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 Amadori products to yield corresponding amino acids, glucosone, and H2O2. We previously reported the purification and characterization of two amadoriase isozymes from Aspergillus sp. that degrade both glycated low molecular weight amines and amino acids (Takahashi, M., Pischetsrieder, M., and Monnier, V. M. (1997) J. Biol. Chem. 272, 3437-3443). To identify the primary structure of the enzymes, we have prepared a cDNA library from Aspergillus fumigatus induced with fructosyl propylamine and isolated a clone using polyclonal anti-amadoriase II antibody. The primary structure of the enzyme deduced from the nucleotide sequence comprises 438 amino acid residues with a predicted molecular mass of 48,798 Da. The deduced primary structure exhibits the presence of an ADP-binding motif near the NH2 terminus. The identity of the amadoriase II cDNA was further confirmed by expression in Escherichia coli cells with an inducible expression system. Northern blotting analysis revealed that amadoriase II was induced by fructosyl propylamine in a dose-dependent manner.
Takahashi
M
M
Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Pischetsrieder
M
M
Monnier
V M
VM
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D007527:Isoenzymes / Q000235:genetics*; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D004271:DNA, Fungal; D007527:Isoenzymes; D000594:Amino Acid Oxidoreductases; C103012:amadoriase