Prodigious substrate specificity of AAC(6′)-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci
Prodigious substrate specificity of AAC(6′)-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci
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DOI：
10.1016/s1074-5521(99)80006-4
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发表时间：
1999-02-01
影响因子：
--
通讯作者：
Wright, GD
中科院分区：
文献类型：
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作者：
Daigle, DM;Hughes, DW;Wright, GD
背景：肠球菌和葡萄球菌的高水平庆大霉素耐药性是由 AAC(6')-APH(2") 赋予的，AAC(6')-APH(2") 是一种具有 6'-N-乙酰转移酶和 2"-O-磷酸转移酶活性的酶。致病性革兰氏阳性菌中存在这种酶，阻碍了庆大霉素 C 和大多数其他氨基糖苷类药物作为治疗药物的成功使用。结果：为了了解氨基糖苷类修饰的机制，我们在枯草芽孢杆菌中表达了 AAC(6')-APH(2")。纯化的酶是单体，分子量为 57 kDa，并显示出预期的氨基糖苷类 N-乙酰转移酶和 O-磷酸转移酶活性。用各种方法进行结构功能分析氨基糖苷类底物揭示了一种在两种酶活性中都具有广泛特异性的酶，解释了 AAC(6')-APH(2") 对临床氨基糖苷类治疗的巨大负面影响。青霉素A和巴龙霉素都是缺少6'-氨基的氨基糖苷类药物，均被AAC(6')-APH(2")乙酰化。巴龙霉素乙酰化产物的红外光谱得到了与O-乙酰化一致的信号。新霉素磷酸化产物的质谱和核磁共振分析表明磷酰基转移主要发生在6-氨基己糖环A的3'-OH处，并且一些二磷酸化物质在环 D 的 3'-OH 和 3"'-OH 处也存在磷酸盐，这对于该酶来说都是前所未有的观察结果。此外，青维霉素A的磷酸化位点被确定为戊糖环C的5"-OH。结论：双功能AAC(6')-APH(2")能够通过N-和O-乙酰化以及羟基的磷酸化来灭活几乎所有临床上重要的氨基糖苷类药物。这种酶极其广泛的底物特异性将影响氨基糖苷类药物的未来发展，并对抗生素设计提出重大挑战。
Background: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents,Results: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C.Conclusions: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design.