Patin Delphine, Turk Samo, Barreteau Hélène, Mainardi Jean-Luc, Arthur Michel, Gobec Stanislav, Mengin-Lecreulx Dominique, Blanot Didier
Institut de Biologie Intégrative de la Cellule (I2BC), UMR 9198 CEA/CNRS/Université Paris-Sud, 91405 Orsay, France.
Fakulteta za Farmacijo, Univerza v Ljubljani, Aškerčeva 7, 1000 Ljubljana, Slovenia.
Biochimie. 2016 Feb;121:209-18. doi: 10.1016/j.biochi.2015.12.006. Epub 2015 Dec 14.
Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.
猪红斑丹毒丝菌是一种革兰氏阳性菌，可感染包括人类在内的多种鸟类和哺乳动物。其肽聚糖的主要特征是在肽茎的第3位存在L-丙氨酸。在本研究中，我们从猪红斑丹毒丝菌中克隆了murE基因，并纯化了相应的His6标签形式的蛋白质。酶活性测定表明，猪红斑丹毒丝菌MurE确实是一种添加L-丙氨酸的酶。令人惊讶的是，它也能够添加内消旋二氨基庚二酸（许多革兰氏阴性菌、芽孢杆菌和分枝杆菌中在第3位发现的氨基酸），尽管程度较小。猪红斑丹毒丝菌和大肠杆菌的MurE酶序列比对显示，添加内消旋二氨基庚二酸的酶所特有的DNPR基序被HDNR取代。通过定点诱变实验和同源模型构建证明了后者基序在与L-丙氨酸和内消旋二氨基庚二酸相互作用中的作用。猪红斑丹毒丝菌murE基因在大肠杆菌中的过表达导致肽聚糖肽部分的第3位掺入L-丙氨酸。
