Mechanistic studies of the MenD-catalyzed reaction
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MenD, a thiamin diphosphate (ThDP)-dependent enzyme, catalyzes the reaction from isochorismate (ISC) to 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC), and thus is also called SEPHCHC synthase. This conversion is the first committed step in the classical menaquinone (Vitamin K2) biosynthetic pathway, requiring 2-ketoglutarate (2-KG), ThDP and Mg2+. Since the biosynthesis of menaquinone is essential in some bacterial pathogens, for example Mycobacterium tuberculosis, MenD or the menaquinone pathway could be a target for drug development. The method for the kinetic assay of the MenD-catalyzed reaction was evaluated by comparing UV spectrophotomeric measurements and HPLC analysis. It was validated that the steady-state kinetics of the MenD-catalyzed reaction can be determined by monitoring UV absorbance of ISC at 278 nm and 300 nm. Phosphonate analogues of 2-KG were synthesized and assayed as inhibitors of the MenD reaction. It was found that the phosphonate analogues of 2-KG are competitive inhibitors with varied affinity for MenD. Of the inhibitors, monomethyl succinyl phosphonate (MMSP) was the most effective, with a Ki of 700 nM. However, the potent MenD inhibitors show no effectiveness against mycobacterial growth. An analogue of isochorismate, trans-(±)-5-carboxymethoxy-6-hydroxy-1,3-cyclohexadiene-1-carboxylate ((±)-CHCD), was synthesized. The (+)-CHCD was found to be an alternative substrate for the MenD-catalyzed reaction. When CHCD was utilized in the MenD reaction, 5-carboxymethoxy-2-(3-carboxy-propionyl)-6-hydroxy-cyclohex-2-enecarboxylate (CCHC) was isolated and characterized, which was believed to be the product of spontaneous isomerization of the SEPHCHC-like analogue. The kinetic study of MenD reaction using (±)-CHCD, in association with the kinetics pattern probed by MMSP, demonstrated for the first time that the MenD-catalyzed reaction has a Ping Pong bi bi kinetic mechanism. The analysis of sequence and structure of MenD from E. coli allowed the investigation of the active site residues and their catalytic functions by mutation of the individual residues. S32A, S32D, R33K, R33Q, E55D, R107K, Q118E, K292Q, R293K, S391A, R395A, R395K, R413K and I418L were prepared and assayed kinetically with respect to 2-KG, ISC, (±)-CHCD, ThDP and Mg2+. The values of Kma and kcata/Kma for the mutants, in comparison with that of wild type MenD, provide valuable insight into the catalytic mechanism of MenD.
DegreeDoctor of Philosophy (Ph.D.)
SupervisorPalmer, David R. J.
CommitteeHonek, John F.; Gravel, Michel; Dimmock, J. R.; Sanders, David A. R.; Ward, Dale E.
Copyright DateOctober 2010