Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein fromStaphylococcus aureuspeptidoglycan

Abstract

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases.

First insights of peptidoglycan amidation in Gram-positive bacteria - the high-resolution crystal structure of Staphylococcus aureus glutamine amidotransferase GatD

“…S. aureus GatD was cloned, expressed, purified and crystallized as previously described 33 . Briefly, the PCR amplified sequence of GatD was cloned into the pOPINF vector (Supplementary Table 1 ) using the In-Fusion method 34 .…”

“…The protein was expressed by the auto-induction method 35 in E.coli strain Lemo21(DE3) and purified in two chromatographic steps: IMAC and SEC. The crystallization screens were performed at a high-throughput facility (OPPF-UK) using a Cartesian instrument 33 . Native and Se-Met labeled protein crystals were obtained by vapor diffusion using polyethylene glycol (PEG) as precipitating agent.…”

“…Native and Se-Met labeled protein crystals were obtained by vapor diffusion using polyethylene glycol (PEG) as precipitating agent. The procedure from protein expression to crystallization is described in 33 .…”

“…The data was processed using Xia2 36 , 37 through the automatic software pipeline available at Diamond Light Source. The phase problem was solved by SAD using a Se-Met derivative, as described in 33 . The experimental phases were later used to solve the structure of GatD in the native dataset (at 1.85 Å resolution).…”