Zhang S, Pohnert G, Kongsaeree P, Wilson D B, Clardy J, Ganem B
Section of Biochemistry, Molecular and Cellular Biology, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, USA.
J Biol Chem. 1998 Mar 13;273(11):6248-53. doi: 10.1074/jbc.273.11.6248.
The bifunctional P-protein, which plays a central role in Escherichia coli phenylalanine biosynthesis, contains two catalytic domains (chorismate mutase and prephenate dehydratase activities) as well as one R-domain (for feedback inhibition by phenylalanine). Six genes coding for P-protein domains or subdomains were constructed and successfully expressed. Proteins containing residues 1-285 and residues 1-300 retained full mutase and dehydratase activity, but exhibited no feedback inhibition. Proteins containing residues 101-386 and residues 101-300 retained full dehydratase activity, but lacked mutase activity. Fluorescence emission spectra and binding assays indicated that residues 286-386 were crucial for phenylalanine binding. The mutase (residues 1-109), dehydratase (residues 101-285), and regulatory (residues 286-386) activities were thus shown to reside in discrete domains of the P-protein. Both the mutase domain and the native P-protein formed dimers. Deletion of the mutase domain diminished phenylalanine binding to the regulatory site as well as prephenate binding to the dehydratase domain, both through cooperative effects. Besides eliminating feedback inhibition, removal of the R-domain decreased the affinity of chorismate mutase for chorismate.
在大肠杆菌苯丙氨酸生物合成中起核心作用的双功能P蛋白，包含两个催化结构域（分支酸变位酶和预苯酸脱水酶活性）以及一个R结构域（用于苯丙氨酸的反馈抑制）。构建并成功表达了六个编码P蛋白结构域或亚结构域的基因。含有1 - 285位残基和1 - 300位残基的蛋白质保留了完整的变位酶和脱水酶活性，但没有表现出反馈抑制。含有101 - 386位残基和101 - 300位残基的蛋白质保留了完整的脱水酶活性，但缺乏变位酶活性。荧光发射光谱和结合试验表明，286 - 386位残基对苯丙氨酸结合至关重要。因此，变位酶（1 - 109位残基）、脱水酶（101 - 285位残基）和调节（286 - 386位残基）活性存在于P蛋白的不同结构域中。变位酶结构域和天然P蛋白均形成二聚体。通过协同效应，变位酶结构域的缺失减少了苯丙氨酸与调节位点的结合以及预苯酸与脱水酶结构域的结合。除了消除反馈抑制外，R结构域的去除还降低了分支酸变位酶对分支酸的亲和力。
