Nixon A E, Warren M S, Benkovic S J
Department of Chemistry, Pennsylvania State University, University Park 16802-6300, USA.
Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1069-73. doi: 10.1073/pnas.94.4.1069.
The feasibility of creating new enzyme activities from enzymes of known function has precedence in view of protein evolution based on the concepts of molecular recruitment and exon shuffling. The enzymes encoded by the Escherichia coli genes purU and purN, N10-formyltetrahydrofolate hydrolase and glycinamide ribonucleotide (GAR) transformylase, respectively, catalyze similiar yet distinct reactions. N10-formyltetrahydrofolate hydrolase uses water to cleave N10-formyltetrahydrofolate into tetrahydrofolate and formate, whereas GAR transformylase catalyses the transfer of formyl from N10-formyltetrahydrofolate to GAR to yield formyl-GAR and tetrahydrofolate. The two enzymes show significant homology (approximately 60%) in the carboxyl-terminal region which, from the GAR transformylase crystal structure and labeling studies, is known to be the site of N10-formyltetrahydrofolate binding. Hybrid proteins were created by joining varying length segments of the N-terminal region of the PurN gene (GAR binding region) and the C-terminal (N10-formyltetrahydrofolate binding) region of PurU. Active PurN/PurU hybrids were then selected for by their ability to complement an auxotrophic E. coli strain. Hybrids able to complement the auxotrophs were purified to homogeneity and assayed for activity. The specific activity of two hybrid proteins was within 100- to 1000-fold of the native purN GAR transformylase validating the approach of constructing an enzyme active site from functional parts of others.
基于分子募集和外显子改组的概念，从已知功能的酶创造新的酶活性在蛋白质进化方面是有先例的。大肠杆菌基因purU和purN分别编码的N10-甲酰四氢叶酸水解酶和甘氨酰胺核糖核苷酸（GAR）转甲酰基酶，催化相似但不同的反应。N10-甲酰四氢叶酸水解酶利用水将N10-甲酰四氢叶酸裂解为四氢叶酸和甲酸，而GAR转甲酰基酶催化甲酰基从N10-甲酰四氢叶酸转移到GAR，生成甲酰-GAR和四氢叶酸。这两种酶在羧基末端区域显示出显著的同源性（约60%），从GAR转甲酰基酶的晶体结构和标记研究可知，该区域是N10-甲酰四氢叶酸的结合位点。通过连接PurN基因N端区域（GAR结合区域）不同长度的片段和PurU的C端（N10-甲酰四氢叶酸结合）区域，构建了杂合蛋白。然后根据它们补充营养缺陷型大肠杆菌菌株的能力，筛选出有活性的PurN/PurU杂合体。能够补充营养缺陷型的杂合体被纯化至同质，并进行活性测定。两种杂合蛋白的比活性在天然purN GAR转甲酰基酶的100到1000倍范围内，验证了从其他酶的功能部分构建酶活性位点的方法。
