Leiros Hanna-Kirsti S, Brandsdal Bjørn Olav, McSweeney Seán M
The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, University of Tromsø, N-9037 Tromsø, Norway.
Biochim Biophys Acta. 2010 Apr;1804(4):967-76. doi: 10.1016/j.bbapap.2010.01.010. Epub 2010 Jan 21.
Metronidazole (MTZ) is an antibiotic commonly used to treat anaerobic bacterial infections in humans and animals. Antibiotic resistance toward this class of 5-nitroimidazole (5-Ni) drug derivatives has been related to the Nim genes thought to encode a reductase. Here we report the biophysical characteristics of the NimA protein from Deinococcus radiodurans (DrNimA) binding to MTZ and three other 5-Ni drugs. The interaction energies of the protein and antibiotic are studied by isothermal titration calorimetry (ITC) and with free energy and linear interaction energy (LIE) calculations, where the latter method revealed that the antibiotic binding is mainly of hydrophobic character. ITC measurements further found that one DrNimA dimer has two antibiotic binding sites which were not affected by mutation of the reactive His71. The observed association constants (K(a)) were in the range of 5.1-4910(4)M(-1) and the enthalpy release upon binding to DrNimA for the four drugs studied was relatively low (approximately -1 kJ/mol) but still measurable. The drug binding is mainly entropy driven and along with the hydrophobic drug binding site found by crystallography, this possibly explains the low observed enthalpy values. The effect of the His71 mutation and the presence of MTZ were studied by differential scanning calorimetry (DSC). Native DrNimA is a yellow colored protein where the interaction from His71 to the cofactor is thought to be responsible for the coloring. Mutations of His71 to Ala, Ser, Leu or Asp all gave transparent, colorless protein solutions, and the two mutant crystal structures of DrNimA-H71A and DrNimA-H71S presented revealed no cofactor binding.
甲硝唑（MTZ）是一种常用于治疗人和动物厌氧细菌感染的抗生素。对这类5-硝基咪唑（5-Ni）药物衍生物的抗生素耐药性与被认为编码还原酶的Nim基因有关。在此，我们报告了来自耐辐射球菌（DrNimA）的NimA蛋白与MTZ及其他三种5-Ni药物结合的生物物理特性。通过等温滴定量热法（ITC）以及自由能和线性相互作用能（LIE）计算研究了蛋白质与抗生素的相互作用能，后一种方法表明抗生素结合主要具有疏水性质。ITC测量进一步发现一个DrNimA二聚体有两个抗生素结合位点，这两个位点不受活性His71突变的影响。观察到的缔合常数（K(a)）在5.1 - 4910(4)M(-1)范围内，所研究的四种药物与DrNimA结合时的焓释放相对较低（约 -1 kJ/mol）但仍可测量。药物结合主要由熵驱动，连同晶体学发现的疏水药物结合位点，这可能解释了观察到的低焓值。通过差示扫描量热法（DSC）研究了His71突变和MTZ存在的影响。天然的DrNimA是一种黄色蛋白质，其中His71与辅因子的相互作用被认为是产生颜色的原因。His71突变为Ala、Ser、Leu或Asp均产生透明、无色的蛋白质溶液，并且所呈现的DrNimA - H71A和DrNimA - H71S的两种突变晶体结构均未显示辅因子结合。
