构建杂化酶体系解析对羟基苯乙酸脱羧酶小亚基的功能

对羟基苯乙酸(HPA)脱羧酶是一类甘氨酸自由基脱羧酶,催化前体对羟基苯乙酸或吲哚乙酸的脱羧反应,形成终产物对羟基甲苯和对甲基吲哚。由于其蛋白质结构中小亚基(HpdC)的存在,使其显示了一些不同于其他已知的几种甘氨酸自由基体系的特性,被认为是一类新型的甘氨基自由基酶体系。前期的研究结果初步显示了HpdC在整个脱羧酶体系中的重要性,但缺乏系统的实验验证。通过构建系列杂化脱羧酶(包括组合杂化酶体系与嵌合杂化酶体系)高表达质粒,对相应的编码蛋白质进行分离纯化以及酶活性检测等,探究了小亚基在整个脱羧酶体系中的功能。研究结果证明,小亚基HpdC的存在,一方面直接影响整个编码蛋白质的溶解性及复合物的稳定性,另一方面则通过介导脱羧酶高级聚合状态的形成,决定着酶的催化活性。

Hybrid decarboxylasesdefining the role of the small subunit

4-hydroxyphenylacetate decarboxylase (HPA) is a kind of glycyl radical enzymes (GREs), which catalyze the formation of p-cresol or skatole. It is characterized by a small subunit (HpdC), and shows unique properties that have not been observed in other GREs. Thus HPA decarboxylase is considered as a new kind of GRE. The existing researches show the importance of HpdC in the decarboxylase family, but its importance hasn''t been experimentally verified. To elucidate the functions of HpdC, a series of over-expression plasmids for the hybrid decarboxylases were established and the corresponding encoded proteins were purified and analyzed. The final physio-chemical analysis and the enzyme activity test results prove that the small subunit is directly involved in the regulation of the enzymes'' oligomeric state and activity.