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

对四个亚细胞组分的比较蛋白质组学分析显示，在已鉴定的约3000个组分中，有14个组分是在真营养罗氏菌H16中特异的PHB颗粒相关蛋白(PGAPs)。除了先前描述的10个PGAPs外，还鉴定了4个以前没有与PHB代谢结合在一起的蛋白质：一个是功能未知的假想蛋白质，两个可能是具有PHB合成酶/解聚酶特征的a/b水解酶，第四个是Patatin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-水解酶是一种新型的PHB解聚酶，可以通过硫代分解来裂解PHB。该产物3-羟丁酰辅酶A可通过磷酸转乙酰酶(Pta1)和醋酸激酶(AckA)产生ATP，其基因在一个操纵子中与两个新的a/b-水解酶之一一起表达和鉴定。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PHB表面的体内催化方向。四个基因缺失表型的测定，从wt和突变体中纯化的蛋白和分离的phb颗粒的生化分析，以及通过GFP与wt和突变株中特定的磷脂结合蛋白的融合来定位磷脂，将被用来鉴定新蛋白的功能。这个项目将回答在活体中PHB颗粒中是否存在磷脂层的问题，并希望证实PHB通过硫解和底物水平磷酸化形成ATP的假设，这是PHB细胞器尚未描述的功能。

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.