高活性亚磷酸酯脱氢酶突变体的机理研究及其在NADPH再生中的应用。,The FEBS Journal

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高活性亚磷酸酯脱氢酶突变体的机理研究及其在NADPH再生中的应用。
The FEBS Journal ( IF 5.5 ) Pub Date : 2005 Aug , DOI: 10.1111/j.1742-4658.2005.04788.x
Ryan Woodyer , Huimin Zhao , Wilfred A Van Der Donk

NAD（P）H再生对于需要这些昂贵的辅因子的生物催化反应非常重要。有效利用NAD和NADP的突变体亚磷酸酯脱氢酶（PTDH-E175A / A176R）是用于NAD（P）H再生的非常有前途的系统。在这项工作中，对PTDH-E175A / A176R的动力学机理和实际应用进行了研究，以更好地理解该酶并为将来的优化提供基础。使用PTDH-E175A / A176R进行的动力学同位素效应研究表明，氢化物转移步骤是（部分）通过NAD和NADP进行速率测定，分别得出（D）V值为2.2和1.7，以及（D）V / K（m亚磷酸酯）值分别为1.9和1.7。为了更好地理解松弛的辅因子特异性，利用色氨酸固有荧光猝灭法测定辅因子解离常数。NAD和NADP与PTDH-E175A / A176R的解离常数分别为53和1.9微米，而产品NADH和NADPH的解离常数分别为17.4和1.22微米。使用亚硫酸盐作为底物模拟物，建立了结合顺序，辅因子首先结合，第二亚硫酸盐结合。辅因子产物NADPH的低解离常数与（D）V和k（cat）的降低值结合在一起，表明产物释放可能部分决定速率。但是，在模型系统中，通过NADH依赖性木糖还原酶将木糖转化为木糖醇，产品抑制作用不会阻止PTDH-E175A / A176R进行的PTNA-E175A / A176R高效原位NADPH再生。PTDH-E175A / A176R的原位再生速度比WT PTDH或NADP特异性假单胞菌sp快四倍。

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Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration.

NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving (D)V values of 2.2 and 1.7, respectively, and (D)V/K(m,phosphite) values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 microm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 microm, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for (D)V and k(cat) implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH-E175A/A176R in a model system in which xylose was converted into xylitol by NADP-dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH-E175A/A176R than with either WT PTDH or a NADP-specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.

更新日期：2017-01-31

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