Purification, crystallization and preliminary X-ray analysis of Enterococcus faecium aminoglycoside-2''-phosphotransferase-Ib [APH(2'')-Ib].
Bacterial resistance to the 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, APH(2'')-Ib, has been cloned and the protein (comprising 299 amino-acid residues) expressed in Escherichia coli, purified and crystallized in the presence of 16%(w/v) PEG 3350 and gentamicin. The crystals belong to the monoclinic space group P2(1), with approximate unit-cell parameters a = 79.7, b = 58.8, c = 81.4 A, beta = 98.4 degrees, and preliminary X-ray diffraction analysis is consistent with the presence of two molecules in the asymmetric unit. Synchrotron diffraction data to approximately 2.65 A resolution were collected from a native APH(2'')-Ib crystal at beamline BL9-2 at SSRL (Stanford, CA, USA). Selenium-substituted crystals have also been produced and structure determination is proceeding.
Walanj
Rupa
R
Laboratory of Structural Biology, School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Young
Paul
P
Baker
Heather M
HM
Baker
Edward N
EN
Metcalf
Peter
P
Chow
Joseph W
JW
Lerner
Stephen
S
Vakulenko
Sergei
S
Smith
Clyde A
CA
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D003460:Crystallization / Q000379:methods; D016984:Enterococcus faecium / Q000737:chemistry*; D005839:Gentamicins; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics; D011092:Polyethylene Glycols; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D005839:Gentamicins; D011092:Polyethylene Glycols; D017853:Phosphotransferases (Alcohol Group Acceptor); C415966:aph(2')-Ib protein, Enterococcus faecium