嗜酸热脂环酸杆菌中甘露聚糖酶活性位点的确立

Identification of the catalytic residues of mannanase from Alicyclobacillus acidocaldarius

摘要:

摘要：【目的】通过定点突变确定嗜酸热脂环酸杆菌中甘露聚糖酶的活性催化位点。【方法】根据序列比对和GH53家族的结构信息选择可能的催化活性位点，利用重叠PCR法构建定点突变体，采用薄层层析（TLC）法和3,5－二硝基水杨酸（DNS）法检测各酶蛋白活性。【结果】通过重叠PCR法成功构建了7个位点的突变体，其中第150和159位的氨基酸突变对活性改变甚少或几乎没有，而第151和231位谷氨酸的羧基基团的改变以及双位点突变体E2Q则导致其对各种底物催化活性的丧失，说明位于β4和β7折叠的C末端的E151和E231

Abstract:

Abstract: [Objective] To identify the catalytic residues of mannanase AaManA from Alicyclobacillus acidocaldarius. [Methods] Based on the sequence alignment by ClustalX and ESPript and the structure information of GH-53 family, the possible catalytic residues were selected and mutated by overlap extension PCR. The protein of wild type and mutant were expressed in E. coli BL21(DE3) and ordinal purified by Ni-NTA affinity chromatography, gel-filtrate chromatography and ion-exchange chromatography. The purified protein was analyzed by thin layer chromatography (TLC) and the dinitrosalicylic acid (DNS) methods for enzyme assay. [Results] Seven mutants, E151A, E159A, E231A, C150A, E151Q, E231Q and double mutation E151Q&E231Q were successful constructed. Mutant E159A showed similar activities with wild type, and C150A mutation resulted in only a 3-fold reduction in the activities, but mutations E151A, E231A, E151Q, E231Q and E151Q&E231Q resulted in sharp decreases or loss in the activities, indicating that Glu151 and Glu231 play critical roles in AaManA activity. Furthermore, the presence of Glu151 at the C terminus of β4 and Glu231 at the C terminus of β7 was entirely consistent with the positions of the acid/base catalyst and the nucleophile catalyst of a GH-A enzyme, respectively. [Conclusion] By combining the results of TLC and enzyme assay of those mutants and the structural comparisons, it was confirmed that Glu151 and Glu231 fulfilled the roles of an acid/base catalyst and nucleophile catalyst in AaManA, respectively.