Crystalization And Function Exploration Of Novel Dipiptidyl Peptidase Ⅲ From Corallococcus Coralloides EGB

Dipeptidyl peptidase Ⅲ(DPPⅢ),is a zinc-dependent aminopeptidase,identified from mammals,plants,fungi,and bacteria which hydrolyses dipeptides from the N-terminal of oligopeptides ranging from three to ten amino acid residues.A number of studies investigated its contribution in terminal stages of protein turnover.DPPⅢs from eukaryotes involved in pain modulation,blood pressure regulation and inflammation.But less research on bacterial DPPⅢ was carried out.In this study,we isolated and purified a hypothetical protein from the culture supernatant of Corallococcus sp.EGB by the way of glycogen absorption,which was identified as DPPⅢ.The gene sequence of dppⅢ was obtained by analysis of its peptide fingerprints and genome of Corallococcus coralloides 2259.DPPⅢ sequence showed low identity with those functionally verified in mammal,fungi,plant and bacteria.dppⅢ was heterologously expressed and studied on the properties,crystalization and potential biological functions.mDPPⅢ was determined as a dimer with a molecular weight of 117.7 kDa by molecular sieve.The en2ymatic properties of the enzyme showed that the optimum temperature and pH was 50℃ and 7.0,and the enzyme has good stability at 30-70℃.0.1 mM Mg2+,Mn2+,Co2+,Ba2+,and Ca2+can stimulate recombinase activity,but Cu2+and Ni2+inhibite mDPPⅢ.EDTA strongly inhibits the activity of the enzyme,and the addition of Zn2+,Co2+,and Mn2+ions can restore enzyme activity.Native-mDPPⅢ and Se-mDPPⅢ with high purity and suitability for crystallization was obtained through cation exchange chromatography.After preliminary screening and optimized screening,an appropriate condition for high-quality protein crystal growth was established.The growth condition of native-mDPPⅢ protein crystal was 0.1 M sodium acetate pH 4.59,23.5%PEG 3350,0.1%n-Octyl-β-D-glucoside as well as native-mDPPⅢprotein with 2.2 mg/ml.The condition of Se-mDPPⅢ protein crystal was 0.1 M sodium acetate pH 4.59,24%PEG 3350,0.01 M TCEP hydrochioride and Se-mDPPⅢ with 5.45 mg/ml.One week after the crystallization experiment,a crystal with a diffraction ability of 1.9 A was obtained,and the crystal structure of mDPPⅢ was successfully resolved by Se anomalous scattering.The structures contain two domains,an upper domain rich in a-helices and a lower domain contains a five-stranded β-sheet barrel.Compared with the hDPPⅢ,the key catalytic site amino acids(H384A,E385A,H388A,E417A,H465A)were mutated,and the same catalytic mechanism of mDPPⅢ and hDPPⅢ was determined.The mDPPⅢ was successfully expressed in M.xanthus DK1622.Comparing the developmental processes of the constructing strain with M.xanthus DK1622 demonstrated that mDPPⅢ could advance the development of M.xanthus DK1622,confirming the physiological function of mDPPⅢ in M.xanthus DK1622.Through the preparation of A signal factor from strains DK1622-mDPPⅢ and DK1622 was added to the suspension of DK1622 bacteria,analysis of fruit body development showed that they had no significant effect on the development of DK1622.Whether mDPPⅢ degrades cell surface proteins by peptidase activity,enhances the production of A signal molecules,and further affects the development of myxobacteria through the A signaling pathway remains to be further verified.