Journal of Biochemistry Advance Access originally published online on January 22, 2008
Journal of Biochemistry 2008 143(4):487-495; 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
1Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, Niigata 950-2181; 2Department of Advanced Bioscience, Kinki University, 3327-204 Nakamachi, Nara 631-8505; 3Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602; and 4School of Pharmacy, Iwate Medical University, Iwate 028-3694, Japan
*To whom correspondence should be addressed. 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
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Abstract |
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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 towards (GlcNAc)6 but also towards 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
Abbreviations: OsChia1a, OsChia1b and OsChia1c, Oryza sativa L., var Nipponbare class I chitinase, respectively; GH, glycoside hydrolases; CBM, carbohydrate-binding modules; ChBD, chitin binding domain; CatD, catalytic domain; IPTG, isopropyl-β-D-thiogalactopyranoside; GlcNAc, 2-deoxy-2-acetamido-D-glucose; Red-(GlcNAc)5, reduced N-acetylchitopentaose; GC, glycol chitin; CC, colloidal chitin