Polyhydroxyalkanoates: A paradigm for non-template dependent polymerizations

描述（由申请人提供）：聚羟基烷酸酯（PHAs）是由3-羟基烷酸辅酶a在营养有限的条件下由许多微生物合成的多氧酯。多达80%的细胞干重可以是pha。当环境提供营养物质时，pha被降解并用作碳和能源。这种聚合通常是非模板依赖聚合过程的代表，其中可溶性底物转化为不溶性聚合物，以颗粒的形式沉积在细胞内。我们选择了产聚羟基丁酸酯（PHBs）的富营养化毛藻H-16作为范例来理解这些非模板依赖系统。pha具有从热塑性塑料到弹性体（取决于衬底）的各种特性，并且是可生物降解的，因此受到普遍关注。我们的论文是，了解PHB稳态（生物合成和降解）对于设计新材料至关重要，这些新材料目前正在研究中，除其他外，用于心脏瓣膜和组织工程的支架。令人惊讶的是，我们对PHB合成酶PhaC的起始、延伸、终止、再起始和调控机制知之甚少。最近我们已经获得证据（体内和体外）PhaC也催化PHB链终止并留下一个重新引物合酶。支持这一模型的证据和详细的延伸机制将被追求。通过对PhaC、PhaP（覆盖颗粒表面的一种相蛋白）、PhaR（调节PhaP产生的一种转录因子）、PhaZIa、PhaZlb、PhaZIc和PhaZ2（推定的解聚合酶）抗体的透射电子显微镜和定量Western分析研究，已经产生了一种新的颗粒起始模型。将使用一系列技术来区分胶束模型、质膜出芽模型和颗粒形成的新中介元素模型。“解聚合酶”可能在结构上与合酶相似，这一发现表明可能需要对phaz进行功能重新分配。将继续对PhaZIa进行研究。在代谢水平和转录水平上对PHB产生和降解的调节也将被检查。对PhaC、PhaP和PhaZIa的作用的理解将随着对PHB结构的了解和对PHB稳态相关蛋白的结晶的努力而大大增强。

DESCRIPTION (provided by applicant): Polyhydroxyalkanoates (PHAs) are polyoxoesters synthesized from 3-hydroxyalkanoate-CoA under nutrient limited conditions by many microorganisms. As much as 80% of the cell dry weight can be PHAs. When the environment makes nutrients available, the PHAs are degraded and used as carbon and energy sources. This polymerization is representative of non-template dependent polymerization processes in general in which a soluble substrate is converted into an insoluble polymer deposited in the form of granules inside the cells. We have chosen Wautersia eutropha H-16 which makes polyhydroxybutyrates (PHBs), as a paradigm to understand these non-template dependent systems. PHAs are of general interest as they possess properties that range from thermoplastics to elastomers (depending on the substrate) and are biodegradable. Our thesis is that understanding PHB homeostasis (biosynthesis and degradation) is essential to engineering new materials which are currently being examined for use, among other things, in heart valves and as scaffolds for tissue engineering. Surprisingly little is known about the mechanisms of initiation, elongation, termination, re-initiation, and regulation of the PHB synthase, PhaC. Recently we have obtained evidence (in vivo and in vitro) PhaC also catalyzes PHB chain termination and leaves a re-primed synthase. Evidence in support of this model and the detailed mechanism of elongation will be pursued. Studies using transmission electron microscopy and quantitative Western analyses with antibodies to PhaC, PhaP (a phasin protein the covers the surface of the granules), PhaR (a transcription factor that regulates PhaP production), PhaZIa, PhaZlb, PhaZIc and PhaZ2 (putative depolymerases), have resulted in a new model for granule initiation. Methods to distinguish between a micelle model, the plasma membrane budding model, and the new mediation element model for granule formation, will be pursued using a range of techniques. The discovery that the "depolymerases" are likely to be structurally similar to the synthase, suggests that functional re-assignments to the PhaZs may need to be made. Studies on PhaZIa will be pursued. The regulation of PHB production and degradation at the metabolic level and at the level of transcription will also be examined. Understanding the roles of PhaC, PhaP and PhaZIa would be greatly enhanced with structural insight and efforts to crystallize proteins involved in PHB homeostasis will be actively pursued.