Identification And Functional Analysis Of Deinoxanthin Synthesis Enzyme CrtI And Stress Response Protein Dps-2 In Deinococcus Radiodurans

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.