Polyhydroxyalkanoates: A paradigm for non-template dependent polymerizations

聚羟基脂肪酸酯：非模板依赖性聚合的范例

• 批准号: 7044471
• 负责人: JOANNE STUBBE
• 金额: $ 35.85万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7044471
• 关键词: biodegradable product; butyrates; enzyme activity; enzyme mechanism; enzyme structure; high performance liquid chromatography; hydrolase; laboratory rabbit; mass spectrometry; microorganism metabolism; polyhydroxy compound; polymerization; polymers; protein structure function; transcription factor

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.

描述（由申请人提供）：聚羟基链烷酸酯（PHA）是由许多微生物在营养限制条件下由3-羟基链烷酸-CoA合成的聚氧酸酯。多达80%的细胞干重可以是PHA。当环境提供养分时，PHA被降解并用作碳和能源。这种聚合通常代表非模板依赖性聚合过程，其中可溶性底物转化为以颗粒形式沉积在细胞内的不溶性聚合物。我们选择了产生聚羟基丁酸酯（PHBs）的Wautersia eutropha H-16作为理解这些非模板依赖性系统的范例。PHA是普遍感兴趣的，因为它们具有从热塑性塑料到弹性体（取决于基材）的性质并且是可生物降解的。我们的论文是，了解聚羟基丁酸稳态（生物合成和降解）是必不可少的工程新材料，目前正在审查使用，除其他外，在心脏瓣膜和组织工程的支架。令人惊讶的是，很少有人知道的起始，延伸，终止，再启动，和调节的机制的PHB合酶，PhaC。最近，我们已经获得的证据（在体内和体外）PhaC也催化PHB链终止，并留下一个重新启动的合酶。支持这一模型的证据和详细的伸长机制将被追究。使用透射电子显微镜和定量Western分析的研究与抗体PhaC，PhaP（一个相位蛋白的颗粒的表面覆盖），PhaR（一个转录因子，调节PhaP生产），PhaZIa，PhaZIb，PhaZIc和PhaZ 2（推定的解聚酶），导致了一个新的模型的颗粒启动。将使用一系列技术来区分胶束模型、质膜出芽模型和用于颗粒形成的新的中介元素模型的方法。发现“解聚酶”可能在结构上类似于合酶，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。还将检查在代谢水平和转录水平上对PHB产生和降解的调节。了解PhaC，PhaP和PhaZIa的作用将大大增强与结构的洞察力和努力结晶蛋白质参与PHB稳态将积极追求。