J Michael Sauder - Computation-facilitated assignment of the function in the enolase superfamily: a regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis

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      Publication Details (including relevant citation   information):

      Biochemistry (2009) 48: 11546-11558.

      Rakus JF, Kalyanaraman C, Fedorov AA, Fedorov EV, Mills-Groninger   FP, Toro R, Bonanno J, Bain K, Sauder JM, Burley SK, Almo SC,   Jacobson MP, Gerlt JA

      Abstract:

      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.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi901731c