沙雷氏菌灵菌红素的前体MBC合成途径中相关蛋白结构解析及反应机制的研究

Prodigiosins are a family of red pigments which contain a pyrrolylpyrromethane as skeleton. Various bacteria such as Serratia marcescens, Streptomyces coelicolor A3(2) and some marine bacteria could produce this kind pigments. Because of their anticancer, antibacterial, antimalarial and antifungal activities, prodigiosins become a promising drug and are attracting increasing research interests especially for its anticancer activity. In vitro, prodigiosins have been shown to primarily target the cancer cells with little or no effect on normal cells. Recently, one synthetic prodigiosin (obatoclax) has shown promising activity in both preclinical and clinical trials and encouraging results in phase Ⅱ clinical trials. Because of the lower efficiency and much more pollutant during synthesizing, biosynthesis of prodigiosin has attracted intense interest. Results show that 2-methyl-3-n-amyl-pyrrole (MAP) and 4-methoxy-2,2-bipyrrole-5-carbaldehyde (MBC) were separately synthesized by two different pathways and then condensed to form prodigiosin. Although the synthesis pathway of MAP is totally different between synthesis of prodigiosin and undecylprodigiosin, MBC synthetic pathway is conserved in Serratia spp and Streptomyces coelicolor A3(2) and shares some similarity with non-ribosomal peptide synthetic pathway(NRPSs) and the polyketide synthetic pathway, but the catalytic mechanism for MBC synthesis is still unclear. This project will focus on the determination of the crystal structures of the enzymes involving in the MBC pathway, especially for some important proteins such as PigI, PigH, PigJ, and to elucidate the synthetic mechanism by combining the crystal structures with mutation analysis. The crystal structures of the proteins in MBC synthetic pathway in Serratia will not only provide insight for the synthetic mechanism of MBC in other bacteria, but also help to understand the difference between the enzymes involved in MBC synthesis and the corresponding enzymes which participate in the synthesis of NRPs and fatty acid. All the results may suggest several strategies for engineering the biosynthesis of novel prodiginines or NRPs for potential medical use.

灵菌红素(Prodigiosin)是一类以吡咯基吡咯甲烷为骨架的天然红色色素，具有抗癌，抗微生物，抗疟疾，抗霉菌，溶锥虫等功能。研究发现其是经MBC和MAP途径先合成前体，然后经缩合而成，其中MBC的合成途径非常保守，并与非核糖体肽以及饱和脂肪酸的生物合成反应具有一定的相似性，但具体的机制不明确。本项目拟运用结构生物学等方法解析MBC合成途径中的重要酶类（PigI，PigH及PigJ等）的晶体结构，并对一些重要蛋白的活性中心进行突变研究，从而明确其催化机制。这些蛋白的三维结构解析，不仅可以阐明沙雷氏菌中MBC合成中的具体反应机制，为诠释其它微生物MBC的合成机理提供科学依据，同时还能够明确其与NRPs和饱和脂肪酸合成起始反应中相关蛋白的结构及催化机制的异同点，从而为今后该类蛋白可能的人工改造，进而为将来可能合成更多的一些具有药用价值的灵菌红素或多肽类次生代谢物等新的有用产物提供理论依据。

灵菌红素（prodigiosins, PG）是一类具有三吡咯环结构的暗红色的脂溶性色素，是由放线菌、沙雷氏菌及一些其他细菌产生的次级代谢产物，具有免疫抑制、抗细菌、抗真菌和抗疟疾等多种生物活性。灵菌红素的生物合成由MAP（2-methyl-3-n-amyl-pyrrole）和MBC（4-methoxy-2,2’-bipyrrole-5-carbaldehyde）两条分支途径组成。为了明确沙雷氏菌中MBC合成途径中的具体反应机制，本研究运用蛋白质晶体结构生物学及分子遗传学等方法对沙雷氏菌中MBC合成途径中相关蛋白的三维结构，功能及催化机制进行了解析。.本研究运用分子置换的方法解析了PigG蛋白晶体的三维结构，并对PigG的功能以及PigG的第36位的丝氨酸，第40位的苯丙氨酸及第59位的异亮氨酸进行了研究，结果发现， pigG的缺失，或将上述这些氨基酸突变都会导致灵菌红素不再合成，因此，PigG对灵菌红素合成至关重要，Ser36，Phe40及Ile59均在PigG发挥功能中起重要作用。.本研究获得了PigI蛋白晶体并用分子置换方法解析了其三维结构，通过在结晶条件中添加不同的底物、底物类似物等，解析了PigI与其底物L-脯氨酸，D-脯氨酸，L-脯氨酸及AMP，L-脯氨酸与ATP，L-脯氨酸与AMPPCP以及L-脯氨酸与ATP及镁离子等的复合物的晶体结构，这些结果为明确PigI的催化机制以及其底物选择机制奠定了基础。.通过分子置换方法解析了PigA蛋白晶体结构。PigA蛋白的晶体结构中大致有三个结构域，包含六个α-螺旋（α1-α6）的N端结构域，中间的β-折叠结构域（β1-β7）和C-端结构域。辅酶FAD以伸展的构象结合在C-端和N-端结构域的形成的凹穴处，主要是通过氢键和范德华力牢固地与蛋白结合。.通过表达硒代甲硫氨酸取代甲硫氨酸的PigF蛋白，获得晶体并收集衍射数据后，运用SAD方法解析了PigF的晶体结构。在PigF蛋白中加入SAH后重新筛选结晶条件后，获得PigF与SAH的复合体晶体，收集衍射数据后，用野生型PigF为模板，解析了PigF与SAH复合体晶体结构。PigF晶体结构与PigF的SAH复合体结构相比，催化亚基有很大的构象改变，其催化属于典型的诱导契合模式，该结构的解析为明确其甲基化的机制奠定了基础。