biosynthesis of the quinaldic acid moiety of thiostrepton. It is annotated as a cobalamin-dependent radical S -adenosylmethionine (SAM) methylase; however, TsrM does not reductively cleave SAM to the universal 5ʹ-deoxyadenosyl 5ʹ-radical intermediate, a hallmark of radical-SAM (RS) enzymes. Herein, we report structures of TsrM from Kitasatospora setae , the first of a cobalamin-dependent radical SAM methylase. Unexpectedly, the structures show an essential arginine residue that resides in the proximal coordination sphere of the cobalamin cofactor and a [4Fe–4S] cluster that is ligated by a glutamyl residue and three cysteines in a canonical CxxxCxxC RS motif. Structures in the presence of substrates suggest a substrate-assisted mechanism of catalysis, wherein the carboxylate group of SAM serves as a general base to deprotonate N1 of the tryptophan substrate, facilitating formation of a C2 carbanion.

Structural basis for carbapenamC-alkylation by TokK, a B12-dependent radical SAM enzyme

“…Interestingly, despite low sequence identity (~29%) between CysS and TokK or ThnK, all three proteins are located within the same node of a sequence similarity network (SSN) composed of uses cobalamin in an unknown fashion to catalyze a complex ring contraction of 2'deoxyadenosine monophosphate (dAMP) (Figure S3) (14). TsrM methylates an sp 2 -hybridized carbon, C2, of L-tryptophan (Trp) by a polar mechanism (Figure S3) (11). TsrM is distinctive among all RS enzymes because it does not catalyze the formation of 5'-dA• during catalysis (11).…”

“…TsrM methylates an sp 2 -hybridized carbon, C2, of L-tryptophan (Trp) by a polar mechanism (Figure S3) (11). TsrM is distinctive among all RS enzymes because it does not catalyze the formation of 5'-dA• during catalysis (11).…”

“…Instead, TsrM uses SAM's carboxylate moiety as an acceptor of the N1 proton of Trp during C2 electrophilic substitution by MeCbl (11,15). Additionally, the structures of TsrM and OxsB have limitations that prevent full understanding of Cbl-dependent RS catalysis.…”

Computational Approaches: An Underutilized Tool in the Quest to Elucidate Radical SAM Dynamics

“…Co-expression of cellular machinery for Fe/S-cluster assembly along with careful handling under anoxic conditions can secure the canonical [4Fe-4S] cluster. The incorporation and instability of methylcobalamin (MeCbl), on the other hand, has proven more problematic, and recently emerged as the primary impediment to structural characterization of a true reactant complex for tryptophan 2C methyltransferase (TsrM), annotated as a rSAM enzyme [ 12 ]. TsrM catalyzes the methylation of L-tryptophan (Trp) at the C2 position during the biosynthesis of the macrocyclic peptide antibiotic thiostrepton A ( Figure 1 B) [ 13 ].…”

“…Experimental structure elucidation of the TsrM•Trp complex depicts an awkwardly oriented substrate molecule, the C2 position of which is too distant (7 Å) for methyl transfer from the cobalt ion ( Figure 1 C). To account for this inconsistency, the authors suggest that the observed binding mode is likely due to degradation of the MeCbl cofactor to aquocobalamin during crystallization and/or data collection [ 12 ]. They utilized the Glide docking program from the Schrödinger software suite to test this hypothesis following manual replacement of the observed cofactor by MeCbl ( Table 1 ) [ 14 ].…”