作者：江永亮#、余维丽#、张钧玮、Cecile Frolet, Anne-Marie Di Guilmi, 周丛照、Thierry Vernet*、陈宇星*。（#共第一作者，*通信作者）

 

Abstract：

The β-N-acetyl-hexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (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/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 (NanA) and β-galactosidase (BgaA), respectively. StrH is the only reported GH20 enzyme which 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 Glu175-Lys642) 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 (pNp-NAG) as the substrate demonstrated that GH20-1 had an enzymatic activity (kcat/Km) of one-fourth compared to GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active-site Cys469 of GH20-1 to the counterpart Tyr903 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 Trp443 and Tyr482 at subsite +1 of GH20-1 (Trp876 and Tyr914 of GH20-2) that might determine the β(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity towards the β(1,2)-linked β-N-acetylglucosides.