Dataset Information

Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase.

ABSTRACT: The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy- l-mannonate) has the "best" kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg (2+); the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy- l-rhamnonate (obtained by reduction of the product with NaBH 4). Like other members of the enolase superfamily, RhamD contains an N-terminal alpha + beta capping domain and a C-terminal (beta/alpha) 7beta-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining "20s loop" in the capping domain is extended in length and the "50s loop" is truncated. The ligands for the Mg (2+) are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth beta-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth beta-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg (2+)-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.

SUBMITTER: Rakus JF

PROVIDER: S-EPMC2562705 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase.

Rakus John F JF Fedorov Alexander A AA Fedorov Elena V EV Glasner Margaret E ME Hubbard Brian K BK Delli Joseph D JD Babbitt Patricia C PC Almo Steven C SC Gerlt John A JA

Biochemistry 20080829 38

The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy- l-mannonate) has the "best" kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from bo ...[more]

PMID: 18754693

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Project description: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.

Dataset Information

Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase.

Publications

Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase.

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