来自大肠杆菌周质的聚-3-羟基丁酸酯合酶。
Poly-3-hydroxybutyrate synthase from the periplasm of Escherichia coli.
作者信息
Dai Dongsheng, Reusch Rosetta N
机构信息
Department of Microbiology and Molecular Genetics, Michigan State University, 6163 Biomedical Biophysical Science, East Lansing, MI 48824, USA.
出版信息
Biochem Biophys Res Commun. 2008 Sep 26;374(3):485-9. doi: 10.1016/j.bbrc.2008.07.043. Epub 2008 Jul 18.
This is the first report of a poly-3-hydroxybutyrate (PHB) synthase in Escherichia coli. The enzyme was isolated from the periplasm using ammonium sulfate fractionation, hydrophobic, and size-exclusion chromatography and identified by LC/MS/MS as YdcS, a component of a putative ABC transporter. Green Fluorescent Protein-tagged ydcS, purified by 2D native gel electrophoresis, also exhibited PHB synthase activity. Optimal conditions for enzyme activity were 37 degrees C, pH 6.8-7.5, 100 mM KCl. K(m) was 0.14 mM and V(max) was 18.7 nmol/mg protein/min. The periplasms of deletion mutants displayed <25% of the activity of the parent strain. Deletion mutants exhibited approximately 25% less growth in M9 medium, glucose, and contained approximately 30% less PHB complexed to proteins (cPHB) in the outer membranes, but the same concentration of chloroform-extractable PHB as wild-type cells. The primary sequence of YdcS suggests it may belong to the alpha-/beta-hydrolase superfamily which includes polyhydroxybutyrate (PHB) synthases, lipases, and esterases.
这是关于大肠杆菌中聚-3-羟基丁酸酯（PHB）合酶的首次报道。该酶通过硫酸铵分级分离、疏水色谱和尺寸排阻色谱从周质中分离出来，并通过液相色谱/串联质谱鉴定为YdcS，它是一种假定的ABC转运蛋白的组成部分。通过二维天然凝胶电泳纯化的绿色荧光蛋白标记的ydcS也表现出PHB合酶活性。酶活性的最佳条件为37℃、pH 6.8 - 7.5、100 mM KCl。米氏常数（K(m)）为0.14 mM，最大反应速度（V(max)）为18.7 nmol/mg蛋白质/分钟。缺失突变体的周质显示出亲本菌株活性的25%以下。缺失突变体在M9培养基、葡萄糖中生长减少约25%，外膜中与蛋白质复合的PHB（cPHB）含量减少约30%，但氯仿可提取的PHB浓度与野生型细胞相同。YdcS的一级序列表明它可能属于α/β水解酶超家族，该超家族包括聚羟基丁酸酯（PHB）合酶、脂肪酶和酯酶。
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