Recombinant production, crystallization and X-ray crystallographic structure determination of peptidyl-tRNA hydrolase fromSalmonella typhimurium

Abstract

Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacteriumSalmonella typhimuriumhas been cloned, expressed inEscherichia coliand crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 Mcitric acid, 0.05 Mbis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space groupP212121with unit-cell parametersa= 62.1,b= 64.9,c= 110.5 Å, α = β = γ = 90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å3 Da−1. The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.