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-fructosyl lysine. Apparent K. 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 35degreesC in 0.1 m potassium phosphate, but more than 80% of its activity was lost at 40degreesC 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.