Institute of Microbiology, University of Stuttgart, Stuttgart, Germany.
Western University, London, Ontario, Canada.
Appl Environ Microbiol. 2018 Jun 18;84(13). doi: 10.1128/AEM.00604-18. Print 2018 Jul 1.
In this study, we screened poly(3-hydroxybutyrate) (PHB) synthase PhaC1 and PHB depolymerase PhaZa1 of for the presence of phosphorylated residues during the PHB accumulation and PHB degradation phases. Thr373 of PHB synthase PhaC1 was phosphorylated during the stationary growth phase but was not modified during the exponential and PHB accumulation phases. Ser35 of PHB depolymerase PhaZa1 was identified in the phosphorylated form during both the exponential and stationary growth phases. Additional phosphosites were identified for both proteins in sample-dependent forms. Site-directed mutagenesis of the codon for Thr373 and other phosphosites of PhaC1 revealed a strong negative impact on PHB synthase activity. Modifications of Thr26 and Ser35 of PhaZa1 reduced the ability of to mobilize PHB in the stationary growth phase. Our results show that phosphorylation of PhaC1 and PhaZa1 can be important for the modulation of the activities of PHB synthase and PHB depolymerase. Poly(3-hydroxybutyrate) (PHB) and related polyhydroxyalkanoates (PHAs) are important intracellular carbon and energy storage compounds in many prokaryotes. The accumulation of PHB or PHAs increases the fitness of cells during periods of starvation and under other stress conditions. The simultaneous presence of PHB synthase (PhaC1) and PHB depolymerase (PhaZa1) on synthesized PHB granules in (alternative designation, ) was previously shown in several laboratories. These findings imply that the activities of PHB synthase and PHB depolymerase should be regulated to avoid a futile cycle of simultaneous synthesis and degradation of PHB. Here, we addressed this question by identifying the phosphorylation sites on PhaC1 and PhaZa1 and by site-directed mutagenesis of the identified residues. Furthermore, we conducted and analyses of PHB synthase activity and PHB contents.
在这项研究中，我们筛选了 中的聚 3-羟基丁酸酯（PHB）合酶 PhaC1 和 PHB 解聚酶 PhaZa1，以检测 PHB 积累和 PHB 降解阶段磷酸化残基的存在。PHB 合酶 PhaC1 的 Thr373 在静止生长阶段发生磷酸化，但在指数和 PHB 积累阶段未被修饰。PHB 解聚酶 PhaZa1 的 Ser35 在指数和静止生长阶段均以磷酸化形式存在。这两种蛋白质在依赖样本的形式中还鉴定出了其他磷酸化位点。对 PhaC1 的 Thr373 密码子和其他磷酸化位点进行定点突变，发现对 PHB 合酶活性有很强的负面影响。PhaZa1 的 Thr26 和 Ser35 的修饰降低了 在静止生长阶段动员 PHB 的能力。我们的结果表明，PhaC1 和 PhaZa1 的磷酸化可能对 PHB 合酶和 PHB 解聚酶活性的调节很重要。聚 3-羟基丁酸酯（PHB）和相关的聚羟基烷酸酯（PHA）是许多原核生物中重要的细胞内碳和能量储存化合物。在饥饿和其他应激条件下，PHB 或 PHA 的积累增加了细胞的适应性。以前在几个实验室中已经证明， 在合成的 PHB 颗粒上同时存在 PHB 合酶（PhaC1）和 PHB 解聚酶（PhaZa1）。这些发现意味着 PHB 合酶和 PHB 解聚酶的活性应该受到调节，以避免 PHB 的同时合成和降解的无效循环。在这里，我们通过鉴定 PhaC1 和 PhaZa1 上的磷酸化位点并对鉴定出的残基进行定点突变来解决这个问题。此外，我们还进行了 PHB 合酶活性和 PHB 含量的 和 分析。
