Purification, crystallization and preliminary X-ray analysis of the aminoglycoside-6'-acetyltransferase AAC(6')-Im

Abstract

Bacterial resistance to the aminoglycoside antibiotics is primarily the result of enzymatic deactivation of the drugs. The aminoglycoside N-acetyltransferases (AACs) are a large family of bacterial enzymes that are responsible for coenzyme-A-facilitated acetylation of aminoglycosides. The gene encoding one of these enzymes, AAC(6')-Im, has been cloned and the protein (comprising 178 amino-acid residues) was expressed in Escherichia coli, purified and crystallized as the kanamycin complex. Synchrotron diffraction data to approximately 2.0 Å resolution were collected from a crystal of this complex on beamline BL12-2 at SSRL (Stanford, California, USA). The crystals belonged to the hexagonal space group P6(5), with approximate unit-cell parameters a = 107.75, c = 37.33 Å, and contained one molecule in the asymmetric unit. Structure determination is under way using molecular replacement.

Figure 1

Sequence alignment between AAC(6′)-Im (AAC-Im) and AAC(6′)-Ie (AAC-Ie). Residues which are identical in the two sequences are designated by asterisks and residues which are similar are designated by colons.

Figure 2

12% SDS–PAGE of AAC(6′)-Im under reducing conditions. Lane 1, molecular-weight marker (labeled in kDa); lanes 2 and 3, soluble proteins from cell lysate; lanes 4, 5 and 6, unbound proteins (flowthrough fractions) during purification by affinity chromatography; lane 7, purified AAC(6′)-Im.

Figure 3

The four crystal forms of AAC(6′)-Im: (a) form I crystals, (b) form II crystals, (c) form III crystals, (d) form IV crystals. The four panels are on the same scale; a scale bar is shown in (a).

Figure 4

Diffraction image of the kanamycin complex of AAC(6′)-Im, crystal form III. The resolution circle is at approximately 2.0 Å resolution.