【作者】 余维丽；

【导师】 陈宇星； 周丛照；

【作者基本信息】 中国科学技术大学 ， 生物化学与分子生物学， 2013， 博士

【摘要】 1.肺炎链球菌表面p-N-乙酰氨基己糖苷酶StrH的结构与功能研究当肺炎链球菌侵染宿主时,会遇到宿主细胞的一些糖复合物组分,包括粘蛋白、防御分子以及连接在人上皮细胞表面的糖组分。肺炎链球菌利用一些糖基水解酶来水解这些分子的糖基组分,三种外切糖基水解酶：唾液酸苷酶NanA、β-半乳糖苷酶BgaA以及β-N-乙酰氨基己糖苷酶StrH可以按顺序地分别水解掉宿主细胞防御分子外面的唾液酸Neu5Ac,半乳糖Gal以及N-乙酰氨基葡萄糖NAG暴露出多糖组分的末端糖单元甘露糖,从而改变宿主防御分子的清除功能。肺炎链球菌R6菌株表面StrH是糖基水解酶20家族的一个β(1,2)-N-乙酰氨基己糖苷酶(EC3.21.52),能够从糖复合物的非还原端水解产生N-乙酰氨基葡萄糖NAG单元。它具有两个GH20催化结构域,其序列同源性达53%(GH20-1和GH20-2结构域)。我们解析了StrH(Glu175-Lys642)与NAG复合物的结构,分辨率为2.1A。其整体结构是一个(β/α)8的桶状结构,与GH20家族糖苷酶相似。其活性位点位于β-barrel凸面中心。利用4-硝基-N-乙酰-p-D-氨基葡萄糖作为底物对StrH进行酶学分析,我们发现GH20-1结构域的(kcat/Km)只有GH20-2结构域的1/4。这主要是由于GH20-1中Cys-469取代了GH20-2结构域中相应的Tyr-903。通过底物NAGβ (1,2)Man与StrH复合物模型结合定点突变的酶学实验,发现了两个芳香族氨基酸残基对于StrH的p(1,2)底物特异性起关键性作用,它们分别是在GH20-1结构域中的Trp443和Tyr482和GH20-2结构域中的Trp876和Tyr914。这是第一个特异性水解p(1,2)连接的p-N-乙酰氨基葡萄糖水解酶的结构,结构和功能的研究帮助我们理解了这一糖苷酶的底物特异性机制。2.肺炎链球菌6-磷酸β-葡萄糖苷酶BglA-2的结构与功能研究糖基水解酶GH-1家族的6-磷酸-p-葡萄糖苷酶BglA-2(EC3.2.1.86)能够催化水解p(1,4)连接的纤维二糖(cellibiose)产生葡萄糖和葡萄糖-6-磷酸。这两种反应产物都能进一步进入产生能量的糖酵解途径中。我们解析了肺炎链球菌6-磷酸β-葡萄糖苷酶BglA-2的apo结构以及它和底物cellobiose-6’P类似物(硫代纤维二糖-6-磷酸thiocellobiose-6’P)复合物的的晶体结构,分辨率分别为2.0A和2.4A。与其他GH-1家族糖苷酶类似,Bg1A-2整体结构是一个(β/α)8的桶状结构,其活性位点位于β-barrel凸面中心。结构分析结合定点突变的酶学实验发现BglA-2中的三个芳香族的氨基酸Tyr126, Tyr303和Trp338决定(1,4)连接的6-磷酸-p-葡萄糖的糖苷酶底物特异性,而三个氨基酸Ser424, Lys430和Tyr432决定了BglA-2水解磷酸化底物的特异性。同时,结构比较发现一个色氨酸特异性识别GH-1家族中6-磷酸-半乳糖苷酶的-1位底物为6-磷酸-半乳糖而非6-磷酸-葡萄糖。这是第一次解析了6-磷酸p-葡萄糖苷酶BglA-2与磷酸化底物的复合物结构,研究结果阐明了这一糖苷酶的底物特异性机制。更多还原

【Abstract】 1. Structure and function of a novel β-N-acetyl-hexosaminidase StrH from Streptococcus pneumoniaeThe β-N-acetyl-hexosaminidase (EC3.2.1.52) from glycoside hydrolase family20(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/Spr0057from Streptococcus pneumoniae R6was 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 GH20enzyme which contains a tandem repeat of two53%sequence-identical catalytic domains (designated as GH20-1and GH20-2, respectively). Here, we present the2.1A crystal structure of the N-terminal domain of StrH (residues Glul75-Lys642) complexed with NAG. It adopts an overall structure similar to other GH20enzymes:a (β/α)8TIM-barrel with the active site residing at the center of the β-barrel convex side. The kinetic investigation using4-nitrophenyl N-acetyl-β-D-glucosaminide (pNp-NAG) as the substrate demonstrated that GH20-1had an enzymatic activity (kcat/Km) of one-fourth compared to GH20-2. The lower activity of GH20-1could be attributed to thesubstitution of Cys469of GH20-1to the counterpart Tyr903of GH20-2at the active site. A putative substrate entrance tunnel and the modeling of NAG β (1,2)Man at the active site characterized two key residues Trp443and Tyr482at+1subsite of GH20-1that might determine the β (1,2) substrate specificity. Mutation of Trp443and/or the counterpart residue Trp876in GH20-2to alanine resulted in significant reduction of the activity towards NAGβ (1,2)Man, whereas mutation of Tyr482and/or the counterpart Tyr914in GH20-2to alanine almost abolished the activity. Taken together, these findings shed light on the mechanism of catalytic specificity towards the β (1,2)-linked β-N-acetylglucosides. 2. Structure and function of a6-phospho-p-glucosidase BglA-2from Streptococcus pneumoniaeThe6-phospho-β-glucosidase BglA-2(EC3.2.1.86) from glycoside hydrolase family1(GH-1) catalyzes the hydrolysis of β (1,4)-linked cellobiose-6-phosphate (cellobiose-6’P) to yield glucose and glucose-6-phosphate (G6P). Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo-and complex-forms of BglA-2with thiocellobiose-6’P (a non-metabolizable analog of cellobiose-6’P) at2.0A and2.4A resolution, respectively. Similar to other GH-1enzymes, the overall structure of BglA-2from Streptococcus pneumoniae adopts a typical (β/α)8TIM-barrel, with the active site located at the center of the convex surface of the β-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues:Tyr126, Tyr303and Trp338at subsite+1of BglA-2, determine substrate specificity with respect to (1,4)-linked6-phospho-β-glucosides. Moreover, three additional residues:Ser424, Lys430and Tyr432of BglA-2, were found to play important roles in the hydrolytic selectivity towards phosphorylated, rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite-1may contribute to the catalytic and substrate differences between the structurally similar6-phospho-β-galactosidases and6-phospho-β-glucosidases assigned to GH-1family.更多还原