Study On The Properties And Crystal Structure Of Prolyl Endopeptidase From Abalone(Haliotis Discus Hannai)

Prolyl endopeptidase(PEP,EC 3.4.21.26),also known as prolyl oligopeptidase(POP),is a type of hydrolase in the serine protease family.The enzyme hydrolyzes small peptides(<33amino acids)at the carboxyl terminus of proline residues.In this study,recombinant PEP(Hdh-PEP)from Haliotis discus hannai was selected as the research object,and its enzymatic,structural and functional characteristics were studied.Fluorescence quantitative PCR analysis showed that Hdh-PEP was distributed in all tissues of abalone,and its expression was high in gonad,muscle and blood.Using Suc-Gly-Pro-MCA as substrate,the enzyme activity of Hdh-PEP in different tissues of abalone was detected by fluorescence spectrophotometer at 380 nm excitation wavelength and 450 nm emission wavelength.The results showed that the highest enzyme activity was detected in gonad.High concentration of recombinant Hdh-PEP was obtained by culturing the expressed strain,and its specific activity was 6.52 U/mg.Circular dichroism analysis showed that random coil(47.3%)accounted for the most in the secondary structure of Hdh-PEP,and ?-sheet and ?-helix accounted for 31.6% and 21.1% respectively.Analysis of the crystal structure of Hdh-PEP showed that it consist of two parts: catalytic domain and ?-propeller domain.One side of the two regions is connected by hinges,and they are close to each other by hydrogen bond,salt bridge and hydrophobic interaction.In addition,the co-crystal structure of Hdh-PEP together with SUAM-14746 was analyzed.The results revealed that SUAM-14746 stably bound to the active center of Hdh-PEP by forming two hydrogen bonds with 639-Arg and 594-Trp.The substrate channel is a tunnel connecting the surface of the protease and the active site,and it is the channel through which the substrate enters the active center.By studying the substrate channel of Hdh-PEP,its mechanism of action with small molecules and the influence of various microenvironments on its activity and specificity can be identified.In this paper,the substrate channel is analyzed by the software of caver analyst.The results show that there are two most likely substrate channels,one is located in the center of the ?-propeller domain(Channel-1)with length of 39.43 ?,and the inlet radius is 1.09 ?.The other channel(Channel-2)is Loop A(193-209)between the two domains,with a length of 28.35 ? and the inlet radius of1.74 ?.MD simulation results show that when SUAM-14746 is close to Hdh-PEP,the conformation of Loop A(193-209)changes from tight to loose.Therefore,Loop A(193-209)is regarded as an important part of substrate channel gating mechanism.Trypsin hydrolysis experiment further verified the hypothesis that Loop A(193-209)drives gating.In order to study the effect of Hdh-PEP on collagen,the degradation of Hdh-PEP on three collagen peptides with different lengths was analyzed by High Performance Liquid Chromatography-Electrospray Ionization Mass Spectrometry(HPLC-ESI-MS).The results showed that Hdh-PEP could degrade the three collagen peptides efficiently,suggesting Hdh-PEP is involved in the metabolism of abalone collagen.In conclusion,this study analyzed the distribution and gene expression of Hdh-PEP in abalone tissues.Hdh-PEP is widely distributed and had the highest expression and enzyme activity in gonad.The crystal culture and structural analysis of Hdh-PEP were completed,and the structural characteristics of Hdh-PEP were obtained.By analyzing the crystal structure of Hdh-PEP-inhibitor complex,the inhibition mechanism of specific inhibitor SUAM-14746 on Hdh-PEP was clarified.The substrate channels of Hdh-PEP were studied,and the entry and path of small molecules into the catalytic activity region of Hdh-PEP were analyzed by molecular dynamics.Degradation effect of Hdh-PEP on collagen peptides showed that Hdh-PEP is involved in the metabolism of abalone collagen.The present results provided experimental basis and theoretical reference for the study of catalytic mechanism and functional characteristics of Hdh-PEP.