Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth
Expression of the Staphylococcus aureus UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase in Escherichia coli and effects on peptidoglycan biosynthesis and cell growth
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DOI：
10.1128/jb.181.19.5909-5914.1999
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发表时间：
1999-10-01
影响因子：
3.2
通讯作者：
Chopra, I
中科院分区：
文献类型：
--
作者：
Mengin-Lecreulx, D;Falla, T;Chopra, I
大肠杆菌和金黄色葡萄球菌细胞壁肽聚糖中的单体单元的不同之处在于L-丙氨酰-γ-D-谷氨酰-X-D-丙氨酰-D-丙氨酸侧链中的第三个氨基酸的性质，其中X分别是内消旋二氨基庚二酸或L-赖氨酸。鉴定来自金黄色葡萄球菌的 murE 基因，编码 UDP-N-乙酰胞壁酰基-L-丙氨酰-D-谷氨酸：L-赖氨酸连接酶，并将其克隆到质粒载体中。在大肠杆菌中诱导其过度表达会迅速导致异常形态变化和随后的细胞裂解。在诱导细胞中观察到肽聚糖含量减少了 28%，并且对肽聚糖组成和结构的分析表明，约。 50% 的内消旋二氨基庚二酸残基被 L-赖氨酸取代。在单体和二聚体片段中均检测到赖氨酸，但后者的受体单元仅含有内消旋二氨基庚二酸，这表明L-赖氨酸的E-氨基和D-丙氨酸的α-羧基之间不会发生转肽作用。大分子的整体交联仅略有下降。对含有内消旋二氨基庚二酸和 L-赖氨酸的肽聚糖前体的检测和分析证实了在 MurE 连接酶催化的反应后添加的含有氨基酸的前体中存在 L-赖氨酸，并提供了有关参与这些后一过程的酶的特异性的附加信息。
The monomer units in the Escherichia coli and Staphylococcus aureus cell wall peptidoglycans differ in the nature of the third amino acid in the L-alanyl-gamma-D-glutamyl-X-D-alanyl-D-alanine side chain, where X is meso-diaminopimelic acid or L-lysine, respectively. The murE gene from S. aureus encoding the UDP-N-acetylmuramoyl-L-alanyl-D-glutamate: L-lysine ligase was identified and cloned into plasmid vectors. Induction of its overexpression in E. coli rapidly results in abnormal morphological changes and subsequent cell lysis. A reduction of 28% in the peptidoglycan content was observed in induced cells, and analysis of the peptidoglycan composition and structure showed that ca. 50% of the meso-diaminopimelic acid residues were replaced by L-lysine. Lysine was detected in both monomer and dimer fragments, but the acceptor units from the latter contained exclusively meso-diaminopimelic acid, suggesting that no transpeptidation could occur between the E-amino group of L-lysine and the a-carboxyl group of D-alanine. The overall cross-linking of the macromolecule was only slightly decreased. Detection and analysis of meso-diaminopimelic acid- and L-lysine-containing peptidoglycan precursors confirmed the presence of L-lysine in precursors containing amino acids added after the reaction catalyzed by the MurE ligase and provided additional information about the specificity of the enzymes involved in these latter processes.