A new l-arginine oxidase engineered from l-glutamate oxidase

Protein Sci. 2021 May;30(5):1044-1055. doi: 10.1002/pro.4070. Epub 2021 Apr 3.

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.

Keywords: l-arginine oxidase; l-glutamate oxidase; crystal structure; modification of substrate specificity; substrate recognition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Oxidoreductases* / chemistry
  • Amino Acid Oxidoreductases* / genetics
  • Bacterial Proteins* / chemistry
  • Bacterial Proteins* / genetics
  • Protein Engineering*
  • Pseudomonas* / enzymology
  • Pseudomonas* / genetics
  • Streptomyces* / enzymology
  • Streptomyces* / genetics

Substances

  • Bacterial Proteins
  • Amino Acid Oxidoreductases
  • arginine oxidase
  • L-glutamate oxidase