真菌来源7-木糖紫杉烷糖基水解酶结构与功能关系研究

The glycoside hydrolases (GH) LXYL-P1-1 and LXYL-P1-2 are two highly similar variants of bifunctional β-xylosidases/β-glucosidases. These two enzymes are cloned from the fungus Lentinula edodes. They can specifically remove the D-xylosyl group from 7-β-xylosyltaxanes, forming the corresponding 7-hydroxyltaxanes which can be used for the semisynthesis of anti-cancer drug paclitaxel. The novel enzymes belong to GH3 family, and have been successfully expressed by the yeast host (Pichia pastoris).The method for obtaining the recombinant proteins with high purity has been established. Such β-xylosidases with the specific activity and the detailed information including the amino acid sequences have not yet been reported. Therefore, the studies on the relationship of structure and function of the enzymes have original innovation. This project is mainly focused on the LXYL-P1-2 enzyme and will utilize the following techniques: site-directed mutagenesis, circular dichroism (CD) spectra, as well as protein crystallography and X-ray diffraction to study the relationship of structures (from primary to three dimentional structure) and function, determine the active sites and the key amino acids which affect the activity, and find out the characteristics of the three dimentional structure. Based on these results, the reasonable elucidation will be put forward to the mechanism of how these novel enzymes specifically act on 7-β-xylosyltaxanes and hydrolyze xylose from the aglycons.

糖基水解酶（GH）LXYL-P1-1和LXYL-P1-2是来源于真菌香菇、且高度同源的双功能β-木糖苷酶/β-葡萄糖苷酶，能特异性水解去除7-木糖-10-去乙酰紫杉醇等紫杉烷的木糖基，生成可用于半合成抗癌药物紫杉醇的7位羟基化产物，该酶隶属于糖基水解酶第3家族，是未知蛋白，已通过酵母系统实现重组表达，高纯度的重组蛋白制备方法已经建立。鉴于能完成上述反应、源自香菇或其它生物的β-木糖苷酶的氨基酸序列等信息均未见其它报道，LXYL-P1-1和LXYL-P1-2结构与功能关系研究具有原始创新性。本项目以LXYL-P1-2为重点，应用定点突变、圆二色谱、蛋白质X-射线单晶衍射等技术，开展该酶的一级结构至三维空间结构与其功能关系研究，确定该酶的活性位点以及影响酶活性的其他重要氨基酸残基和三维结构特征，对其特异性地催化7-木糖紫杉烷为7-羟基紫杉烷的作用机制给出较为合理的诠释。

糖基水解酶GH3家族目前超过13,000个成员，但获得蛋白晶体并解析出三维结构者甚少。LXYL-P1-2是隶属于GH3家族的新酶，能特异性水解7-木糖紫杉醇(XDT)为10-去乙酰紫杉醇(DT)。本项目开展其结构与功能研究，结果如下：.⑴ 预测到前体蛋白存在前导肽序列，包括Met1~Ala19信号肽(识别位点Ala17Leu18Ala19↓)和KEX2蛋白加工信号(Ile32Phe33Arg34Arg35↓)，成熟蛋白从Asp36开始。构建了一系列N-端突变体，研究发现：KEX2位点不仅决定成熟蛋白起始位置，而且确保RNA正常转录。前体蛋白第32位氨基酸之前的前导肽对于将前体蛋白转运到蛋白质加工场所不可或缺，部分缺失则导致成熟蛋白生成减少。若缺失KEX2位点则成熟蛋白从信号肽酶切割位点处起始，且该信号肽甚至整个前导肽能被异源信号肽所取代。.⑵ 通过序列比对、定点突变和圆二色谱技术确定了保守的底物结合位点Asn172-Gly173-Arg174、Lys207-His208与催化位点Asp300和Glu529。还结合三维结构分析及定点突变方法确定Asp109、Ser466、Leu158、Gly465、Tyr268、Phe324和Leu328在LXYL-P1-2水解XDT过程中具重要作用，其中Ser466、Leu158、Gly465和Phe324对于XDT具有更高的底物特异性。.⑶ 获得了高分辨的LXYL-P1-2晶体和LXYL-P1-2/XDT复合物晶体，解析出三维结构。发现该酶晶体为四聚体，每个单体均由3个结构域组成，酶活性腔由Domain 1和Domain 2围成，上述位点均参与活性腔的组成。LXYL-P1-2三维结构与同为GH3家族、同样含有3个结构域的细菌TnBgl3B有很大的不同；与真菌HjCel3A等也有很大差异。该酶活性腔入口处较为开放，有助于容纳XDT底物，而其外围存在的4段特异性loop(Asn219Leu234、Gly321Ala327、Thr378Thr387和Gly445Ser449)可能也是决定该酶特异性结合XDT的重要因素之一。.上述结果较合理地诠释了LXYL-P1-2特异性水解XDT作用机理，丰富了本领域蛋白质结构与功能研究的科学信息、尤其是更新了三维结构方面的科学数据，对于该酶定向进化研究进而获得性能更优的突变蛋白也具有指导意义。