Substrate recognition and modification by the nosiheptide resistance methyltransferase.
Substrate recognition and modification by the nosiheptide resistance methyltransferase.
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DOI：
10.1371/journal.pone.0122972
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Murchie AI
中科院分区：
文献类型：
--
作者：
Yin S;Jiang H;Chen D;Murchie AI
抗生素耐药病原体的扩散对普通公众构成越来越大的威胁。耐药性可由多种机制引起，包括抗生素结合部位的共价或突变修饰、药物的共价修饰或外排泵的过度表达。诺西肽抗性甲基转移酶通过23SRRNA在A1067位核苷酸的2ʹO-甲基化，在诺西肽产生菌精算链霉菌中对噻唑类抗生素诺西肽产生抗药性。尽管NHR的晶体结构以及与辅因子S-腺苷-L-蛋氨酸形成的复合体中与之密切相关的硫链霉菌甲基转移酶(TSR)的晶体结构是已知的，但NHR底物识别和催化的原理仍不清楚。我们用凝胶电泳迁移率改变分析(EMSA)和荧光各向异性分析了NHR与模型58和29核苷酸底物RNA的结合作用。我们证明了这种酶以二聚体的形式与RNA结合。通过构建由一个野生型亚基和一个失活突变体NHR-R135A亚基组成的异源二聚体复合体，我们证明了NHR同源二聚体只需要一个功能亚基就能发挥其酶活性。突变分析表明，相邻碱基(G1068和U1066)与A1067之间的相互作用在甲基转移活性中起着重要作用，从而使脱氧糖间隔区(5ʹ)替代目标核苷酸达到接近野生型甲基化水平。底物腺嘌呤上特定位置的一系列原子取代表明，相邻碱基之间的局部碱基-碱基相互作用对甲基化很重要。综上所述，这些数据表明，局部碱基-碱基相互作用在对准A1067底物2‘羟基进行甲基转移中起着重要作用。核酸的甲基化在基本的生物学过程中发挥着越来越重要的作用，我们预计这篇手稿中概述的方法可能对研究其他类型的核酸甲基转移酶有用。
The proliferation of antibiotic resistant pathogens is an increasing threat to the general public. Resistance may be conferred by a number of mechanisms including covalent or mutational modification of the antibiotic binding site, covalent modification of the drug, or the over-expression of efflux pumps. The nosiheptide resistance methyltransferase (NHR) confers resistance to the thiazole antibiotic nosiheptide in the nosiheptide producer organism Streptomyces actuosus through 2ʹO-methylation of 23S rRNA at the nucleotide A1067. Although the crystal structures of NHR and the closely related thiostrepton-resistance methyltransferase (TSR) in complex with the cofactor S-Adenosyl-L-methionine (SAM) are available, the principles behind NHR substrate recognition and catalysis remain unclear. We have analyzed the binding interactions between NHR and model 58 and 29 nucleotide substrate RNAs by gel electrophoresis mobility shift assays (EMSA) and fluorescence anisotropy. We show that the enzyme binds to RNA as a dimer. By constructing a hetero-dimer complex composed of one wild-type subunit and one inactive mutant NHR-R135A subunit, we show that only one functional subunit of the NHR homodimer is required for its enzymatic activity. Mutational analysis suggests that the interactions between neighbouring bases (G1068 and U1066) and A1067 have an important role in methyltransfer activity, such that the substitution of a deoxy sugar spacer (5ʹ) to the target nucleotide achieved near wild-type levels of methylation. A series of atomic substitutions at specific positions on the substrate adenine show that local base-base interactions between neighbouring bases are important for methylation. Taken together these data suggest that local base-base interactions play an important role in aligning the substrate 2’ hydroxyl group of A1067 for methyl group transfer. Methylation of nucleic acids is playing an increasingly important role in fundamental biological processes and we anticipate that the approach outlined in this manuscript may be useful for investigating other classes of nucleic acid methyltransferases.
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