Perlin M H, Brown S A, Dholakia J N
Department of Biology, University of Louisville, Louisville, KY 40292 USA.
Front Biosci. 1999 Jan 1;4:D63-71. doi: 10.2741/perlin.
The aminoglycoside (AG) 3'-phosphotransferases [APH(3')s] are an important class of modifying enzymes which confer high-level resistance to those AGs actively modified by the enzymes. They catalyze the transfer of the terminal phosphate from ATP to the drug, thus preventing the AG s action at the 70S ribosome. These enzymes, which utilize ATP as a co-substrate, appear from amino acid alignments to be part of a much larger superfamily of kinases and ATP-binding proteins. Structure-function analyses have been initiated in our laboratory for APH(3')-II, whose gene was derived from transposon Tn5. Site-directed mutagenesis of the cloned APH(3')-II gene was used to genetically examine the residues in two highly-conserved motifs proposed to participate in ATP binding. Several of these residues, in fact, were shown to affect the enzyme s affinity for ATP. We have also initiated studies using photoaffinity labelling of APH(3')-II with the ATP analogs, 8-azido-ATP and 2-azido-ATP. We have shown that 8-N3ATP and 2-N3ATP can be substituted for ATP in the APH(3')-II catalyzed phosphorylation of kanamycin; such findings indicate that the interaction of these photoaffinity analogs of ATP with APH(3')-II is biologically relevant. One of the best-characterized of the APH(3') enzymes is APH(3')-IIIa, the first of the group whose structure has been analyzed by x- ray crystallography. Several studies have demonstrated that this enzyme functions by a Theorell-Chance mechanism. Moreover, the architecture of the enzyme, crystallized in the presence of ADP has revealed residues in the ATP-binding pocket which are likely to play important roles in catalysis. Once the results from biochemical analyses can be correlated with those from mutagenesis studies and x-ray crystallography, a clearer picture of the active site will be provided for an important class of AG-modifying enzymes and phosphotransferases. This picture will also allow a better understanding of these enzymes within the greater context of kinases and nucleotide-binding proteins.
氨基糖苷类（AG）3'-磷酸转移酶[APH(3')s]是一类重要的修饰酶，可赋予对那些被该酶主动修饰的AG高水平耐药性。它们催化ATP的末端磷酸基团转移至药物上，从而阻止AG在70S核糖体上发挥作用。这些以ATP作为共底物的酶，从氨基酸序列比对来看，似乎是一个更大的激酶和ATP结合蛋白超家族的一部分。我们实验室已针对APH(3')-II开展了结构-功能分析，其基因源自转座子Tn5。对克隆的APH(3')-II基因进行定点诱变，用于从遗传学角度研究两个被认为参与ATP结合的高度保守基序中的残基。事实上，其中几个残基已被证明会影响该酶对ATP的亲和力。我们还启动了使用ATP类似物8-叠氮基-ATP和2-叠氮基-ATP对APH(3')-II进行光亲和标记的研究。我们已表明，在APH(3')-II催化的卡那霉素磷酸化反应中，8-N3ATP和2-N3ATP可替代ATP；这些发现表明，这些ATP的光亲和类似物与APH(3')-II的相互作用具有生物学相关性。APH(3')酶中特征最明确的之一是APH(3')-IIIa，它是该组中第一个其结构已通过X射线晶体学分析的酶。多项研究表明，该酶通过Theorell-Chance机制发挥作用。此外，在ADP存在下结晶的该酶结构已揭示出ATP结合口袋中的残基，这些残基可能在催化中发挥重要作用。一旦生化分析结果能与诱变研究和X射线晶体学结果相关联，就能为一类重要的AG修饰酶和磷酸转移酶提供更清晰的活性位点图景。这一图景也将有助于在更大的激酶和核苷酸结合蛋白背景下更好地理解这些酶。
