Cloning of amadoriase I isoenzyme from Aspergillus sp.: Evidence of FAD covalently linked to Cys342

Amadoriases are a novel class of FAD enzymes which catalyze the oxidative d eglycation of glycated amino acids to yield corresponding amino acids, gluc osone, and H2O2. We previously reported the purification and characterizati on of two amadoriase isoenzymes from Aspergillus fumigatus and the molecula r cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a cl one using a probe amplified by polymerase chain reaction with primers desig ned according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence compr ises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofacto r, which is covalently linked to Cys342, as determined by mutagenesis analy sis, matrix-assisted laser desorption/ionization time-of-flight mass spectr ometry, and electrospray ionization-collisional-activated dissociation tand em mass spectrometry. Sequence alignment studies show that amadoriase I has 22% homology with monomeric sarcosine oxidase in which FAD is also linked to a homologous Cys residue. Amadoriases are of potential importance as too ls for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.