5ejm | pdb_00005ejm
ThDP-Mn2+ complex of R413A variant of EcMenD soaked with 2-ketoglutarate for 35 min
Structural highlights
FunctionMEND_ECOLI Catalyzes the thiamine diphosphate-dependent decarboxylation of 2-oxoglutarate and the subsequent addition of the resulting succinic semialdehyde-thiamine pyrophosphate anion to isochorismate to yield 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC).[1] [2] Publication Abstract from PubMedThe bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active-inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active-site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C2-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent, with the C2-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180 degrees relative to the native intermediate, resulting in reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a nonnative mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the nonnative binding site of the inactive MenD intermediate uncovered here provides a new target for development of antibiotics. Two active site arginines are critical determinants of substrate binding and catalysis in MenD, a thiamine-dependent enzyme in menaquinone biosynthesis.,Qin M, Song H, Dai X, Chen Y, Guo Z Biochem J. 2018 Oct 19. pii: BCJ20180548. doi: 10.1042/BCJ20180548. PMID:30341164[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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