Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland.
Nucleic Acids Res. 2010 Mar;38(5):1652-63. doi: 10.1093/nar/gkp1142. Epub 2009 Dec 10.
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX(3)CX(2)C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity.
Cfr 甲基转移酶通过催化 23S rRNA 核苷酸 A2503 的 C-8 位甲基化，赋予细菌核糖体肽基转移酶中心结合的五类抗生素的联合耐药性。该核苷酸是管家甲基转移酶 RlmN 的靶标，其将 C-2 位甲基化。用 Cfr 序列进行的数据库搜索揭示了来自所有生命领域的一大组密切相关的序列，它们包含特征性的激进 S-腺苷甲硫氨酸 (SAM) 酶的 CX(3)CX(2)C 基序。Cfr/RlmN 家族的系统发育分析表明，RlmN 亚家族可能是原始形式，而 Cfr 亚家族是通过复制和水平基因转移产生的。已经计算了 Cfr 的结构模型，并将其用作丙氨酸诱变研究的指南，该研究证实了模型预测的 4Fe-4S 簇、与铁-硫簇（SAM1）配位的 SAM 分子以及假定的甲基供体（SAM2）的存在。在预测的功能位点的所有突变都显著影响 Cfr 的活性，如抗生素敏感性测试和引物延伸分析所示。该研究确定了必需氨基酸和反应机制改变的 Cfr 变体，这是理解 Cfr 活性结构基础的第一步。
