Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3")-Ii/AAC(6′)-IId from Serratia marcescens
Characterization of the bifunctional aminoglycoside-modifying enzyme ANT(3")-Ii/AAC(6′)-IId from Serratia marcescens
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DOI：
10.1021/bi060723g
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发表时间：
2006-07-11
期刊：
影响因子：
2.9
通讯作者：
Mobashery, Shahriar
中科院分区：
文献类型：
--
作者：
Kim, Choonkeun;Hesek, Dusan;Mobashery, Shahriar
粘质沙雷氏菌新发现的双功能抗生素耐药酶催化氨基糖苷类抗生素的腺苷酸化和乙酰化。酶解产物的结构鉴定表明，卡那霉素A的6 ′-氨基发生乙酰化，链霉素和大观霉素的3 ″-羟基和9-羟基分别发生腺苷化。腺苷酰转移酶结构域似乎是高度特异性的壮观霉素和链霉素，而乙酰转移酶结构域显示了广泛的底物配置文件。初始速度模式表明，这两个域遵循顺序动力学机制。使用死端和产物抑制、溶剂同位素效应和溶剂粘度效应揭示了腺苷酰转移酶结构域通过Theorell-Chance动力学机制催化反应，其中ATP在氨基糖苷之前与酶结合，并且修饰的抗生素是最后释放的产物。乙酰转移酶结构域遵循有序的双-双动力学机制，其中抗生素是结合活性位点的第一底物，CoASH在修饰的氨基糖苷类之前释放。两个基因的合并，创造双功能耐药酶与扩大的配置文件，现在已经记录在四个实例，包括本报告中的研究主题，这表明一个新的趋势，在出现耐氨基糖苷类抗生素的病原体。
A newly discovered bifunctional antibiotic resistance enzyme from Serratia marcescens catalyzes adenylation and acetylation of aminoglycoside antibiotics. The structure assignment of the enzymic products indicated that acetylation takes place on the 6'-amine of kanamycin A and the adenylation on 3"- and 9-hydroxyl groups of streptomycin and spectinomycin, respectively. The adenyltransferase domain appears to be highly specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition, the solvent isotope effect, and the solvent viscosity effect reveals that the adenyltransferase domain catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released. The acetyltransferase domain follows an ordered bi-bi kinetic mechanism, in which the antibiotic is the first substrate that binds to the active site and CoASH is released prior to the modified aminoglycoside. The merging of two genes to create bifunctional resistance enzymes with expanded profiles has now been documented in four instances, including the subject of study in this report, which suggests a new trend in the emergence of resistance to aminoglycoside antibiotics among pathogens.