title: Heterologous expression of l-lysine α-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme.
abstract: Fish have a complex self-defense mechanism against microbial invasion. Recently, l-lysine α-oxidases have been identified from a number of fish species as a novel type of antibacterial protein in the integument. These enzymes exhibit strict substrate specificity for l-lysine, but the underlying mechanisms and details of their catalytic properties remain unknown. In this study, a synthetic gene coding for Scomber japonicus l-lysine α-oxidase, originally termed AIP (for apoptosis-inducing protein), was expressed in Pichia pastoris, and the recombinant enzyme (rAIP) was purified and characterized. rAIP exhibited essentially the same substrate specificity as the native enzyme, catalyzing the oxidative deamination of l-lysine as an exclusive substrate. rAIP was N-glycosylated and remained active over a wide range of pH, with an optimal pH of 7.5. The enzyme was stable in the pH range from 4.5 to 10.0 and was thermally stable up to 60°C. A molecular modelling of rAIP and a comparative structure/sequence analysis with homologous enzymes indicate that Asp(220) and Asp(320) are the substrate-binding residues that are likely to confer exclusive substrate specificity for l-lysine on the fish enzymes.
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Tani
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Omatsu
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Shigeki
authors.initials: S
authors.lastname: Saito
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Ryoma
authors.initials: R
authors.lastname: Miyake
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawabata
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Ueda
authors.affiliation: College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; R-GIRO, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan; Mitsubishi Chemical Group Science and Technology Research Center, Inc., Yokohama, Kanagawa 227-8502, Japan; and API Corporation, Yokohama, Kanagawa 227-8502, Japan mihara@fc.ritsumei.ac.jp.
authors.forename: Hisaaki
authors.initials: H
authors.lastname: Mihara
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D020134:Catalytic Domain; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005399:Fishes / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; C041123:L-lysine oxidase
keywords: Pichia pastoris; Scomber japonicus; apoptosis-inducing protein; flavoprotein; l-lysine oxidase