Directed evolution of ()8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase -subunit
Directed evolution of ()8-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase -subunit
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DOI：
10.1093/protein/gzs015
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发表时间：
2012-06-01
影响因子：
2.4
通讯作者：
Sterner, Reinhard
中科院分区：
文献类型：
--
作者：
Evran, Serap;Telefoncu, Azmi;Sterner, Reinhard
磷酸核糖基氨基苯甲酸（PRA）异构酶（TrpF）和色氨酸合成酶亚基（TrpA）是参与色氨酸生物合成的（±）（8）-桶酶.它们含有一个保守的磷酸盐结合位点，这表明它们具有共同的进化起源。为了通过实验支持这一假设，我们已经使用蛋白质工程在来自鼠伤寒沙门氏菌的TrpA的支架上建立了TrpF活性。基于晶体结构与结合配体的叠加，使用定点突变将TrpA活性位点中的两个残基替换为TrpF的催化残基。使用易错PCR对该TrpA变体以及野生型TrpA各自进行随机诱变。两个所得的trpA基因文库用于转化营养缺陷型大肠杆菌trpF缺失菌株，并通过体内互补分离具有PRA异构化活性的TrpA变体。通过DNA改组重组所选TrpA变体的氨基酸取代，然后再次进行体内互补。在E.大肠杆菌中表达，并纯化，采用稳态酶动力学法测定其体外催化TrpF活性。我们的研究结果支持，TrpA和TrpF已经演变的基因复制和多样化从对方或一个共同的前身，并提供了深入了解PRA异构化催化的最低要求。
Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase -subunit (TrpA) are ()(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.