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Journal of Biochemistry Advance Access published online on January 22, 2008
Journal of Biochemistry, doi:10.1093/jb/mvn004
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© 2008 The Japanese Biochemical Society

Role of the Loop Structure of the Catalytic Domain in Rice Class I Chitinase

Ryoji Mizuno1, Tamo Fukamizo2, Shinichi Sugiyama1, Yoko Nishizawa3, Yuichiro Kezuka4, Takamasa Nonaka4, Kazushi Suzuki1 and Takeshi Watanabe1,{dagger}

1Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan
2Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505, Japan
3Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
4School of Pharmacy, Iwate Medical University, Iwate 028-3694, Japan

{dagger}To whom correspondence should be addressed: Dr. Takeshi Watanabe: Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Nishi-ku, Niigata 950-2181, Japan. Tel: +81-25-262-6647, Fax: +81-25-262-6854, E-mail: wata{at}agr.niigata-u.ac.jp

Received November 3, 2007; Accepted December 13, 2007


  Abstract

In the three-dimensional structure of a rice class I chitinase (OsChia1b) determined recently, a loop structure (loop II) is located at the end of the substrate-binding cleft, and is thus suggested to be involved in substrate binding. In order to test this assumption, deletion of the loop II region from the catalytic domain of OsChia1b and replacement of Trp159 in loop II with Ala were carried out. The loop II deletion and the W159A mutation increased hydrolytic activity not only toward (GlcNAc)6 but also toward polysaccharide substrates. Similar results were obtained for kcat/Km values determined for substrate reduced-(GlcNAc)5. The two mutations shifted the splitting positions in (GlcNAc)6 to the reducing end side, but the shift was less intensive in the Trp mutant. Theoretical analysis of the reaction time course indicated that sugar residue affinity at the +3 subsite was reduced from –2 kcal/mol to +0.5 kcal/mol by loop II deletion. Reduced affinity at the +3 subsite might enhance the release of product fragments, resulting in higher turnover and higher enzymatic activities. Thus, we concluded that loop II is involved in sugar residue binding at the +3 subsite, but that Trp159 itself appears to contribute only partly to sugar residue interaction at the subsite.

Key Words: family 19 chitinase, loop structure, Oryza sativa


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