X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction

Abstract

Current enzymatic systems for quantifying glycated hemoglobin are based on the FAD-containing enzyme fructosyl peptide oxidase (FPOX). FPOX has substrate specificity for fructosyl-α N-valyl-histidine derived from proteolytic digestion of the N-terminus of the HbA1c β-chain. This study reports the X-ray structures of the wild-type and Asn56Ala (N56A) mutant of Phaeosphaeria nodorum fructosyl peptide oxidase (PnFPOX) to elucidate the residues responsible for the oxidative half-reaction. N56A showed decreased oxidase activity compared to the wild -type, while its dye-mediated dehydrogenase activity was higher than that of wild type. In wild-type PnFPOX, Asn56 forms a hydrogen bond with Lys274, thereby preventing it from forming a salt bridge with Asp54. By contrast, Lys274 of PnFPOX N56A moves toward Asp54, and they approach each other to form a salt bridge at a distance of 2.92–3.35 Å. Site-directed mutagenesis studies and protein channel analysis suggest that Asp54 assists in accepting oxygen properly at the position of the bound water molecule in the main oxygen channel. These results reveal that Asn56 in PnFPOX is essential for maintaining an effective oxygen accession path, and support the role of Asp54 as a gate keeper that cooperates with Lys274 to enable oxygen to reach the active site properly.

Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases

“…The FPOx Asn56Ala mutant decreased the oxygen interference effect on the electrochemical system with an artificial electron mediator compared with the wild-type. Shimasaki et al reported the X-ray structures of FPOx wild-type and Asn56Ala mutant [53], revealing that the main reason for the alteration of electron acceptor preference toward oxygen was due to the formation of novel hydrogen bonds which might eliminate the accessibility of oxygen toward the short pathway, thereby resulting in a drastic decrease in oxidase activity compared with dye-mediated dehydrogenase activity.…”

Creation of haemoglobin A1c direct oxidase from fructosyl peptide oxidase by combined structure-based site specific mutagenesis and random mutagenesis

“…The structure of AnFPOX-15 resembled the known structures of other FPOX enzymes (Table S3). Recently, the crystal structures of group I FPOX, namely Eupenicillium terrenum FPOX (EtFPOX) 17 and Phaeosphaeria nodorum FPOX (PnFPOX) 18 , were reported. They both shared high amino acid sequence identity with AnFPOX-15 (Fig.…”

Engineering an efficient mutant of Eupenicillium terrenum fructosyl peptide oxidase for the specific determination of hemoglobin A1c

“…Ferri and his coworkers created a mutant variant of P. anodorum FPOX with 17-fold greater activity against hexapeptide compared to the wild-type enzyme (Ferri et al 2013). Shimasaki designed a mutant variant of FPOX from P. nodorum with improved dehydrogenase activity with K m value of 0.67 mM (Shimasaki et al 2017). Since the substitution of Tyr in E. terrenum FPOX with a more hydrophobic residue (Trp) resulted in enhanced activity toward Fru-ValHis, it was supposed that hydrophobic interactions may play an important role in substrate recognition.…”

“…HbA1c is formed by glycation of N-terminal valine residue of the hemoglobin ß-subunit, while albumin results from the glycation of -amino groups of lysine residues. The levels of these glycated proteins reflect the average blood glucose concentration in the periods corresponding to the half-life of the protein (Liu et al 2008;Shimasaki et al 2017). Since FPOXs are unable to oxidize glycated proteins directly, the enzymatic assay of HbA1c or glycated albumin must be preceded by proteolytic digestion.…”

“…Also, FPOX from Eupenicillium terrenum shows high activity toward Fru-ValHis (Xing et al 2013;Hirokawa et al 2003b) and is expected to be applicable in HbA1c measurement (Gan et al 2015). However, its substrate specificity is a weak point in diagnostic applications, and as a result enzyme engineering is required in the specific determination of HbA1c (Shimasaki et al 2017). Here, we describe the design, production, purification, and functional characterization of a mutant with improved specificity toward Fru-ValHis for more efficient HbA1c measurement.…”