中子与X-光衍射相结合解析木聚糖酶突变体N44D晶体结构  

Combination of Neutron and X-ray Crystallography for Structural Analysis of N44D Xylanase Mutant

作　　者：万群[1] 孟浩[1] 祝娉婷[1] 高亚雄[1] 卜平[1] 

机构地区：[1]扬州大学医学院,江苏扬州225001

出　　处：《中国生物化学与分子生物学报》2014年第12期1223-1230,共8页Chinese Journal of Biochemistry and Molecular Biology

基　　金：扬州大学科技培育基金(No.2013CXJ083);教育部留学人员科研启动基金资助~~

摘　　要：木聚糖酶(xylanase)是降解木质纤维素中半纤维素的特定酶,在酶法生产生物能源的过程中有重要应用.木质纤维素在降解时需要用酸或碱预处理,而木聚糖酶反应的最适p H值为中性.因此,获得在酸或碱性条件下酶活力仍然很高的木聚糖酶,是生物能源生产中的重要课题.木聚糖酶活性中心的天冬酰胺突变为天冬氨酸(N44D)后,木聚糖酶的最适p H值从5.7下调到4.6,酶活提高20%.本研究首次获得了分辨率为2.20的木聚糖酶突变体N44D的晶体在291 K的中子衍射数据.同时,分别获得了分辨率为1.70和1.07的在291 K和100 K的X-光衍射晶体数据.通过解析以上数据,获得了木聚糖酶中几乎所有原子的空间位置.以上结果将有助于在原子水平研究这种突变是如何影响酶反应最适p H值,并进一步为酶蛋白的改性提供了结构生物学的依据.Xylanase is the specific enzyme to hydrolyze hemicellulose from lignocellulosic biomass. For better hydrolysis efficiency, pretreatments to destroy hydrogen bonds linking cellulosic fibers are necessary, such as exposure to dilute acid or ammonia fiber explosion, which conflict with the optimal pH of xylanase reaction approximately at neutral. Therefore, obtaining a highly active xylanase in the acidic or alkaline condition is desirable in the bioenergy industry. When Asn44 in the active site was mutated to Asp （N44D） , its optimal pH was decreased from 5.7 to 4.6 and increased the activity by 20%. The crystal of mutant diffracted to 2.20A°resolution at 291 K using neutron source, and to 1.70A° （291 K） and 1.07 A° （100 K） using X-ray. By refining the above diffraction data, the full structure of N44D was solved, including the hydrogen atoms. The results will help to learn how mutation to decrease of optimum pH of an enzyme and facilitate future protein engineering.

关 键 词：中子衍射晶体结构 木聚糖酶 氢原子空间位置 

分 类 号：Q71[生物学—分子生物学]