Pedersen L C, Benning M M, Holden H M
Institute for Enzyme Research, Graduate School, University of Wisconsin, Madison 53705, USA.
Biochemistry. 1995 Oct 17;34(41):13305-11. doi: 10.1021/bi00041a005.
Kanamycin nucleotidyltransferase (KNTase) is a plasmid-coded enzyme responsible for some types of bacterial resistance to aminoglycosides. The enzyme deactivates various antibiotics by transferring a nucleoside monophosphate group from ATP to the 4'-hydroxyl group of the drug. Detailed knowledge of the interactions between the protein and the substrates may lead to the design of aminoglycosides less susceptible to bacterial deactivation. Here we describe the structure of KNTase complexed with both the nonhydrolyzable nucleotide analog AMPCPP and kanamycin. Crystals employed in the investigation were grown from poly(ethylene glycol) solutions and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 57.3 A, b = 102.2 A, c = 101.8 A, and one dimer in the asymmetric unit. Least-squares refinement of the model at 2.5 A resolution reduced the crystallographic R factor to 16.8%. The binding pockets for both the nucleotide and the antibiotic are extensively exposed to the solvent and are composed of amino acid residues contributed by both subunits in the dimer. There are few specific interactions between the protein and the adenine ring of the nucleotide; rather the AMPCPP molecule is locked into position by extensive hydrogen bonding between the alpha-, beta-, and gamma-phosphates and protein side chains. This, in part, may explain the observation that the enzyme can utilize other nucleotides such as GTP and UTP. The 4'-hydroxyl group of the antibiotic is approximately 5 A from the alpha-phosphorus of the nucleotide and is in the proper orientation for a single in-line displacement attack at the phosphorus.(ABSTRACT TRUNCATED AT 250 WORDS)
卡那霉素核苷酸转移酶（KNTase）是一种由质粒编码的酶，它导致某些类型的细菌对氨基糖苷类抗生素产生耐药性。该酶通过将三磷酸腺苷（ATP）的核苷单磷酸基团转移到药物的4'-羟基上，使各种抗生素失活。深入了解该蛋白质与底物之间的相互作用，可能会有助于设计出不易被细菌失活的氨基糖苷类抗生素。在此，我们描述了KNTase与不可水解的核苷酸类似物AMPCPP和卡那霉素形成的复合物的结构。用于研究的晶体是从聚乙二醇溶液中生长出来的，属于空间群P2(1)2(1)2(1)，晶胞参数为a = 57.3 Å，b = 102.2 Å，c = 101.8 Å，不对称单位中有一个二聚体。在2.5 Å分辨率下对模型进行最小二乘精修，将晶体学R因子降低到了16.8%。核苷酸和抗生素的结合口袋都广泛暴露于溶剂中，由二聚体中两个亚基贡献的氨基酸残基组成。蛋白质与核苷酸的腺嘌呤环之间几乎没有特异性相互作用；相反，AMPCPP分子通过α-、β-和γ-磷酸基团与蛋白质侧链之间广泛的氢键作用而固定在其位置上。这在一定程度上可能解释了该酶能够利用其他核苷酸如鸟苷三磷酸（GTP）和尿苷三磷酸（UTP）这一现象。抗生素的4'-羟基距离核苷酸的α-磷约5 Å，且处于对磷进行单一线性取代攻击的合适方向。（摘要截短于250字）
