Discovery of Function in the Enolase Superfamily: d-Mannonate and d-Gluconate Dehydratases in the d-Mannona

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• 作者：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 A. Gerlt
• 刊名：Biochemistry
• 出版年：2014
• 出版时间：April 29, 2014
• 年：2014
• 卷：53
• 期：16
• 页码：2722-2731
• 全文大小：478K
• 年卷期：v.53,no.16(April 29, 2014)
• ISSN：1520-4995

文摘

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), d>e1000605d>). 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鈥揻unction space in the ManD subgroup. These were found to differ in both catalytic efficiency and substrate specificity: (1) high efficiency (k_cat/K_M = 10³ to 10⁴ M^鈥? s^鈥?) for dehydration of d-mannonate, (2) low efficiency (k_cat/K_M = 10¹ to 10² M^鈥? s^鈥?) 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鈥?/span>136) (Ahmed et al. Biochem. J. 2005, 390, 529鈥?/span>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.