State Key Laboratory of Chemical Resources Engineering, Beijing University of Chemical Technology, Beijing, 100029, People's Republic of China.
Appl Microbiol Biotechnol. 2016 Nov;100(22):9543-9553. doi: 10.1007/s00253-016-7643-0. Epub 2016 Jun 2.
2-Azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) is an important precursor of many carbocyclic nucleoside analogs and pharmaceuticals. (-)-γ-Lactam has attracted much attention because of its role as an intermediate of antiviral drugs such as abacavir and carbovir. (+)-γ-Lactamase can be used for the kinetic resolution of γ-lactam to obtain (-)-γ-lactam. In this study, a novel (+)-γ-lactamase (Mh33H4-5540) was discovered from the gene library of Microbacterium hydrocarbonoxydans based on a colorimetric high-throughput screening method and it could be used to enantioselectively catalyze the bioresolution of racemic γ-lactam with high enantiomeric excess (ee) (>99 %) and yield (>49 %). An unexpected finding was that Mh33H4-5540 was unrelated to other known γ-lactamases (5.7, 4.8, 7.2, and 5.4 % similarities in amino sequence with (+)-γ-lactamase from Comamonas acidovorans, Bradyrhizobium japonicum, Aeropyrum pernix, and Sulfolobus solfataricus, respectively) but rather related to isochorismatases. The homolog analysis of Mh33H4-5540 revealed that it was similar in structure with bacterial isochorismatases (an isochorismatase from Pseudomonas putida (PDB number 4H17) and a putative isochorismatase from Oleispira antarctica (PDB number 3LQY)). Thus, Mh33H4-5540 represented another type of (+)-γ-lactamase. Mh33H4-5540 was overexpressed in E. coli Rosetta (DE3), purified to homogeneity and functionally characterized. The enzyme displayed optimal activity at 25 °C and pH 8.0. The activity showed a 5.5-fold increase in the presence of 0.5 M Ni or Co. Mh33H4-5540 displayed much higher (+)-γ-lactamase activity than any other biochemically characterized (+)-γ-lactamases. Overall, we discovered a novel (+)-γ-lactamase Mh33H4-5540 which displayed the highest activity. It could be a promising candidate of biocatalyst for industrial applications of highly valuable chiral pharmaceutical chemicals.
2-氮杂双环[2.2.1]庚-5-烯-3-酮（γ-内酰胺）是许多碳环核苷类似物和药物的重要前体。（-）-γ-内酰胺因其作为抗病毒药物如阿巴卡韦和卡波韦的中间体而备受关注。（+）-γ-内酰胺酶可用于动力学拆分γ-内酰胺以获得（-）-γ-内酰胺。在这项研究中，基于比色高通量筛选方法，从烃氧化微杆菌的基因文库中发现了一种新型（+）-γ-内酰胺酶（Mh33H4-5540），可用于对映选择性地催化外消旋γ-内酰胺的生物拆分，ee 值（>99%）和产率（>49%）均很高。一个意外的发现是，Mh33H4-5540与其他已知的γ-内酰胺酶（与来自 Comamonas acidovorans、Bradyrhizobium japonicum、Aeropyrum pernix 和 Sulfolobus solfataricus 的（+）-γ-内酰胺酶的氨基酸序列相似度分别为 5.7%、4.8%、7.2%和 5.4%）无关，而是与异柠檬酸裂解酶有关。Mh33H4-5540 的同源分析表明，它与细菌异柠檬酸裂解酶结构相似（来自 Pseudomonas putida 的异柠檬酸裂解酶（PDB 编号 4H17）和来自南极油杆菌的假定异柠檬酸裂解酶（PDB 编号 3LQY））。因此，Mh33H4-5540 代表了另一种类型的（+）-γ-内酰胺酶。Mh33H4-5540 在大肠杆菌 Rosetta（DE3）中过表达，纯化至均一性并进行功能表征。该酶在 25°C 和 pH 8.0 下表现出最佳活性。在存在 0.5 M Ni 或 Co 的情况下，活性增加了 5.5 倍。Mh33H4-5540 的（+）-γ-内酰胺酶活性明显高于任何其他生化表征的（+）-γ-内酰胺酶。总的来说，我们发现了一种新型（+）-γ-内酰胺酶 Mh33H4-5540，它表现出最高的活性。它可能是一种有前途的生物催化剂候选物，可用于工业应用中高价值手性药物化学品的生产。
