title: Reconstitution and Substrate Specificity of the Radical S-Adenosyl-methionine Thiazole C-Methyltransferase in Thiomuracin Biosynthesis.
abstract: Thiomuracin is a thiopeptide antibiotic with potent activity toward Gram-positive drug-resistant bacteria. Thiomuracin is biosynthesized from a precursor peptide, TbtA, by a complex array of posttranslational modifications. One of several intriguing transformations is the C-methylation of thiazole, occurring at an unactivated sp2carbon. Herein, we report the in vitro reconstitution of TbtI, the responsible radical S-adenosyl-methionine (rSAM) C-methyltransferase, which catalyzes the formation of 5-methylthiazole at a single site. Our studies demonstrate that a linear hexazole-bearing intermediate of TbtA is a substrate for TbtI whereas macrocyclized thiomuracin GZ is not. In determining the minimal substrate for TbtI, we found that the enzyme is functional when most of the leader peptide has been removed. The in vitro reconstitution of TbtI, a class C rSAM methyltransferase, further adds to the chemical versatility of rSAM enzymes, and informs on the complexity of thiomuracin biosynthesis.
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States.
authors.forename: Nilkamal
authors.initials: N
authors.lastname: Mahanta
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Zhengan
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Graham A
authors.initials: GA
authors.lastname: Hudson
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States.|Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
authors.forename: Wilfred A
authors.identifier: 0000-0002-5467-7071
authors.initials: WA
authors.lastname: van der Donk
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.|Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign , 1206 West Gregory Drive, Urbana, Illinois 61801, United States.
authors.forename: Douglas A
authors.identifier: 0000-0002-9564-0953
authors.initials: DA
authors.lastname: Mitchell
mesh_terms: D005609:Free Radicals / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000737:chemistry; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013844:Thiazoles / Q000737:chemistry
chemical_list: D005609:Free Radicals; D010456:Peptides, Cyclic; D013844:Thiazoles; C576049:thiomuracin A; D012436:S-Adenosylmethionine; D008780:Methyltransferases