Cox Georgina, Stogios Peter J, Savchenko Alexei, Wright Gerard D
Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada.
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, Canada Center for Structural Genomics of Infectious Diseases (CSGID).
mBio. 2015 Jan 6;6(1):e02180-14. doi: 10.1128/mBio.02180-14.
The aminoglycosides are highly effective broad-spectrum antimicrobial agents. However, their efficacy is diminished due to enzyme-mediated covalent modification, which reduces affinity of the drug for the target ribosome. One of the most prevalent aminoglycoside resistance enzymes in Gram-negative pathogens is the adenylyltransferase ANT(2″)-Ia, which confers resistance to gentamicin, tobramycin, and kanamycin. Despite the importance of this enzyme in drug resistance, its structure and molecular mechanism have been elusive. This study describes the structural and mechanistic basis for adenylylation of aminoglycosides by the ANT(2″)-Ia enzyme. ANT(2″)-Ia confers resistance by magnesium-dependent transfer of a nucleoside monophosphate (AMP) to the 2″-hydroxyl of aminoglycoside substrates containing a 2-deoxystreptamine core. The catalyzed reaction follows a direct AMP transfer mechanism from ATP to the substrate antibiotic. Central to catalysis is the coordination of two Mg(2+) ions, positioning of the modifiable substrate ring, and the presence of a catalytic base (Asp86). Comparative structural analysis revealed that ANT(2″)-Ia has a two-domain structure with an N-terminal active-site architecture that is conserved among other antibiotic nucleotidyltransferases, including Lnu(A), LinB, ANT(4')-Ia, ANT(4″)-Ib, and ANT(6)-Ia. There is also similarity between the nucleotidyltransferase fold of ANT(2″)-Ia and DNA polymerase β. This similarity is consistent with evolution from a common ancestor, with the nucleotidyltransferase fold having adapted for activity against chemically distinct molecules. IMPORTANCE : To successfully manage the threat associated with multidrug-resistant infectious diseases, innovative therapeutic strategies need to be developed. One such approach involves the enhancement or potentiation of existing antibiotics against resistant strains of bacteria. The reduction in clinical usefulness of the aminoglycosides is a particular problem among Gram-negative human pathogens, since there are very few therapeutic options for infections caused by these organisms. In order to successfully circumvent or inhibit the activity of aminoglycoside-modifying enzymes, and to thus rejuvenate the activity of the aminoglycoside antibiotics against Gram-negative pathogens, structural and mechanistic information is crucial. This study reveals the structure of a clinically prevalent aminoglycoside resistance enzyme [ANT(2″)-Ia] and depicts the molecular basis underlying modification of antibiotic substrates. Combined, these findings provide the groundwork for the development of broad-spectrum inhibitors against antibiotic nucleotidyltransferases.
氨基糖苷类是高效的广谱抗菌剂。然而，由于酶介导的共价修饰，其效力会降低，这种修饰会降低药物对靶核糖体的亲和力。革兰氏阴性病原体中最常见的氨基糖苷类耐药酶之一是腺苷酸转移酶ANT(2″)-Ia，它赋予对庆大霉素、妥布霉素和卡那霉素的耐药性。尽管这种酶在耐药性中很重要，但其结构和分子机制一直难以捉摸。本研究描述了ANT(2″)-Ia酶对氨基糖苷类进行腺苷酸化的结构和机制基础。ANT(2″)-Ia通过将核苷单磷酸（AMP）以镁依赖的方式转移到含有2-脱氧链霉胺核心的氨基糖苷类底物的2″-羟基上来赋予耐药性。催化反应遵循从ATP到底物抗生素的直接AMP转移机制。催化的核心是两个Mg(2+)离子的配位、可修饰底物环的定位以及催化碱基（Asp86）的存在。比较结构分析表明，ANT(2″)-Ia具有两结构域结构，其N端活性位点结构在其他抗生素核苷酸转移酶中保守，包括Lnu(A)、LinB、ANT(4')-Ia、ANT(4″)-Ib和ANT(6)-Ia。ANT(2″)-Ia的核苷酸转移酶折叠与DNA聚合酶β之间也存在相似性。这种相似性与从共同祖先进化而来一致，核苷酸转移酶折叠已适应针对化学性质不同的分子的活性。重要性：为了成功应对与多重耐药传染病相关的威胁，需要制定创新的治疗策略。一种方法涉及增强或提高现有抗生素对耐药菌株的效力。氨基糖苷类临床效用的降低在革兰氏阴性人类病原体中是一个特别的问题，因为针对这些生物体引起的感染的治疗选择非常少。为了成功规避或抑制氨基糖苷类修饰酶的活性，从而恢复氨基糖苷类抗生素对革兰氏阴性病原体的活性，结构和机制信息至关重要。本研究揭示了一种临床常见的氨基糖苷类耐药酶[ANT(2″)-Ia]的结构，并描述了抗生素底物修饰的分子基础。综合起来，这些发现为开发针对抗生素核苷酸转移酶的广谱抑制剂奠定了基础。
