Zhu P P, Nosworthy N, Ginsburg A, Miyata M, Seok Y J, Peterkofsky A
Laboratory of Biochemical Genetics, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Biochemistry. 1997 Jun 10;36(23):6947-53. doi: 10.1021/bi963090m.
The gene encoding enzyme IIA(glc) (EIIA) of the phosphoenolpyruvate:sugar phosphotransferase system of Mycoplasma capricolum was cloned into a regulated expression vector. The purified protein product of the overexpressed gene was characterized as an active phosphoacceptor from HPr with a higher pI than previously described EIIAs. M. capricolum EIIA was unreactive with antibodies directed against the corresponding proteins from either Gram-positive or Gram-negative bacteria. Enzyme IIA(glc) behaved as a homogeneous, monomeric species of 16,700 Mr in analytical ultracentrifugation. The circular dichroism far-UV spectrum of EIIA reflects a low alpha-helical content and predominantly beta-sheet structural content: temperature-induced changes in ellipticity at 205 nm showed that the protein undergoes reversible, two-state thermal unfolding with Tm = 70.0 +/- 0.3 degrees C and a van't Hoff deltaH of 90 kcal/mol. Enzyme I (64,600 Mr) from M. capricolum exhibited a monomer-dimer-tetramer association at 4 and 20 degrees C with dimerization constants of log K(A) = 5.6 and 5.1 [M(-1)], respectively, in sedimentation equilibrium experiments. A new vector, capable of introducing an N-terminal His tag on a protein, was developed in order to generate highly purified heat-stable protein (HPr). No significant interaction of EIIA with HPr was detected by gel-filtration chromatography, intrinsic tryptophanyl residue fluorescence changes, titration calorimetry, biomolecular interaction, or sedimentation equilibrium studies. While Escherichia coli EIIA inhibits Gram-negative glycerol kinase activity, the M. capricolum EIIA has no effect on the homologous glycerol kinase. The probable regulator of sugar transport systems, HPr(Ser) kinase, was demonstrated in extracts of M. capricolum and Mycoplasma genitalium. Gene mapping studies demonstrated that, in contrast to the clustered arrangement of genes encoding HPr and enzyme I in E. coli, these genes are located diametrically opposite in the M. capricolum chromosome.
糖磷酸转移酶系统中编码酶IIA（glc）（EIIA）的基因克隆到一个调控表达载体中。过表达基因的纯化蛋白产物被鉴定为一种来自HPr的活性磷酸受体，其pI值高于先前描述的EIIA。山羊支原体EIIA与针对革兰氏阳性或革兰氏阴性细菌相应蛋白的抗体无反应。在分析超速离心中，酶IIA（glc）表现为一种均一的、16700 Mr的单体。EIIA的圆二色远紫外光谱反映出低α-螺旋含量和主要的β-折叠结构含量：温度诱导的205 nm处椭圆率变化表明该蛋白经历可逆的两态热解折叠，Tm = 70.0 +/- 0.3℃，范特霍夫焓变ΔH为90 kcal/mol。山羊支原体的酶I（64600 Mr）在4℃和20℃时表现出单体-二聚体-四聚体缔合，在沉降平衡实验中，二聚化常数的对数K(A)分别为5.6和5.1 [M(-1)]。为了产生高度纯化的热稳定蛋白（HPr），开发了一种能够在蛋白上引入N端His标签的新载体。通过凝胶过滤色谱、内在色氨酸残基荧光变化、滴定热分析、生物分子相互作用或沉降平衡研究，未检测到EIIA与HPr之间有显著相互作用。虽然大肠杆菌EIIA抑制革兰氏阴性甘油激酶活性，但山羊支原体EIIA对同源甘油激酶没有影响。糖转运系统的可能调节因子HPr（Ser）激酶在山羊支原体和生殖支原体的提取物中得到证实。基因图谱研究表明，与大肠杆菌中编码HPr和酶I的基因成簇排列不同，这些基因在山羊支原体染色体上位于直径相对的位置。
