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Discovery of function in the enolase superfamily: D-mannonate and D-gluconate dehydratases in the D-mannonate dehydratase subgroup.

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) Biochemistry 53, 2722-31. PMCID: PMC4007978

Exploration of the mannonate dehydratase (ManD) subgroup of the Enolase Superfamily, entailing the characterization of 42 mis-/unannotated proteins, brings resolution to the relationship between catalytic efficiency and substrate specificity, which may vary dramatically between proteins that share as much as 70% sequence identity. The following study addressly broadly impactful concepts, such as determinants of physiologically relevant values of catalytic efficiency.

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 = 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.

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Abstract Image

Figure 1: “Denotes the structural features of the ManDs (PDB 2QJJ). On the left, the “150–180s” loop (blue), TIM barrel (red), and capping domain (green) are displayed. The right inset shows the active site residues: metal binding Asp210, Glu236, Glu262 (green); Tyr157-Arg149 catalytic dyad (magenta); acidic His212 (blue); and conserved His315 at the end of the 7th β-strand (red). The d-mannonate ligand from the 2QJM structure is shown in light blue.

Scheme

Figure 2 “Sequence similarity networks (SSNs) of the ManD subgroup at several e-value thresholds to illustrate the effect of increasing stringency on clustering. Panel A, 10–80, 35% identity. Panel B, 10–120, 45% identity. Panel C, 10–190, 75% identity. Pink coloring indicates proteins predicted to be ManDs by the Structure Function Linkage Database. Green coloring indicates proteins that were purified and subjected to activity screening.

Figure 3: “SSN (e-value threshold of at 10–190) showing the distribution of high- (green), low- (blue), and no-activity (red) proteins along with substrate specificities (M, d-mannonate; G, d-gluconate; M/G, d-mannonate and d-gluconate). Proteins for which structures were determined are marked with asterisks. The Pro and Ala residues associated with different substrate specificities for d-mannonate and d-gluconate are located in separate clusters: clusters 1, 4, 5, 6, 8, 9, and 10 contain Pro; clusters 2 and 7 contain Ala; and cluster 3 contains both. Pro-containing clusters exhibit low or no dehydration activity; Ala-containing clusters exhibit high dehydration activity with d-mannonate.

Figure 4: A superposition of a structure with d-mannonate bound in the active site (2QJM, NaManD) with one with d-gluconate bound in the active site (3TWB; CsManD). In 2QJM, Tyr 161 is the general base that abstracts the 2-proton and His 215 is the general acid that catalyzes the departure of the 3-OH group from d-mannonate. In 3TWB, His 315 is proposed to be the general base that abstracts the 2-proton from d-gluconate or hydrogen bonds with the C5 hydroxyl of d-mannonate. The ε-nitrogen of His 315 is 3.0 Å from the C5 hydroxyl of d-mannonate and 3.1 Å from C2 of d-gluconate. Both distances are appropriate for proton abstraction or hydrogen bonding.

Figure 5: A superposition of a structure with d-mannonate bound in the active site (2QJM, NaManD) with one with d-gluconate bound in the active site (3TWB; CsManD). In 2QJM, Tyr 161 is the general base that abstracts the 2-proton and His 215 is the general acid that catalyzes the departure of the 3-OH group from d-mannonate. In 3TWB, His 315 is proposed to be the general base that abstracts the 2-proton from d-gluconate or hydrogen bonds with the C5 hydroxyl of d-mannonate. The ε-nitrogen of His 315 is 3.0 Å from the C5 hydroxyl of d-mannonate and 3.1 Å from C2 of d-gluconate. Both distances are appropriate for proton abstraction or hydrogen bonding.

Figure 6: An overlay of CsManD (4F4R, blue ribbon, orange loop) and NaManD (2QJJ, tan ribbon, green loop) showing the regions of their “150–180s” loops that were mutated. The sequences of the loops are given with the area of mutagenesis highlighted with yellow.

Figure 7: An overlay of the active sites of NaManD (2QJJ, high-activity, d-mannonate specific - red), CsManD (4F4R, low-activity, promiscuous for d-mannonate/d-gluconate - blue), Uniprot ID D4GJ14 (3T6C, low-activity, d-gluconate specific - green), and Uniprot ID A4W7D6 (3TJI, no-activity - magenta). The metal binding and acid/base residues are superimposable. The Pro/Ala dimorphism is also shown. The ligands are d-mannonate from 2QJM (red) and d-gluconate from 3T6C (green).

Reprinted with permission from Biochemistry. Copyright 2014 American Chemical Society.