Shmara A, Weinsetel N, Dery K J, Chavideh R, Tolmasky M E
Institute of Molecular Biology and Nutrition, Department of Biological Science, College of Natural Sciences and Mathematics, California State University Fullerton, Fullerton, California 92834-6850, USA.
Antimicrob Agents Chemother. 2001 Dec;45(12):3287-92. doi: 10.1128/AAC.45.12.3287-3292.2001.
Alanine-scanning mutagenesis was applied to the aminoglycoside 6'-N-acetyltransferase type Ib 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 degrees C (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.
丙氨酸扫描诱变应用于氨基糖苷类6'-N-乙酰基转移酶Ib型保守基序B，并通过测定卡那霉素（KAN）及其半合成衍生物阿米卡星（AMK）的最低抑菌浓度（MIC）来分析替换的效果。几个替换导致MIC没有重大变化。E167A和F171A产生的衍生物失去了赋予对KAN和AMK耐药性的能力。P155A、P157A、N159A、L160A、I163A、K168A和G170A赋予中等水平的耐药性。Y166A产生了一种具有改变特异性的酶衍生物；它赋予对KAN的高水平耐药性，但失去了赋予对AMK耐药性的能力。尽管不那么明显，但N159A和G170A替换赋予的耐药谱与Y166A赋予的相关。这些表型，连同先前的结果表明当在42℃进行测定时突变体F171L不能催化AMK的乙酰化（D. Panaite和M. Tolmasky，《质粒》39:123 - 133，1998），表明基序B的一些氨基酸在受体底物特异性中起直接或间接作用。对于携带替换C165A的细胞，AMK和KAN的MIC很高，这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发生，而不是通过包括乙酰基向半胱氨酸残基的瞬时转移的乒乓机制。C末端片段的缺失表明最多可删除10个氨基酸而不丧失活性。
