State Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
J Org Chem. 2010 Nov 19;75(22):7559-64. doi: 10.1021/jo101541n. Epub 2010 Oct 22.
A carbonyl reductase from Sporobolomyces salmonicolor reduced para-substituted acetophenones with low enantioselectivity. Enzyme-substrate docking studies revealed that residues M242 and Q245 were in close proximity to the para-substituent of acetophenones in the substrate binding site. Site-saturation mutagenesis of M242 or Q245, and double mutation of M242 and Q245 were performed in order to enhance the enzyme's enantioselectivity toward the reduction of para-substituted acetophenones. Three Q245 mutants were obtained, which inverted the enantiopreference of product alcohols from (R)- to (S)-configuration with high ee values (Org. Lett. 2008, 10, 525-528). Four M242 mutant enzymes also showed greater preference for the formation of (S)-enantiomeric alcohols than the wild-type enzyme, but to a much less extent than Q245 mutants. M242/Q245 double variations not only greatly affect the enantiomeric purity of the product alcohols, but also invert the enantiopreference, demonstrating that these residues play a critical role in determining the enantioselectivity of these ketone reductions. The kinetic parameters of these mutant enzymes indicated that residues 242 and 245 also exert an effect on the catalytic activity of this carbonyl reductase. Highly enantioselective mutant carbonyl reductases were created by site-saturation mutagenesis, among which the one bearing double mutations, M242L/Q245P, showed the highest enantioselectivity that catalyzed the reduction of the tested para-substituted acetophenones to give (S)-enantiomeric products in ≥99% ee with only one exception of p-fluoroacetophenone (92% ee).
三文鱼色球孢酵母的羰基还原酶对对位取代苯乙酮的还原具有低对映选择性。酶-底物对接研究表明，在底物结合位点中，残基 M242 和 Q245 与苯乙酮的对位取代基非常接近。为了提高酶对对位取代苯乙酮还原的对映选择性，对 M242 或 Q245 进行了定点饱和突变，以及 M242 和 Q245 的双重突变。获得了三个 Q245 突变体，它们将产物醇的对映体偏好从（R）-构型反转为（S）-构型，具有高 ee 值（Org. Lett. 2008, 10, 525-528）。四个 M242 突变酶也显示出比野生型酶更大的形成（S）-对映体醇的偏好，但程度远低于 Q245 突变体。M242/Q245 双变异不仅极大地影响产物醇的对映体纯度，而且反转对映体偏好，表明这些残基在确定这些酮还原的对映选择性方面起着关键作用。这些突变酶的动力学参数表明，残基 242 和 245 也对该羰基还原酶的催化活性有影响。通过定点饱和突变产生了高度对映选择性的突变羰基还原酶，其中双突变体 M242L/Q245P 表现出最高的对映选择性，在测试的对位取代苯乙酮还原中，仅对 p-氟苯乙酮（92%ee）有一个例外，以大于 99%ee 的对映体纯产物（S）-构型催化。
