Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6′)-Iy aminoglycoside N-acetyltransferase
Kinetic and mutagenic characterization of the chromosomally encoded Salmonella enterica AAC(6′)-Iy aminoglycoside N-acetyltransferase
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DOI：
10.1021/bi002736e
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发表时间：
2001-03-27
期刊：
影响因子：
2.9
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Magnet, S;Lambert, T;Blanchard, JS
肠道沙门氏菌染色体编码的氨基糖苷类N-乙酰转移酶AAC（6 '）-Iy赋予对许多氨基糖苷类抗生素的耐药性。结构基因被克隆和表达，纯化的酶在溶液中以ca. 17 000 Da单体。乙酰辅酶A是优选的酰基供体，并且大多数治疗上重要的氨基糖苷类是乙酰化的底物。这些是具有6 ′-羟基取代基的氨基糖苷类（例如，利维多霉素）。因此，该酶对6 ′-氨基糖苷类具有区域选择性和专一性的乙酰转移酶活性。该酶对某些氨基糖苷类底物表现出米氏动力学，但对其他底物表现出“底物活化”。动力学研究支持酶的随机动力学机制。碘乙酰胺以两相方式灭活酶，其中一半活性迅速丧失，另一半活性丧失得更慢。妥布霉素，而不是乙酰辅酶A，防止失活。野生型酶中的三个半胱氨酸残基（C70、C109、C145）中的每一个都被碘乙酰胺羧酰胺甲基化。AAC（6 '）-Iy中的半胱氨酸109在主要I类亚家族的12种AAC（6'）酶序列中是保守的。令人惊讶的是，该残基突变为丙氨酸既不消除活性，也不改变碘乙酰胺的双相失活。最大速度和V/K值的一些氨基糖苷类在这个单一的突变体中升高，并表现出线性与非线性动力学底物的动力学行为被逆转。AAC（6 '）-Iy中的半胱氨酸70是主要I类亚家族的所有12种AAC（6'）酶中的半胱氨酸或苏氨酸残基。双突变体C109 A/C70 A不能被碘乙酰胺灭活。双突变体的乙酰辅酶A和氨基糖苷类底物的Km值均大幅增加，所有氨基糖苷类底物均表现出Michaelis-Menten动力学。溶剂动力学同位素对V/K的影响是正常的WT酶和反向的双突变体。我们讨论了化学机制和可能的限速步骤的野生型和突变形式的酶。
The chromosomally encoded aminoglycoside N-acetyltransferase, AAC(6')-Iy, from Salmonella enterica confers resistance toward a number of aminoglycoside antibiotics. The structural gene was cloned and expressed and the purified enzyme existed in solution as a dimer of ca. 17 000 Da monomers. Acetyl-CoA was the preferred acyl donor, and most therapeutically important aminoglycosides were substrates for acetylation. Exceptions are those aminoglycosides that possess a 6'-hydroxyl substituent (e.g., lividomycin). Thus, the enzyme exhibited regioselective and exclusive acetyltransferase activity to 6'-amine-containing aminoglycosides. The enzyme exhibited Michaelis-Menten kinetics for some aminoglycoside substrates but "substrate activation" with others. Kinetic studies supported a random kinetic mechanism for the enzyme. The enzyme was inactivated by iodoacetamide in a biphasic manner, with half of the activity being lost rapidly and the other half more slowly. Tobramycin, but not acetyl-CoA, protected against inactivation. Each of the three cysteine residues (C70, C109, C145) in the wild-type enzyme were carboxamidomethylated by iodoacetamide. Cysteine 109 in AAC(6')-Iy is conserved in 12 AAC(6') enzyme sequences of the major class I subfamily. Surprisingly, mutation of this residue to alanine neither abolished activity nor altered the biphasic inactivation by iodoacetamide. The maximum velocity and V/K values for a number of aminoglycosides were elevated in this single mutant, and the kinetic behavior of substrates exhibiting linear vs nonlinear kinetics was reversed. Cysteine 70 in AAC(6')-Iy is either a cysteine or a threonine residue in all 12 AAC(6') enzymes of the major class I subfamily. The double mutant, C109A/C70A, was not inactivated by iodoacetamide. The double mutant exhibited large increases in the K-m values for both acetyl-CoA and aminoglycoside substrates, and all aminoglycoside substrates exhibited Michaelis-Menten kinetics. Solvent kinetic isotope effects on V/K were normal for the WT enzyme and inverse for the double mutant. We discuss a chemical mechanism and the likely rate-limiting steps for both the wild-type and mutant forms of the enzyme.