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. 2006 Jun 1;62(Pt 6):593-6.
doi: 10.1107/S1744309106017829. Epub 2006 May 31.

Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae

Zhu Yang  1 , Cong-Zhao Zhou
Affiliations

Purification, crystallization and preliminary X-ray analysis of glutathione peroxidase Gpx3 from Saccharomyces cerevisiae

Zhu Yang et al. Acta Crystallogr Sect F Struct Biol Cryst Commun. .

Abstract

The glutathione peroxidase Gpx3 from the yeast Saccharomyces cerevisiae has been overexpressed, purified and crystallized. Both gel-filtration and dynamic light-scattering (DLS) results indicate that Gpx3 is a monomer in solution at a concentration of about 2 mg ml(-1), whereas glutathione peroxidases are normally tetrameric or dimeric. X-ray diffraction data from a single crystal of Gpx3 have been collected to 2.6 A resolution. The crystals are triclinic and belong to space group P1, with unit-cell parameters a = 38.187, b = 43.372, c = 56.870 A, alpha = 71.405, beta = 73.376, gamma = 89.633 degrees. There are two Gpx3 monomers in a crystallographic asymmetric unit. Preliminary analyses show that the yeast Gpx3 is quite different from those of mammals.

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Figures

Figure 1
Figure 1
(a) Diffraction-quality crystal of Gpx3. (b) X-ray diffraction pattern.
Figure 2
Figure 2
(a) Results of gel filtration of Gpx3. The flow rate was 1.5 ml min−1. (b) The results of gel filtration of Gpx3 and standard proteins. The flow rate was 1.5 ml min−1 and the proteins in the fractions were monitored at 280 nm. Peak 1, albumin, 2 mg ml−1. MW 67 kDa; peak 2, ovalbumin, 2 mg ml−1, MW 43 kDa; peak 3, dual-specificity protein phosphatase VHR, 1 mg ml−1, 21.2 kDa; peak 4, Gpx3, 1 mg ml−1; peak 5, lysozyme, 4 mg ml−1, MW 14.4 kDa. (c) The standard curve for molecular weight. V e of Gpx3 is 77 ml.
Figure 3
Figure 3
The monomodal size-distribution histogram of Gpx3. x axis of histogram, discrete particle sizes; y axis, percentage intensity (relative amount of light scattered by each bin). The histogram has one peak (a monomodal size distribution). The peak is defined by the mean value (2.1 nm) and polydispersity (13.0%).
Figure 4
Figure 4
Alignment of yeast Gpxs (Gpx1, Gpx2 and Gpx3) with bovine Gpx (PDB code 1gp1) and the selenocysteine to glycine mutant of human Gpx1 (PDB code 2f8a). The position numbering of the 1gp1 sequence is used throughout. Residues conserved in the three types of Gpx are shaded. The secondary structure of 1gp1 is shown on the top line. The Cys36 which is the active site of Gpx3 is labelled and the X in 1gp1 represents SeCys.
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