【作者】 许镇坚；

【导师】 华跃进；

【作者基本信息】 浙江大学 ， 生物物理学， 2008， 博士

【摘要】 耐辐射球菌（Deinococcus radiodurans，简称DR）是地球上辐射抗性最强的生物之一，它以对电离辐射和干燥等表现强耐受力而著称。已有研究显示，DR菌极端生存能力是其保护、耐受和修复三方面协同作用的结果，而高效的抗氧化机制和DNA损伤修复是当前研究热点。最新研究认为，电离辐射产生的DNA损伤约80％由辐射水解产生的活性氧自由基（reactive oxygen species，ROS）攻击而间接导致，这表明抗氧化损伤保护机制在DR菌中可能扮演重要角色。因此，鉴定和研究DR菌中高效抗氧化物质，对阐明抗氧化保护机制在DR菌中的贡献及解释其极端抗性机制具有重要意义。类胡萝卜素能高效猝灭单线态氧（¹O₂），并清除多种ROS对生物体造成的伤害。DR菌体内大量合成以deinoxanthin为主要产物的类胡萝卜素，但至今尚缺乏对它们在DR菌中的功能及其代谢的分子机理进行深入的研究。本文主要对deinoxanthin等类胡萝卜素功能及其合成酶CrtI进行鉴定和分析。研究结果如下：1．以高效液相色谱（HPLC）和薄层层析（TLC）方法分离获得高纯度deinoxanthin色素。化学发光法和体外Fenton反应分析deinoxanthin抗氧化能力，结果表明deinoxanthin具有比lycopene和β-carotene更高效的¹O₂和H₂O₂清除能力，能有效保护质粒DNA并降低其氧化损伤程度。2．生物信息学方法筛选基因组获得控制deinoxanthin合成的关键蛋白DR0861，蛋白N-端和C-端分别具有保守βαβ折叠Motif和八氢番茄红素脱氢酶（PDS）信号序列。DR0861全基因敲除和N-端功能域（19-69aa）缺失均导致红色DR菌变为无色菌株，HPLC显示DR菌deinoxanthin等类胡萝卜素随DR0861突变而消失，而欧文氏菌（E.uredovora）crtI基因补偿可以使无色突变株恢复颜色。表明DR0861基因参与了DR菌中deinoxanthin的代谢路径，具有催化合成有色类胡萝卜素功能。DR0861缺失影响突变株对γ射线和H₂O₂抗性，导致细胞提取物对¹O₂和H₂O₂清除能力降低。表明DR0861蛋白催化合成的类胡萝卜素参与了DR菌体内的抗氧化保护体系，可能对其极端抗性机制有重要贡献。3．大肠杆菌（E.coli）过表达和纯化获得DR0861蛋白，体内和体外生化反应产物HPLC分析显示，DR0861能通过多步脱氢反应，催化无色八氢番茄红素（phytoene）底物生成红色的番茄红素（lycopene）产物，表明DR菌DR0861基因编码CrtI类型而非CrtP／Q型PDS。通过对E.coli色素工程菌研究还发现，lycopene等类胡萝卜素的生成可使E.coli产生浅红色，并明显提高E.coli对低剂量电离辐射和H₂O₂抗性。除研究非酶类抗氧化物（类胡萝卜素）之外，我们还对DR菌抗氧化体系重要组成部分——抗氧化功能蛋白Dps-2进行鉴定和研究。Dps-2（DRB0092）序列含多个赖氨酸保守位点，其N-端前沿包含一段特殊信号肽序列，C-端存在保守亚铁氧化酶（ferroxidase）功能域。dps-2全基因缺失导致DR菌生长受到明显影响，而且细胞对电离辐射、紫外线、干燥和H₂O₂等均表现敏感，并导致蛋白提取物对自由基清除能力降低。E.coli表达和纯化分别获得Dps-2完整蛋白WDps、N-端信号肽突变蛋白N₃₀Dps和C-端ferroxidase突变蛋白C₂₀Dps。SDS-PAGE结果表明，三种蛋白均能形成多聚体，但产生高聚体的比例受盐离子浓度等影响；信号肽突变提高N₃₀Dps对不同温度（37-60℃）的热稳定性，而C-端结构域缺失导致C₂₀Dps蛋白活性对温度异常敏感。Native-PAGE活性染色和亚铁氧化动力学结果表明，WDps和N₃₀Dps蛋白具有催化Fe²⁺生成Fe³⁺功能，而C₂₀Dps亚铁氧化酶活性完全丧失。体外DNA结合和Fenton反应结果显示，突变蛋白C₂₀Dps不具有DNA结合功能，不能保护DNA免受自由基攻击；而N₃₀Dps具有结合DNA的能力，它和WDps均能降低Fenton反应自由基攻击导致的DNA氧化损伤。综上所述，耐辐射球菌DR0861基因编码CrtI类型PDS，它以phytoene为底物，在DR菌体内参与催化合成deinoxanthin。抗氧化保护蛋白Dps-2具有亚铁氧化酶活性和非特异DNA结合等功能，催化毒性Fe²⁺生成无害Fe³⁺并能结合生成的Fe³⁺，降低细胞内环境毒性，并与染色体形成致密Dps-DNA复合体，保护DNA降低自由基损伤程度，它与类胡萝卜素等物质共同参与了DR菌体内抗氧化体系，对该菌的极端抗性机制具有重要贡献。更多还原

【Abstract】 Deinococcus radiodurans is one of the most radiation-resistant organisms on the earth. This bacterium is famous for its extraordinary tolerance toγ-ray, oxidizing agents and desiccation. The remarkable capability of D. radiodurans to survive the harmful damage was attributed to three mechanisms: protection, tolerance and repair. The current research focus on its highly efficient anti-oxidative effect and the complex network of DNA repair mechanism. Although the mechanisms underlying the extraordinary resistance of this bacterium are still poorly understood, it was figured out that about 80% of DNA damage is caused indirectly by irradiation-induced reactive oxygen species （ROS）, the remaining～20% by direct interaction betweenγ-photons and DNA. It shows that anti-oxidative and protective mechanism may act on a very important role in D. radiodurans. Therefore, it’s essential to identify and demonstrate the protective character of these antioxidative metabolite and functional proteins which involve in DNA protection and response to environmental stress in D. radiodurans for understanding of the extreme radiation-resistant mechanism.Carotenoids are well known for their single oxygen （¹O₂） and free radical scavenging activities. The protective role of carotenoids against oxidative damage is essential to various organisms. D. radiodurans R1 was reported to synthesize a unique carotenoid that was identified as deinoxanthin. However, no investigations concerning the antioxidant effects of deinoxanthin have been done so far, and its biosynthetic pathway remained unclear. In the present study, detailed analysis of the function of deinoxanthin in D. radiodurans and identification of the pivotal gene involved in deinoxanthin synthesis were carried out.1. The main product of carotenoid （deinoxanthin） in D. radiodurans was separated and purified by thin layer chromatography （TLC） and high performance liquid chromatography （HPLC）. The deinoxanthin was evaluated on its ROS scavenging activity using chemiluminescence method in vitro and agarose gel electrophoresis analysis after Fenton reaction induced DNA damage. Results showed purified deinoxanthin could quench most of singlet oxygen （¹O₂） and peroxide hydrogen （H₂O₂） rapidly, and showed more obviously effective protection to plasmid DNA than well-known antioxidants lycopene andβ-carotene.2. On the basis of sequence alignment and conserved amino acid analysis, DR0861 exhibited the highest similarity to the sequence of phytoene desaturase from the cyanobacterium Gloeobacter violaceus. Two highly conserved regions, the putative dinucleotide-binding motif （βαβfold） at N-terminus and the ’bacterial-type phytoene desaturase signature’ at C-terminus were detected in DR0861. Using gene inactivation strategy and in vivo complementation, we found that D. radiodurans lost its red pigment after DR0861 deletion （M61） or N-terminal domain mutation （MD61）, and HPLC results exhibited that most carotenoids including deinoxanthin in mutant were disappeared, while complementation of DR0861 or crtI from Erwinia uredovora could resort its carotenoids component. The M61 and MD61 mutant became more sensitive than wild-type strain after exposed toγ-ray and H₂O₂. Furthermore, the ROS scavenging activity of cellular extraction from M61 decreased obviously compared with R1 strain. These results indicated that DR0861 worked as a key enzyme involved in deinoxanthin biosynthesis in D. radiodurans.3. Purified DR0861 protein was obtained after gene heterologous expression in E. coli. Based on in vitro and in vivo enzyme activity assays, followed by HPLC analysis, the results showed that DR0861 could catalyze the substrate 15, 15’-cis-phytoene to lycopene both in vivo and in vitro. Moreover, lycopene generated in E. coli transformant could convert the colorless cells to pink, and the amount of carotenoids in the transformant was also determined. The accumulated carotenoid showed obviously protective effect against oxidative damage and led to a significant enhancement of its resistance to H₂O₂ andγ-ray. Based on these observations, it was obviously that DR0861 gene function as a typical bacterial-type phytoene desaturase （CrtI）, which catalyzed the colorless phytoene to pigmented lycopene through four-step desaturation reaction.Furthermore, we identified and analyzed the function of the stress tolerance and antioxidative protein Dps-2 （DRB0092） in D. radiodurans. Based on bioinformatics methods, it was found that Dps-2 had a unique signal peptide in its forefront and contained multi-lysine residues in N-terminal region which was assumed to be involved in DNA binding, and a conserved ferritin-like domain constituted by several positive glutamic acid and aspartic acid appeared in its C-terminus. The dps-2 deletion mutant M92 was constructed by replacing the target gene with kanamycin resistance cassette. The results showed that the growth curve of M92 was influenced obviously in normal condition, and the survival rate of the mutant was greatly decreased after exposure toγ-ray, UV, desiccation and H₂O₂. The ROS scavenging activity of whole cellular extraction from M92 was reduced evidently compared with wild-type R1. These phenomena demonstrated that dps-2 take a protective role in D. radiodurans under different environmental stress.The purified Dps-2 whole protein WDps, N-terminal singal peptide deleted protein N₃₀Dps and C-terminal ferroxidase domin deleted protein C₂₀Dps was obtained by over-expression in E. coli. SDS-PAGE showed that C₂₀Dps is sensitive to temperature while N₃₀Dps exhibited thermal stability from 42℃to 60℃. Native-PAGE demonstrated that WDps monomer could assembled to different conformation of polymers, N-terminal or C-terminal deletion didn’t influence polymerization of N₃₀Dps and C₂₀Dps. However the ratio of high polymers in solution was affected by the concentration of sodium chloride. Based on Native-PAGE, iron staining and spectroscopic analysis, we found that WDps and N₃₀Dps could catalyze Fe²⁺ to Fe³⁺, while C₂₀Dps didn’t show ferroxidase activity. Dps-2 monomer could not interact with plasmid DNA while polymer Dps-2 showed linear and supercoiled DNA binding activity. N₃₀Dps exhibited prominent DNA binding activity as WDps protein and protected DNA against hydroxyl radical-mediated DNA cleavage caused by Fenton reaction in vitro. However, C-terminal deleted protein C₂₀Dps didn’t show any DNA binding ability or protective ability to plasmid DNA against Fenton reaction.In conclusion, our results demonstrated that carotenoid （deinoxanthin） synthesis pathway contributed to the radioresistance and oxidative stress tolerance of D. radiodurans, and its biosynthesis is controlled by a pivotal enzyme DR0861 which acts as a bacterial-type phytoene desaturase （CrtI）. Dps-2 is a multi-functional protein which has the capacity of DNA binding, iron binding and DNA protection that involved in protecting cell from multiple stresses. Both of the carotenoid metabolite and functional protein Dps-2 play a significant role in antioxidant and protection system in D. radiodurans, which may contribute a great deal to its extremely resistant mechanism.更多还原