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. ? ?
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