Structural And Biochemical Studies Of Key Enzyme Of Polyphosphate Metabolism In Deincoccus Radiodurans

Deinococcus radiodurans is an extreme bacterium which has unparalleled resistance to stresses including ionizing radiation,ultraviolet(UV)radiation and desiccation.As an ideal model organism for studying microbial resistance to extreme stresses,investigation on the resistant mechanism of D.radiodurans is important to understand the tolerance of life and nature law under stresses.In this study,we identified the Polypphosphate metabolic system and enzymes by using biochemical and molecular biological methods.The mechanism of Polypphosphate degradation was elucidated in D.radiodurans.We also investigated the iron transport related protein DR1440.Their roles in the resistance of D.radiodurans coping with oxdative damage stress were evaluated.First,we predicted and identified the gene for encoding Polypphosphate kinase(DrPPK)and exopolyphosphatase(DrPPX)in D.radiodurans using bioinformatic methods and biochemical methods including DAPI staining and Urea PAGE?The deficiency in synthesis PolyP had effect on cell growth of D.radiodurans.Under stress of H2O2,the transcription and protein expression level of drppk and drppx were upregulated,and the increasing level of drppx was greater than drppk.Mutation of drppk resulted in lackness of Polypphosphate in cells and increased celluar tolerance to oxdative stress.For the drppx mutant,level of intracellular Polyphosphate increased under oxidative stress compared with the wild type strain,leading to decreased level of intracellular free Mn2+ which possess antioxidant activity due to the binding of PolyP on large amount of free Mn2+,thus resulted in the decreased tolerance of the drppx mutant to oxdative stress.These results suggest that drppx and its product might be involved in the tolerance of D.radiodurans to oxdative stress.Second,by analysing the crystal structure of DrPPX,we found that DrPPX was a novel type of dimer which different from the strong dimer structure of EcPPX in E.coli,and exhibiting a looser protein structure and a more "opening" active center compared with the EcPPX.DrPPX can be dimerized and activated in the presence of Mn2+.The product of PolyP catalyzed by DrPPX,in the form of Mn2++ Pi complex,might contribute to the oxidative stress-resistance of D.radiodurans,and Amino acid E114 is the key site for catalytic activity of DrPPX.We first analyzed the structure of DrPPX-PolyP complex?Amino acids(H14?K35?R37?8271),which contain positive charge,and T10?S12?T82?S141?S212,which contain hydroxyl groups were the binding sites to PolyP in N domain.C-terminal was essential for dimeration of DrPPX.Delection of C-terminal resulted in decreased enzyme activity.Four amino acids(H340?H377?E378?D453)were involved in protein binding to Mn2+.Among them,D453 is the key amino acid site,since mutation of D453 resulted in no longer dimeration and decreased enzyme activity.Results from structure analysis of DrPPX mutant with a deletion of C-terminal supposed that the C-terminal was possibly used to enhance the enzyme activity by binding Mn2+ to form space force on N terminal.DrPPX plays an important role in the tolerance of D.radiodurans to oxidative stress based on survival assays of C-terminal deletion and E114 site mutants of DrPPX under oxidative stress.DrPPK and DrPPX participated in oxidative stress response by controlling the concentration of intracellular free Mn2+ and intracellular Mn/Fe ratio has play an important role in the tolerance of D.radiodurans to oxidative stress.So,in addition to the Mn2+ balance,metabolism of Fe2+ is also critical for the oxidative resistance of D.radiodurans.By using biochemistry and molecular biological methods,we identified a potential iron efflux protein-DR1440.DR1440 was predicted as a typical PIB-type ATPases from P-type ATPase family by using sequence analysis,which are involved in transport of heavy metal ion.Mutation of dr1440 resulted in cellular accumulation of iron ions and the dr1440 mutant displayed higher sensitivity to iron ions and oxidative stresses including hydrogen peroxide and gamma-ray irradiation compared with the wild-type strain.ROS accumulation in dr1440 mutant led to more serious protein carbonylation,suggesting that DR1440 might contribute to intracellular protein protection against reactive oxygen species(ROS)generated from ferrous ion-mediated Fenton-reaction through efflux iron ion.Finally,mutations of S297 and C299 led to intracellular accumulation of iron and cell sentitive to H2O2 stress,indicating that S297 and C299 might be important functional residues of DR1440.In sum,we studied the key enzymes of PolyP metabolism and their functions in D.radiourans.It was first discovered that the special dimer regulation mechanism of DrPPX and its molecular mechanism of enzyme catalysis,and revealed the relationship of PolyP metabolism and oxidative stress resistance.Moreover,we identified DR 1440 as an important protein involved in iron transport and investigated its function in cellular tolerance to oxidative stress.These results broaden the understanding of the catalytic and regulatory mechanisms of exopolyphosphatase involved in polyphosphate metabolism,and further deepen the comprehensive understanding of the resistance of polyphosphate,Mn and Fe to oxidative stress stress,and provided new ideas for elucidating the survival mechanism of D.radiodurans under extreme stresses.