Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) fromStaphylococcus aureusMSSA476

Abstract

The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li 2 SO 4 , 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 Å resolution using a Rigaku MicroMax-007 HF Cu K X-ray generator and a Rigaku R-AXIS IV ++ detector. The diffraction data were consistent with the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 Å , = = = 90, and the crystal contained two molecules in the asymmetric unit.

Structural elucidation of the binding site and mode of inhibition of Li+ and Mg2+ in inositol monophosphatase

“…Recombinant staphylococcal inositol monophosphatase I (SaIMPase I, SAS2203 from Staphylococcus aureus MSSA476) was purified as described previously . In brief, N–terminally hexahistidine‐tagged recombinant SaIMPase I was purified by Ni–NTA affinity chromatography followed by gel filtration chromatography.…”