CRYSTAL STRUCTURE OF THERMOSTABLE ENDO-1,5-a-L-ARABINASE FROM BACILLUS THERMODENITRIFICANS TS-3

Asako Yamaguchi,^a Toshiji Tada,^a Tetsuko Nakaniwa,^a Makoto Takao,^b Takuo Sakai,^b and Keiichiro Nishimura^a

^aResearch Institute for Advanced Science and Technology, Osaka Prefecture University, Sakai, Osaka 599-8570, Japan; ^bIGA Bioresearch, Amagasaki, Hyogo 660-0805, Japan (asako-y@biochem.osakafu-u.ac.jp)

Arabinan is a common structural component of plant cell walls. It consists of a backbone of a-1,5-linked L-arabinofuranosyl residues, some of which are substituted with a-1,3- and a-1,2-linked L-arabinosyl side-chains in the furanose conformation. End-1,5-a-L-arabinase (ABN) hydorolyzes the a-1,5-L-arabinofuranoside linkage of arabinan. Thermostable ABN (ABN-TS) from Bacillus thermodenitrificans TS-3 showed optimal activity at 343 K, and its thermostability was characterized by a half-life of 4 h at 348 K. We have initiated an analysis for the crystal structure of ABN-TS to clarify the structural features participating in thermostability of ABN.

The recombinant ABN-TS was overexpressed in B. subtilis MI112. The purified enzyme was crystallized by using sodium citrate as a precipitant. The crystals were soaked in a cryo-protectant solution containing 40% sucrose and frozen in a nitrogen-gas stream at 100 K. A native data set was collected to 1.9 Å resolution from a frozen crystal using synchrotron radiation of wavelength 0.9 Å at SPring-8. A total of 88876 observed reflections were scaled and reduced to yield a data set containing 22751 unique reflections with an R_merge of 8.6%. The data set was 99.2% complete. The crystals belong to the orthorhombic space group P2₁2₁2₁ with unit-cell parameters a = 40.2, b = 77.8 and c = 89.2 Å. Molecular-replacement calculations were carried out with the program AmoRe using the structure of a-L-arabinase 43A from Cellvibrio japonicus as a search model (46% homology). A clear peak was found with a correlation coefficient of 35.2 and R-factor of 47.3% after translation-function calculations in the space group P2₁2₁2₁. Refinement of the model was performed, resulting in an R-factor of 36.7% and an R_free of 42.1%. Manual modifications of the model structure are currently in progress.