2X7J

Structure of the menaquinone biosynthesis protein MenD from Bacillus subtilis

Summary for 2X7J

• Entry DOI: 10.2210/pdb2x7j/pdb
• Descriptor: 2-SUCCINYL-5-ENOLPYRUVYL-6-HYDROXY-3-CYCLOHEXENE -1-CARBOXYLATE SYNTHASE, THIAMINE DIPHOSPHATE, MANGANESE (II) ION, ... (7 entities in total)
• Functional Keywords: transferase, metal-binding
• Biological source: BACILLUS SUBTILIS
• Total number of polymer chains: 4
• Total formula weight: 270849.09

Authors

Dawson, A.,Chen, M.,Fyfe, P.K.,Guo, Z.,Hunter, W.N. (deposition date: 2010-03-01, release date: 2010-07-14, Last modification date: 2024-05-08)

Primary citation

Dawson, A.,Chen, M.,Fyfe, P.K.,Guo, Z.,Hunter, W.N.
Structure and Reactivity of Bacillus Subtilis Mend Catalyzing the First Committed Step in Menaquinone Biosynthesis.
J.Mol.Biol., 401:253-, 2010

PubMed Abstract: The first committed step in the classical biosynthetic route to menaquinone (vitamin K(2)) is a Stetter-like conjugate addition of alpha-ketoglutarate with isochorismate. This reaction is catalyzed by the thiamine diphosphate and metal-ion-dependent 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate synthase (MenD). The medium-resolution (2.35 A) crystal structure of Bacillus subtilis MenD with cofactor and Mn(2+) has been determined. Based on structure-sequence comparisons and modeling, a two-stage mechanism that is primarily driven by the chemical properties of the cofactor is proposed. Hypotheses for the molecular determinants of substrate recognition were formulated. Five basic residues (Arg32, Arg106, Arg409, Arg428, and Lys299) are postulated to interact with carboxylate and hydroxyl groups to align substrates for catalysis in combination with a cluster of non-polar residues (Ile489, Phe490, and Leu493) on one side of the active site. The powerful combination of site-directed mutagenesis, where each of the eight residues is replaced by alanine, and steady-state kinetic measurements has been exploited to address these hypotheses. Arg409 plays a significant role in binding both substrates while Arg428 contributes mainly to binding of alpha-ketoglutarate. Arg32 and in particular Arg106 are critical for recognition of isochorismate. Mutagenesis of Phe490 and Ile489 has the most profound influence on catalytic efficiency, indicating that these two residues are important for binding of isochorismate and for stabilizing the cofactor position. These data allow for a detailed description of the structure-reactivity relationship that governs MenD function and refinement of the model for the catalytic intermediate that supports the Stetter-like conjugate addition.

PubMed: 20600129
DOI: 10.1016/J.JMB.2010.06.025

Experimental method

X-RAY DIFFRACTION (2.35 Å)