| Enolase has multiple cellular regulation activities and plays important roles in physiology and biochemistry functions.Furthermore,the differential expression of the enzyme is considered as a marker of some pathological states,such as cancer,Alzheimer’s disease,rheumatoid arthritis and so on.In this paper,the total cDNA from drosophila was used as template to clone the enolase gene and the sequence analysis showed that the obtained gene was identical with that of GenBank.Then the expression vector containing the gene was constructed and transferred into E.coli.BL21,the results showed that the target gene was expressed in BL21,the size of the protein was 47 KD,and this result was consistent with our expection.After affinity chromatography,the protein was further purified by the method of desalination and gel filtration chromatography.The purified protein can meet the requirements of crystallization.Then the large-scale crystal screening of the enolase protein was carried out by the method of gas phase diffusion,and the primary screening condition was obtained,i.e.10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4.After optimization,the optimal crystallization conditions to obtain the single crystal with large size and good growth were 10 mM CoCl2,0.1 M MES pH6.5,1.9 M(NH4)2SO4,0.1 M CdCl2.A preliminary analysis of the optimized crystal was carried out using the method of X-ray crystal diffraction.Diffraction data were collected uing beamline BL17 U at Shanghai Synchrotron Radiation Facility(SSRF)of China and the data was analysized using the HKL2000.The results showed that the resolution ratio of the enzyme crystallization was 2.1 ? and it belonged to space group C121 with unit-cell parameters a = 168.746 ?,b = 119.210,c = 103.191 ?,α = γ = 90.00°,and β = 114.28°.We studied the structure of enolase from drosophila by the method of molecular replacement,and the results showed that DmENO(enolase of Drosophila melanogaster)formed a dimer by ionic bonds,hydrogen bonds and hydrophobic interactions,which were the interaction of conserved residues in the interface.DmENO had an active structural domain with an unstable L1 and a conserved catalytic domain.The active site of DmENO was occupied by one cadmium ion,two cobalt ions and one sulfate ion.The sulfate occupied the position of phosphate group in enolase and formed salt bridge and hydrogen bond with residues Ala106,Arg440 and Ser441.Unlike other enolases,DmENO has an extra long region at its N-terminal and it may involve in a regulatory activity for DmENO.In this paper,the structural analysis of the enolase from Drosophila melanogaster(69-500)was carried out.To the best of our knowledge,this is the first report that illuminates the structure of enolase.This study lays the foundation for further study on the function of enolase. |
