Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase.
Crystallization and preliminary crystallographic analysis of a thermostable mutant of kanamycin nucleotidyltransferase.
复制标题
卡那霉素核苷酸转移酶热稳定性突变体的结晶和初步晶体学分析。
DOI：
10.1016/0003-9861(92)90479-g
复制
发表时间：
1992
影响因子：
3.9
通讯作者：
Rayment,I
中科院分区：
文献类型：
--
作者：
Kanikula,AM;Liao,HH;Sakon,J;Holden,HM;Rayment,I
从嗜热脂肪芽孢杆菌中克隆选育出一株耐卡那霉素突变株，在70℃条件下对卡那霉素核苷酰转移酶进行了筛选，得到一株耐热的卡那霉素核苷酰转移酶突变体，其晶化形式适合高分辨衍射分析。这种酶催化核苷酸基团从三磷酸腺苷等核苷三磷酸转移到各种氨基糖苷类的羟基上，从而使抗生素失活。利用穿梭载体将金黄色葡萄球菌pUB110上编码的卡那霉素核苷酸转移酶基因转移到嗜热脂肪嗜热菌中，从70℃培养的卡那霉素抗性菌落中分离到携带D80Y和T130K取代位的突变株。从19%(w/v)的PEG8000和200mmNaC l中生长的第一类晶体属于正交晶系C222 1空间群，晶胞尺寸a=128.4，b=156.8，c=155.8 Å，衍射率至少达到2.4A。由10%的聚乙二醇8000、200mmKCl3和3mmCoCl2生长的二类晶体属于四方空间群P4 1 2 1 2或P4 3 2 1 2，晶胞尺寸a=b=78.9，c=220.4 Å；这些晶体的衍射率至少达到2.5？分辨率。
A thermostable mutant of kanamycin nucleotidyltransferase isolated by cloning and selection for kanamycin resistance in Bacillus stearothermophilus at 70° 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° 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 m m 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-Å resolution. The type II form of the crystals were grown from 10% PEG8000, 200 m m KCl, and 3 m m CoCl 2, 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-Å resolution.