TY - JOUR
T1 - Mechanism of a Class C Radical S-Adenosyl- l -methionine Thiazole Methyl Transferase
AU - Zhang, Zhengan
AU - Mahanta, Nilkamal
AU - Hudson, Graham A.
AU - Mitchell, Douglas A.
AU - Van Der Donk, Wilfred A.
N1 - Funding Information:
This work was supported by the Howard Hughes Medical Institute (to W.A.V.), the National Institutes of Health (GM097142 to D.A.M.), and a Seemon Pines Fellowship from the Department of Chemistry at the University of Illinois at Urbana−Champaign (to G.A.H.). Constructs for expression of flavodoxin/flavodoxin reductase were generously provided by the laboratory of Squire J. Booker (Penn State University), and constructs for SAM synthetase were provided by Vahe Bandarian (University of Utah).
Publisher Copyright:
© 2017 American Chemical Society.
PY - 2017/12/27
Y1 - 2017/12/27
N2 - 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.
AB - 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.
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U2 - 10.1021/jacs.7b10203
DO - 10.1021/jacs.7b10203
M3 - Article
C2 - 29190095
AN - SCOPUS:85038583739
SN - 0002-7863
VL - 139
SP - 18623
EP - 18631
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 51
ER -