Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone

Abstract

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK, 8-MMK and 7,8-DMMK were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.

Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea

“…S1). The latter is named after the biochemically characterized coproporphyrinogen III oxidase HemN, which catalyzes the oxygen-independent decarboxylation of two propionate side chains in haem biosynthesis (Layer et al, 2003(Layer et al, , 2006Broderick et al, 2014;Hein et al, 2018;Jin et al, 2018). Thus, in contrast to class C RSMTs, HemN does not catalyse a methylation reaction.…”

“…MqnK/MenK and MenK2 catalyze similar reactions but methylate the naphthoquinone group of MK at different positions. MqnK/MenK enzymes have been generally suggested to methylate menaquinone specifically at position C-8 to obtain 8-MMK, while MenK2 has been envisaged to specifically methylate MK at position C-7 to produce 7-MMK (Hein et al, 2017(Hein et al, , 2018. Previously, the MenK proteins from Adlercreutzia equolifaciens and Shewanella oneidensis were individually produced in Escherichia coli and shown to promote the production of 8-MMK 8 and 8-methyl-2-demethylmenaquinone (8-MDMK 8 ) (Hein et al, 2017).…”

“…Composition and function of microbial quinone/quinol pools Membranous quinones are essential to the majority of life forms and are firmly established as an important biomarker in microbial taxonomy (Collins and Jones, 1981;Nowicka and Kruk, 2010). In anaerobic environments, the most important quinones are 2-demethylmenaquinone (DMK), menaquinone (MK) and its methylated derivatives 7-methylmenaquinone (7-MMK), 8-methylmenaquinone (8-MMK) and 7,8-dimethylmenaquinone (7,8-DMMK) (Hein et al, 2018). A single microorganism can synthesize a variety of quinones that collectively form the quinone/quinol pool in the membrane.…”

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

“…[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.…”