Department of Biological Science, Osaka Prefecture University^* Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Japan^** Graduate School of Engineering, Osaka University, Japan / SOSHO Inc., Japan / CREST, JST, Japan^*** The Institute of Scientific and Industrial Research, Osaka University, Japan / SOSHO Inc., Japan / CREST, JST, Japan^****
○Yuri Sogabe^* Takayoshi Kinoshita^* Asako Yamaguchi^* Tomoya Kitatani^** Tatsuji Sakamoto^** Hideshi Ihara^* Hiroaki Adachi^*** Hiroyoshi Matsumura^*** Kazufumi Takano^*** Satoshi Murakami^**** Tsuyoshi Inoue^*** Yusuke Mori^*** Toshiji Tada^*

Arabinanase hydrolyzes the α-1,5-L-arabinofuranosyide linkage of arabinan which is widely distribute in plant walls. The crystal structures of three arabinanases have revealed that the enzymes have a common unique fold consisting of five β-sheets, each of which is made up of four antiparallel β-strands. Penicillium chrysogenum exo-arabinanase (Abnx) releases arabinobiose from the nonreducing terminus of arabinan. Abnx has a completely different primary structure from other arabinanses so far isolated. We have initiated an X-ray structure analysis of Abnx to clarify the three-dimensional structure and molecular mechanisms of the novel enzyme.
The recombinant Abnx was expressed in E.coli. The purified enzyme was crystallized by 2.0 M ammonium sulfate as a precipitant using the hanging-drop vapor diffusion method. The crystals diffracted to a maximum resolution of 3.2 Å and belonged to the tetragonal system, P4₁22 with unit cell parameters of a = b = 166.4 and c = 401.2 Å. The crystal was estimated to include over eight Abnx molecules in the asymmetric unit. Thus, we have attempted to improve the diffraction quality of the crystals and/or to obtain new crystal forms by the mutational surface engineering and the laser-irradiation technique. The latter technique gave a new crystal in 30% MPD solution. The crystal diffracted beyond 1.5 Å resolution and belonged to the orthorhombic system, P2₁2₁2₁ with unit cell parameters of a = 66.6, b = 76.9 and c = 79.3 Å. Assuming one protein molecule in the asymmetric unit, the V_M value is 2.4 ųDa^-1. A search for heavy-atom derivatives is currently under way for structure determination by isomorphous replacement method.