Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.
Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.
复制标题
通过卤代醇脱卤酶的定向进化增强阿托伐他汀关键中间体的生物催化生产
DOI：
10.1038/srep42064
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发表时间：
2017-02-06
影响因子：
4.6
通讯作者：
Zhang J
中科院分区：
文献类型：
--
作者：
Luo Y;Chen Y;Ma H;Tian Z;Zhang Y;Zhang J
卤代醇脱卤酶（HHDH）由于其形成C-C、C-N、C-O和C-S键的能力而在生物催化方面令人感兴趣。HHDH最重要的应用之一是HheC（来自放射性土壤杆菌AD 1的卤代醇脱卤酶）的蛋白质工程，用于（R）-4-氰基-3-羟基丁酸乙酯（HN）的工业生产，（R）-4-氰基-3-羟基丁酸乙酯（HN）是降胆固醇药物阿托伐他汀的关键手性合成子。在我们开发用于阿托伐他汀中间体的化学-酶促制备的替代的、更有效的和经济的途径期间，我们发现先前报道的用于HN制造的HheC 2360对于（3R，5S）-6-氰基-3，5-二羟基己酸叔丁酯（A7）的氰解生产没有足够的活性。在此，我们提出了集中的定向进化HheC 2360具有更高的活性和增强的生物催化性能，使用活性位点诱变。通过将产物A7对接到HheC 2360的晶体结构中，选择6个残基用于组合活性位点测试（CASTing）。在文库筛选后，变体V84 G/W86 F被鉴定为具有15倍的活性增加。由该变体催化的氰解反应的时程分析显示，与HheC 2360相比，时空产率增加了2倍。这些结果证明了变体V84 G/W86 F作为生物催化剂用于有效和实际生产阿托伐他汀中间体的适用性。
Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.