Kanikula A M, Liao H H, Sakon J, Holden H M, Rayment I
Biotechnology Center, University of Wisconsin, Madison 53705.
Arch Biochem Biophys. 1992 May 15;295(1):1-4. doi: 10.1016/0003-9861(92)90479-g.
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.
通过克隆并在嗜热脂肪芽孢杆菌中于70℃下筛选卡那霉素抗性而分离得到的卡那霉素核苷酸转移酶的热稳定突变体，已结晶成适合高分辨率衍射分析的形式。该酶催化核苷三磷酸（如ATP）的核苷酸基团转移至各种氨基糖苷的羟基上，从而使抗生素失活。卡那霉素核苷酸转移酶基因最初编码于嗜温菌金黄色葡萄球菌的质粒pUB110上，通过穿梭质粒转移至嗜热菌嗜热脂肪芽孢杆菌，并且从在70℃下生长的卡那霉素抗性菌落中分离出携带D80Y和T130K替换的突变体。使用批量和气相扩散方法，从聚乙二醇8000（PEG8000）溶液中以两种形式使热稳定酶结晶。从19%（w/v）PEG8000和200 mM NaCl中生长的I型晶体属于正交晶系空间群C222(1)，晶胞尺寸为a = 128.4，b = 156.8，c = 155.8 Å，并且衍射至至少2.4 Å分辨率。II型晶体从10% PEG8000、200 mM KCl和3 mM CoCl2中生长，属于四方晶系空间群P4(1)2(1)2或P4(3)2(1)2，晶胞尺寸为a = b = 78.9，c = 220.4 Å；这些晶体衍射至至少2.5 Å分辨率。
