Recombinant Agrobacterium AgaE-like protein with fructosyl amino acid oxidase activity

• Published in: Bioscience, biotechnology, and biochemistry biotechnology, and biochemistry, 2002-11, Vol.66 (11), p.2323-2329
• Main Authors: Hirokawa, K. (Kikkoman Corp., Noda, Chiba (Japan)), Kajiyama, N
• Format: Article
• Language: English
• Subjects: AGROBACTERIUM; Agrobacterium tumefaciens - enzymology; Agrobacterium tumefaciens - genetics; Agrobacterium tumefaciens C58; Amino Acid Motifs; Amino Acid Oxidoreductases - genetics; Amino Acid Oxidoreductases - metabolism; Amino Acid Sequence; AMINO ACIDS; Bacteriology; Biological and medical sciences; Cloning, Molecular; Corynebacterium - enzymology; Corynebacterium - genetics; enzymatic measurement of HbA; ENZYME ACTIVITY; Escherichia coli - genetics; ESTERS; FRUCTOSE; fructosyl amino acid oxidase; Fundamental and applied biological sciences. Psychology; glycated protein; Hydrogen-Ion Concentration; Mannitol - analogs & derivatives; Mannitol - metabolism; mannityl opine; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; OXIDOREDUCTASES; RECOMBINANT PROTEINS; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Homology, Amino Acid; Spectrophotometry - methods; Substrate Specificity; Temperature
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.66.2323

Agrobacterium tumefaciens AgaE-like protein had a similar sequence to that of a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1. To characterize the AgaE-like protein, we produced the enzyme in Escherichia coli, and purified it to homogeneity. The molecular mass of recombinant AgaE-like protein was 42 kDa on SDS-PAGE and 85 kDa on gel filtration. The protein acted on N-fructosyl valine and N-fructosyl glycine as substrates, but not on glycated protein or N ε -fructosyl lysine. Apparent K m for N-fructosyl valine and N-fructosyl glycine were 1.64 and 0.31 mM, respectively. The AgaE-like protein had maximum activity at pH 7.8 and 35°C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40°C or more. In contrast to eukaryotic fructosyl amino acid oxidases, the AgaE-like protein contained noncovalently bound FAD as a cofactor and was inactive against N ε -fructosyl N α -Z(benzyloxycarbonyl)-lysine. These characteristics were similar to a fructosyl amino acid oxidase from Corynebacterium sp. strain 2-4-1, suggesting that these prokaryotic enzymes comprise a new family of fructosyl amino acid oxidases.