Oberholzer Anselm Erich, Bumann Mario, Schneider Philipp, Bächler Christoph, Siebold Christian, Baumann Ulrich, Erni Bernhard
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Bern, Switzerland.
J Mol Biol. 2005 Feb 18;346(2):521-32. doi: 10.1016/j.jmb.2004.11.077. Epub 2004 Dec 22.
Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.
酶I（EI）是磷酸烯醇丙酮酸（PEP）：糖磷酸转移酶系统（PTS）的第一个组分，由一个N端蛋白结合结构域（EIN）和一个C端PEP结合结构域（EIC）组成。EI通过EIN上的一个组氨酸残基以双取代方式将磷酸从PEP转移至通用磷酸载体蛋白HPr。在此，我们报道了嗜热栖热放线菌（一种在75℃下生长最佳的解糖真细菌）同二聚体EIC结构域的1.82Å晶体结构。EIC折叠成一个（βα）8桶状结构，在β2/α2、β3/α3和β6/α6之间有三个大的螺旋插入片段。大的两亲性二聚体界面每个单体掩埋3750Å2的可及表面积。与丙酮酸磷酸二激酶（PPDK）的比较表明，EIC的空PEP结合位点和PPDK的结合PEP的位点中的活性位点残基具有几乎相同的构象，表明活性位点结构刚性。EIC与氯代PEP的Z和E异构体复合物的计算机模拟为它们作为EI底物和抑制剂的差异提供了合理的解释。EIC结构域与大肠杆菌的氨基酸序列同一性为54%，与枯草芽孢杆菌EIC的为60%，具有相同的氨基酸组成，但比大肠杆菌EIC的同源模型含有更多的盐桥和更复杂的盐桥网络。EIC易于结晶表明嗜热栖热放线菌可作为对结晶来说过于不稳定的嗜温蛋白稳定同源物的来源。
