Computation-facilitated assignment of the function in the enolase superfamily

A regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis

John F. Rakus, Chakrapani Kalyanaraman, Alexander A. Fedorov, Elena V. Fedorov, Fiona P. Mills-Groninger, Rafael Toro, Jeffrey Bonanno, Kevin Bain, J. Michael Sauder, Stephen K. Burley, Steven C. Almo, Matthew P. Jacobson, John Alan Gerlt

Research output: Contribution to journalArticle

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 Mg2+ ions located 10.4 Å from one another, with one located in the canonical position in the (β/α)7β-barrel domain (although the ligand at the end of the fifth β-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 (kcat=6.8 s-1, KM=620 μM, k cat/KM= 1.1 × 104 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 Mg2+ and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the α-proton and Tyr 90 as the acid that facilitates departure of the β-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.

Original languageEnglish (US)
Pages (from-to)11546-11558
Number of pages13
JournalBiochemistry
Volume48
Issue number48
DOIs
StatePublished - Dec 8 2009

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Hydro-Lyases
Phosphopyruvate Hydratase
Sugars
Catalytic Domain
Sugar Acids
Enantiomers
Substrates
Enzymes
Protons
Screening
Genes
Ions
Genomics
Ligands
Computer Simulation
Libraries
Acids
Cats
Genome
Research

ASJC Scopus subject areas

  • Biochemistry

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Computation-facilitated assignment of the function in the enolase superfamily : A regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis. / Rakus, John F.; Kalyanaraman, Chakrapani; Fedorov, Alexander A.; Fedorov, Elena V.; Mills-Groninger, Fiona P.; Toro, Rafael; Bonanno, Jeffrey; Bain, Kevin; Sauder, J. Michael; Burley, Stephen K.; Almo, Steven C.; Jacobson, Matthew P.; Gerlt, John Alan.

In: Biochemistry, Vol. 48, No. 48, 08.12.2009, p. 11546-11558.

Research output: Contribution to journalArticle

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 2009, 'Computation-facilitated assignment of the function in the enolase superfamily: A regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis', Biochemistry, vol. 48, no. 48, pp. 11546-11558. https://doi.org/10.1021/bi901731c
Rakus, John F. ; Kalyanaraman, Chakrapani ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Mills-Groninger, Fiona P. ; Toro, Rafael ; Bonanno, Jeffrey ; Bain, Kevin ; Sauder, J. Michael ; Burley, Stephen K. ; Almo, Steven C. ; Jacobson, Matthew P. ; Gerlt, John Alan. / Computation-facilitated assignment of the function in the enolase superfamily : A regiochemically distinct galactarate dehydratase from Oceanobacillus iheyensis. In: Biochemistry. 2009 ; Vol. 48, No. 48. pp. 11546-11558.
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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 Mg2+ ions located 10.4 {\AA} from one another, with one located in the canonical position in the (β/α)7β-barrel domain (although the ligand at the end of the fifth β-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 (kcat=6.8 s-1, KM=620 μM, k cat/KM= 1.1 × 104 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 Mg2+ and galactarate allowed identification of a Tyr 164-Arg 162 dyad as the base that initiates the reaction by abstraction of the α-proton and Tyr 90 as the acid that facilitates departure of the β-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.",
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