Discovery of function in the enolase superfamily

D-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup

Daniel J. Wichelecki, Bryan M. Balthazor, Anthony C. Chau, Matthew W. Vetting, Alexander A. Fedorov, Elena V. Fedorov, Tiit Lukk, Yury V. Patskovsky, Mark B. Stead, Brandan S. Hillerich, Ronald D. Seidel, Steven C. Almo, John Alan Gerlt

Research output: Contribution to journalArticle

Abstract

The continued increase in the size of the protein sequence databases as a result of advances in genome sequencing technology is overwhelming the ability to perform experimental characterization of function. Consequently, functions are assigned to the vast majority of proteins via automated, homology-based methods, with the result that as many as 50% are incorrectly annotated or unannotated (Schnoes et al. PLoS Comput. Biol. 2009, 5 (12), e1000605). This manuscript describes a study of the d-mannonate dehydratase (ManD) subgroup of the enolase superfamily (ENS) to investigate how function diverges as sequence diverges. Previously, one member of the subgroup had been experimentally characterized as ManD [dehydration of d-mannonate to 2-keto-3-deoxy-d-mannonate (equivalently, 2-keto-3-deoxy-d-gluconate)]. In this study, 42 additional members were characterized to sample sequence-function space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (kcat/KM = 103 to 104 M-1 s-1) for dehydration of d-mannonate, (2) low efficiency (kcat/KM = 101 to 102 M-1 s-1) for dehydration of d-mannonate and/or D-gluconate, and 3) no-activity with either d-mannonate or d-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes d-gluconate dehydratases (GlcDs) that are divergent from the GlcDs that have been characterized in the mandelate racemase subgroup of the ENS (Lamble et al. FEBS Lett. 2004, 576, 133-136) (Ahmed et al. Biochem. J. 2005, 390, 529-540). These observations signal caution for functional assignment based on sequence homology and lay the foundation for the studies of the physiological functions of the GlcDs and the promiscuous ManDs/GlcDs.

Original languageEnglish (US)
Pages (from-to)2722-2731
Number of pages10
JournalBiochemistry
Volume53
Issue number16
DOIs
StatePublished - Apr 29 2014

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gluconate dehydratase
Phosphopyruvate Hydratase
Dehydration
mandelate racemase
Sugar Acids
Protein Databases
Sequence Homology
Substrate Specificity
Proteins
Genes
mannonate dehydratase
mannonate
Genome
Technology

ASJC Scopus subject areas

  • Biochemistry

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Discovery of function in the enolase superfamily : D-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup. / Wichelecki, Daniel J.; Balthazor, Bryan M.; Chau, Anthony C.; Vetting, Matthew W.; Fedorov, Alexander A.; Fedorov, Elena V.; Lukk, Tiit; Patskovsky, Yury V.; Stead, Mark B.; Hillerich, Brandan S.; Seidel, Ronald D.; Almo, Steven C.; Gerlt, John Alan.

In: Biochemistry, Vol. 53, No. 16, 29.04.2014, p. 2722-2731.

Research output: Contribution to journalArticle

Wichelecki, DJ, Balthazor, BM, Chau, AC, Vetting, MW, Fedorov, AA, Fedorov, EV, Lukk, T, Patskovsky, YV, Stead, MB, Hillerich, BS, Seidel, RD, Almo, SC & Gerlt, JA 2014, 'Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup', Biochemistry, vol. 53, no. 16, pp. 2722-2731. https://doi.org/10.1021/bi500264p
Wichelecki, Daniel J. ; Balthazor, Bryan M. ; Chau, Anthony C. ; Vetting, Matthew W. ; Fedorov, Alexander A. ; Fedorov, Elena V. ; Lukk, Tiit ; Patskovsky, Yury V. ; Stead, Mark B. ; Hillerich, Brandan S. ; Seidel, Ronald D. ; Almo, Steven C. ; Gerlt, John Alan. / Discovery of function in the enolase superfamily : D-mannonate and d-gluconate dehydratases in the d-mannonate dehydratase subgroup. In: Biochemistry. 2014 ; Vol. 53, No. 16. pp. 2722-2731.
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AU - Balthazor, Bryan M.

AU - Chau, Anthony C.

AU - Vetting, Matthew W.

AU - Fedorov, Alexander A.

AU - Fedorov, Elena V.

AU - Lukk, Tiit

AU - Patskovsky, Yury V.

AU - Stead, Mark B.

AU - Hillerich, Brandan S.

AU - Seidel, Ronald D.

AU - Almo, Steven C.

AU - Gerlt, John Alan

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