Publications

• Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2.

  Yew W.S., Fedorov A.A., Fedorov E.V., Almo S.C., Gerlt J.A.

  We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like ... >> More

  We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture. << Less

  Biochemistry 46:9564-9577(2007) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Computation-facilitated assignment of the function in the enolase superfamily: a regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis.

  Rakus J.F., Kalyanaraman C., Fedorov A.A., Fedorov E.V., Mills-Groninger F.P., Toro R., Bonanno J., Bain K., Sauder J.M., Burley S.K., Almo S.C., Jacobson M.P., Gerlt J.A.

  The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI 23100298, IMG locus tag Ob2843, PDB entry 2OQY ) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg(2+) ions located 10.4 A from ... >> More

  The structure of an uncharacterized member of the enolase superfamily from Oceanobacillus iheyensis (GI 23100298, IMG locus tag Ob2843, PDB entry 2OQY ) was determined by the New York SGX Research Center for Structural Genomics (NYSGXRC). The structure contained two Mg(2+) ions located 10.4 A from one another, with one located in the canonical position in the (beta/alpha)(7)beta-barrel domain (although the ligand at the end of the fifth beta-strand is His, unprecedented in structurally characterized members of the superfamily); the second is located in a novel site within the capping domain. In silico docking of a library of mono- and diacid sugars to the active site predicted a diacid sugar as a likely substrate. Activity screening of a physical library of acid sugars identified galactarate as the substrate (k(cat) = 6.8 s(-1), K(M) = 620 microM, k(cat)/K(M) = 1.1 x 10(4) M(-1) s(-1)), allowing functional assignment of Ob2843 as galactarate dehydratase (GalrD-II). The structure of a complex of the catalytically impaired Y90F mutant with Mg(2+) and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the alpha-proton and Tyr 90 as the acid that facilitates departure of the beta-OH leaving group. The enzyme product is 2-keto-d-threo-4,5-dihydroxyadipate, the enantiomer of the product obtained in the GalrD reaction catalyzed by a previously characterized bifunctional l-talarate/galactarate dehydratase (TalrD/GalrD). On the basis of the different active site structures and different regiochemistries, we recognize that these functions represent an example of apparent, not actual, convergent evolution of function. The structure of GalrD-II and its active site architecture allow identification of the seventh functionally and structurally characterized subgroup in the enolase superfamily. This study provides an additional example in which an integrated sequence- and structure-based strategy employing computational approaches is a viable approach for directing functional assignment of unknown enzymes discovered in genome projects. << Less

  Biochemistry 48:11546-11558(2009) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Evolution of enzymatic activities in the enolase superfamily: characterization of the (D)-glucarate/galactarate catabolic pathway in Escherichia coli.

  Hubbard B.K., Koch M., Palmer D.R., Babbitt P.C., Gerlt J.A.

  The genes encoding the enzymes in the (D)-glucarate/galactarate catabolic pathway have been identified in the Escherichia coli genome. These encode, in three transcriptional units, (D)-glucarate dehydratase (GlucD), galactarate dehydratase, 5-keto-4-deoxy-(D)-glucarate aldolase, tartronate semiald ... >> More

  The genes encoding the enzymes in the (D)-glucarate/galactarate catabolic pathway have been identified in the Escherichia coli genome. These encode, in three transcriptional units, (D)-glucarate dehydratase (GlucD), galactarate dehydratase, 5-keto-4-deoxy-(D)-glucarate aldolase, tartronate semialdehyde reductase, a glycerate kinase that generates 2-phosphoglycerate as product, and two hexaric acid transporters. We also have identified a gene proximal to that encoding GlucD that encodes a protein that is 72% identical in primary sequence to GlucD (GlucD-related protein or GlucDRP). However, whereas GlucD catalyzes the efficient dehydration of both (D)-glucarate and (L)-idarate as well as their epimerization, GlucDRP is significantly impaired in both reactions. Perhaps GlucDRP is an example of gene duplication and evolution in progress in the E. coli chromosome. << Less

  Biochemistry 37:14369-14375(1998) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.