【作者】 孙宗涛；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理， 2010， 博士

【摘要】 耐辐射球菌(Deinococcus radiodurans, DR)以对电离辐射,UV辐射,干燥以及DNA损伤试剂具有超强的抗性而著称,它能在几个小时内准确地修复由辐射产生的几十个双链DNA碎片(double-strand breaks, DSBs)。耐辐射球菌所具有的抗性能力主要归功于其体内高效的DNA修复系统和抗氧化系统。电离辐射所产生的约80%DNA损伤是由辐射水解形成的活性氧自由基(reactive oxygen species,ROS)攻击DNA造成的,负责清除活性氧自由基的抗氧化系统对DR菌的辐射抗性有着重要的贡献。类胡萝卜素作为抗氧化系统非酶类的一种,能有效的清除单线态氧(1O2)等活性氧自由基,而DR菌的主要类胡萝卜素产物deinoxanthin中的特殊活性基团如C-3’,4’双键、C-1′羟基、C-4酮基对其活性功能有着重要的影响。因此,对这些活性基团合成酶的研究能够帮助我们阐明DR菌类胡萝卜素的合成途径,并更好地理解特殊活性类胡萝卜素对DR菌极端抗性的贡献。本文研究主要是围着类胡萝卜素活性基团合成酶的功能展开的研究,主要内容和研究结果如下：1.通过生物信息学方法我们发现DR菌中存在编码甲氧基链孢红素脱氢酶(CrtD)的同源物DR2250,并利用PCR方法构建了DR2250的缺失突变株R1ΔcrtD。通过比较突变株R1ΔcrtD与野生型R1的类胡萝卜素组成发现,R1ΔcrtD主要色素产物为3’,4’-dihydrodeinoxanthin,与R1中主要产物deinoxanthin比较,在C-3’,4’位各多了一个氢原子,这表明DR2250编码C3’,4’-脱氢酶。进一步利用质粒共转化方法检验DR2250蛋白具体的功能,结果得到DR2250所催化的底物是具有选择性的,它不能催化Ψ末端的番茄红素,而只能催化经过羟基化修饰的Ψ末端。这也证实在耐辐射球菌体内DR2250催化的C-3’,4’位的脱氢反应是发生在C-1′的羟基化之后。对比R1和R1ΔcrtD在氧化胁迫条件下的生存率得知,突变株的抗氧化能力要比野生型低,并且无论是否在氧化处理条件下突变株R1ΔcrtD中ROS水平要高于野生型R1。比较R1和R1ΔcrtD中主要色素产物自由基清除能力的结果表明,3’,4’-dihydrodeinoxanthin作为突变株体内主要的产物比野生型中deinoxanthin的自由基清除能力低。这些结果证明DR2250负责的C-3’,4’位的脱氢反应对deinoxanthin的抗氧化活性是有影响的。2.研究表明类胡萝卜素链末端的C-1羟基基团对色素的抗氧化能力具有重要的作用。我们通过分析参与色素合成基因的临近编码基因发现了一种新类型的C-1′,2′-水合酶DR0091(来自D. radiodurans)和Dgeo2309(来自D. geothermalis)。质粒共转化实验表明它们都能以链孢红素、番茄红素和γ-胡萝卜素为底物,产生羟基化的色素产物。通过构建DR0091缺失突变株R1ΔcruF发现,突变株中类胡萝卜素组成产生了三种非C-1′羟基化产物,进一步证实了DR0091在DR菌体内编码C1′,2′-水合酶。进化树分析DR0091及其同源物与已知的CrtC类型水合酶发现,它们不同于CrtC类型水合酶。3. Deinoxanthin是一种酮基化的类胡萝卜素,它的合成需要在β环C4位置经过酮基化酶修饰得到。已有研究报道表明DR0093编码β-胡萝卜素酮基化酶,它能将β-胡萝卜素转化为4-酮基-β-胡萝卜素和4,4′-二酮基-β-胡萝卜素。但是DR菌中类胡萝卜素都是单环的,所以DR0093在耐辐射球菌体内具体的功能尚不清楚。我们利用DNA重组技术构建了DR0093缺失突变体R1ΔcrtO,分析其体内的色素组成发了明显的变化,产生了三种非酮基化类胡萝卜素：1-OH-γ-胡萝卜素,1’-OH-3’,4’-双脱氢-γ-胡萝卜素,2,1’-OH-3’,4’-双脱氢-γ-胡萝卜素。这表明DR0093的缺失导致了deinoxanthin合成途径受到阻断,产生了非酮基化的色素。构建dr0093的补偿质粒到突变株R1ΔcrtO中,补偿株又产生和R1同样的色素组成,这意味着突变株中色素组成的变化完全是由DR0093缺失造成的。在正常生长状态下,野生型R1和突变株R1ΔcrtO并没有差异,而在氧化胁迫条件下,R1ΔcrtO的生存率却比R1发生了明显的下降。比较R1和R1ΔcrtO体内类胡萝卜素的自由基清除能力,我们发现R1ΔcrtO的色素产物DPPH’清除能力要比野生型中的色素低。由此可以看出,DR0093所负责的酮基化反应对于deinoxanthin的抗氧化活性有着重要的作用。综上所述,deinoxanthin结构中的活性基团对其抗氧化能力具有重要的贡献,同时,这些活性基团合成酶的研究有助于我们对DR菌中类胡萝卜素生物合成途径的了解。更多还原

【Abstract】 Deinococcus radiodurans is a red-pigmented and nonphotosynthetic bacterium well known for its resistance to y-ray, ultraviolet radiation and desiccation. Its extremely resistance was attributted to its highly efficient antioxidative effect and the complex network of DNA repair mechanism. Most of the damaging effects of ionizing radiation on biological macromolecules are due to the reactive oxygen species (ROS) produced by water radiolysis. Among non-enzymic antioxidants, carotenoids are efficient scavengers of ROS, especially of singlet oxygen. The major carotenoid in D. radiodurans is a unique hydroxylated ketocarotenoid (deinoxanthin), which shows stronger ROS scavenging ability than lycopene andβ-carotene and contributes to the cell resistance of D. radiodurans under oxidative stress. The active groups of deinoxanthin, including C-3’,4’double bond, C-1’hydroxyl group and C-4 keto group, may be important for the antioxidant ability of deinoxanthin. So, investigations of the carotenoid synthetases for these active groups can help us to reveal the biosynthetic pathway of deinoxanthin and understand the role of carotenoids in the cell resistance of DR. This Ph.D thesis focuses on identification of carotenoid synthetases for the active groups. The main results are as follows:1. The biosynthetic pathway of deinoxanthin is unclear, although several enzymes are presumed to be involved. The gene (dr2250) was predicted by gene homologue analysis to encode carotenoid 3’,4’-desaturase (CrtD). This putative gene was deleted to investigate its function. Carotenoid analysis of the resultant mutant verified that DR2250 encodes the carotenoid 3’,4’-desaturase, which catalyses the C3’,4’-desaturation of the monocyclic precursor of deinoxanthin but not acyclic carotenoids. The co-transformed plamids experiments confirmed that DR2250 can not catalyze the non-modifiedψend, but only catalyze theψend with hydroxylation at C1’. The lack of CrtD decreased the antioxidant capacity of the mutant of dr2250 compared with the wild-type, indicating that the C3’,4’-desaturation step contributes to the antioxidant capacity of deinoxanthin in D. radiodurans.2. Carotenoid 1,2-hydratase is required to catalyse the synthesis of deinoxanthin by hydration at the C-1’,2’double bond. A novel carotenoid 1,2-hydratase (CruF) responsible for the C-1’,2’hydration of y-carotene was identified in the non-photosynthetic bacteria D. radiodurans R1 and D. geothermalis DSM 11300. Gene expression and disruption experiments demonstrated that dr0091 and dgeo2309 encode CruF in D. radiodurans and D. geothermalis, respectively. Their homologues were also found in the genomes of cyanobacteria, and exhibited little homology to the hydroxyneurosporene synthase (CrtC) proteins found mainly in photosynthetic bacteria. Phylogenetic analysis showed that CruF homologues form a separate family, which is evolutionarily distant from the known CrtC family.3. D. radiodurans strain R1 synthesizes the unique ketocarotenoid deinoxanthin. It was reported that expression of DR0093 in the E. coli strain which accumulated P-carotene resulted in the synthesis of canthaxanthin and echinenone. However, the bicyclic carotenoids were not the intermediate product of the deinoxanthin biosynthetic pathway, in which only monocyclic carotenoids were detected. Thus, the function of DR0093 and its catalyzing step need to be elucidated in the native host. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans. Analysis of the carotenoids in the resultant mutant RIΔcrtO demonstrated that dr0093 encodesγ-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position ofγ-carotene derivatives to form ketolated carotenoids. The mutant R1ΔcrtO became more sensitive to H2O2 treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans. Carotenoid extracts from mutant R1ΔcrtO exhibited lower DPPH radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.In sum, our results demonstrated that the active groups of deinoxanthin were important for its antioxidant capability. The carotenoid biosynthetic enzymes identified from D. radiodurans can also be used, via genetic engineering, for the production of novel carotenoids with high activities.更多还原