Heterologous expression of l-lysine α-oxidase from Scomber japonicus in Pichia pastoris and functional characterization of the recombinant enzyme

• Published in: Journal of biochemistry (Tokyo) Vol. 157; no. 4; pp. 201 - 210
• Main Authors: Tani, Yasushi, Omatsu, Koichiro, Saito, Shigeki, Miyake, Ryoma, Kawabata, Hiroshi, Ueda, Makoto, Mihara, Hisaaki
• Format: Journal Article
• Language: English
• Published: England 01.04.2015
• Subjects: Amino Acid Oxidoreductases - genetics; Amino Acid Oxidoreductases - metabolism; Amino Acid Sequence; Animals; Base Sequence; Catalytic Domain; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Fishes - genetics; Hydrogen-Ion Concentration; Molecular Sequence Data; Pichia - genetics; Pichia pastoris; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Scomber japonicus; Sequence Alignment; Substrate Specificity; Temperature; apoptosis-inducing protein; l-lysine oxidase; Pichia pastoris; Scomber japonicus; flavoprotein
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvu064

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.