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J Biol Chem, Vol. 274, Issue 53, 37533-37537, December 31, 1999

Stabilization of Pseudomonas aeruginosa Cytochrome c551 by Systematic Amino Acid Substitutions Based on the Structure of Thermophilic Hydrogenobacter thermophilus Cytochrome c552*

Jun HasegawaDagger §, Hideto Shimahara, Masayuki Mizutani∥, Susumu Uchiyama, Hiroyuki Arai∥**, Masaharu Ishii∥, Yuji Kobayashi, Stuart J. FergusonDagger Dagger , Yoshihiro Sambongi∥Dagger Dagger §§, and Yasuo Igarashi∥

From the Dagger  Daiichi Pharmaceutical Co., Ltd., Edogawa-ku, Tokyo 134-8630, the  Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, the ∥ Department of Biotechnology, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan, and the Dagger Dagger  Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c551 (PA c551) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c552 (HT c552), which has similar main chain folding to PA c551, but is more stable to heat. Thermodynamic properties of PA c551 with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c551 with HT c552. In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c552. We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c552. The higher stability of HT c552 may in part be attributed to some of these substitutions.

* This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence and requests for reprints may be addressed: Daiichi Pharmaceutical Co. Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134-8630, Japan. Fax: 81-3-5696-8336; E-mail: haseg7li@daiichipharm.co.jp.

** Supported by a fellowship from the Japan Society for the Promotion of Science.

§§ Supported by a fellowship from the Japan Society for the Promotion of Science and by grants from the Naito Foundation and the Wellcome Trust. To whom correspondence and requests for reprints may be addressed. Present address: Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; E-mail: sambongi@sanken.osaka-u.ac.jp.

Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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