| Summary: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006. === Includes bibliographical references. === Polyhydroxybutyrate (PHB) is a polymer accumulated by many bacterial species to store carbon and reducing equivalents. PHB is packaged intracellularly in inclusions termed "granules" that contain the polymer in an amorphous state, surrounded by a protein coat. The molecular weight (Mw) of PHB is typically in the range of 1-2 x 106 Da, and it shows the striking characteristic of low polydispersity, (narrow Mw distribution), implying that PHB synthesizing organisms have a mechanism to control PHB Mw. Several approaches have been undertaken in this work to uncover this mechanism of Mw control. An in vitro investigation was conducted, using purified PHA synthase, the native substrate (hydroxybutyryl-coenzyme A), and substrate analogs. The size of PHB synthesized in vitro was found to be dependent on the size of the substrate cofactor and that when hydroxybutyryl-N-acetylcysteamine was used as a substrate, the product was PHB covalently linked to N-acetylcysteamine. Preliminary experiments supported a model whereby substrate cofactor in thiolate form (e.g. -S-CoA) participates in a chain transfer reaction at an internal PHB ester within the enzyme, and the accessibility of the site at which this reaction takes place changes during the course of polymerization. === (cont.) In an attempt to understand what this change was and how its timing was governed, a variety of in vivo studies were performed. Transcriptional profiling of the genes known to be involved in PHB biosynthesis was performed in order to identify candidates that may be involved in these processes, and phaP, known to be involved in granule structure formation was singled out for further investigation. Analysis of a PhaP overproducing C. necator strain revealed that it was indeed important for Mw determination, which was underscored by observations in recombinant Escherichia coli harboring phaP in combination with the PHB synthesis genes. Additionally, it was found that levels of the PHB polymerizing enzyme (PHB synthase), PHB Mw and granule formation were linked in recombinant E. coli. These results led to a model of a physical mechanism whereby the size of the elongating PHB chain governed accessibility to the site of the chain transfer reaction. === by Adam G. Lawrence. === Ph.D.
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