Novel functions associated with poly(3-hydroxybutyrate) (PHB) granules

四个亚细胞组分的比较蛋白质组分析显示存在14个（约。3000鉴定），特别是在Ralstonia eutropha H16中的PHB颗粒相关蛋白（PGAP）。除了10个先前描述的PGAP，还鉴定了四种蛋白质，它们之前没有与PHB代谢结合在一起：一种是功能未知的假设蛋白质，两种是具有PHB合酶/解聚酶特征的推定a/b-水解酶，第四种是马铃薯糖蛋白样磷脂酶。在这个项目中，我们将确定这些蛋白质的功能。我们推测这两个a/b-水解酶是通过硫解裂解PHB的新型PHB解聚酶。产物3-羟基丁酰辅酶A可用于通过磷酸转乙酰酶（Pta 1）和乙酸激酶（AckA）产生ATP，其基因与两种新的a/b-水解酶中的一种一起在操纵子中表达和鉴定。磷脂酶可以负责从PHB颗粒表面裂解磷脂或合成磷脂，这取决于疏水性PHB表面上的体内催化方向。确定的四个基因的缺失，纯化的蛋白质和分离的聚羟基丁酸颗粒从野生型和突变体的生化分析的表型，连同本地化的磷脂与特定的磷脂结合蛋白在野生型和突变株的GFP融合将被用来确定新的蛋白质的功能。该项目将回答50年前的问题，即在体内的PHB颗粒中是否存在磷脂层，并且我们希望证实假设的通过硫解和底物水平磷酸化从PHB形成ATP，这是一种尚未描述的PHB细胞器功能。

Comparative proteome analysis of four subcellular fractions revealed the presence of 14 (out of ca. 3000 identified) specifically PHB granule associated proteins (PGAPs) in Ralstonia eutropha H16. In addition to 10 previously described PGAPs four proteins were identified that have not been pulled together with PHB metabolism before: one is a hypothetical protein of unknown function, two are putative a/b-hydrolases with PHB synthase/depolymerase signatures and the fourth is a patatin-like phospholipase. In this project we will determine the function of these proteins. We hypothesize that the two a/b-hydrolases are novel PHB depolymerases that cleave PHB by thiolysis. The product, 3-hydroxybutyryl-CoA, could be used to generate ATP via phospho-transacetylase (Pta1) and acetate kinase (AckA), the genes of which were expressed and identified in an operon together with one of the two new a/b-hydrolases. The phospholipase could be responsible for either cleaving phospholipids from the PHB granule surface or for synthesis of phospholipids depending on the in vivo direction of catalysis on the hydrophobic PHB surface. Determination of the phenotypes of deletions of the four genes, biochemical analysis of the purified proteins and of isolated PHB granules from wt and mutants, together with localization of phospholipids by GFP fusions with specific phospholipid-binding proteins in wt and mutant strains will be used to identify the functions of the new proteins. This project will answer the 50 years old question on the absence or presence of a phospholipid layer in PHB granules in vivo, and we hope to confirm the postulated formation of ATP from PHB via thiolysis and substrate level phosphorylation, a not yet described function of PHB organelles.