Structural Basis for the Substrate Specificity of a Novel β-N-Acetylhexosaminidase StrH Protein from Streptococcus pneumoniae R6

The beta-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the beta-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of beta(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and beta-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 angstrom crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (beta/alpha)(8) TIM barrel with the active site residing at the center of the beta-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-beta-D-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k(cat)/K-m) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAG beta(1,2) Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the beta(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the beta(1,2)-inked beta-N-acetylglucosides.