Alanine-scanning mutagenesis of HM-1 killer toxin and the essential residues for killing activity.
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.
Miyamoto
Masahiko
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, 5-13-2 Kamishinei-cho, Niigata 950-2081. miyamoto@niigata-pharm.ac.jp
Han
Gi Dong
GD
Kimura
Tetsuya
T
Furuichi
Yasuhiro
Y
Komiyama
Tadazumi
T
D000409:Alanine / Q000235:genetics*; D001483:Base Sequence; D020128:Inhibitory Concentration 50; D055646:Killer Factors, Yeast; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics
D055646:Killer Factors, Yeast; D009183:Mycotoxins; D000409:Alanine