Open AccessPurification, crystallization and preliminary X‐ray analysis of aminoglycoside‐2′′‐phosphotransferase‐Ic [APH(2′′)‐Ic] from Enterococcus gallinarum
Author(s) -
Byrnes Laura J.,
Badarau Adriana,
Vakulenko Sergei B.,
Smith Clyde A.
Publication year - 2008
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309108001450
Subject(s) - aminoglycoside , phosphotransferases , phosphotransferase , escherichia coli , mutant , gentamicin , monoclinic crystal system , chemistry , enzyme , enterococcus faecalis , pep group translocation , crystallization , biology , biochemistry , microbiology and biotechnology , stereochemistry , crystallography , gene , antibiotics , crystal structure , organic chemistry
Bacterial resistance to aminoglycoside antibiotics is primarily the result of deactivation of the drugs. Three families of enzymes are responsible for this activity, with one such family being the aminoglycoside phosphotransferases (APHs). The gene encoding one of these enzymes, aminoglycoside‐2′′‐phosphotransferase‐Ic [APH(2′′)‐Ic] from Enterococcus gallinarum , has been cloned and the wild‐type protein (comprising 308 amino‐acid residues) and three mutants that showed elevated minimum inhibitory concentrations towards gentamicin (F108L, H258L and a double mutant F108L/H258L) were expressed in Escherichia coli and subsequently purified. All APH(2′′)‐Ic variants were crystallized in the presence of 14–20%( w / v ) PEG 4000, 0.25 M MgCl 2 , 0.1 M Tris–HCl pH 8.5 and 1 m M Mg 2 GTP. The crystals belong to the monoclinic space group C 2, with one molecule in the asymmetric unit. The approximate unit‐cell parameters are a = 82.4, b = 54.2, c = 77.0 Å, β = 108.8°. X‐ray diffraction data were collected to approximately 2.15 Å resolution from an F108L crystal at beamline BL9‐2 at SSRL, Stanford, California, USA.