Characterization of trimethoprim resistant E-coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates
Characterization of trimethoprim resistant E-coli dihydrofolate reductase mutants by mass spectrometry and inhibition by propargyl-linked antifolates
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DOI：
10.1039/c6sc05235e
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发表时间：
2017-05-01
期刊：
影响因子：
8.4
通讯作者：
Brodbelt, Jennifer S.
中科院分区：
文献类型：
--
作者：
Cammarata, Michael;Thyer, Ross;Brodbelt, Jennifer S.
致病性大肠杆菌是尿路感染的主要原因之一，对最常用的抗生素甲氧苄啶（TMP）具有显著的耐药性，TMP可抑制二氢叶酸还原酶（DHFR）。DHFR的单点突变可调节耐药性。通过测量Michaelis-Menten和抑制动力学评价了两种临床相关突变P21 L和W30 R对DHFR活性的影响，并通过紫外光解离（UVPD）的天然质谱法进行了结构表征。与WT-DHFR相比，P21 L和W30 R突变体在辅因子NADPH存在下与TMP产生较不稳定的复合物，如在ESI质谱中观察到的复合物的相对丰度所证明的。此外，基于通过二元和三元DHFR复合物的UVPD质谱法获得的片段化模式的变化，显著的结构变化定位于W30 R的底物结合口袋和M20环区域以及含有P21 L突变体的必需G-H功能环的C-末端部分。结果表明，突变通过独特的机制赋予抗性。一种新的炔丙基连接的抗叶酸化合物1038被证明是一种合理有效的P21 L突变体抑制剂。
Pathogenic Escherichia coli, one of the primary causes of urinary tract infections, has shown significant resistance to the most popular antibiotic, trimethoprim (TMP), which inhibits dihydrofolate reductase (DHFR). The resistance is modulated by single point mutations of DHFR. The impact of two clinically relevant mutations, P21L and W30R, on the activity of DHFR was evaluated via measurement of Michaelis-Menten and inhibitory kinetics, and structural characterization was undertaken by native mass spectrometry with ultraviolet photodissociation (UVPD). Compared to WT-DHFR, both P21L and W30R mutants produced less stable complexes with TMP in the presence of co-factor NADPH as evidenced by the relative abundances of complexes observed in ESI mass spectra. Moreover, based on variations in the fragmentation patterns obtained by UVPD mass spectrometry of binary and ternary DHFR complexes, notable structural changes were localized to the substrate binding pocket for W30R and to the M20 loop region as well as the C-terminal portion containing the essential G-H functional loop for the P21L mutant. The results suggest that the mutations confer resistance through distinctive mechanisms. A novel propargyl-linked antifolate compound 1038 was shown to be a reasonably effective inhibitor of the P21L mutant.