Substrate specificity of amadoriase I from Aspergillus fumigatus.
Generation of Amadori products is the major single modification by the Maillard reaction in vivo and a source of biologically active glycoxidation products leading to protein cross-linking in biological tissues. Amadoriase I from Aspergillus fumigatus cleaves Amadori products into deoxyglucosone, hydrogen peroxide, and the corresponding primary amine. It has been reported that Amadori products formed on free amino acids are a good substrate for amadoriase I, whereas the enzyme is unable to cleave Amadori products formed on whole proteins. This work aims to investigate the affinity of amadoriase I for oligopeptides and small proteins. Recombinant amadoriase I was expressed in E. coli and purified by Ni His-tag affinity chromatography. Di-, tri-, and tetrapeptides were derivatized with glucose, and the corresponding Amadori products were purified by TLC and HPLC. Glycated beta-lactoglobulin was also used as a substrate. In both cases the formation of Amadori products was monitored by electrospray ionization-mass spectrometry (ESI-MS). The Km of amadoriase for glycated-L-lysine was 4.2 mM, which is in accordance with the literature. Km decreases with the length of peptide, being slightly reduced for dipeptides, and is around 10 mM for tri- and tetrapeptides. Glycated proteins are not substrates of the enzyme; but when amadoriase I was added during the glycation reaction, a significant reduction of Amadori product formation was observed on both peptides and proteins. Data confirm the hypothesis that steric hindrance is critical for amadoriase I activity, indicating that oligopeptides up to four amino acids in length are good substrates. Moreover, these data show that, at least in some experimental conditions, amadoriase I is able to reduce the formation of protein-bound Amadori product.
Mennella
Carmela
C
Dipartimento di Scienza degli Alimenti, University of Napoli "Federico II," via Università 100, 80055 Portici, Italy.
Borrelli
Rosa Cinzia
RC
Vinale
Francesco
F
Ruocco
Michelina
M
Fogliano
Vincenzo
V
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D001234:Aspergillus niger / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D013058:Mass Spectrometry; D010455:Peptides / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D010455:Peptides; D000594:Amino Acid Oxidoreductases; C103012:amadoriase