Recombinant agrobacterium AgaE-like protein with fructosyl amino acid oxidase activity.
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(epsilon)-fructosyl lysine. Apparent Km 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 degrees C in 0.1 M potassium phosphate, but more than 80% of its activity was lost at 40 degrees 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(epsilon)-fructosyl N(alpha)-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.
Hirokawa
Kozo
K
Research and Development Division, Kikkoman Corporation, 399 Noda, Noda City, Chiba 278-0037, Japan. khirokawa@mail.kikkoman.co.jp
Kajiyama
Naoki
N
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D020816:Amino Acid Motifs; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000031:analogs & derivatives* / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; D008353:Mannitol; C041352:mannopine; D000594:Amino Acid Oxidoreductases