5EJM

ThDP-Mn2+ complex of R413A variant of EcMenD soaked with 2-ketoglutarate for 35 min

Summary for 5EJM

• Entry DOI: 10.2210/pdb5ejm/pdb
• Related: 5EJ4 5EJ5 5EJ6 5EJ7 5EJ8 5EJ9 5EJA
• Descriptor: 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase, (4~{R})-4-[3-[(4-azanyl-2-methyl-pyrimidin-5-yl)methyl]-4-methyl-5-[2-[oxidanyl(phosphonooxy)phosphoryl]oxyethyl]-1,3-thiazol-3-ium-2-yl]-4-oxidanyl-butanoic acid, MANGANESE (II) ION, ... (6 entities in total)
• Functional Keywords: post-decarboxylation intermediate of r413a variant of ecmend, transferase
• Biological source: Escherichia coli K12
• Total number of polymer chains: 8
• Total formula weight: 496673.34

Authors

Song, H.G.,Dong, C.,Chen, Y.Z.,Sun, Y.R.,Guo, Z.H. (deposition date: 2015-11-02, release date: 2016-11-02, Last modification date: 2024-03-20)

Primary citation

Qin, M.M.,Song, H.G.,Dai, X.,Chen, Y.Z.,Guo, Z.H.
Two active site arginines are critical determinants of substrate binding and catalysis in MenD, a thiamine-dependent enzyme in menaquinone biosynthesis.
Biochem. J., 2018

PubMed Abstract: The 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 C-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 C-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° relative to the native intermediate, resulting in the 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 non-native mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.

PubMed: 30341164
DOI: 10.1042/BCJ20180548

Experimental method

X-RAY DIFFRACTION (1.72 Å)