Purification, crystallization and preliminary X-ray analysis of aminoglycoside-2''-phosphotransferase-Ic [APH(2'')-Ic] from Enterococcus gallinarum.
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 mM Mg(2)GTP. The crystals belong to the monoclinic space group C2, with one molecule in the asymmetric unit. The approximate unit-cell parameters are a = 82.4, b = 54.2, c = 77.0 A, beta = 108.8 degrees. X-ray diffraction data were collected to approximately 2.15 A resolution from an F108L crystal at beamline BL9-2 at SSRL, Stanford, California, USA.
Byrnes
Laura J
LJ
Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, CA 94025, USA.
Badarau
Adriana
A
Vakulenko
Sergei B
SB
Smith
Clyde A
CA
D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D016983:Enterococcus / Q000201:enzymology*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C048564:aminoglycoside 2''-phosphotransferase