Identification of essential residues in the Erm(B) rRNA methyltransferase of Clostridium perfringens
Identification of essential residues in the Erm(B) rRNA methyltransferase of Clostridium perfringens
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DOI：
10.1128/aac.46.5.1253-1261.2002
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发表时间：
2002-05-01
影响因子：
4.9
通讯作者：
Rood, JI
中科院分区：
文献类型：
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作者：
Farrow, KA;Lyras, D;Rood, JI
大环内酯-林可酰胺-链菌素B耐药现象普遍存在，在许多细菌病原体中检测到对红霉素等抗生素耐药的决定因素。抗性最常见的途径是产生一种Erm蛋白，一种23S rRNA甲基转移酶。我们对产气荚膜梭菌的Erm(B)蛋白进行了突变分析，目的是为了更好地了解该蛋白的作用机制。通过体外羟胺诱变或通过突变菌株XL1-Red，对携带erm(B)基因的重组质粒进行了突变。鉴定出28个独立衍生的突变体，其中9个在erm(B)基因中有单点突变。这些突变体产生稳定但无功能的Erm(B)蛋白，并且在保守的甲基转移酶基序中都有氨基酸变化，这对底物结合或催化都是重要的。对产气荚膜梭菌Erm(B)蛋白的模拟证实，点突变都涉及对该rRNA甲基转移酶的结构和/或功能重要的残基。因此，这些蛋白质区域是合理开发甲基转移酶抑制剂的潜在靶点。
Macrolide-lincosamide-streptogramin B resistance is widespread, with the determinants encoding resistance to antibiotics such as erythromycin being detected in many bacterial pathogens. Resistance is most commonly mediated by the production of an Erm protein, a 23S rRNA methyltransferase. We have undertaken a mutational analysis of the Erm(B) protein from Clostridium perfringens with the objective of developing a greater understanding of the mechanism of action of this protein. A recombinant plasmid that carried the erm(B) gene was mutated by either in vitro hydroxylamine mutagenesis or passage through the mutator strain XL1-Red. Twenty-eight independently derived mutants were identified, nine of which had single point mutations in the erm(B) gene. These mutants produced stable but nonfunctional Erm(B) proteins, and all had amino acid changes within conserved methyltransferase motifs that were important for either substrate binding or catalysis. Modeling of the C. perfringens Erm(B) protein confirmed that the point mutations all involved residues important for the structure and/or function of this rRNA methyltransferase. These regions of the protein therefore represent potential targets for the rational development of methyltransferase inhibitors.