Abstract:

Objective To investigate the three-dimensional structure of oligoribonuclease from Pseudomonas aeruginosa （P.aeruginosa） Orn （PaOrn），and to verify the active sites of PaOrn，and explore the effect of PaOrn on the intracellular Bis-（3'-5'） cyclic diguanylic acid（c-di-GMP） levels and virulence of P. aeruginosa. Methods A fusion protein with a histidine （His） tag and a glutathione （GST） tag was obtained by constructing the recombinant plasmid pET 32M3C-PaOrn that was expressed by the Escherichia coli（E. coli） BL21.The protein was purified with multistage purifiation method； after Ni affinity chromatography， GST affinity chromatography， and anion exchange chromatography， and finally yielding the PaOrn target protein with high purity and high homogeneity were obtained.The crystals were screened with commercial crystallization reagents and sitting drops， and they were further optimized according to the crystal state. The quality of crystal was verified by X-ray diffraction method， and resolution above 3 ? was used for structure resolution.The activity of PaOrn degradation 5'-phosphoguanylyl-（3'，5'）-guanosine（pGpG） was verified by using high resolution mass spectrometry Q TOF， and the GMP product was used as an indicator to verify the activity. The hydrolytic activities of the PaOrn and the mutant PaOrn proteins （Orn^D11A， Orn^E13A， Orn^D111A， Orn^H157A， and Orn^D162A） against the 10 nt RNA was verified by using Urea-PAGE， and the degradation bands were used as analytical indicators for the normal activity. Results The target protein PaOrn was obtained with purity up to 95% after prokaryotic expression and multistage purification. Good quality single crystals were grown at Wizard Ⅰ # 12［1.0 mmol·L^－1（NH₄） ₂HPO₄， 0.1 mmol·L^－1 Imidazole］ pH 8.0， the resolution of crystal PaOrn was 1.85 ?，and the resolutions of PaOrn-GMP complex and PaOrn^D11A-pGpG complex were 1.90 ? and 1.70 ?， respectively.The Urea-PAGE results showed that PaOrn hydrolyzed the substrate pGpG to GMP. The urea polyacrylamide gel electrophoresis results revealed that PaOrn could degrade up to 10 nt of RNA， and after mutation of the active sites of PaOrn （Asp11， Glu13， Asp111， His157， and Asp162），the activity of PaOrn was lost，and failed to degrade the substrate pGpG and RNA（10 nt）. Conclusion PaOrn is involved in the regulation of intracellular c-di-GMP and RNA levels through degradation of oligonucleotides，and affects transcription initiation and the multiple cellular biological behaviors.

Key words: Pseudomonas aeruginosa, Oligoribonuclease, c-di-GMP, Protein purification, Crystal screening