Purification, crystallization and preliminary X-ray characterization ofBacillus cereusarylamineN-acetyltransferase 3 [(BACCR)NAT3]

Abstract

Arylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes (XMEs) that catalyze the acetylation of arylamines. All functional NATs described to date possess a strictly conserved Cys-His-Asp catalytic triad. Here, the purification, crystallization and preliminary X-ray characterization of Bacillus cereus arylamine N-acetyltransferase 3 [(BACCR)NAT3], a putative NAT isoenzyme that possesses a unique catalytic triad containing a glutamate residue, is reported. The crystal diffracted to 2.42 Å resolution and belonged to the monoclinic space group C121, with unit-cell parameters a = 90.44, b = 44.52, c = 132.98 Å , = 103.8 .

Structural and Biochemical Characterization of an Active Arylamine N-Acetyltransferase Possessing a Non-canonical Cys-His-Glu Catalytic Triad

“…Cloning of B. cereus nat3 ORF and Construction of an E123D Mutant-Cloning of the ORF coding for (BACCR)NAT3 wild type (WT) was reported in Kubiak et al (13). The (BACCR)NAT3 E123D mutant was generated by site-directed mutagenesis.…”

“…Finally, proteins were reduced with 10 mM dithiothreitol (DTT) and dialyzed overnight against 25 mM Tris-HCl, pH 7.5. Proteins were digested with thrombin and further purified by ion exchange chromatography (Mono Q column, Ä KTA system, GE Healthcare) as described previously (13). Purification yield was estimated by SDS-PAGE, and protein concentration was determined by absorbance at 280 nm.…”

“…Crystal Structure Determination-Crystallization and data collection of the (BACCR)NAT3 wild-type protein were reported previously (13). Briefly, crystals were obtained with the hanging drop-vapor diffusion method with a 1:1 mixture of 30 mg⅐ml Ϫ1 (BACCR)NAT3 protein with 1.6 M sodium citrate, pH 6.5, 0.28 M NDSB-221 as additive (Hampton Research) at 18°C.…”

“…The best crystals were flash cooled in liquid nitrogen with a 1:1 volume ratio of Paratone and paraffin oil as cryoprotectant before diffraction data collection at the Swiss Light Source synchrotron (Beamline PX3, Paul Scherrer Institute, Switzerland). The crystallographic parameters and data statistics were reported in Kubiak et al (13). Briefly, data collection was achieved at 0.984 Å with a 1°oscillation per image on a chargecoupled device mar225 detector.…”

Arylamine N‐acetyltransferases: a structural perspective. Comments regarding the BJP paper by Zhou et al., 2013

“…In particular, Zhou et al, (2013) overlooked three crystal structures that were published in the last 5 years: Nocardia farcinica NAT [PDB 3D9W, (Martins et al, 2008)], Mycobacterium marinum NAT in complex with hydralazine drug [PDB 3LTW, (Abuhammad et al, 2010)] and Bacillus anthracis NAT1 in complex with coenzyme A [PDB 3LNB, (Pluvinage et al, 2011)]. In addition, the structure of an NAT isoform from Bacillus cereus has been deposited in the PDB (4DMO) and its coordinates will be available in May 2013; the crystallization of this NAT enzyme was published early last year (Kubiak et al, 2012).…”