title: Cloning of amadoriase I isoenzyme from Aspergillus sp.: evidence of FAD covalently linked to Cys342.
abstract: Amadoriases are a novel class of FAD enzymes which catalyze the oxidative deglycation of glycated amino acids to yield corresponding amino acids, glucosone, and H(2)O(2). We previously reported the purification and characterization of two amadoriase isoenzymes from Aspergillus fumigatus and the molecular cloning of amadoriase II. To identify the primary structure of amadoriase I, we prepared a cDNA library from Aspergillus fumigatus and isolated a clone using a probe amplified by polymerase chain reaction with primers designed according to the partial amino acid sequences from peptide mapping. The primary structure of the enzyme deduced from the nucleotide sequence comprises 445 amino acid residues. The enzyme contains 1 mol of FAD as a cofactor, which is covalently linked to Cys342, as determined by mutagenesis analysis, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and electrospray ionization-collisional-activated dissociation tandem 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 tools for uncoupling hyperglycemia and glycation reactions that are thought to play a role in diabetic complications.
authors.affiliation: Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: S G
authors.initials: SG
authors.lastname: Chen
authors.forename: V M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005415:Flavins / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D005415:Flavins; D007527:Isoenzymes; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D000594:Amino Acid Oxidoreductases; C103012:amadoriase; D003545:Cysteine