Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.
Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.
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DOI：
10.1093/nar/gkp1142
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发表时间：
2010-03
影响因子：
14.9
通讯作者：
Long KS
中科院分区：
文献类型：
--
作者：
Kaminska KH;Purta E;Hansen LH;Bujnicki JM;Vester B;Long KS
Cfr甲基转移酶通过催化23 S rRNA核苷酸A2503的C-8位的甲基化，赋予对结合到细菌核糖体的肽基转移酶中心的五类抗生素的组合抗性。相同的核苷酸被使C-2位置甲基化的管家甲基转移酶RlmN靶向。用Cfr序列进行的数据库搜索已经揭示了来自所有生命领域的一大组密切相关的序列，这些序列含有自由基S-腺苷-L-甲硫氨酸（SAM）酶的保守CX 3CX 2C基序特征。对Cfr/RlmN家族的系统发育分析表明，RlmN亚家族可能是祖先的形式，而Cfr亚家族通过重复和水平基因转移而产生。Cfr的结构模型已被计算，并用作指导丙氨酸诱变研究，证实了基于模型的预测的4Fe-4S簇，SAM分子协调的铁-硫簇（SAM 1）和SAM分子，这是推定的甲基供体（SAM 2）。通过抗生素敏感性测试和引物延伸分析，预测功能位点的所有突变显著影响Cfr活性。这项研究已经确定了必需氨基酸和Cfr变体与改变的反应机制，并代表了第一步了解Cfr活性的结构基础。
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 CX3CX2C 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.