Unité des Agents Antibactériens, Institut Pasteur, F-75724 Paris Cedex 15, France.
RNA. 2011 Feb;17(2):251-62. doi: 10.1261/rna.2233511. Epub 2010 Dec 15.
Aminoglycosides are ribosome-targeting antibiotics and a major drug group of choice in the treatment of serious enterococcal infections. Here we show that aminoglycoside resistance in Enterococcus faecium strain CIP 54-32 is conferred by the chromosomal gene efmM, encoding the E. faecium methyltransferase, as well as by the previously characterized aac(6')-Ii that encodes a 6'-N-aminoglycoside acetyltransferase. Inactivation of efmM in E. faecium increases susceptibility to the aminoglycosides kanamycin and tobramycin, and, conversely, expression of a recombinant version of efmM in Escherichia coli confers resistance to these drugs. The EfmM protein shows significant sequence similarity to E. coli RsmF (previously called YebU), which is a 5-methylcytidine (m⁵C) methyltransferase modifying 16S rRNA nucleotide C1407. The target for EfmM is shown by mass spectrometry to be a neighboring 16S rRNA nucleotide at C1404. EfmM uses the methyl group donor S-adenosyl-L-methionine to catalyze formation of m⁵C1404 on the 30S ribosomal subunit, whereas naked 16S rRNA and the 70S ribosome are not substrates. Addition of the 5-methyl to C1404 sterically hinders aminoglycoside binding. Crystallographic structure determination of EfmM at 2.28 Å resolution reveals an N-terminal domain connected to a central methyltransferase domain that is linked by a flexible lysine-rich region to two C-terminal subdomains. Mutagenesis of the methyltransferase domain established that two cysteines at specific tertiary locations are required for catalysis. The tertiary structure of EfmM is highly similar to that of RsmF, consistent with m⁵C formation at adjacent sites on the 30S subunit, while distinctive structural features account for the enzymes' respective specificities for nucleotides C1404 and C1407.
氨基糖苷类抗生素是一类靶向核糖体的抗生素，也是治疗严重肠球菌感染的主要药物。在这里，我们表明屎肠球菌 CIP 54-32 中的氨基糖苷类耐药性是由染色体基因 efmM 赋予的，该基因编码屎肠球菌甲基转移酶，以及先前表征的 aac(6')-Ii，它编码 6'-N-氨基糖苷乙酰转移酶。在屎肠球菌中失活 efmM 会增加对氨基糖苷类药物卡那霉素和妥布霉素的敏感性，反之，在大肠杆菌中表达重组版的 efmM 会赋予这些药物的抗性。EfmM 蛋白与大肠杆菌 RsmF（以前称为 YebU）具有显著的序列相似性，后者是一种修饰 16S rRNA 核苷酸 C1407 的 5-甲基胞嘧啶（m⁵C）甲基转移酶。通过质谱法显示，EfmM 的靶标是 30S 核糖体亚基上的邻近核苷酸 C1404。EfmM 使用甲基供体 S-腺苷-L-甲硫氨酸催化在 30S 核糖体亚基上形成 m⁵C1404，而裸露的 16S rRNA 和 70S 核糖体不是底物。C1404 上的 5-甲基的添加阻碍了氨基糖苷类药物的结合。EfmM 的晶体结构测定分辨率为 2.28 Å，揭示了一个连接到中央甲基转移酶结构域的 N 端结构域，该结构域通过富含赖氨酸的区域连接到两个 C 端亚结构域。对甲基转移酶结构域的突变建立了两个特定三级位置的半胱氨酸是催化所必需的。EfmM 的三级结构与 RsmF 高度相似，这与在 30S 亚基上相邻位置形成 m⁵C 一致，而独特的结构特征解释了这些酶对核苷酸 C1404 和 C1407 的各自特异性。
