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.
J Biol Chem. 2011 Dec 16;286(50):43004-12. doi: 10.1074/jbc.M111.256578. Epub 2011 Oct 19.
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.
β-N-乙酰氨基葡萄糖苷酶（EC 3.2.1.52）属于糖苷水解酶家族 20（GH20），能够从各种糖缀合物的非还原端水解β-N-乙酰氨基葡萄糖（NAG）基团。肺炎链球菌 R6 中假定的表面暴露的 N-乙酰氨基葡萄糖苷酶 StrH/Spr0057 通过神经氨酸酶和β-半乳糖苷酶分别切割唾液酸和半乳糖后，从宿主防御分子上除去β(1,2)-连接的 NAG，从而被证明有助于毒力。StrH 是唯一报道的含有两个 53%序列相同的催化结构域（分别指定为 GH20-1 和 GH20-2）串联重复的 GH20 酶。在这里，我们展示了 StrH（Glu-175 到 Lys-642）与 NAG 结合的 N 端结构域的 2.1 Å 晶体结构。它采用与其他 GH20 酶相似的整体结构：一个（β/α）(8) TIM 桶，其活性位点位于β-桶凸面的中心。使用 4-硝基苯基 N-乙酰-β-d-葡萄糖胺作为底物进行的动力学研究表明，与 GH20-2 相比，GH20-1 的酶活性（k(cat)/K(m)）降低了四分之一。GH20-1 活性较低的原因可能归因于 GH20-1 的活性位点半胱氨酸 469 被 GH20-2 的对应酪氨酸 903 取代。在 GH20-1 的活性位点结合 NAGβ(1,2)Man 的复合物模型以及相应的定点突变体的活性测定结果表明，GH20-1 的 +1 亚位点的两个关键残基色氨酸 443 和酪氨酸 482（GH20-2 的色氨酸 876 和酪氨酸 914）可能决定了β(1,2)底物特异性。总之，这些发现揭示了对β(1,2)-连接的β-N-乙酰葡萄糖苷催化特异性的机制。
