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 N ⑀ -fructosyl N ␣ -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-SerThr-Glu-Ser-Ser-Ile-Ile-Val-Ile-Gly-Ala-Gly-Thr-TrpGly-) and Ic except that the first 5 amino acids were truncated. The sequence of enzyme II was different (AlaVal-Thr-Lys-Ser-Ser-Ser-Leu-Leu-Ile-Val-Gly-Ala-GlyThr-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.

Lens ageing as an indicator of tissue damage associated with smoking and non-enzymatic glycation - a twin study

“…Fluorophore formation in the lens is believed to originate from non-enzymatic glycation of lens proteins. Hypothetically, the genetic influence could be caused by variations in the activity of enzymes modulating the glycated proteins as it has been observed in non-mammalian species [19,20]. A study showed that longevity in mammalian species was closely related to the rate of formation of advanced glycation end products and that species able to withstand the tissue damage caused by non-enzymatic glycation had longer life-spans [21].…”

Aging: Drugs to Eliminate Methylglyoxal, a Reactive Glucose Metabolite, and Advanced Glycation Endproducts

“…Amadori adducts, formed in the intermediate stages of AGEs formation can either be quenched by compounds such as aminoguanidine, or degraded enzymatically by enzymes such as amadoriase and human fructosamine-3-kinase, which belong to this group [149,150]. Amadoriases have not been detected in higher organisms [151,152].…”

Biomolecular Engineering of Biosensing Molecules —The Challenges in Creating Sensing Molecules for Glycated Protein Biosensing—

“…The enzyme's reduced FAD is then reoxidized by molecular oxygen with the release of hydrogen peroxide. Although all FAODs are unable to oxidize large glycated peptides or intact glycated proteins, some FAODs have recently been shown to have relatively high activity towards fructosyl valyl histidine (f-A Val-His), 11 which corresponds to the N-terminal fructosyl dipeptide derived from HbA1c. These enzymes are therefore referred to as fructosyl peptide oxidase (FPOX).…”

“…FAODs/FPOXs have been isolated from a number of diverse microorganisms, including bacteria, 1214 filamentous fungi, 11,1519 and marine yeast 20 (Table 1). FAODs can be subdivided into different groups according to origins and substrate specificity.…”