Structural Insights into the Substrate Specificity of a 6-Phospho-β-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4

Abstract

The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr126, Tyr303, and Trp338, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser424, Lys430, and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family.Background: Streptococcus pneumoniae BglA-2 is a GH-1 6-phospho-β-glucosidase with specificity toward 1,4-linked 6-phospho-β-glucosides.Results: BglA-2 and other GH-1 members adopt a similar overall structure and catalytic mechanism.Conclusion: Tyr126, Tyr303, and Trp338 determine substrate specificity, and Ser424, Lys430, and Tyr432 discriminate phosphorylated from non-phosphorylated substrate. A tryptophan residue discriminates 6-phospho-β-glucosidase from 6-phospho-β-galactosidase activities.Significance: BglA-2 structures provide new insight into characteristics and substrate specificity of 6-phospho-β-glucosidase. The 6-phospho-β-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of β-1,4-linked cellobiose 6-phosphate (cellobiose-6′P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6′P (a non-metabolizable analog of cellobiose-6′P) at 2.0 and 2.4 Å resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (β/α)8 TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr126, Tyr303, and Trp338, at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-β-glucosides. Moreover, three additional residues, Ser424, Lys430, and Tyr432 of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite −1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-β-galactosidases and 6-phospho-β-glucosidases assigned to the GH-1 family. Background: Streptococcus pneumoniae BglA-2 is a GH-1 6-phospho-β-glucosidase with specificity toward 1,4-linked 6-phospho-β-glucosides. Results: BglA-2 and other GH-1 members adopt a similar overall structure and catalytic mechanism. Conclusion: Tyr126, Tyr303, and Trp338 determine substrate specificity, and Ser424, Lys430, and Tyr432 discriminate phosphorylated from non-phosphorylated substrate. A tryptophan residue discriminates 6-phospho-β-glucosidase from 6-phospho-β-galactosidase activities. Significance: BglA-2 structures provide new insight into characteristics and substrate specificity of 6-phospho-β-glucosidase.