title: Isolation, purification, and characterization of amadoriase isoenzymes (fructosyl amine-oxygen oxidoreductase EC 1.5.3) from Aspergillus sp.
abstract: Four "amadoriase" enzyme fractions, which oxidatively degrade glycated low molecular weight amines and amino acids under formation of hydrogen peroxide and glucosone, were isolated from an Aspergillus sp. soil strain selected on fructosyl adamantanamine as sole carbon source. The enzymes were purified to homogeneity using a combination of ion exchange, hydroxyapatite, gel filtration, and Mono Q column chromatography. Molecular masses of amadoriase enzymes Ia, Ib, and Ic were 51 kDa, and 49 kDa for amadoriase II. Apparent kinetic constants for Nepsilon-fructosyl Nalpha-t-butoxycarbonyl lysine and fructosyl adamantanamine were almost identical for enzymes Ia, Ib, and Ic, but corresponding values for enzyme II were significantly different. FAD was identified in all enzymes based on its typical absorption spectrum. N-terminal sequence was identical for enzymes Ia and Ib (Ala-Pro-Ser-Ile-Leu-Ser-Thr-Glu-Ser-Ser-Ile-Ile-Val-Ile-Gly-Ala-Gly- Thr-Trp-Gly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different (Ala-Val-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-Gly-Thr-Trp-Gly- Thr-Ser-Thr-). All enzymes had the FAD cofactor-binding consensus sequence Gly-X-Gly-X-X-Gly within the N-terminal sequence. In summary, these data show the presence of two distinct amadoriase enzymes in the Aspergillus sp. soil strain selected on fructosyl adamantanamine and induced by fructosyl propylamine. In contrast to previous described enzymes, these novel amadoriase enzymes can deglycate both glycated amines and amino acids.
authors.affiliation: Institute of Pathology, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA.
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: M
authors.initials: M
authors.lastname: Pischetsrieder
authors.forename: V M
authors.initials: VM
authors.lastname: Monnier
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D000594:Amino Acid Oxidoreductases; C103012:amadoriase