MOLECULAR-CLONING AND EXPRESSION OF AMADORIASE ISOENZYME (FRUCTOSYL AMINE-OXYGEN OXIDOREDUCTASE, EC-1.5.3) FROM ASPERGILLUS-FUMIGATUS

Amadoriase is an enzyme catalyzing the oxidative deglycation of Amador i products to yield corresponding amino acids, glucosone, and H2O2. We previously reported the purification and characterization of two amad oriase isozymes from Aspergillus sp, that degrade both glycated low mo lecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V. M. (1997) J. Biol, Chem. 272, 3437-3443), To ident ify the primary structure of the enzymes, we have prepared a cDNA libr ary from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody, The pri mary structure of the enzyme deduced from the nucleotide sequence comp rises 438 amino acid residues with a predicted molecular mass of 48,79 8 Da. The deduced primary structure exhibits the presence of an ADP-bi nding motif near the NH2 terminus. The identity of the amadoriase II c DNA was further confirmed by expression in Escherichia coli cells with an inducible expression system, Northern blotting analysis revealed t hat amadoriase II was induced by fructosyl propylamine in a dose-depen dent manner.