title: Steady-state kinetics and molecular evolution of Escherichia coli MenD [(1R,6R)-2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase], an anomalous thiamin diphosphate-dependent decarboxylase-carboligase.
abstract: (1R,6R)-2-Succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase, or MenD, catalyzes the thiamin diphosphate- (ThDP-) dependent decarboxylation of 2-oxoglutarate, the subsequent addition of the resulting succinyl-ThDP moiety to isochorismate, and the delta-elimination of pyruvate to yield SHCHC, pyruvate, and carbon dioxide. The enzyme is part of a superfamily of ThDP-dependent 2-oxo acid decarboxylases that includes pyruvate decarboxylase, benzoylformate decarboxylase, and acetohydroxy acid synthase, among others. However, this is the only enzyme known to catalyze a Stetter-like 1,4-addition of a ThDP adduct to the beta-carbon of an unsaturated carboxylate. Herein we report properties of the MenD protein from Escherichia coli, including the results of the first steady-state kinetic studies of the SHCHC synthase reaction. The protein is a dimer and shows cooperativity with respect to both substrates. The enzyme prefers divalent manganese as its metal ion cofactor and shows no dependence on FAD. MenD, required for biosynthesis of menaquinone and phylloquinone, is found in the genomes of a wide range of bacteria, as well as that of the archaeon Halobacterium sp. NRC-1 and the eukaryote Arabidopsis thaliana. Sequence alignments with other members of the superfamily are used to predict amino acid residues likely to be important in the binding and activation of ThDP. A site-directed mutant that replaces the conserved glutamic acid residue (E55), predicted to interact with N1' of the aminopyrimidine ring, with glutamine was generated, with catastrophic results for catalysis. There is no evidence for the release of succinate semialdehyde as a product; therefore, EC 4.1.1.71 should not be used for this enzyme.
authors: Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada.
authors: Milan
authors: M
authors: Bhasin
authors: Jennifer L
authors: JL
authors: Billinsky
authors: David R J
authors: DR
authors: Palmer
mesh_terms: D000595:Amino Acid Sequence; D002827:Chorismic Acid / Q000737:chemistry / Q000378:metabolism; D053138:Cyclohexenes; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D019143:Evolution, Molecular; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D053138:Cyclohexenes; D029968:Escherichia coli Proteins; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; C052985:isochorismic acid; D008345:Manganese; C060510:2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase; D007652:Oxo-Acid-Lyases; D002827:Chorismic Acid; D013835:Thiamine Pyrophosphate