A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase
A modified consensus approach to mutagenesis inverts the cofactor specificity of Bacillus stearothermophilus lactate dehydrogenase
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DOI：
10.1093/protein/gzi043
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发表时间：
2005-08-01
影响因子：
2.4
通讯作者：
Ellington, AD
中科院分区：
文献类型：
--
作者：
Flores, H;Ellington, AD
来自嗜热脂肪芽孢杆菌的乳酸脱氢酶对NAD（+）具有特异性。已经有几种尝试来改变这种酶的辅因子特异性，但是这些已经产生了具有相对低活性的酶，其仍然在很大程度上偏好NAD（+）。使用改良的共有方法来创建位于辅因子结合位点附近的遗传学上优选的氨基酸的文库，并筛选变体利用NMN（+）的能力。发现了三重突变体（Mut31），其被证明比野生型更有催化效率。Mut31在利用NAD（+）方面也优于野生型酶，并且对NADP（+）和NMN（+）具有弱活性。单个氨基酸取代的分析表明，所有三个突变以协调一致的方式工作，以产生强大的辅因子利用。当两个先前鉴定的氨基酸取代被引入Mut31背景中时，所得到的五重取代的酶不仅比野生型酶更好地利用NADP（+），而且实际上颠倒了其对NAD（+）和NADP（+）的偏好。
Lactate dehydrogenase from Bacillus stearothermophilus is specific for NAD(+). There have been several attempts to alter the cofactor specificity of this enzyme, but these have yielded enzymes with relatively low activities that still largely prefer NAD(+). A modified consensus approach was used to create a library of phylogenetically preferred amino acids situated near the cofactor binding site, and variants were screened for their ability to utilize NMN(+). A triple mutant (Mut31) was discovered that proved to be more catalytically efficient than wild-type. Mut31 was also better at utilizing NAD(+) than the wild-type enzyme and was weakly active with NADP(+) and NMN(+). An analysis of single amino acid substitutions suggested that all three mutations worked in a concerted fashion to yield robust cofactor utilization. When two previously identified amino acid substitutions were introduced into the Mut31 background, the resultant quintuply substituted enzyme not only utilized NADP(+) far better than the wild-type enzyme, it actually inverted its preference for NAD(+) and NADP(+).