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.

Expanding the Chemistry of the Class C Radical SAM Methyltransferase NosN by Using an Allyl Analogue of SAM

“…Characterization of the enzymecatalyzed tAdoH alkylation by HSL explained the production of SAH by NosN and several other class CR SMTs. [7,8,12] We would also like to point out that according to our DFT calculations using model chemicals,t he dAdo-radical-mediated hydrogen abstraction from the SAM methyl group appears to be only slightly endothermic ( Figure S10), and as imilar observation was also shown in an early report by Hioe and Zipse. [16] Hence although the sulfonium radical 7 is not involved in the NosN reaction, the possibility of its involvement in other radical-SAM-dependent reactions cannot be excluded.…”

“…[3] In contrast to the vast majority of SAM-dependent methylation reactions,w hich proceed through an S N 2m echanism, radical-SAM-dependent methyltransferases (RSMTs) utilize diverse radical-based mechanisms and are grouped into several classes.T he class AR SMTs,r epresented by the ribosome methyltransferases RlmN and Cfr,t ransfer the SAM methyl group to ap rotein-based Cys residue and use this Cys-linked methyl group as the direct methyl donor in the reaction. [3a,d] Thus far,biochemically characterized class C RSMT members include YtkT,w hich is involved in yatakemycin biosynthesis, [6] ChuW,w hich is involved in heme degradation, [7] TbtI, which is involved in thiomuracin biosynthesis, [8] MenK and MenK2, which are involved in methylmenaquinone biosynthesis, [9] and NosN,w hich is involved in nosiheptide biosynthesis. [3e,f,5] Theclass CRSMTs are an emerging subfamily,w hich share significant sequence similarities with the coproporphyrinogen III oxidase HemN involved in Heme biosynthesis.…”

“…[3e,f,5] Theclass CRSMTs are an emerging subfamily,w hich share significant sequence similarities with the coproporphyrinogen III oxidase HemN involved in Heme biosynthesis. [3a,d] Thus far,biochemically characterized class C RSMT members include YtkT,w hich is involved in yatakemycin biosynthesis, [6] ChuW,w hich is involved in heme degradation, [7] TbtI, which is involved in thiomuracin biosynthesis, [8] MenK and MenK2, which are involved in methylmenaquinone biosynthesis, [9] and NosN,w hich is involved in nosiheptide biosynthesis. [10] Unlike most members of the radical SAM superfamily,these enzymes likely bind two SAM molecules simultaneously in the active site,u sing one SAM molecule to produce ad Ado radical and the second as amethyl donor.T he fact that HemN binds two SAMs in the active site is consistent with this proposal.…”

Hybridization Biosensors Relying on Electrical Properties of Nucleic Acids

“…One of the most widely used non-specific indicators of DNAh ybridization is negatively charged [Fe(CN) 6 ] 3À/4À : it is electrostatically repelled by the negatively charged ssDNAm oleculesa nd DNAh ybridization producesa dditional repulsion of ferricyanide from the DNA-modified electrode surface and slowsd own its electrochemical transformation.T he positively charged ruthenium hexamine [Ru(NH 3 ) 6 ] 3 + reacts with DNAi nt he opposite way, replacingt he monovalent charge compensating anions from the sugar-phosphate backbone of DNAa nd binding stoichiometrically to DNAsp hosphates.T his binding forms the basis for quantitative chronocoulometric analysis of surface amounts of DNA, G DNA (mol cm À2 ), attached to the electrodes urface [5a] where m is the numbero fD NA basesi nt he sequence, z is the redox indicator charge,a nd the surface concentration G RuHex of Ru hexamine boundt oD NA is deduced by separating the double-layer capacitivea nd redox indicator diffusionc harges from the total chargea ssociated with Ru hexamine.T herewith, [Ru(NH 3 ) 6 ] 3 + can form an electronic wire on the DNAs urfacec apable of shuttling electrons with ET rates exceeding 600 s À1 [21],a nd one of possibly the best works exploiting wiring properties of Ru hexamine is af emtomolar DNAs andwich assayw ith DNA-modified Au nanoparticles as ar eporter [22].…”

Bio‐Derived Co2P Nanoparticles Supported on Nitrogen‐Doped Carbon as Promising Oxygen Reduction Reaction Electrocatalyst for Anion Exchange Membrane Fuel Cells

“…Recently, synthesis of such composite materials using various biomaterials and their performance as electrocatalysts have been reported. [19][20][21][22] Bean sprouts, as a living plant, contain rich protein and cellulose which can be precursor for carbon matrix. In addition, various intrinsic heteroatoms of the organism make capable homogenous doping into the carbon matrix to enhance the catalytic activity.…”