Role Of Xanthine Dehydrogenase Gene In Oxidative Stress Response Of Deinococcus Radiodurans R1

Xanthine dehydrogenase（XDH）is a key enzyme in the catabolism pathway of purines.It can catalyze xanthine and hypoxanthine to produce uric acid and degrade aldehydes,and it is related to nitrogen assimilation,hormone metabolism,aging and the production of reactive oxygen species.Now,the research on the functions of this enzyme and its encoding genes are mainly focused on animals and plants,while there are few reports on the functions of the gene（xdhABC）,especially in the response of biological oxidation stress.Deinococcus radiodurans R1 was taken as the research object.In this study,the mutation strain construction,transcriptional expression analysis,molecular interaction and other assay were performed to study the function of xdhA,the xanthine dehydrogenase encoding gene,and to investigate the role of xanthine dehydrogenase gene in oxidative stress response.The main results are described as follows:1.Bioinformatics results showed that the xdhABC gene was located in the same operon.Homologous comparison showed that XdhA protein had 71%similarity（58%Identities）with the iron-sulfur binding subunit of xanthine dehydrogenase in Streptomyces coelicolor.xdhA encodes functional xanthine dehydrogenase with xdhB and xdhC.The results of co-transcriptional assay further confirmed that xdhA,xdhB and xdhC shared a co-transcriptional unit.2.In order to confirm that the xdhABC gene product has the characteristic of xanthine dehydrogenase,a xdhA deletion mutant strain（ΔxdhA）was constructed in this study.It was found that the deletion of the xdhA gene did not affect the growth of the strain under normal growth condition.Adenine or formaldehyde toxicity tests showed that the deletion of the xdhA gene led to the sensitivity of strains to exogenous purine and formaldehyde.The results of qRT-PCR showed that xdhABC was not expressed inΔxdhA.xdhB and xdhC genes could not be expressed in the complementary strain containing intact xdhA gene.In contrast,the complementary strain with intact xdhABC can completely restore the phenotype ofΔxdhA.The above results indicated that the deletion of xdhA caused xdhABC gene encoding xanthine dehydrogenase to be not expressed,and its product inactivated.3.Bioinformatics prediction and transcriptional expression analysis of xdhABC gene showed that xdhA might be a target gene of ncRNA OsiR.OsiR is an ncRNA induced by oxidative stress response.The deletion of osiR led to the decrease of H₂O₂ stress tolerance and total intracellular antioxidant capacity during oxidative stress response.The results of MST confirmed the interaction between OsiR and xdhA mRNA.The results of mRNA half-life determination showed that osiR mutation significantly reduced the half-life of xdhA mRNA,indicating that OsiR enhanced the stability of xdhA mRNA through paired combination with xdhA mRNA.4.To investigate the role of xdhA in response to oxidative stress,the results showed that the expression of xdhA was up-regulated about 8 times after H₂O₂ shock treatment,indicating that the expression of xdhA was induced by oxidative stress.The H₂O₂ stress tolerance of xdhA mutants and the total intracellular antioxidant capacity decreased significantly under oxidative stress condition.The complementary strain cmxdhABC could restore the relevant phenotype.At the same time,it was found that adding uric acid to the medium could restore the phenotype of xdhA mutant which was sensitive to oxidative stress.It is speculated that xanthine dehydrogenase in D.radiodurans R1 can produce uric acid through purine metabolism and participate in response to oxidative stress.In summary,OsiR can enhance the stability of xdhA mRNA and the activity of xanthine dehydrogenase,which catalyzes purine to produce the antioxidant（uric acid）,and then improving the oxidation resistance.It might be one of the ways of D.radiodurans R1 in response to oxidative stress.