Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability
Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability
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DOI：
10.1007/s00253-012-4142-9
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发表时间：
2013-03-01
影响因子：
5
通讯作者：
Weuster-Botz, Dirk
中科院分区：
文献类型：
--
作者：
Hoelsch, Kathrin;Suehrer, Ilka;Weuster-Botz, Dirk
甲酸脱氢酶(FDHS)在NAD(P)H依赖的氧化还原酶的生物转化中常用于辅因子的再生。大多数本地外籍家庭佣工的主要缺点是对NAD(+)的强烈偏好，以及在存在活性有机化合物(如α-卤代酮)的情况下操作稳定性较低。本研究对母牛分枝杆菌N10的脱氢酶(MycFDH)进行了改造，以获得一种不仅能再生NADPH而且对α-卤代酮-4-氯乙酰乙酸乙酯(ECAA)稳定的酶。为了改变辅因子的特异性，对保守的NAD(+)结合基序中的氨基酸进行了突变。其中，MycFDH A198G/D221Q对NADP(+)的催化效率最高(k(CAT)/K(M))。另外两个半胱氨酸(C145S/C255V)的替代不仅赋予了对ECAA的高度抗性，而且使催化效率提高了6倍。得到的四重突变株MycFDHC145S/A198G/D221Q/C255V在30℃、pH 7的温度下比活力为4.00+/-0.13U mg(-1)，K(m，NADP)(+)为0.147+/-0.020 mm。相应的三重突变体MycFDH C145S/D221Q/C255V对接受NADP(+)的FDH(v(Max)，10.25A+/-1.63U mg(-1))具有最高的比活力。而NADP(+)(K(m，NADP)(+)，0.92A+/-0.10 mM)的半饱和常数比四倍体高一个数量级。根据反应体系的不同，这两个新的MycFDH变体都可以用依赖于NADPH的酮还原酶不对称还原ECAA来合成手性合成子(S)-4-氯-3-羟基丁酸乙酯[(S)-ECHB]。
Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD(+) and their low operational stability in the presence of reactive organic compounds such as alpha-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the alpha-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD(+) binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k (cat)/K (m)) with NADP(+). The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 +/- 0.13 U mg(-1) and a K (m, NADP) (+) of 0.147 +/- 0.020 mM at 30 A degrees C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP(+)-accepting FDH (v (max), 10.25 A +/- 1.63 U mg(-1)). However, the half-saturation constant for NADP(+) (K (m, NADP) (+) , 0.92 A +/- 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.