Inhibiting MenD : an essential enzyme in menaquinone biosynthesis in mycobacterium tuberculosis. (2020)
Type of ContentTheses / Dissertations
Degree NameMaster of Science
PublisherUniversity of Canterbury
AuthorsO’Rourke, Connorshow all
Menaquinone (MQ) is an important electron carrier for electron transport across the membrane, which as a result is essential for energy generation for many bacteria in anaerobic and aerobic respiration. MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate [SEPHCHC] synthase) is the enzyme responsible for the first committed step in the classical MQ biosynthesis pathway and as such is a central element for investigation into the inhibition of the classical MQ pathway.
The aim of this thesis was to purify a newly developed construct of Smeg-MenD that could have the His-tag cleaved, and to use this and MenD enzymes from E. coli (purified in this thesis) and S. aureus (obtained purified) to expand upon previous work from the Johnston laboratory characterising the MenD from M. tuberculosis. This thesis details the results from two different bioanalytical techniques (intrinsic protein fluorescence and differential scanning fluorimetry) to probe the MenD interaction with a variety of well-known natural ligands (both allosteric- and active-site binders) and a small set of potential inhibitors of the MenD enzyme.
Differential scanning fluorimetry proved to be the most useful technique for investigation of the MenD enzymes, with the small volumes required, ability of replicates, and the broad conditions of ligands that were tested proving invaluable from this technique. DHNA, the ligand that binds to the allosteric site in Mtb-MenD was found to have an effect on MenD from all three species investigated in this work, suggesting the potential for an allosteric site to be present in all MenD species. Three of the potential inhibitors screened also displayed potential as inhibitors and warrant further investigation into the effectiveness of these ligands.