Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: Identification of a ligase superfamily
Conditionally lethal Escherichia coli murein mutants contain point defects that map to regions conserved among murein and folyl poly-gamma-glutamate ligases: Identification of a ligase superfamily
复制标题
DOI：
10.1021/bi9701078
复制
发表时间：
1997-05-20
期刊：
影响因子：
2.9
通讯作者：
Anderson, MS
中科院分区：
文献类型：
--
作者：
Eveland, SS;Pompliano, DL;Anderson, MS
细菌肽聚糖生物合成包括四个酶促反应，其中连续的氨基酸残基与尿苷二磷酸-N-乙酰胞壁酸（UDP-MurNAc）连接。通过比较来自不同细菌属的MurC、-D、-E和-F蛋白的氨基酸序列，鉴定了四个同源区域。对Swissprot的相关序列的概况搜索显示，这些区域相似性存在于叶酰-γ-聚谷氨酸连接酶中。这些序列同源性似乎与催化功能有关：两种酶家族都通过有序的动力学机制进行，并通过酰基磷酸中间体形成产物。区域II中的两个高度保守的残基通过来自大肠杆菌（murF; E158和H188）的胞壁素D-丙氨酰-D-丙氨酸添加酶的定点诱变来检查。所有突变都对活性高度有害，酶比活性降低200-4500倍，验证了这些残基的关键性质。对3株E.携带murC 3（G344 D）、murE 1（G344 K、A495 S）和murF 2（A288 T）突变的大肠杆菌突变体揭示了与这些比对区域中的第四个密切相关的点突变的存在。murF 2等位基因，表达和纯化的谷胱甘肽S-转移酶：：MurF 2融合，是181倍的催化活性在30摄氏度，并进一步减少在非允许的温度（42摄氏度）。因此，murF 2温度敏感表型产生于该蛋白质家族内高度保守区域内的点突变。这些数据表明，这些蛋白质包括一个超家族的三个底物酰胺连接酶，共享显着的结构和催化同源性。
Bacterial peptidoglycan biosynthesis includes four enzymatic reactions in which successive amino acid residues are ligated to uridine diphospho-N-acetylmuramic acid (UDP-MurNAc). By comparing the amino acid sequences of MurC, -D, -E, and -F proteins from various bacterial genera, four regions of homology were identified. A profile search of Swissprot for related sequences revealed that these regional similarities were present in the folyl-gamma-polyglutamate ligases. These sequence homologies appear to track with catalytic function: both enzyme families proceed through an ordered kinetic mechanism and form product via an acyl phosphate intermediate. Two highly conserved residues in region II were examined through site-directed mutagenesis of the murein D-alanyl-D-alanine-adding enzyme from Escherichia coli (murF; E158 and H188). All mutations were highly detrimental to activity with enzyme specific activity reductions of 200-4500-fold, validating the critical nature of these residues. DNA sequence analysis from three E. coli mutants harboring the murC3 (G344D), murE1 (G344K, A495S), and murF2 (A288T) mutations revealed the presence of point mutation(s) closely associated with the fourth of these aligned regions. The murF2 allele, expressed and purified as a glutathione S-transferase::MurF2 fusion, was 181-fold less catalytically active at 30 degrees C and was further reduced at the nonpermissive temperature (42 degrees C). Thus the murF2 temperature-sensitive phenotype arises from a point mutation within a highly conserved region within this protein family. These data argue that these proteins comprise a superfamily of three substrate amide ligases that share significant structural and catalytic homologies.