Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase.
A thermostable mutant of kanamycin nucleotidyltransferase isolated by cloning and selection for kanamycin resistance in Bacillus stearothermophilus at 70 degrees C has been crystallized in a form suitable for high-resolution diffraction analysis. This enzyme catalyzes nucleotidyl group transfer from nucleoside triphosphates such as ATP to hydroxyl groups of various aminoglycosides, thus inactivating the antibiotic. The kanamycin nucleotidyltransferase gene, originally encoded on plasmid pUB110 from the mesophile Staphylococcus aureus, was transferred to the thermophile B. stearothermophilus via shuttle plasmids and the mutant carrying the substitutions D80Y and T130K was isolated from kanamycin-resistant colonies grown at 70 degrees C. The thermostable enzyme was crystallized in two forms from solutions of polyethylene glycol 8000 (PEG8000) using batch and vapor diffusion methods. Type I crystals grown from 19% (w/v) PEG8000 and 200 mM NaCl belong to the orthorhombic space group C222(1), have unit cell dimensions of a = 128.4, b = 156.8, c = 155.8 A, and diffract to at least 2.4-A resolution. The type II form of the crystals were grown from 10% PEG8000, 200 mM KCl, and 3 mM CoCl2, and belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with unit cell dimensions of a = b = 78.9, and c = 220.4 A; these crystals diffract to at least 2.5-A resolution.
Kanikula
A M
AM
Biotechnology Center, University of Wisconsin, Madison 53705.
Liao
H H
HH
Sakon
J
J
Holden
H M
HM
Rayment
I
I
D003460:Crystallization; D003461:Crystallography; D001411:Geobacillus stearothermophilus / Q000737:chemistry*; D007613:Kanamycin Resistance / Q000235:genetics; D009154:Mutation; D009713:Nucleotidyltransferases / Q000737:chemistry*
D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase