Structural basis for the substrate specificity of a novel β-N-acetylhexosaminidase StrH protein from Streptococcus pneumoniae R6.
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.
Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
Yong-Liang
YL
Jiang
Wei-Li
WL
Yu
Jun-Wei
JW
Zhang
Cecile
C
Frolet
Anne-Marie
AM
Di Guilmi
Cong-Zhao
CZ
Zhou
Thierry
T
Vernet
Yuxing
Y
Chen
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D017433:Protein Structure, Secondary; D013296:Streptococcus pneumoniae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D001619:beta-N-Acetylhexosaminidases