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

Dipeptidyl peptidase ?(DPP?),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.DPP?s from eukaryotes involved in pain modulation,blood pressure regulation and inflammation.But less research on bacterial DPP? 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 DPP?.The gene sequence of dpp? was obtained by analysis of its peptide fingerprints and genome of Corallococcus coralloides 2259.DPP? sequence showed low identity with those functionally verified in mammal,fungi,plant and bacteria.dpp? was heterologously expressed and studied on the properties,crystalization and potential biological functions.mDPP? 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 mDPP?.EDTA strongly inhibits the activity of the enzyme,and the addition of Zn2+,Co2+,and Mn2+ions can restore enzyme activity.Native-mDPP? and Se-mDPP? 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-mDPP? protein crystal was 0.1 M sodium acetate pH 4.59,23.5%PEG 3350,0.1%n-Octyl-?-D-glucoside as well as native-mDPP?protein with 2.2 mg/ml.The condition of Se-mDPP? protein crystal was 0.1 M sodium acetate pH 4.59,24%PEG 3350,0.01 M TCEP hydrochioride and Se-mDPP? 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 mDPP? 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 ?-sheet barrel.Compared with the hDPP?,the key catalytic site amino acids(H384A,E385A,H388A,E417A,H465A)were mutated,and the same catalytic mechanism of mDPP? and hDPP? was determined.The mDPP? was successfully expressed in M.xanthus DK1622.Comparing the developmental processes of the constructing strain with M.xanthus DK1622 demonstrated that mDPP? could advance the development of M.xanthus DK1622,confirming the physiological function of mDPP? in M.xanthus DK1622.Through the preparation of A signal factor from strains DK1622-mDPP? 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 mDPP? 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.