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Substrate Specificity of Amadoriase I from Aspergillus fumigatus
Author(s) -
MENNELLA CARMELA,
BORRELLI ROSA CINZIA,
VINALE FRANCESCO,
RUOCCO MICHELINA,
FOGLIANO VINCENZO
Publication year - 2005
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1333.096
Subject(s) - amadori rearrangement , chemistry , maillard reaction , aspergillus fumigatus , glycation , biochemistry , substrate (aquarium) , lysine , chromatography , enzyme , affinity chromatography , peptide , amino acid , oceanography , receptor , microbiology and biotechnology , biology , geology
A bstract : 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 β‐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 K m of amadoriase for glycated‐l‐lysine was 4.2 mM, which is in accordance with the literature. K m 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.