Protein and Peptide Derived Cofactors

描述（由申请人提供）：该项目侧重于产生肽和蛋白质衍生的醌辅因子、吡咯并喹啉醌（PQQ）和三羟基苯丙氨酸醌（TPQ）的需氧和非需氧工艺。所描述的研究探索了一组反应，其特征在于前所未有或非常不寻常的机制和结构功能关系。PQQ生物合成是导致酶辅因子的途径中了解最少的途径之一，并且正在研究PQQ生产中编码必需蛋白质的五个保守开放阅读框架中的每一个。这些包括肽底物（PqqA），可能的加氧酶（PqqB），自由基SAM酶（PqqE）和一种功能尚未确定的蛋白质（PqqD）。除了对分离酶的计划研究外，还将对蛋白质/蛋白质复合物形成的重要性进行详细研究。这些研究有望揭示PQQ途径如何能够整合必须厌氧（PqqE）或需要摄取分子氧（PqqC和PqqB）的反应。PqqC是一种氧化酶，在没有任何金属或有机辅因子的情况下发挥作用，通过八电子，八质子氧化催化PQQ形成的最后一步。我们已经开发了一种酸淬灭试验，使我们能够表征PqqC反应路径上沿着发生的许多化学中间体。这种新的试验与定点诱变结合使用，以检查特定氨基酸侧链（i）在产生O2结合/反应性口袋中的作用，（ii）在逐步提取八个 电子和质子从衬底，和（iii）在创建一个路径，允许抽象的质子被“引导”向活性部位氧化剂。PqqC/产物复合物的结构研究暗示了控制O2反应性的大的构象变化，以及在分子内质子转移导管中使用底物本身。本项目的最终目的是利用表达的前体酵母铜胺氧化酶研究TPQ的生物发生。与PQQ生产相反，TPQ是通过自处理机制形成的。这些研究使用非天然氨基酸4-氨基苯丙氨酸（p-AF），该氨基酸已成功插入酶活性位点内的三个关键位置。p-AF的性质与取代的酪氨酸有关，揭示了辅因子生物合成和酶催化的重要特征。

DESCRIPTION (provided by applicant): This project is focused on the aerobic and non-aerobic processes that create the peptide and protein- derived quino-cofactors, pyrroloquinoline quinone (PQQ) and trihydroxyphenylalanine quinone (TPQ). The described studies explore a cohort of reactions that are characterized by both unprecedented or highly unusual mechanisms and structure function relationships. PQQ biosynthesis is one of the least well understood among the pathways that lead to enzymatic cofactors, and each of the five conserved open reading frames that encode for essential proteins in PQQ production is being studied. These include the peptide substrate (PqqA), a likely oxygenase (PqqB,) a radical SAM enzyme (PqqE), and a protein with, as yet, no defined function (PqqD). In addition to the planned studies on isolated enzymes, the importance of protein/protein complex formation will undergo detailed investigation. These studies are expected to reveal how the PQQ pathway is able to integrate reactions that either must be anaerobic (PqqE) or require the uptake of molecular oxygen (PqqC and PqqB). PqqC is an oxidase that functions in the absence of any metal or organic cofactor, catalyzing the final step in PQQ formation via an eight-electron, eight-proton oxidation. We have developed an acid quench assay that allows us to characterize many of the chemical intermediates that occur along the PqqC reaction path. This new assay is being used in conjunction with site-specific mutagenesis, to examine the role of specific amino acid side chains (i) in generating an O2 binding/reactivity pocket, (ii) in the stepwise abstraction of eight electrons and protons from the substrate, and (iii) in the creation of a pathway that permits the abstracted protons to be "guided" toward the active site oxidant. Structural studies of the PqqC/product complex implicate a large conformational change that controls O2 reactivity, and the use of substrate itself in the intra-molecular proton transfer conduit. The final aim of this project focuses on the biogenesis of TPQ, using an expressed, precursor yeast copper amine oxidase. In contrast to PQQ production, TPQ is formed via a self-processing mechanism. These studies use an unnatural amino acid, 4-amino-phenylalanine (p-AF) that has been successfully inserted at three key positions within the enzyme active site. The properties of p-AF, in relation to the replaced tyrosines, are uncovering important features of both cofactor biosynthesis and enzymatic catalysis.