Menaquinone (MK) serves as an essential membranous redox mediator in several electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The class C radical SAM methyltransferases (RSMTs) MenK, MqnK and MenK2 have been shown to facilitate specific menaquinone methylation reactions at position C-8 (MenK/MqnK) or C-7 (MenK2) to synthesise 8-methylmenaquinone, 7-methylmenaquinone or 7,8-dimethylmenaquinone [1-3]. In addition, primary structure motifs of the MenK/MqnK/MenK2 family allowed a functional categorisation [4].

Here, the functional MenK and MenK2 enzymes from Collinsella tanakaei were produced in Escherichia coli. (Methyl)menaquinone species from the respective cells were separated and identified by UV/Vis spectroscopy and mass spectrometry. The activity of purified and Fe/S centre-reconstituted MenK and MenK2 was investigated using a specific in vitro MK methylation assay. The biochemical analysis was supported by AlphaFold structure prediction, which suggested the architecture of the catalytic site and a putative MK entry cavity, thus helping to predict a universal reaction mechanism of MK methylation.

References

1 Wilkens, D. and Simon, J. (2023) Biosynthesis and function of microbial methylmenaquinones. Adv. Microb. Physiol. 83, 1-58.

2 Hein, S., von Irmer, J., Gallei, M., Meusinger, R. and Simon, J. (2018) Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone. Biochim. Biophys. Acta 1859, 300-308

3 Hein, S., Klimmek, O., Polly, M., Kern, M. and Simon, J. (2017) A class C radical S-adenosylmethionine methyltransferase synthesizes 8-methylmenaquinone. Mol. Microbiol. 104, 449-462.

4 Wilkens, D., Meusinger, R., Hein, S. and Simon, J. (2021) Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in Bacteria and Archaea. Environ. Microbiol. 23, 1407-1421.