Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase
Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase
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DOI：
10.1073/pnas.0504033102
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发表时间：
2005-06-28
影响因子：
11.1
通讯作者：
Wong, CH
中科院分区：
文献类型：
--
作者：
Hsu, CC;Hong, ZY;Wong, CH
以大肠杆菌D-Neu5Ac （n -乙酰神经氨酸，d -唾液酸）醛缩酶为基础，定向进化制备了高效的l -3-脱氧甘露糖-2-辛糖酸醛缩酶。进行5轮易出错PCR和迭代筛选，每轮取样103个菌落。天然糖L-KDO的特异性常数（k(cat)/ k -m）被提高到与天然底物D-Neu5Ac的野生型d-唾液酸醛缩酶相当的水平。最终进化的酶的特异性常数[kcat/Km (L-KDO)]/[k(cat)/ k -m （d -唾液酸）]的比值提高了1000倍。进化后的醇解酶蛋白序列与原酶相比发生了8个氨基酸的变化，且这些变化均发生在活性位点之外。我们的努力表明，酶可以快速改变，接受对映体底物，筛选一小群菌落迭代到目标底物，提高催化效率。这项工作为对映体糖的合成和对映体催化受远程突变影响的研究提供了一种方法。
An efficient L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase was created by directed evolution from the Escherichia coli D-Neu5Ac (N-acetylneuraminic acid, D-sialic acid) aldolase. Five rounds of error-prone PCR and iterative screening were performed with sampling of 103 colonies per round. The specificity constant (k(cat)/K-m) of the unnatural sugar L-KDO is improved to a level equivalent to the wild-type D-Sialic acid aldolase for its natural substrate, D-Neu5Ac. The final evolved enzyme exhibits a > 1,000 fold improved ratio of the specificity constant [kcat/Km (L-KDO)]/[k(cat)/K-m (D-sialic acid)]. The protein sequence of the evolved alclolase showed eight amino acid changes from the native enzyme, with all of the observed changes occurring outside of the active site. Our effort demonstrates that an enzyme can be rapidly altered to accept enantiomeric substrates with screening of a small population of colonies iteratively toward the target substrate with improved catalytic efficiency. This work provides a method for the synthesis of enantiomeric sugars and for the study of enantiomeric catalysis affected by remote mutations.