摘要
为了进一步获得具有更高活力的细胞色素P450 BM-3(F87V/A74G/L188Q)突变酶,利用饱和突变技术对该突变酶的4个氨基酸位点D168、A225、K434、E435分别进行单一的随机突变,通过对其羟基化吲哚生成靛蓝的催化性能表征,发现P450 BM-3氨基酸残基的168、434和435位均位于蛋白功能区域,而225位则位于非功能区域,同时发现突变酶D168H具有更高的结合辅酶NADPH的能力,其消耗NADPH的能力几乎是亲本酶的8倍,但生成靛蓝的能力却只是亲本酶的3倍,说明该位点极有可能承担了将电子从黄素单核苷酸(FMN)氧化还原酶区域传递到亚铁血红素区域的任务,在P450 BM-3结构与功能的关系中扮演着重要的角色.
Mutant libraries created by saturation mutagenesis of each single amino acid site D168, A225, K434 and E435 were screened to further improve the capability of cytochrome P450 BM-3（A74G/F87V/ L188Q）mutant from Bacillus Megaterium which can hydroxylate indole into indigo. Results showed that D168, K434 and E435 are located at the functional domain of P450 BM-3 protein while A225 is sited at the unfunctional domain. The NADPH turnover rate of the mutant D168H is almost eight times that of the parental mutant P450 BM-3（F87V/A74G/L188Q）, but indigo formation rate is only about three times that of the parental mutant. Exchange at position 168 aspartic acid replaced by hisitidine could influence the interaction between the monooxygenase domain and the FMN-binding reductase domain, which plays a key role in the relationship between the structure and function of P450 BM-3.
出处
《浙江大学学报（工学版）》
EI
CAS
CSCD
北大核心
2007年第7期1214-1218,共5页
Journal of Zhejiang University：Engineering Science
基金
国家自然科学基金资助项目(30570411)
CSC-DAAD中德合作PPP资助项目
