5Z2P
ThDP-Mn2+ complex of R413K variant of EcMenD soaked with 2-ketoglutarate for 5 min
Summary for 5Z2P
| Entry DOI | 10.2210/pdb5z2p/pdb |
| Descriptor | 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase, (4S)-4-{3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-(2-{[(S)-hydroxy(phosphonooxy)phosphoryl]oxy}ethyl)-4-methyl-1,3lambda~5~-thiazol-2-yl}-4-hydroxybutanoic acid, MANGANESE (II) ION, ... (6 entities in total) |
| Functional Keywords | complex, transferase |
| Biological source | Escherichia coli (strain K12) |
| Total number of polymer chains | 8 |
| Total formula weight | 497147.62 |
| Authors | |
| 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., 475:3651-3667, 2018 Cited by 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: 30341164DOI: 10.1042/BCJ20180548 PDB entries with the same primary citation |
| Experimental method | X-RAY DIFFRACTION (2.3 Å) |
Structure validation
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