Molecular mechanisms of improvement of hydrolytic antibody 6D9 by site-directed mutagenesis.
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 k(cat)/k(uncat) and 9.8-fold higher k(cat)/K(m) 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 k(cat)/k(uncat) and k(cat)/K(m) 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.
Takahashi-Ando
Naoko
N
Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc. 6-2, Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512. ntando@postman.riken.jp
Shimazaki
Kazuko
K
Kakinuma
Hiroyuki
H
Fujii
Ikuo
I
Nishi
Yoshisuke
Y
D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics; D019020:Directed Molecular Evolution; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
D017151:Antibodies, Catalytic; D011994:Recombinant Proteins