Abstract
Quantum chemical calculations are used to investigate the detailed reaction mechanism of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase (also known as MenD), a thiamin diphosphate-dependent decarboxylase that catalyzes the formation of SEPHCHC from 2-ketoglutarate and isochorismate. This enzyme is involved in the menaquinone biosynthesis pathway in M. tuberculosis and is thought of as a potential drug target for anti-tuberculosis therapeutics. In addition, MenD shows promise as a biocatalyst for the synthesis of 1,4-functionalized compounds. Models of the active site are constructed on the basis of available X-ray structures, and the intermediates and transition states involved in the reaction mechanism are optimized and characterized. The calculated mechanism is in good agreement with prior kinetic studies and gives new insights into the mode of action of the enzyme. In particular, the structure and role of the tetrahedral post-decarboxylation intermediate observed in X-ray structures are discussed.
Highlights
MenD, formally known as 2-succinyl-5-enolpyruvyl-6-hydroxy3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase, is a thiamine diphosphate (ThDP)-dependent enzyme found in the menaquinone biosynthetic pathway that has attracted interest for more than 30 years
It has been proposed that selective inhibition of the menaquinone biosynthesis pathway in M. tuberculosis may provide a novel approach to anti-tuberculosis therapeutics.[9,10]
The results show that this structure is only 2 kcal/mol higher in energy than the unconstrained Int2-TdH+ intermediate and still more than 10 kcal/mol more stable than the IP forms of HSThDP
Summary
MenD, formally known as 2-succinyl-5-enolpyruvyl-6-hydroxy3-cyclohexene-1-carboxylic-acid (SEPHCHC) synthase, is a thiamine diphosphate (ThDP)-dependent enzyme found in the menaquinone biosynthetic pathway that has attracted interest for more than 30 years. MenD was reported to have unusual bifunctional activity, catalyzing both the 1,4addition of 2-ketoglutarate (2KG) to isochorismate (IC) and the subsequent δ-elimination of pyruvate to form 2-succinyl-6hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC).[1,2] Some time later, the pyruvate elimination was shown to require a second enzyme, MenH,[3−5] and it is accepted that the native reaction catalyzed by MenD yields only SEPHCHC (Scheme 1). It has been proposed that selective inhibition of the menaquinone biosynthesis pathway in M. tuberculosis may provide a novel approach to anti-tuberculosis therapeutics.[9,10] The fact that it catalyzes the first committed step in that pathway, when combined with the knockout results, has seen a number of studies focusing on the structure and mechanism of MenD as an aid to inhibitor design
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