Structural and Biochemical Analysis of the Pentapeptide Repeat Protein EfsQnr, a Potent DNA Gyrase Inhibitor
Structural and Biochemical Analysis of the Pentapeptide Repeat Protein EfsQnr, a Potent DNA Gyrase Inhibitor
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DOI：
10.1128/aac.01158-10
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发表时间：
2011-01-01
影响因子：
4.9
通讯作者：
Blanchard, John S.
中科院分区：
文献类型：
--
作者：
Hegde, Subray S.;Vetting, Matthew W.;Blanchard, John S.
粪肠球菌（Enterococcus faecalis）的染色体编码的Qnr同源蛋白（EfsQnr）在表达时赋予其宿主降低的对喹诺酮类药物的敏感性，并且主要由串联重复序列组成，这与属于五肽重复序列蛋白家族（PRPs）一致。用N-末端6x His标签克隆EfsQnr并纯化至同质。EfsQnr在低至20 nM的浓度下部分保护DNA促旋酶免受氟喹诺酮抑制。EfsQnr抑制DNA促旋酶的ATP依赖性超螺旋活性，50%抑制浓度（IC 50）为1.2 μ M，而没有观察到ATP非依赖性松弛活性的显著抑制。EfsQnr在大肠杆菌中过表达时具有细胞毒性，导致细胞凝集和活力丧失。EfsQnr的X射线晶体结构被确定为1.6埃分辨率。EfsQnr表现出典型的PRP的右手四边形β-螺旋折叠，其特征更类似于MfpA（分枝杆菌氟喹诺酮抗性五肽）而不是通常在蓝藻中发现的PRP。
The chromosomally encoded Qnr homolog protein from Enterococcus faecalis (EfsQnr), when expressed, confers to its host a decreased susceptibility to quinolones and consists mainly of tandem repeats, which is consistent with belonging to the pentapeptide repeat family of proteins (PRPs). EfsQnr was cloned with an N-terminal 6 x His tag and purified to homogeneity. EfsQnr partially protected DNA gyrase from fluoroquinolone inhibition at concentrations as low as 20 nM. EfsQnr inhibited the ATP-dependent supercoiling activity of DNA gyrase with a 50% inhibitory concentration (IC50) of 1.2 mu M, while no significant inhibition of ATP-independent relaxation activity was observed. EfsQnr was cytotoxic when overexpressed in Escherichia coli, resulting in the clumping of cells and a loss of viability. The X-ray crystal structure of EfsQnr was determined to 1.6-angstrom resolution. EfsQnr exhibits the right-handed quadrilateral beta-helical fold typical of PRPs, with features more analogous to MfpA (mycobacterium fluoroquinolone resistance pentapeptide) than to the PRPs commonly found in cyanobacteria.