| As one of three domains of life,archaea has an important role in the evolution of life.It is thought that,compared with bacteria,the archaea is more similar to eukaryotes.Compared with eukaryotes,the simplified metabolic pathways make archaea as an ideal model,especial for the researches on metabolic pathways of nucleic acid and nucleotides derivatives.Pyrococcus are thermophilic archaea that inhabit in environments with temperature ranging from 70 to 100?,such as deep-sea hydrothermal vents.The DHH phosphoesterase superfamily possesses a diagnostic motif consisting of three consecutive conserved DHH residues and commonly exists in eukaryotic,bacterial and archaeal genomes.They play many functions in cells,such as participating in DNA repair,hydrolysing cyclic nucleoside messengers,and recycling RNA.High temperature is a threat to the genome of hyperthermophiles.Many proteins are involved in maintaining the genomic stability in thermophilic archaea.Besides,the signaling transduction pathway is also crucial for the strains to interact with environment.To address the possible functions of DHH phosphoesterase in hyperthermophiles,two strains from Pyrococcus Genus,Pyrococcus yayanosii and Pyrococcus furiosus were selected to elucidate the potential functions of DHH phosphoesterase during DNA repair and nucleoside messenger metabolic pathway.The nucleotide-based signaling molecules and its metabolic mechanism were explored in P.yayanosii CH1.Our results reveal that,for the first time,the signaling molecule c-di-AMP also exists in the archaeal cells,and the intracellular concentration is about 3.742 ?M.Phosphodiesterase(PDE)can degrade the nucleoside messenger molecule,such as c-di-AMP,in cells.Then,the enzyme responsible for degrading c-di-AMP was identified and biochemically characterized in P.yayanosii.P.yayanosii PDE(PyaPDE)can hydrolyze nucleotide-based molecules such as c-di-AMP,c-di-GMP and cGAMP,and c-di-AMP is the preferred substrate.PyaPDE has obvious ribose dependence with a preference to ribose-type substrate in comparison with deoxyribose-type substrate.Further analysis of substrate specificity indicates that the linear molecules pApA/pGpG of c-di-AMP/c-di-GMP are the preferred substrates,while ApAp/GpGp and ApA/GpG are not the substrates.PyaPDE is a multidomain phosphodiesterase consisting of N-terminal NAD+ binding domain and C-terminal DHH-DHHA1 domain.The results indicate that C-terminal DHH-DHHA1 domain is the essential domain for phosphodiesterase activity,and the NAD+ binding domain promotes the catalytic activity of PyaPDE via binding substrates.Sequence conservation analysis and amino acid site-directed mutagenesis experiments identified the essential residues involved in binding substrates and divalent metal ions.After biochemical characterization of PyaPDE,the physiological role of the pde gene was studied in vivo in Haloferax volcanii.The results show that the Strain becomes sensitive to the oxidative reagent(0.5 mM H2O2)when the pde gene was deleted,indicating that the pde gene may be involved in resisting oxidative stress in H.volcanii.We also explored the enzymatic characterization and biological role of Hef-associated nuclease(HAN),a member of DHH superfamily phosphoesterases,during DNA damage repair in P.furiosus.Bioinformatic analysis indicates that HAN,which is highly conserved in Euryarchaeota,is a DHH superfamily protein with additional N-terminal domains.P.furiosus HAN(PfuHAN)is consisted of C-terminal DHH-DHHA1 nuclease domain and N-terminal zinc finger structure(Zn-finger),OB-Fold and S1-like ribosomal domain.Biochemical characterization of nuclease activity shows that PfuHAN can digest ssDNA in 3'-5'direction.The exonuclease activity of PfuHAN is dependent on bivalent ions,manganese and magnesium.The optimal reaction temperature range is 55-70?,and the optimum pH is 8.0.PfuHAN can hydrolyze both ssDNA and ssRNA without ribose selectivity.The ssDNA has a preference length of 6-30 nt and ssRNA preference length of 4-16 nt.Besides,the 3' terminal phosphate group significantly inhibits the exonuclease activity.PfuHAN can only hydrolyze dsDNA with a 3' single-stranded structure and finally digest into single nucleotides.We confirm that the C-terminal DHH-DHHA1 nuclease domain(DND)is required for exonuclease activity through truncating the N-terminal domains of PfuHAN.Meanwhile the domains of zinc finger structure(Zn-finger),OB-Fold and S1-like ribosomal protein increase the exonuclease activity of the C-terminal DND via promoting substrate binding capability.The crystal structure of the C-terminal DHH-DHHA1 nuclease domain(PfuHAN_DND)was solved and exists in a trimetric form,and the interaction force between each subunit is presumed be the salt bridge and hydrogen bond.Destruction of the trimer structure results in a significant decrease in exonuclease activity.The catalytic core of the PfuHAN_DND possesses a divalent metal ion Mn2+ coordinated by several conserved residues shared by the DHH superfamily protein.The molecular dynamic simulation of the PfuHAN_DND during binding ssDNA further identifies the key amino acid residues for substrate binding.Site-directed mutations of conserved residues responsible for binding Mn2+ and the substrate result in loss or large decrease of exonuclease activity of PfuHAN.To determine the possible function of HAN in DNA repair,the gene han was deleted in H.volcanii.The tolerance of the ?han strain to the DNA damage reagent MMS becomes weaker than wt strain,and the accumulation of methylated adenine(N3-methyadenine)was detected in ?han strain,indicating that the gene han may be involved in repairing DNA damage caused by MMS.The further research confirms that this physiological function is mainly dependent on the C-terminal DND domain.Our researches provide important evidence for the roles of HAN in DNA repair,and deepen the understanding of the metabolic pathway of c-di-AMP and related second messenger molecules in thermophilic archaea. |
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