Smith Amber Marie, Brown William Clay, Harms Etti, Smith Janet L
From the Department of Biological Chemistry, Life Sciences Institute.
Life Sciences Institute, Center for Structural Biology, University of Michigan, Ann Arbor, Michigan 48109 and.
J Biol Chem. 2015 Feb 27;290(9):5226-39. doi: 10.1074/jbc.M114.626382. Epub 2015 Jan 7.
PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5'-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.
磷酸吡哆醛合酶（PLPS）是一种非凡的单酶生物合成途径，可从谷氨酰胺、5-磷酸核糖和3-磷酸甘油醛生成5'-磷酸吡哆醛（PLP）。完整的酶包括12个合酶亚基和12个谷氨酰胺酶亚基。PLP的合成通过一种复杂的机制在合酶活性位点发生，该机制涉及催化赖氨酸处的至少两个共价中间体。第一个中间体与5-磷酸核糖形成。谷氨酰胺酶亚基是一种谷氨酰胺酰胺转移酶，可水解谷氨酰胺并将氨输送到合酶活性位点。氨对第一个共价中间体的攻击形成第二个中间体。3-磷酸甘油醛与第二个中间体反应形成PLP。为了研究合酶亚基的机制，获得了嗜热栖热放线菌完整PLPS或其合酶亚基三种中间状态的晶体结构。这些结构捕捉了合酶活性位点在其复杂催化循环中的三个不同步骤，深入了解了难以捉摸的机制，并说明了合酶亚基内将催化状态分开的协同运动。在谷氨酰胺酶活性位点具有类似米氏中间体的完整PLPS中，合酶的第一个共价中间体通过一个通常无序的20个残基C末端尾巴的有序排列而完全隔离在酶内。加入氨后，合酶活性位点打开并接纳参与第二个中间体和PLP形成的Lys-149侧链。确定了保守的Asp-24在第一个中间体形成中的作用以及保守的Arg-147在第一个中间体向第二个中间体转化中的作用。
