Methods for studying ThDP-dependent MenD enzymes

Bacterial MenD enzymes (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC) synthases), involved in the biosynthesis of the redox carrier molecule menaquinone (vitamin K₂), belong to the decarboxylase family of thiamine diphosphate (ThDP)-dependent enzymes. MenD is of interest for antimicrobial design because menaquinone plays an essential role in bacterial energy generation. It is also a valuable bioengineering target for creating biocatalysts, and for improving fermentative vitamin K₂ production, due to the high utility of ThDP-dependent enzymes and the importance of menaquinone as a dietary-derived cofactor in human blood clotting and bone metabolism. A range of tools and methods have been deployed to study wildtype and bioengineered (mutant) MenD enzymes to understand and manipulate their activity, structure, and allosteric regulation. Allosteric regulation is best characterized in mycobacterial enzymes, such as MenD from the slow-growing pathogen Mycobacterium tuberculosis (Mtb-MenD), for which there are added challenges in obtaining soluble and stable protein. This chapter focuses on selected methods used for the production and purification of Mtb-MenD in the Mycobacterium smegmatis expression system, characterization using intrinsic fluorescence quenching for allosteric inhibitor binding, and assays for measuring activity and inhibition. Where applicable, extension of these methods to the study of other MenD enzymes is also described.