Department of Botany and Plant Biology, University of Geneva, Geneva, Switzerland.
PLoS One. 2011 Jan 21;6(1):e16042. doi: 10.1371/journal.pone.0016042.
The prevalent de novo biosynthetic pathway of vitamin B6 involves only two enzymes (Pdx1 and Pdx2) that form an ornate multisubunit complex functioning as a glutamine amidotransferase. The synthase subunit, Pdx1, utilizes ribose 5-phosphate and glyceraldehyde 3-phosphate, as well as ammonia derived from the glutaminase activity of Pdx2 to directly form the cofactor vitamer, pyridoxal 5'-phosphate. Given the fact that a single enzyme performs the majority of the chemistry behind this reaction, a complicated mechanism is anticipated. Recently, the individual steps along the reaction co-ordinate are beginning to be unraveled. In particular, the binding of the pentose substrate and the first steps of the reaction have been elucidated but it is not known if the latter part of the chemistry, involving the triose sugar, takes place in the same or a disparate site. Here, we demonstrate through the use of enzyme assays, enzyme kinetics, and mutagenesis studies that indeed a second site is involved in binding the triose sugar and moreover, is the location of the final vitamin product, pyridoxal 5'-phosphate. Furthermore, we show that product release is triggered by the presence of a PLP-dependent enzyme. Finally, we provide evidence that a single arginine residue of the C terminus of Pdx1 is responsible for coordinating co-operativity in this elaborate protein machinery.
维生素 B6 的主要从头生物合成途径仅涉及两种酶（Pdx1 和 Pdx2），它们形成一个复杂的多亚基复合物，作为谷氨酰胺酰胺转移酶发挥作用。合成酶亚基 Pdx1 利用核糖 5-磷酸和甘油醛 3-磷酸以及来自 Pdx2 的谷氨酰胺酶活性衍生的氨，直接形成辅酶维生素 B6 的前体吡哆醛 5'-磷酸。鉴于单个酶执行该反应背后的大部分化学反应，预计会有一个复杂的机制。最近，反应协调的各个步骤开始被揭示。特别是，已经阐明了戊糖底物的结合以及反应的第一步，但尚不清楚涉及三碳糖的化学的后一部分是在同一还是不同的部位进行。在这里，我们通过使用酶测定、酶动力学和突变研究证明，确实存在第二个结合三碳糖的结合位点，而且该位点是最终维生素产物吡哆醛 5'-磷酸的位置。此外，我们表明产物释放是由 PLP 依赖性酶的存在触发的。最后，我们提供的证据表明，Pdx1 的 C 末端的单个精氨酸残基负责协调这种复杂的蛋白质机器中的协同作用。
