Mechanism of a Class C Radical S-Adenosyl-l-methionine Thiazole Methyl Transferase

Abstract

The past decade has seen the discovery of four different classes of radical S-adenosylmethionine (rSAM) methyltransferases that methylate unactivated carbon centers. Whereas the mechanism of class A is well understood, the molecular details of methylation by classes B-D are not. In this study, we present detailed mechanistic investigations of the class C rSAM methyltransferase TbtI involved in the biosynthesis of the potent thiopeptide antibiotic thiomuracin. TbtI C-methylates a Cys-derived thiazole during posttranslational maturation. Product analysis demonstrates that two SAM molecules are required for methylation and that one SAM (SAM1) is converted to 5'-deoxyadenosine and the second SAM (SAM2) is converted to S-adenosyl-l-homocysteine (SAH). Isotope labeling studies show that a hydrogen is transferred from the methyl group of SAM2 to the 5'-deoxyadenosine of SAM1 and the other two hydrogens of the methyl group of SAM2 appear in the methylated product. In addition, a hydrogen appears to be transferred from the β-position of the thiazole to the methyl group in the product. We also show that the methyl protons in the product can exchange with solvent. A mechanism consistent with these observations is presented that differs from other characterized radical SAM methyltransferases.

Figure 1

Product analysis for the methyl transfer reaction catalyzed by TbtI. (a) Structure of thiomuracin A1. The methyl group that is introduced by TbtI is shown in red. (b) Regioselective methylation of the thiazole at position 4 of the TbtA core peptide by TbtI. The Val-Gly-Ala sequence at the N-terminus of peptide 1 originates from the leader peptide. (c) Time-dependent formation of the methylated TbtA peptide, 5′-dA, and SAH. Reaction mixtures included the following components: TbtA hexazole (50 μM), [4Fe–4S] cluster reconstituted TbtI (10 μM), SAM (1 mM), flavodoxin (10 μM), flavodoxin reductase (10 μM), NAPDH (2 mM) in reaction buffer (50 mM Tris–HCl, pH 7.5).

Figure 2

Proposed potential reaction mechanisms for TbtI. 5′-dA• is generated from SAM1 in a process mediated by the reduced [4Fe–4S] cluster. The 5′-dA• then abstracts a hydrogen atom from the methyl group of SAM2, and the resulting radical adds to the thiazole. An active site base deprotonates radical 3, leading to the elimination of SAH, yielding radical 4. Three different pathways (a–c) can provide the product and reset the enzyme. In pathway a, the radical is reduced to the anion 5 and protonated. In pathway b, radical 4 abstracts a hydrogen atom from an active site amino acid (X–H). X–H could be the protonated base B–H. In pathway c, radical 4 abstracts a hydrogen atom from the methyl group of 5′-dA. Ade = adenine.

Figure 3

ESI mass spectra to investigate the origin of the methyl protons. ¹³C- and ¹⁵N-depleted substrate peptides were obtained as described (see the Experimental Section). (a) Spectra showing the doubly charged ion for the hexazole-containing core peptide substrate 1 (red) and the methylated product obtained with CD₃-SAM in H₂O after 1 h (blue) or 16 h (black) reactions. (b) Spectra showing substrate (red) and product obtained with CD₃-SAM in D₂O (blue) and CD₃-5′,5′,4′,3′-D₄-SAM in D₂O (black) after 1 h. See Table S2 for calculated and observed masses.

Figure 4

A solvent exchangeable proton migrates from the β-position of thiazole 4 to the methyl group in the product. (a) Spectra showing the doubly charged core peptide of the TbtA hexazole substrate 2 that is deuterated at the β-carbon of each thiazole (red) and the corresponding product obtained with CD₃-SAM in D₂O (blue) in a single turnover reaction. (b) Spectra showing the doubly charged core peptide of the TbtA hexazole substrate 1 (red) and the corresponding product obtained with CD₃-SAM in D₂O (blue) in a TbtI-catalyzed single turnover reaction. See Table S2 for calculated and observed masses.