Discovery of Function in the Enolase Superfamily: d-Mannonate and d-Gluconate Dehydratases in the d-Mannonate Dehydratase Subgroup
2014
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 A.
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.
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