Discovery of function in the enolase superfamily: D-mannonate and d-gluconate dehydratases in the D-mannonate dehydratase subgroup.
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 = 10(3) to 10(4) M(-1) s(-1)) for dehydration of D-mannonate, (2) low efficiency (kcat/KM = 10(1) to 10(2) 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.
Departments of Biochemistry and Chemistry and Institute for Genomic Biology, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.
Daniel J
DJ
Wichelecki
Bryan M
BM
Balthazor
Anthony C
AC
Chau
Matthew W
MW
Vetting
Alexander A
AA
Fedorov
Elena V
EV
Fedorov
Tiit
T
Lukk
Yury V
YV
Patskovsky
Mark B
MB
Stead
Brandan S
BS
Hillerich
Ronald D
RD
Seidel
Steven C
SC
Almo
John A
JA
Gerlt
D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005942:Gluconates / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013400:Sugar Acids / Q000378:metabolism
D005942:Gluconates; D013400:Sugar Acids; C014078:mannonate; D006836:Hydro-Lyases; D010751:Phosphopyruvate Hydratase; C020576:gluconate dehydratase; C021545:mannonate dehydratase; C030691:gluconic acid