Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase
Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase
复制标题
NADH 依赖性铁氧还蛋白还原酶的完全吡啶核苷酸特异性转化
DOI：
10.1042/bj20140384
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发表时间：
2014
影响因子：
4.1
通讯作者：
Kimura S
中科院分区：
文献类型：
--
作者：
Nishizawa A;Harada A;Senda M;Tachihara Y;Muramatsu D;Kishigami S;Mori S;Sugiyama K;Senda T;Kimura S
酶的辅酶特异性是利用细菌工程生产生物化合物的关键参数之一。由于NADPH在光合生物中大量产生，因此将nadh特异性酶转化为NADPH特异性酶是光合生物中有效的碳中性生物化合物生产的有用方法。在本研究中，一种nadh特异性的铁氧还蛋白还原酶组分，联苯双加氧酶BphA的BphA4。采用基于结构的系统突变和定点随机突变相结合的方法，将菌株KKS102转变为nadph依赖的形式。由此产生的CRG突变体将野生型BphA4中NADH识别环的glu175 - thr176 - gln177替换为Cys175-Arg176-Gly177，对NADPH具有高度特异性和活性，其对NADPH的生化和结构特性与野生型BphA4的NADH几乎相同。此外，通过突变效应的半经验预测方法对该突变项目进行了评估，结果表明，CRG突变体是最好的nadph特异性突变体之一。
The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA fromAcidovoraxsp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.