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.

Figure 1

Reaction scheme of non-enzymatic HbA1c-formation reaction, the catalytic reaction of FPOX, and representative principle of HbA1c measurement using FPOX. (a) Non-enzymatic reaction between glucose and the N-terminal valine of the hemoglobin β subunit, resulting in HbA1c. (b) Oxidation of fructosyl-^α N-valyl-histidine (f-^αVal-His) is catalyzed by FPOX. The C-N bond linking the C1 of the fructosyl moiety and nitrogen of the amino group is oxidized. The resulting Schiff base is rapidly hydrolyzed and glucosone and valyl-histidine are produced. (c) HbA1c in the blood is denatured and then digested by protease to release f-^αVal-His. f-^αVal-His is oxidized by FPOX.

Figure 2

Structural comparison between wild-type PnFPOX, EtFPOX, and FAOXs. (a) Overall structure of wild-type PnFPOX. (b) Overall structure of EtFPOX (PDB: 4RSL, light blue) superimposed onto that of wild-type PnFPOX. Slight differences between the two models are colored in yellow in wild-type PnFPOX. The FAD of PnFPOX is shown as a stick model. (c) Overall structures of amadoriase I (PDB: 4XYZ, light green) and amadoriase II (PDB: 3DJE, light gray) superimposed onto wild-type PnFPOX. Remarkably different regions between the three models are colored in yellow in wild-type PnFPOX. FAD is shown as a stick model. The figure on the right is rotated 180 degrees along the y axis.

Figure 3

Substrate binding-site comparison of wild-type PnFPOX, N56A, EtFPOX, amadoriase I with bound BDF-Lys, and amadoriase II with bound FSA. The structures of (a) wild-type PnFPOX (yellow stick) with bound acetate (light pink stick with dots) and N56A (cyan stick), (b) wild-type PnFPOX with bound acetate and EtFPOX (purple stick) (PDB: 4RSL), (c) wild-type PnFPOX with bound acetate and amadoriase I (green stick) with bound fructosyllysine (white stick with dots) (PDB: 4XWZ), and (d) wild-type PnFPOX with bound acetate and amadoriase II (magenta stick) with bound FSA (orange stick with dots) (PDB: 3DJE). BDF: beta-D-fructopyranose. Lys: Lysine. FSA: 1-s-(carboxymethyl)-1-thio-beta-D-fructopyranose.

Figure 4

Comparison of structures around FAD at the si-face of wild-type PnFPOX, N56A, and the oxygen binding site of monomeric sarcosine oxidase (MSOX) with bound chloride. (a) Structure of wild-type PnFPOX. Red spheres indicate water molecules. (b) Structure of PnFPOX N56A. Light pink spheres indicate water molecules. (c) Structure of MSOX with bound chloride (PDB: 3QSM). Light green sphere indicates chloride. Magenta spheres indicate water molecules. (d) The structures of (b) and (c) were superimposed onto that of (a). FAD and covalently bonded Cys, and the structure of MSOX are shown as a line model. Selected hydrogen bonds and salt bridge interactions (<3.53 Å) are indicated by dotted lines. Dotted lines indicate the distance between selected atoms.