Active site recruitment in an ancestral, bifunctional ProFAR/PRA isomerase

两种类似的(b/a)8桶酶(HISA，TrpF)分别催化组氨酸和色氨酸生物合成中的异构化反应。然而，一些微生物只包含这两个基因中的一个，互补实验表明对应的基因产物PriA具有双底物特异性，与先前假设的HISA/TrpF前体酶的性质相匹配。我们建议模仿酶多样化导致单一底物专一性的进化过程。在第一步中，我们将通过稳态动力学来确定PriA的双功能催化性质，在底物类似物存在和不存在的情况下确定其X射线结构，并通过定点突变来探索几个活性残基对扩大底物专一性的贡献。在项目的第二步，我们将通过应用随机突变方法，使用对组氨酸或色氨酸具有营养缺陷性的大肠杆菌菌株，分别筛选类似HISA和TrpF的PriA单底物变体。

Two similar (b/a)8 barrel enzymes (HisA, TrpF) catalyse isomerisation reactions in the histidine and tryptophan biosynthesis, respectively. Some microorganisms, however, comprise only one of the two genes, and complementation experiments have suggested dual substrate specificity of the corresponding gene product PriA, matching the properties of a previously hypothesized promiscuous HisA/TrpF precursor enzyme. We are proposing to mimic the evolutionary process of enzyme diversification leading to single substrate specificity. In the first step, we will determine the bifunctional catalytic properties of PriA by steady-state kinetics, determine its X-ray structure in the absence and presence of substrate analogues, and probe the contributions of several active residues to broadened substrate specificity by site directed mutagenesis. In the second step of the project, we will separately screen for HisA-like and TrpF-like PriA single substrate variants by the application of random mutagenesis methods, using E. coli strains that are auxotroph either for histidine or tryptophan.