Toth Marta, Vakulenko Sergei, Smith Clyde A
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Jan 1;66(Pt 1):81-4. doi: 10.1107/S1744309109050039. Epub 2009 Dec 25.
The deactivation of aminoglycoside antibiotics by chemical modification is one of the major sources of bacterial resistance to this family of therapeutic compounds, which includes the clinically relevant drugs streptomycin, kanamycin and gentamicin. The aminoglycoside phosphotransferases (APHs) form one such family of enzymes responsible for this resistance. The gene encoding one of these enzymes, aminoglycoside-2''-phosphotransferase-IVa [APH(2'')-IVa] from Enterococcus casseliflavus, has been cloned and the protein (comprising 306 amino-acid residues) has been expressed in Escherichia coli and purified. The enzyme was crystallized in three substrate-free forms. Two of the crystal forms belonged to the orthorhombic space group P2(1)2(1)2(1) with similar unit-cell parameters, although one of the crystal forms had a unit-cell volume that was approximately 13% smaller than the other and a very low solvent content of around 38%. The third crystal form belonged to the monoclinic space group P2(1) and preliminary X-ray diffraction analysis was consistent with the presence of two molecules in the asymmetric unit. The orthorhombic crystal forms of apo APH(2'')-IVa both diffracted to 2.2 A resolution and the monoclinic crystal form diffracted to 2.4 A resolution; synchrotron diffraction data were collected from these crystals at SSRL (Stanford, California, USA). Structure determination by molecular replacement using the structure of the related enzyme APH(2'')-IIa is proceeding.
通过化学修饰使氨基糖苷类抗生素失活是细菌对这类治疗性化合物产生耐药性的主要来源之一，这类化合物包括具有临床相关性的药物链霉素、卡那霉素和庆大霉素。氨基糖苷磷酸转移酶（APHs）就是造成这种耐药性的一类酶。编码其中一种酶——来自格氏肠球菌的氨基糖苷-2''-磷酸转移酶-IVa [APH(2'')-IVa] 的基因已被克隆，其蛋白质（由306个氨基酸残基组成）已在大肠杆菌中表达并纯化。该酶以三种无底物形式结晶。其中两种晶体形式属于正交晶系空间群P2(1)2(1)2(1)，具有相似的晶胞参数，不过其中一种晶体形式的晶胞体积比另一种小约13%，溶剂含量极低，约为38%。第三种晶体形式属于单斜晶系空间群P2(1)，初步X射线衍射分析表明不对称单元中存在两个分子。脱辅基APH(2'')-IVa的正交晶体形式均衍射至2.2 Å分辨率，单斜晶体形式衍射至2.4 Å分辨率；在美国加利福尼亚州斯坦福的SSRL从这些晶体收集了同步辐射衍射数据。使用相关酶APH(2'')-IIa的结构通过分子置换进行结构测定正在进行中。
