Studies On Crystal Structure Of Glycosyltransferase GT1 And Endoglucanase An5

The crystal structure of β-1,4-endoglucanase AnCel5 A and glycosyl-transferaser MoeGT1 was studied by X-ray diffraction in this paper.β-1,4-endoglucanase(An Cel5A) from Aspergillus niger is a cellulase which is classified to glycoside hydrolases subfamily GH5, it hydrolysises β-1,4-glycosidic bond-linked in the amorphous region of cellulose moleculesand and plays a key role in degrading cellulose. Better crystals of AnCel5 A were obtained after optimization. The crystal of mutant type AnCel5A(E267A) was obtained at the similar condition with wide type, and crystal of the cellotetraose-bound complex was obtained by soaking the mutant type crystal in 10 mmol·L-1 cellotetraose. The X-ray diffraction datasets of the wild type-apo, mutant type-apo and the complex(mutant type AnCel5A-cellotetraose) crystals were collected at beam line BL15A1 of the National Synchrotron Radiation Research Center(NSRRC, Hsinchu, Taiwan). To generate the initial electron cloud density map by using HKL2000 and CCP4 i software. Phaser information was definited by using MR method with a template structure of TaCel5A(which is from the same subfamily GH-A6 with AnCel5 A in 74% sequence identity, PDB code is 1GZJ). The final optimum structures were obtained by using Refmac5 and COOT software alternatively.The structure showed that AnCel5 A was a typical(β/α)8 barrel fold. The active-site of AnCel5 A located in a shallow and long cleft, the catalytic sites in the loop may contribute acting with substrates suggesting its relatively higher activity. In comparison with the bind sites of wild type-apo, mutant type-apo and the complex(mutant type AnCel5A-cellotetraose) crystals, it can be affirmed that substrate binding sites are from-4 to +3. Moreover, the functions of conserved amino acids in the active-site and the retaining mechanism of AnCel5 A have also been analyzed and elucidated.MoeGT1 transfered the first sugar group of moenomycins biosynthesis, plays an irreplaceable role in the biosynthesis process of moenomycins or moenomycins intermediates which have antibacterial activity. Protein G-Sso is a fusion protein of Moe GT1, contains 471 amino acid residues, and the molecular weight of it is about 52.6 kDa. The fusion gene was obtained by Overlaping PCR and was inserted into the express vector pET-46Ek/LIC. The recombinant plasmids were transformed into expression host Escherichia coli BL21TrxB(DE3) after sequenced. The recombinant E. coli was incubated at 37 oC, 200 r·min-1 until OD600 reaches 0.6, and then the culture was induced with 1.0 mmol·L-1 IPTG at 16 oC for 24 h. Purified fusion protein was obtained by using affinity chromatography, ion-exchange and gel-filtration. The crystal screen of G-Sso was completed by using sitting-drop vapor diffusion method and screen kits bought from Hampton Research company, Emerald Biosystems company and Molecular Dimensions Ltd company. Initial crystals were found in the reservoir solution of JCSG-plus 1(15#)å'Œ JCSG-plus 2(25#). Further optimization of initial crystals are underway.