X-RaY ANALYSES OF FAMILY 8 CHITOSANASE FROM BACILLus SP. K17

 

Wataru Adachi, Shinji Shimizu, Tomoko Sunami, Tesuya Fukazawa, Mamie Suzuki,

Rie Yatsunami, Satoshi Nakamura and Akio TakŽnaka

 

Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan (wadachi@bio.titech.ac.jp)

 

 

Glycosylases are classified into 86 families according to the characteristics of their amino acid sequences.  Chitosanase from Bacillus sp. K-17 (C-K17) belongs to the family 8 and differs from other chitosanases in cleavage site specificity.  The family 8 also includes cellulase, xylanase and lichenase.  Sequence comparison was difficult to distinguish the difference of their catalytic site.  The active site of C-K17 must, however, have some difference in their three-dimensional structures.  To understand the structural evolution of the enzymes in the family 8, X-ray analyses of C-K17 have been performed at different pH.

Text Box: Fig. 1. The double-a6-barrel structure of C-K17. The two structures of the active (pH 6.4) and inactive (pH3.7) forms are almost the same except the Glu74 residue that have two conformations at pH3.7, suggesting its inactiveness.  The architecture of the enzyme is composed of a double-a6-barrel structure, the protruded loops from the b-sheets making a large cleft for binding the substrate. 

Text Box: Fig. 2. The active sites superimposed between C-K17(CPK color in gray scale) and CelA(gray). The inserted loop is circled. A structural comparison between cellulase from Clostridium thermocellum (CelA) and C-K17 shows that their overall structures are similar, but different in the cleft regions with insertions of additional b-sheets and loops.  The Asp residue, which acts as a proton acceptor in CelA, is changed to Asn271 in C-K17.  Instead, the inserted Glu261 residue is located close the catalytic site as a proton acceptor.

Sequence alignment based on the above structural feature shows that proteins in the family 8 are classified into the three subfamilies (A, B and C) according to the proton acceptor, chitosanases and lichenases being in the subfamily A, whereas cellulases and xylanases in the subfamily B.  It is thus concluded that the common ancestor of proteins in the family 8 is diverged into three subfamilies by inserting a loop to change the specificity of catalytic reactions.