Crystallization and preliminary crystallographic analysis of two eukaryotic fructosyl peptide oxidases

Abstract

Fructosyl peptide oxidase (FPOX) catalyses the oxidation of α-glycated dipeptides such as N(α)-(1-deoxy-D-fructos-1-yl)-L-valyl-L-histidine (Fru-ValHis) and is used in the diagnosis of diabetes mellitus. Here, two thermostable mutants of FPOX, CFP-T7 and EFP-T5M, were crystallized by the sitting-drop vapour-diffusion method. The crystal of CFP-T7 belonged to the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 110.09, c = 220.48 Å, and that of EFP-T5M belonged to the monoclinic space group P2(1), with unit-cell parameters a = 43.00, b = 230.05, c = 47.27 Å, β = 116.99°. The crystals of CFP-T7 and EFP-T5M diffracted to 1.8 and 1.6 Å resolution, respectively.

Keywords: Coniochaeta sp.; Eupenicillium terrenum; diagnosis of diabetes; fructosyl peptide oxidase; haemoglobin A1c.

Figure 1

SDS–PAGE analysis after gel-filtration chromatography. Lane M contains molecular-mass markers. (a) CFP-T7. 4 µg protein was used in all lanes. The arrow indicates CFP-T7. Fractions 1–4 were collected and used for crystallization. (b) EFP-T5M. 2.5 µg protein was used in all lanes. The arrow indicates EFP-T5M. Fractions 1–13 were collected and used for crystallization.

Figure 2

Crystals of FPOXs in a sitting drop. (a) A crystal of CFP-T7 obtained from 2.0 M potassium/sodium phosphate pH 6.4 with 2.6%(v/v) PEG 400. The crystal dimensions are about 1.0 × 0.2 × 0.1 mm. (b) A crystal of EFP-T5M obtained from 100 mM sodium citrate pH 6.1 with 24%(w/v) PEG 4000 and 10%(v/v) 2-­propanol. The crystal dimensions are about 0.3 × 0.2 × 0.05 mm.