MECHANISM OF THE PQQ-DEPENDENT METHANOL DEHYDROGENASE

除了依赖烟酰胺和黄素的脱氢酶外，还有 是第三类称为藜麦蛋白的脱氢酶，它含有 新型辅因子吡咯喹啉醌(PQQ)而PQQ和藜麦蛋白 最早以甲基营养细菌为特征，现在是 公认在自然界中广泛分布的。除 藜麦蛋白脱氢酶，PQQ已被确定为 偶联原生生物氧化的含铜胺氧化酶 将O2还原为H2O2的胺；含PQQ的胺氧化酶 已在细菌、植物和哺乳动物中发现。此外，最近的数据 提示PQQ是小鼠的一种营养需求。显然，PQQ和 藜麦蛋白在自然界中具有广泛而重要的作用。 在甲基营养细菌中，依赖于PQQ的甲醇脱氢酶(MDH) 直接从甲醇的氧化过程中给出电子 电子传递链到O2；这一过程是化学渗透耦合的 以ADP和PI为原料净合成ATP。PQQ保持酶结合状态 在整个MDH催化循环中，并被认为作为2E-/2H+起作用 载体并直接与伯醇底物相互作用。 然而，关于MDH的催化机理的数据很少。这个 这项建议的总体目标是了解催化机理。 从Extorquens AM1中分离到依赖于PQQ的MDH。具体来说， 这里提出的实验将定义PQQ在 伯醇底物的氧化和化学形态的鉴定 PQQ结合的中间体在该机制中的作用。此程序的执行将 有助于我们对PQQ依赖机制的全面理解 酵素。

In addition to the nicotinamide- and flavin-dependent dehydrogenases, there is a third class of dehydrogenases called quinoproteins that contain the novel cofactor pyrroloquinoline quinone (PQQ). While PQQ and quinoproteins were first characterized in methylotrophic bacteria, they are now recognized to be widely distributed in nature. In addition to the quinoprotein dehydrogenases, PQQ has been identified as the cofactor in the copper-containing amine oxidases which couple the oxidation of primary amines with the reduction of O2 to H2O2; PQQ-containing amine oxidases have been identified in bacteria, plants, and mammals. In addition, recent data indicate that PQQ is a nutritional requirement in mice. Clearly, PQQ and quinoproteins play a diverse and important role in nature. In methylotrophic bacteria, the PQQ-dependent methanol dehydrogenases (MDH) donate electrons from the oxidation of methanol directly through an electron transport chain to O2; this process is coupled chemi-osmotically to the net synthesis of ATP from ADP and Pi. PQQ remains enzyme bound throughout the MDH catalytic cycle and is thought to function as a 2e-/2H+ carrier and to interact directly with the primary alcohol substrate. However, there is little data on the catalytic mechanism of MDH. The overall objective of this proposal is to understand the catalytic mechanism of the PQQ-dependent MDH from Methylbacterium extorquens AM1. Specifically, experiments are proposed here that will define the role of PQQ in the oxidation of the primary alcohol substrate and identify the chemical form of PQQ-bound intermediates in the mechanism. Execution of this program will contribute to our overall understanding of the mechanism of PQQ-dependent enzymes.