MdaB from Escherichia coli: cloning, purification, crystallization and preliminary X-ray analysis

Abstract

The gene mdaB from Escherichia coli encodes an enzyme with activity similar to that of mammalian DT-diaphorase. It has been reported that the protein is able to confer resistance to the antibiotics DMP 840, adriamycin and etoposide. The gene was cloned and overexpressed in E. coli, allowing purification of the protein to homogeneity. The protein co-purified with an unidentified flavin. Suitable crystals for X-ray diffraction experiments were obtained by hanging-drop vapour diffusion. Their space group was triclinic P1, with unit-cell parameters a = 48.664, b = 52.099, c = 86.584 A, alpha = 87.106, beta = 86.889, gamma = 63.526 degrees. X-ray diffraction data were collected to 2.5 A.

Figure 1

Crystals obtained under various conditions from commercially available screens. The crystallization conditions were (a) 0.2 M magnesium acetate tetrahydrate, 0.1 M sodium cacodylate pH 6.5, 20% polyethylene glycol 8000, (b) 0.1 M sodium acetate trihydrate pH 4.6, 8% polyethylene glycol 4000, (c) 0.1 M HEPES sodium salt pH 7.5, 2% polyethylene glycol 400, 2.0 M ammonium sulfate, (d) 0.2 M ammonium sulfate, 0.1 M MES pH 6.5, 30% polyethylene glycol monomethylether 5000, (e) 1.6 M trisodium citrate dihydrate pH 6.5 and (f) the refined condition 0.15 M magnesium acetate tetrahydrate, 0.1 M sodium cacodylate pH 6.5, 20% polyethylene glycol 8000.

Figure 2

(a) Diffraction of native MdaB crystals grown in 0.15 M magnesium acetate, 0.1 M sodium cacodylate pH 6.5, 20% PEG 8000. Diffraction data were indexed and reduced to 2.5 Å with good statistics. However, diffraction is apparent even at 2.3 Å at the edge of the detector (b).