Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli:: Catalytic role of conserved aspartic acid residues
Phospho-N-acetyl-muramyl-pentapeptide translocase from Escherichia coli:: Catalytic role of conserved aspartic acid residues
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DOI：
10.1128/jb.186.6.1747-1757.2004
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发表时间：
2004-03-01
影响因子：
3.2
通讯作者：
Bugg, TDH
中科院分区：
文献类型：
--
作者：
Lloyd, AJ;Brandish, PE;Bugg, TDH
磷酸-N-乙酰基-胞壁酰-五肽移位酶（移位酶1）催化最终组装细菌细胞壁的肽聚糖层的一系列脂质连接步骤中的第一步。这种必需的酶是几种天然产物抗生素的靶点，最近成为抗菌药物发现计划的焦点。易位酶1的催化机制被认为是通过磷酸-N-乙酰基-胞壁酰-五肽和亲核氨基酸残基之间形成的共价中间体进行的。易位酶1家族和相关跨膜磷酸糖转移酶超家族成员的氨基酸序列比对显示，只有三个保守的残基，具有亲核侧链：天冬氨酸残基D115，D116和D267。在这里，我们报告的大肠杆菌易位酶1的表达和部分纯化的C-末端六组氨酸（C-His（6））融合蛋白。构建具有定点突变D115 N、D116 N和D267 N的三种酶，表达并纯化为C-His融合物。酶分析确定，所有三个突变消除了易位酶I的活性，这一发现证实了这些残基的重要作用。通过与异戊烯基转移酶中发现的双天冬氨酸基序的结构环境进行类比，我们提出了一个模型，其中D115和D116螯合镁离子，该镁离子与UDP-N-乙酰基-胞壁酰-五肽底物的焦磷酸桥相协调，因此D267实现了转位酶1活性位点亲核试剂的作用。
Phospho-N-acetyl-muramyl-pentapeptide translocase (translocase 1) catalyzes the first of a sequence of lipid-linked steps that ultimately assemble the peptidoglycan layer of the bacterial cell wall. This essential enzyme is the target of several natural product antibiotics and has recently been the focus of antimicrobial drug discovery programs. The catalytic mechanism of translocase 1 is believed to proceed via a covalent intermediate formed between phospho-N-acetyl-muramyl-pentapeptide and a nucleophilic amino acid residue. Amino acid sequence alignments of the translocase 1 family and members of the related transmembrane phosphosugar transferase superfamily revealed only three conserved residues that possess nucleophilic side chains: the aspartic acid residues D115, D116, and D267. Here we report the expression and partial purification of Escherichia coli translocase 1 as a C-terminal hexahistidine (C-His(6)) fusion protein. Three enzymes with the site-directed mutations D115N, D116N, and D267N were constructed, expressed, and purified as C-His, fusions. Enzymatic analysis established that all three mutations eliminated translocase I activity, and this finding verified the essential role of these residues. By analogy with the structural environment of the double aspartate motif found in prenyl transferases, we propose a model whereby D115 and D116 chelate a magnesium ion that coordinates with the pyrophosphate bridge of the UDP-N-acetyl-muramyl-pentapeptide substrate and in which D267 therefore fulfills the role of the translocase 1 active-site nucleophile.