A new l‐arginine oxidase engineered from l‐glutamate oxidase. (3rd April 2021)

• Record Type: Journal Article
• Authors: Yano, Yoshika; Matsuo, Shinsaku; Ito, Nanako; Tamura, Takashi; Kusakabe, Hitoshi; Inagaki, Kenji; Imada, Katsumi
• Is Part Of: Protein science. Volume 30:Number 5(2021)
• Page Start: 1044
• Page End: 1055
• Publication Date: 2021-04-03
• Subjects: crystal structure -- l‐arginine oxidase -- l‐glutamate oxidase -- modification of substrate specificity -- substrate recognition; Proteins -- Periodicals; 572.6
• DOI: 10.1002/pro.4070
• Languages: English
• ISSNs: 0961-8368

Abstract

Abstract: The alternation of substrate specificity expands the application range of enzymes in industrial, medical, and pharmaceutical fields. l‐ Glutamate oxidase (LGOX) from Streptomyces sp. X‐119‐6 catalyzes the oxidative deamination of l‐ glutamate to produce 2‐ketoglutarate with ammonia and hydrogen peroxide. LGOX shows strict substrate specificity for l‐ glutamate. Previous studies on LGOX revealed that Arg305 in its active site recognizes the side chain of l‐ glutamate, and replacement of Arg305 by other amino acids drastically changes the substrate specificity of LGOX. Here we demonstrate that the R305E mutant variant of LGOX exhibits strict specificity for l‐ arginine. The oxidative deamination activity of LGOX to l‐ arginine is higher than that of l‐ arginine oxidase form from Pseudomonas sp. TPU 7192. X‐ray crystal structure analysis revealed that the guanidino group of l‐ arginine is recognized not only by Glu305 but also Asp433, Trp564, and Glu617, which interact with Arg305 in wild‐type LGOX. Multiple interactions by these residues provide strict specificity and high activity of LGOX R305E toward l‐ arginine. LGOX R305E is a thermostable and pH stable enzyme. The amount of hydrogen peroxide, which is a byproduct of oxidative deamination of l‐ arginine by LGOX R305E, is proportional to the concentration of l‐ arginine in a range from 0 to 100 μM. The linear relationship is maintained around 1 μM of l‐ arginine. Thus, LGOX R305E is suitable for the determination of l‐ arginine. Abstract : PDB Code(s): 7E0C and 7E0D .