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O-GlcNAcase: promiscuous hexosaminidase or key regulator of O-GlcNAc signaling?

Alonso J, Schimpl M, van Aalten DM - J. Biol. Chem. (2014)

Bottom Line: O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins.The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches.We identify several areas of "known unknowns" that would benefit from future research, such as the enigmatic C-terminal domain of OGA.

View Article: PubMed Central - PubMed

Affiliation: From the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit and.

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Crystal structures of CpOGA and OgAT serve as models for the human enzyme. Structures are shown in ribbon representation, with colors as described for Fig. 1. GlcNAc in the active site of CpOGA and acetyl-CoA in the active site of OgAT are shown as sticks with black carbons. The internal surface of the tunnel-like structure in OgAT (gray surface) may form the binding site for an as yet unidentified ligand.
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Figure 2: Crystal structures of CpOGA and OgAT serve as models for the human enzyme. Structures are shown in ribbon representation, with colors as described for Fig. 1. GlcNAc in the active site of CpOGA and acetyl-CoA in the active site of OgAT are shown as sticks with black carbons. The internal surface of the tunnel-like structure in OgAT (gray surface) may form the binding site for an as yet unidentified ligand.

Mentions: Human OGA (hOGA) is a 103-kDa enzyme comprising an N-terminal GH84 catalytic domain and a C-terminal HAT-like domain connected by a 300-amino acid region termed the stalk domain (Fig. 1). Although hOGA can be expressed and purified for biochemical studies (29), there is currently no crystal structure for a eukaryotic OGA; however, valuable insights have been obtained from structures of apparent bacterial orthologs (Figs. 1 and 2). The OGA catalytic domain is structurally and mechanistically related to chitinases, N-acetylhexosaminidases, and hyaluronidases in the GH18, GH20, and GH56 families (39). These enzymes perform glycoside hydrolysis with net retention of the anomeric configuration via a double-displacement reaction with the carbonyl oxygen of the N-acetyl group acting as a nucleophile, a mechanism described as substrate-assisted catalysis or neighboring group participation (40, 41). The active site for the OGA activity of OGA is characterized by a conserved pair of essential aspartic acid residues, Asp-174 and Asp-175, in the human enzyme (41), which constitute the catalytic DD motif. The first insights into the structure of the OGA catalytic domain came from crystal structures of two bacterial OGA orthologs in the GH84 family that were reported in 2006, namely Clostridium perfringens NagJ (CpOGA) (36) and Bacteroides thetaiotaomicron hexosaminidase (BtOGA) (37) (Fig. 1). The catalytic domain consists of a (β/α)8 or TIM (triose-phosphate isomerase) barrel, with the active site located on the C-terminal face of the barrel, where the GlcNAc sugar occupies a deep pocket in the surface of the enzyme (Fig. 2). Both structures were determined in the presence of OGA inhibitors, shedding light on the active site architecture, which then facilitated the targeted generation of catalytically impaired hOGA through the introduction of point mutations. In addition to the D174A/D175A double mutant, which results in loss of catalytic activity, substitution of either aspartic acid with asparagine slows down the reaction and has been employed to trap Michaelis complexes in crystallographic studies (42). The mutation D285A leads to impaired substrate binding. Detailed knowledge of the active site architecture was also exploited for rational design of improved OGA inhibitors, as discussed below. Subsequent work by He et al. (42) made elegant use of various substrate analogs to give detailed information about the reaction coordinate of OGA in the form of crystallographic “snapshots.”


O-GlcNAcase: promiscuous hexosaminidase or key regulator of O-GlcNAc signaling?

Alonso J, Schimpl M, van Aalten DM - J. Biol. Chem. (2014)

Crystal structures of CpOGA and OgAT serve as models for the human enzyme. Structures are shown in ribbon representation, with colors as described for Fig. 1. GlcNAc in the active site of CpOGA and acetyl-CoA in the active site of OgAT are shown as sticks with black carbons. The internal surface of the tunnel-like structure in OgAT (gray surface) may form the binding site for an as yet unidentified ligand.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263850&req=5

Figure 2: Crystal structures of CpOGA and OgAT serve as models for the human enzyme. Structures are shown in ribbon representation, with colors as described for Fig. 1. GlcNAc in the active site of CpOGA and acetyl-CoA in the active site of OgAT are shown as sticks with black carbons. The internal surface of the tunnel-like structure in OgAT (gray surface) may form the binding site for an as yet unidentified ligand.
Mentions: Human OGA (hOGA) is a 103-kDa enzyme comprising an N-terminal GH84 catalytic domain and a C-terminal HAT-like domain connected by a 300-amino acid region termed the stalk domain (Fig. 1). Although hOGA can be expressed and purified for biochemical studies (29), there is currently no crystal structure for a eukaryotic OGA; however, valuable insights have been obtained from structures of apparent bacterial orthologs (Figs. 1 and 2). The OGA catalytic domain is structurally and mechanistically related to chitinases, N-acetylhexosaminidases, and hyaluronidases in the GH18, GH20, and GH56 families (39). These enzymes perform glycoside hydrolysis with net retention of the anomeric configuration via a double-displacement reaction with the carbonyl oxygen of the N-acetyl group acting as a nucleophile, a mechanism described as substrate-assisted catalysis or neighboring group participation (40, 41). The active site for the OGA activity of OGA is characterized by a conserved pair of essential aspartic acid residues, Asp-174 and Asp-175, in the human enzyme (41), which constitute the catalytic DD motif. The first insights into the structure of the OGA catalytic domain came from crystal structures of two bacterial OGA orthologs in the GH84 family that were reported in 2006, namely Clostridium perfringens NagJ (CpOGA) (36) and Bacteroides thetaiotaomicron hexosaminidase (BtOGA) (37) (Fig. 1). The catalytic domain consists of a (β/α)8 or TIM (triose-phosphate isomerase) barrel, with the active site located on the C-terminal face of the barrel, where the GlcNAc sugar occupies a deep pocket in the surface of the enzyme (Fig. 2). Both structures were determined in the presence of OGA inhibitors, shedding light on the active site architecture, which then facilitated the targeted generation of catalytically impaired hOGA through the introduction of point mutations. In addition to the D174A/D175A double mutant, which results in loss of catalytic activity, substitution of either aspartic acid with asparagine slows down the reaction and has been employed to trap Michaelis complexes in crystallographic studies (42). The mutation D285A leads to impaired substrate binding. Detailed knowledge of the active site architecture was also exploited for rational design of improved OGA inhibitors, as discussed below. Subsequent work by He et al. (42) made elegant use of various substrate analogs to give detailed information about the reaction coordinate of OGA in the form of crystallographic “snapshots.”

Bottom Line: O-GlcNAc signaling is regulated by an opposing pair of enzymes: O-GlcNAc transferase installs and O-GlcNAcase (OGA) removes the modification from proteins.The dynamics and regulation of this process are only beginning to be understood as the physiological functions of both enzymes are being probed using genetic and pharmacological approaches.We identify several areas of "known unknowns" that would benefit from future research, such as the enigmatic C-terminal domain of OGA.

View Article: PubMed Central - PubMed

Affiliation: From the Medical Research Council Protein Phosphorylation and Ubiquitylation Unit and.

Show MeSH