Molecular cloning and expression of novel fructosyl peptide oxidases and their application for the measurement of glycated protein.
Fructosyl peptide oxidases, enzymes that are active against a model compound of glycated hemoglobin, N(alpha)-fructosyl valyl-histidine, were characterized. To identify the primary structure of fructosyl peptide oxidases, we have prepared cDNA libraries from Eupenicillium terrenum ATCC18547 and Coniochaeta sp. NISL9330. The coding regions, both fungal fructosyl peptide oxidases consisting of 1314-bp, were obtained with degenerated primers based on the amino acid sequences and specific primers by 3(') and 5(') RACE (rapid amplification of cDNA ends). By their sequence similarities and substrate specificities, fructosyl peptide oxidases and their homologs could be categorized into two groups: (A) enzymes that preferably oxidize alpha-glycated molecules and (B) enzymes that preferably oxidize epsilon-glycated molecules. We showed that recombinant fructosyl peptide oxidases could be used to detect protease-treated fructosyl-hexapeptide, a glycated peptide that is released from HbA(1C) by endoproteinase Glu-C, suggesting these enzymes could be useful for the enzymatic measurement of HbA(1C).
Hirokawa
Kozo
K
Research and Development Division, Kikkoman Corporation, Noda-city, Chiba Pref. 278-0037, Japan. khirokawa@mail.kikkoman.co.jp
Gomi
Keiko
K
Kajiyama
Naoki
N
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000145:classification / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000737:chemistry* / Q000145:classification / Q000201:enzymology*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006442:Glycated Hemoglobin / Q000032:analysis* / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D015336:Molecular Probe Techniques; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology; D013379:Substrate Specificity
D000594:Amino Acid Oxidoreductases; D006442:Glycated Hemoglobin; D011994:Recombinant Proteins; C103012:amadoriase