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Journal of Biochemistry Advance Access published online on August 18, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj179
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© 2006 The Japanese Biochemical Society.
Received May 28, 2006
Accepted August 15, 2006

Regular Paper

Molecular Mechanisms of Improvement of Hydrolytic Antibody 6D9 by Site-Directed Mutagenesis

Naoko Takahashi-Ando 1 *, Kazuko Shimazaki 2, Hiroyuki Kakinuma 3, Ikuo Fujii 4, and Yoshisuke Nishi 5

1 Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, JAPAN; Present address: Plant & Microbial Metabolic Engineering Research Unit, RIKEN, 2-1, Hirosawa, Wako-shi, Saitama, 351-0198 JAPAN
2 Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, JAPAN; Present address: Nippon Veterinary and Life Science University, Department of Veterinary, Laboratory of Structural Bioinformatics, 1-7-1, Kyonan-Cho, Musashino-shi, Tokyo 180-8602 JAPAN
3 Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, JAPAN; Present address: Medicinal Chemistry Laboratory, Taisho Pharmaceutical CO., LTD. 1-403, Yoshino-Cho, Saitama-Shi, Saitama 330-8530 JAPAN
4 Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka, 565-0874 JAPAN; Present address: Research Institute of advances Science and Technology, Osaka Prefectural University, 1-2 Gakuen-cho, Sakai, Osaka 599-853, JAPAN
5 Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, JAPAN; Present address: Faculty of Bioscience, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, JAPAN

* To whom correspondence should be addressed.
Naoko Takahashi-Ando, E-mail: ntando{at}postman.riken.jp


  Abstract

We performed a series of site-directed mutagenesis experiments of catalytic antibody, 6D9, which hydrolyzes a prodrug of chloramphenicol, based on our previous directed evolution study [Takahashi et al., (2001) Nat. Biotechnol. 19, 563-567]. Since we previously found that the variants with a mutation of Ser(L27e)Tyr afforded a one order of magnitude increase in catalytic rate, we created a site-directed mutant containing this mutation. The resulting mutant, 6D9-Ser(L27e)Tyr, had 6.5-fold higher kcat/kuncat and 9.8-fold higher kcat/Km than wild-type 6D9. We also created 6D9-Thr(L27a)Pro, since this mutation occurred frequently in the previous directed evolution, and it had 2.1-fold higher kcat/kuncat and kcat/Km than 6D9. Kinetic and computational analyses suggest that Tyr at L27e contributes to transition-state stabilization, while Pro at L27a does not interact with the transition-state structure directly, but obviously contributes to enhanced catalytic activity. Including double mutants that combined favourable substitutions, we created seven site-directed mutants. However, none of them had higher catalytic activities than some of highly improved variants obtained in the previous directed evolution. The present study gives direct evidence that not only a specific amino acid residue which obviously contributes to transition-state stabilization, but also a group of amino acid residues working in concert is important for efficient catalysis of a given transformation.

Keywords: abzyme; antibody engineering; catalytic antibodies; computational analysis; molecular evolution.
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