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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), el000605). 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 n-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 n-mannonate, (2) low efficiency (kcat/KM = 101 to 102 M-1 s-1) for dehydration of n-mannonate and/or D-gluconate, and 3) no-activity with either n-mannonate or n-gluconate (or any other acid sugar tested). Thus, the ManD subgroup is not isofunctional and includes n-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. [ABSTRACT FROM AUTHOR]