title: Engineering fructosyl peptide oxidase to improve activity toward the fructosyl hexapeptide standard for HbA1c measurement.
abstract: The recently discovered fructosyl peptide oxidase from Phaeosphaeria nodorum (PnFPOX) was demonstrated to react with the glycated hexapeptide measurement standard of hemoglobin A1c, fVHLTPE. The highly reactive Coniochaeta FPOX (FPOX-C) showed no detectable activity with the hexapeptide. Two loop regions were identified as having important effects on the enzymatic properties of FPOX. The first loop has a strong influence on the ability to bind larger glycated peptides, while the second loop has a significant effect on catalytic activity. Loop-substitution mutants showed that the highest activity against fVHLTPE resulted from the combination of the first loop from PnFPOX and the second loop from FPOX-C. The most promising engineered FPOX created, which showed 17-fold greater dehydrogenase activity against fVHLTPE than wild-type PnFPOX, was the FPOX-C mutant with a PnFPOX-derived loop 1 region and an Asn56Ala substitution.
authors: Department of Biotechnology, Graduate School of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei-shi, Tokyo 184-8588, Japan. stefano@cc.tuat.ac.jp
authors: Stefano
authors: S
authors: Ferri
authors: Yusuke
authors: Y
authors: Miyamoto
authors: Akane
authors: A
authors: Sakaguchi-Mikami
authors: Wakako
authors: W
authors: Tsugawa
authors: Koji
authors: K
authors: Sode
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D001203:Ascomycota / Q000201:enzymology / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006442:Glycated Hemoglobin; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012015:Reference Standards; D013379:Substrate Specificity
chemical_list: D000594:Amino Acid Oxidoreductases; D005656:Fungal Proteins; D006442:Glycated Hemoglobin; D006020:Glycopeptides; D009842:Oligopeptides; D011994:Recombinant Proteins; C520096:fructosyl-peptide oxidase