Structure And Function Of The Glycosidases StrH And BglA-2from Streptococcus Pneumoniae

1. Structure and function of a novel β-N-acetyl-hexosaminidase StrH from Streptococcus pneumoniaeThe β-N-acetyl-hexosaminidase (EC3.2.1.52) from glycoside hydrolase family20(GH20) catalyzes the hydrolysis of the β-N-acetylglucosamine (NAG) group from the non-reducing end of various glycoconjugates. The putative surface-exposed N-acetyl-hexosaminidase StrH/Spr0057from Streptococcus pneumoniae R6was proved to contribute to the virulence by removal of β (1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase (NanA) and β-galactosidase (BgaA), respectively. StrH is the only reported GH20enzyme which contains a tandem repeat of two53%sequence-identical catalytic domains (designated as GH20-1and GH20-2, respectively). Here, we present the2.1A crystal structure of the N-terminal domain of StrH (residues Glul75-Lys642) complexed with NAG. It adopts an overall structure similar to other GH20enzymes:a (β/α)8TIM-barrel with the active site residing at the center of the β-barrel convex side. The kinetic investigation using4-nitrophenyl N-acetyl-β-D-glucosaminide (pNp-NAG) as the substrate demonstrated that GH20-1had an enzymatic activity (kcat/Km) of one-fourth compared to GH20-2. The lower activity of GH20-1could be attributed to thesubstitution of Cys469of GH20-1to the counterpart Tyr903of GH20-2at the active site. A putative substrate entrance tunnel and the modeling of NAG β (1,2)Man at the active site characterized two key residues Trp443and Tyr482at+1subsite of GH20-1that might determine the β (1,2) substrate specificity. Mutation of Trp443and/or the counterpart residue Trp876in GH20-2to alanine resulted in significant reduction of the activity towards NAGβ (1,2)Man, whereas mutation of Tyr482and/or the counterpart Tyr914in GH20-2to alanine almost abolished the activity. Taken together, these findings shed light on the mechanism of catalytic specificity towards the β (1,2)-linked β-N-acetylglucosides. 2. Structure and function of a6-phospho-p-glucosidase BglA-2from Streptococcus pneumoniaeThe6-phospho-β-glucosidase BglA-2(EC3.2.1.86) from glycoside hydrolase family1(GH-1) catalyzes the hydrolysis of β (1,4)-linked cellobiose-6-phosphate (cellobiose-6’P) to yield glucose and glucose-6-phosphate (G6P). 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-2with thiocellobiose-6’P (a non-metabolizable analog of cellobiose-6’P) at2.0A and2.4A resolution, respectively. Similar to other GH-1enzymes, the overall structure of BglA-2from Streptococcus pneumoniae adopts a typical (β/α)8TIM-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, Tyr303and Trp338at subsite+1of BglA-2, determine substrate specificity with respect to (1,4)-linked6-phospho-β-glucosides. Moreover, three additional residues:Ser424, Lys430and Tyr432of BglA-2, were found to play important roles in the hydrolytic selectivity towards phosphorylated, rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite-1may contribute to the catalytic and substrate differences between the structurally similar6-phospho-β-galactosidases and6-phospho-β-glucosidases assigned to GH-1family.