Purification, crystallization and preliminary crystallographic study of an IDS-epimerase from Agrobacterium tumefaciens BY6.

Abstract

The initial degradation of all stereoisomers of the complexing agent iminodisuccinate (IDS) is enabled by an epimerase in the bacterial strain Agrobacterium tumefaciens BY6. This protein was produced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method. Crystals of IDS-epimerase were obtained under several conditions. The best diffracting crystals were grown in 22% PEG 3350, 0.2 M (NH4)2SO4 and 0.1 M bis-Tris propane pH 7.2 at 293 K. These crystals belong to the monoclinic space group P2(1), with unit-cell parameters a = 55.4, b = 104.2, c = 78.6 angstroms, beta = 103.3 degrees, and diffracted to 1.7 angstroms resolution. They contain two protein molecules per asymmetric unit. In order to solve the structure using the MAD phasing method, crystals of the L-selenomethionine-substituted epimerase were grown in the presence of 20% PEG 3350, 0.2 M Na2SO4 and 0.1 M bis-Tris propane pH 8.5.