吡咯喹啉醌生物合成途径中AHQ中间产物的生成反应机制研究

Pyrroloquinoline quinone (PQQ) has an orthoquinone structure that is directly responsible for the oxidoreduction, and thus it has been evaluated as the third class coenzyme following pyridine nucleotide and flavin in biological oxidoreduction and serves as a new Vitamin B in mammals for physiological importance. In previous investigation, it has been shown that the polypeptide PqqA containing highly conserved Glu and Tyr residues serves as the precursor for PQQ bio-synthesis. However, the mechanism of the polypeptide processing has not been fully elucidated yet. Recently, we have found that the interaction between protein F and polypeptide A was significant observed by a VP-ITC (Isothermal Titration Microcalorimetry). Furthermore, according to the literature, theory analysis, and our precious research work, we propose that the putative peptidase PqqF may catalyze the polypeptide PqqA processing, and the putative SAM protein PqqE may play an essential role in the ligation between Glu residue and Tyr residue. Therefore, the aim of this project is to elucidate the catalytic mechanism of PqqF and PqqE and its molecular basis. Firstly, the intermediate, i.e., the product after the protein F catalyze the polypeptide A in vivo and in vitro, will be isolated and purified. And the reaction catalyzed by protein F will be determined using a VP ITC. This section will yield the thermodynamic and reaction kinetics measurements. Secondly, a fundamental problem is to understand how enzyme work. A combination of biochemical and biophysical methods will be used to determine the relationship between the structure and function of this protein. The interaction between protein F and polypeptide A will resolve the very perplexing question of how protein F catalytic functions and add substantially to our knowledge of peptidase structure and function, in general. Finally, the interaction between protein E and the intermediate containing the Glu and Tyr residues will be carried out on a VP ITC. This section will be helpful to understand how the protein E processes the intermediate. The scientific broader impacts of this project will not only elucidate the mechanism of the polypeptide processing catalyzed by PqqF,but also this demonstrate the function of PqqE. The detailed studies may aid in the development of PQQ biosynthesis and shed light on the noval mechanisms of proteome diversification found in G.oxydans.

吡咯喹啉醌（Pyrroloquinoline quinone,PQQ）是继烟酰胺和核黄素之后第三类辅酶，为B 族维生素新成员，在体内具有增强免疫力、促进细胞生长和抗氧化等重要生理功能。PQQ由基因簇pqqA-F编码的蛋白负责合成，其中PqqA多肽为PQQ合成的前体化合物，但至今该化合物至中间产物AHQ（Acid hydroquinoline）的生成反应机制尚未揭示。根据理论分析和前期研究，我们提出了PqqF和PqqE蛋白在该阶段的催化作用假说。为此，本课题计划通过体内外构建PQQ合成体系，分离与鉴定中间产物，研究PqqF和PqqE两种蛋白与底物的相互作用关系，揭示PqqF对PqqA多肽的剪切机制和PqqE对谷氨酸和酪氨酸之间的环化机制，最终阐明AHQ的生成反应机理。该项研究不仅进一步完善PQQ合成途径，而且开拓体外酶法全合成吡咯喹啉醌类化合物新领域，为规模化制备PQQ打下坚实的理论基础。

该项目详细研究了PQQ在反应液、发酵液中的检测分析技术，以及PQQ的分离技术和表征技术，发现HP-20大孔固相萃取可以减少其他物质在液质中对PQQ的信号干扰，建立了PQQ的检测分析技术。在此基础上，进一步通过对基因簇各个基因的克隆和表达、以及细胞内与PQQ合成有关基因的敲除，运用液相和质谱以及NMR技术，分离、纯化和表征了PQQ合成各阶段的产物，以及各步反应的动力学，揭示了PQQ合成路径及反应条件，即，PqqA首先经PqqE酶和D酶作用，生成环化多肽，后经PqqF酶水解，再经PqqC蛋白氧化合成PQQ，PqqB与合成无关，但与PQQ分泌到发酵液有关，在此过程中，发现了PqqA环化反应为限速步骤，该步反应耗时120小时才能达到产物浓度的最高位点。探索了PQQ无细胞合成体系，在反应液中外加人工合成的前体物PqqA进行合成PQQ条件研究，同时对PqqA多肽里谷氨酸残基和酪氨酸残基的N进行了同位素标记，在终产物PQQ里发现了同位素标记的氨基酸，揭示了PQQ的骨架确实来源于谷氨酸和酪氨酸残基，同时也发现不同电子供体对反应的促进与破坏，发现氧气对合成影响并不大，无机化合物亚硫酸钠对合成反应有破坏作用，而有机化合物FAD或FMN，以及NADH，对合成反应有促进作用。另外，进行了PQQ与SOD酶相互作用研究，阐述了PQQ提高SOD酶捕捉自由基活性的结构变化，同时，也研究了PQQ对促进种子萌发以及提高食品营养方面的研究等。以上研究为全酶法合成PQQ打下了理论基础，同时开拓了PQQ应用领域，为PQQ产业化生产提供理论技术支撑。