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Journal of Biochemistry 2005 137(4):517-522; doi:10.1093/jb/mvi063
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© 2005 The Japanese Biochemical Society

Regular Paper

Alanine-Scanning Mutagenesis of HM-1 Killer Toxin and the Essential Residues for Killing Activity

Masahiko Miyamoto1,*, Gi Dong Han1, Tetsuya Kimura2, Yasuhiro Furuichi3 and Tadazumi Komiyama1

1 Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 5-13-2 Kamishinei-cho, Niigata 950-2081; 2 Faculty of Bioresources, Mie University, Tsu, Mie 514-8507; and 3 GeneCare Research Institute Co., Ltd., Kamakura 247-0063

* To whom correspondence should be addressed. Tel: +81-25-269-3170, Fax: +81-25-268-1230, E-mail: miyamoto{at}niigata-pharm.ac.jp

Each of the aromatic, acidic and basic amino acid residues in HM-1 were separately substituted with alanine by site-directed mutagenesis. The mutant genes were successfully expressed in HM-1 resistant Saccharomyces cerevisiae. HM-1 gene analogues corresponding to the aromatic substitutions resulted in lower production of HM-1 analogues. In the case of the acidic amino acid residue and basic amino acid residue substitutions, some analogues were produced in the same amount as and exhibited similar killing activity to that of the wild type HM-1. But the H35A HM-1 analogue had completely lost the killing activity, and D44A, K21A, K46A, R82A, R85A and R86A HM-1 showed highly decreased killing activities. These results strongly indicate the importance of histidine-35, aspartic acid-44, lysine-21, lysine-46, and C-terminal arginine residues in HM-1 for the killing activity.


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