Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
Bioorg Med Chem. 2011 Nov 1;19(21):6447-53. doi: 10.1016/j.bmc.2011.08.056. Epub 2011 Aug 30.
The use of biological catalysts for industrial scale synthetic chemistry is highly attractive, given their cost effectiveness, high specificity that obviates the need for protecting group chemistry, and the environmentally benign nature of enzymatic procedures. Here we evolve the naturally occurring 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolases from Thermatoga maritima and Escherichia coli, into enzymes that recognize a nonfunctionalized electrophilic substrate, 2-keto-4-hydroxyoctonoate (KHO). Using an in vivo selection based on pyruvate auxotrophy, mutations were identified that lower the K(M) value up to 100-fold in E. coli KDPG aldolase, and that enhance the efficiency of retro-aldol cleavage of KHO by increasing the value of k(cat)/K(M) up to 25-fold in T. maritima KDPG aldolase. These data indicate that numerous mutations distal from the active site contribute to enhanced 'uniform binding' of the substrates, which is the first step in the evolution of novel catalytic activity.
鉴于生物催化剂具有成本效益高、特异性高（无需保护基团化学）以及酶法过程环境友好的特点，将其用于工业规模的合成化学极具吸引力。在这里，我们对来自Thermatoga maritima 和 Escherichia coli 的天然存在的 2-酮-3-脱氧-6-磷酸葡萄糖酸（KDPG）醛缩酶进行了进化，使之成为能够识别非功能化亲电底物 2-酮-4-羟基辛烯酸（KHO）的酶。通过基于丙酮酸营养缺陷型的体内选择，鉴定到了突变，这些突变使大肠杆菌 KDPG 醛缩酶的 K(M)值降低了 100 倍，使 T. maritima KDPG 醛缩酶的 KHO 反向醛裂解效率提高了 25 倍，这是通过增加 k(cat)/K(M) 值来实现的。这些数据表明，许多远离活性位点的突变有助于增强底物的“均匀结合”，这是新催化活性进化的第一步。
