Structural basis for the substrate specificity of a novel ?-N-acetylhexosaminidase StrH protein from Streptococcus pneumoniae R6.

ABSTRACT: The ?-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the ?-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 ?(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and ?-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 Å 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 (?/?)(8) TIM barrel with the active site residing at the center of the ?-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-?-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?(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 ?(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the ?(1,2)-linked ?-N-acetylglucosides.

SUBMITTER: Jiang YL 

PROVIDER: S-EPMC3234876 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature