Crystal structure of endo-1,4--glucanase from Eisenia foetida

シマミミズ由来1,4--エンドグルカナーゼの結晶構造解析

有森 貴夫*; 伊藤 彰紘*; 中澤 昌美*; 上田 光宏*; 玉田 太郎

Arimori, Takao*; Ito, Akihiro*; Nakazawa, Masami*; Ueda, Mitsuhiro*; Tamada, Taro

We cloned the gene for endo-1,4--glucanase from Eisenia foetida (EF-EG2) consisting of 1368 bp encoding 456 amino acid residues. The amino acid sequence of the gene shares sequence homology ( 50%) with endo-1,4--glucanases belonging to glycoside hydrolase family 9. The recombinant EF-EG2 showed highest activity at 40C, and also retained a comparatively high activity at 10C. We purified the recombinant EF-EG2 expressed in Pichia pastoris, and then grew needle shaped EF-EG2 crystals with dimensions of 0.02 0.02 1 mm. Diffraction data of EF-EG2 was collected at beamline 1A, Photon Factory, KEK, and integrated and scaled to 2.1 resolution. The crystals belonged to space group 321 with unit-cell parameters of = = 135 , =54.9 . The location of one EF-EG2 molecule in the asymmetric unit was identified by molecular replacement analysis using the coordinates of endoglucanase from Nasutitermes takasagoensis (NtEgl). The final model of EF-EG2 was refined to a crystallographic -factor of 17.9% (free R-factor of 21.4%) to 2.1 resolution. Overall structure of EF-EG2 is similar to that of NtEgl with an RMSD value of 0.9 for 423 C atoms, however, a slight difference between both structures was confirmed around substrate binding site.