Nalam Pranav, Cook Paul D, Smith Brian A
Department of Chemistry, Grand Valley State University, Allendale, Michigan, USA.
Proteins. 2025 Feb;93(2):413-419. doi: 10.1002/prot.26745. Epub 2024 Sep 9.
Aminoglycoside antibiotics have played a critical role in the treatment of both Gram-negative and Gram-positive bacterial infections. However, antibiotic resistance has severely compromised the efficacy of aminoglycosides. A leading cause of aminoglycoside resistance is mediated by bacterial enzymes that inactivate these drugs via chemical modification. Aminoglycoside nucleotidyltransferase-6 (ANT(6)) enzymes inactivate streptomycin by transferring an adenyl group from ATP to position 6 on the antibiotic. Despite the clinical significance of this activity, ANT(6) enzymes remain relatively uncharacterized. Here, we report the first high resolution x-ray crystallographic structure of ANT(6)-Ib from Campylobacter fetus subsp. fetus bound with streptomycin. Structural modeling and gel filtration chromatography experiments suggest that the enzyme exists as a dimer in which both subunits contribute to the active site. Moreover, superposition of the ANT(6)-Ib structure with the structurally related enzyme lincosamide nucleotidyltransferase B (LinB) permitted the identification of a putative nucleotide binding site. These data also suggest that residues D44 and D46 coordinate essential divalent metal ions and D102 functions as the catalytic base.
氨基糖苷类抗生素在革兰氏阴性菌和革兰氏阳性菌感染的治疗中发挥了关键作用。然而，抗生素耐药性严重损害了氨基糖苷类药物的疗效。氨基糖苷类耐药的一个主要原因是由细菌酶介导的，这些酶通过化学修饰使这些药物失活。氨基糖苷核苷酸转移酶-6（ANT(6)）通过将ATP上的腺苷基团转移到抗生素的第6位，使链霉素失活。尽管这种活性具有临床意义，但ANT(6)酶的特征仍相对不明确。在此，我们报道了来自胎儿弯曲杆菌亚种胎儿的ANT(6)-Ib与链霉素结合的首个高分辨率X射线晶体结构。结构建模和凝胶过滤色谱实验表明，该酶以二聚体形式存在，两个亚基均对活性位点有贡献。此外，将ANT(6)-Ib结构与结构相关的林可酰胺核苷酸转移酶B（LinB）进行叠加，有助于确定一个假定的核苷酸结合位点。这些数据还表明，残基D44和D46配位必需的二价金属离子，而D102作为催化碱基发挥作用。
