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扩展细胞色素p4502a6对大位阻底物的适应性

DOI: 10.3724/SP.J.1145.2009.00059, PP. 59-63

Keywords: 细胞色素p4502a6,cyp2a6,点突变,生物转化,6-苄氧基吲哚

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Abstract:

细胞色素p450酶(p450)的分子改造及其催化的生物转化是当前的两个研究热点.本研究以扩展p4502a6的底物适应性为目标,通过尝试对p4502a6酶进行n-端修饰提高表达量、筛选随机突变库以及点突变改变活性位点重要氨基酸残基等手段,在两个随机突变体中未筛选到功能突变体,但通过点突变重要氨基酸残基获得了新的突变体p4502a6(n297q/i300a).该突变体具有比野生型和已报道的其它突变体更广泛的底物适应性,能够催化氧化大位阻底物6-苄氧基吲哚(6-obzl-indole)生成蓝绿色物质.该物质经质谱测定为靛蓝类二聚体化合物.实验表明,其中两个氨基酸残基的变化都是关键突变,此结果与最新的p4502a6突变体晶体结构研究相符.计算机模拟显示,i300a突变导致活性口袋在铁卟啉环的垂直和水平方向都增大,使其能接纳在两个方向都有位阻的底物;而297位的天冬酰胺是一个功能保守的残基,n297q的变化可能与氢键等弱作用有关.图4表1参29

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