Wang Z, Schmitt M P, Holmes R K
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814.
Infect Immun. 1994 May;62(5):1600-8. doi: 10.1128/iai.62.5.1600-1608.1994.
The diphtheria toxin repressor (DtxR) is an iron-dependent regulator of diphtheria toxin production and iron uptake in Corynebacterium diphtheriae. It is activated in vitro by divalent metal ions including Fe2+, Cd2+, Co2+, Mn2+, Ni2+, and Zn2+. We characterized 20 different mutations in dtxR induced by bisulfite mutagenesis, 18 of which caused single-amino-acid substitutions in DtxR and two of which were chain-terminating mutations. Six of the amino acid replacements were clustered between residues 39 and 52 in a predicted helix-turn-helix motif that exhibits homology with several other repressors and is identified as the putative DNA-binding domain of DtxR. Three substitutions occurred within a predicted alpha-helical region with the sequence His-98-X3-Cys-102-X3-His-106 that resembles metal-binding motifs in several other proteins and is identified as the putative metal-binding site of DtxR. Several purified variants of DtxR with decreased repressor activity failed to bind in gel retardation assays to DNA fragments that contained the tox operator. A quantitative assay for binding of DtxR to 63Ni2+ was also developed. Scatchard analysis revealed that DtxR has a single class of high-affinity 63Ni(2+)-binding sites with a Kd of 2.11 x 10(-6) M and a maximum binding capacity of approximately 1.2 atoms of Ni2+ per DtxR monomer. The P39L, T40I, T44I, and R47H variants of DtxR exhibited normal to slightly decreased 63Ni(2+)-binding activity, but H106Y, which has an amino acid substitution in the presumed metal-binding domain, exhibited markedly decreased 63Ni(2+)-binding activity.
白喉毒素阻遏蛋白（DtxR）是一种铁依赖性调节因子，可调控白喉棒状杆菌中白喉毒素的产生及铁的摄取。在体外，它可被包括Fe2+、Cd2+、Co2+、Mn2+、Ni2+和Zn2+在内的二价金属离子激活。我们对亚硫酸氢盐诱变诱导的dtxR基因中的20种不同突变进行了表征，其中18种导致DtxR中单个氨基酸替换，另外两种为链终止突变。6个氨基酸替换集中在预测的螺旋-转角-螺旋基序中的39至52位残基之间，该基序与其他几种阻遏蛋白具有同源性，被确定为DtxR的推定DNA结合结构域。3个替换发生在一个预测的α-螺旋区域内，其序列为His-98-X3-Cys-102-X3-His-106，类似于其他几种蛋白质中的金属结合基序，被确定为DtxR的推定金属结合位点。几种阻遏活性降低的DtxR纯化变体在凝胶阻滞试验中未能与包含tox操纵子的DNA片段结合。还开发了一种用于检测DtxR与63Ni2+结合的定量分析方法。Scatchard分析表明，DtxR具有一类高亲和力的63Ni(2+)结合位点，解离常数Kd为2.11×10(-6) M，每个DtxR单体的最大结合容量约为1.2个Ni2+原子。DtxR的P39L、T40I、T-44I和R47H变体表现出正常至略有降低的63Ni(2+)结合活性，但在推定的金属结合结构域中存在氨基酸替换的H106Y变体表现出明显降低的63Ni(2+)结合活性。
