Systematic analysis of a conserved region of the aminoglycoside 6′-N-acetyltransferase type Ib
Systematic analysis of a conserved region of the aminoglycoside 6′-N-acetyltransferase type Ib
复制标题
DOI：
10.1128/aac.45.12.3287-3292.2001
复制
发表时间：
2001-12-01
影响因子：
4.9
通讯作者：
Tolmasky, ME
中科院分区：
文献类型：
--
作者：
Shmara, A;Weinsetel, N;Tolmasky, ME
对氨基糖苷类6 '-N-乙酰基转移酶Ib型保守基序B进行丙氨酸扫描突变，并通过测定卡那霉素（KAN）及其半合成衍生物阿米卡星（AMK）的MIC来分析取代的效果。几个取代导致MIC没有重大变化。E167 A和F171 A导致衍生物失去赋予对KAN和AMK的抗性的能力。P155 A、P157 A、N159 A、L160 A、I163 A、K168 A和G170 A赋予中等水平的抗性。Y166 A产生了一种具有修饰特异性的酶衍生物;它赋予了对KAN的高水平抗性，但失去了赋予对AMK的抗性的能力。尽管不明显，但由取代N159 A和G170 A赋予的抗性谱与由Y166 A赋予的抗性谱相关。这些表型与先前的结果一起表明，当在42 ℃下进行测定时，突变体F171 L不能催化AMK的乙酰化（D. Panaite和M. Tolmasky，Plasmid 39：123-133，1998），表明某些基序B氨基酸在受体底物特异性中起直接或间接作用。AMK和KAN对含有C165 A取代的细胞的MIC较高，表明酶的活性形式可能不是通过二硫键形成的二聚体。此外，该结果表明，乙酰化反应通过直接机制而不是乒乓机制发生，乒乓机制包括乙酰基瞬时转移到半胱氨酸残基。在C末端的片段的缺失表明，可以删除多达10个氨基酸而不丧失活性。
Alanine-scanning mutagenesis was applied to the aminoglycoside 6'-N-acetyltransferase type lb conserved motif B, and the effects of the substitutions were analyzed by measuring the MICs of kanamycin (KAN) and its semisynthetic derivative, amikacin (AMK). Several substitutions resulted in no major change in MICs. E167A and F171A resulted in derivatives that lost the ability to confer resistance to KAN and AMK. P155A, P157A, N159A, L160A, I163A, K168A, and G170A conferred intermediate levels of resistance. Y166A resulted in an enzyme derivative with a modified specificity; it conferred a high level of resistance to KAN but lost the ability to confer resistance to AMK. Although not as pronounced, the resistance profiles conferred by substitutions N159A and G170A were related to that conferred by Y166A. These phenotypes, taken together with previous results indicating that mutant F171L could not catalyze acetylation of AMK when the assays were carried out at 42 degreesC (D. Panaite and M. Tolmasky, Plasmid 39:123-133, 1998), suggest that some motif B amino acids play a direct or indirect role in acceptor substrate specificity. MICs of AMK and KAN for cells harboring the substitution C165A were high, suggesting that the active form of the enzyme may not be a dimer formed through a disulfide bond. Furthermore, this result indicated that the acetylation reaction occurs through a direct mechanism rather than a ping-pong mechanism that includes a transient transfer of the acetyl group to a cysteine residue. Deletion of fragments at the C terminus demonstrated that up to 10 amino acids could be deleted without a loss of activity.