Molecular mechanism of aminoglycoside antibiotic kinase APH(3′)-IIIa -: Roles of conserved active site residues
Molecular mechanism of aminoglycoside antibiotic kinase APH(3′)-IIIa -: Roles of conserved active site residues
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DOI：
10.1074/jbc.m100540200
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发表时间：
2001-06-29
影响因子：
4.8
通讯作者：
Wright, GD
中科院分区：
文献类型：
--
作者：
Boehr, DD;Thompson, PR;Wright, GD
氨基糖苷类抗生素激酶(APHs)是临床上一类重要的抗生素耐药酶。APHs与丝氨酸/苏氨酸和酪氨酸激酶在结构和功能上具有同源性，但只有5个氨基酸在两组酶之间是不变的，这些残基都位于蛋白质的核苷酸结合区。我们已经对氨基糖苷激酶APH(3‘)-IIIa中的所有五个保守残基进行了定点突变：参与ATP捕获的Lys(44)和Glu(60)，一个假定的活性碱基，用于去质子化进入的氨基糖苷羟基Asp(190)，以及镁2+配体Asn(195)和Glu(208)，它们配位两个镁离子，即镁1和镁2。先前的结构和突变证据表明，Lys(44)与ATP的磷酸基团直接相互作用；不变Glu(60)的突变表明该残基在识别或催化ATP中不起关键作用；不变Glu(60)与Lys44的epsilon-氨基形成盐桥；Asp(190)的突变结果与在磷酸转移过程中氨基糖苷羟基的正确定位相一致，但不是作为一般碱。研究发现，镁1和镁2配基Asp(208)是酶活性所必需的，而镁2配基Asn(195)对识别镁三磷酸腺苷非常重要。诱变的结果与溶剂同位素、溶剂粘度和二价阳离子的要求一致，符合磷酰化转移的解离机制，其中初始底物去质子化对磷酸盐转移不是必不可少的，而Mg2和Asp(208)可能在类偏磷酸盐过渡态的稳定中发挥关键作用。这些结果为在体内合成可逆转氨基糖苷类抗生素耐药性的过渡态模拟物奠定了基础。
The aminoglycoside antibiotic kinases (APHs) constitute a clinically important group of antibiotic resistance enzymes. APHs share structural and functional homology with Ser/Thr and Tyr kinases, yet only five amino acids are invariant between the two groups of enzymes and these residues are all located within the nucleotide binding regions of the proteins. We have performed site-directed mutagenesis on all five conserved residues in the aminoglycoside kinase APH(3 ')-IIIa: Lys(44) and Glu(60) involved in ATP capture, a putative active site base required for deprotonating the incoming aminoglycoside hydroxyl group Asp(190), and the Mg2+ ligands Asn(195) and Glu(208), which coordinate two Mg2+ ions, Mg1 and Mg2. Previous structural and mutagenesis evidence have demonstrated that Lys(44) interacts directly with the phosphate groups of ATP; mutagenesis of invariant Glu(60), which forms a salt bridge with the epsilon -amino group of Lys44, demonstrated that this residue does not play a critical role in ATP recognition or catalysis, Results of mutagenesis of Asp(190) were consistent with a role in proper positioning of the aminoglycoside hydroxyl during phosphoryl transfer but not as a general base. The Mg1 and Mg2 ligand Asp(208) was found to be absolutely required for enzyme activity and the Mg2 ligand Asn(195) is important for Mg ATP recognition. The mutagenesis results together with solvent isotope, solvent viscosity, and divalent cation requirements are consistent with a dissociative mechanism of phosphoryl transfer where initial substrate deprotonation is not essential for phosphate transfer and where Mg2 and Asp(208) likely play a critical role in stabilization of a metaphosphate-like transition state. These results lay the foundation for the synthesis of transition state mimics that could reverse aminoglycoside antibiotic resistance in vivo.