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

Abstract

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

FIGURE 1.

Domain organization of StrH and overall structure of GH20-1. A, five distinct domains of StrH were drawn by Domain Graph, version 2.0 (40). B, overall structure of GH20-1 (cyan) together with the linker (magenta) and the N-terminal α-helix of GH20-2 (cyan). The secondary structural elements were labeled sequentially. The NAG molecule at the active site of GH20-1 is shown as green sticks.

FIGURE 2.

The active site of GH20-1. A, NAG binding site. The residues are shown as sticks, and the water molecule Wat-1 is shown as a sphere. The polar interactions are indicated by dashed lines. B, active site comparison of GH20-1 (cyan) and SpHex (pink).

FIGURE 3.

The substrate specificity of StrH. A, the putative substrate entrance tunnel calculated by CAVER program, denoted in orange mesh. Trp-443 and Tyr-482 in the vicinity were shown as sticks and labeled. B, a manually built model of NAGβ(1,2)Man in complex with GH20-1.

FIGURE 4.

Conservation of Trp-443 and Tyr-482 in StrH and its homologs. Multiple-sequence alignment of putative GH20 enzymes with β(1,2) substrate specificity. The active site residues at subsite −1 are labeled with green triangles. Residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 were marked with red triangles.