Mechanistic investigation of polyhydroxybutyrate (PHB) synthases and elucidation of PHB biosynthesis and degradation process in Wautersia eutropha H16

Polyhydroxyalkanoate (PHA) synthase from various bacterial organisms is able to catalyze the polymerization of (R)-hydroxyalkanoate-CoAs into high molecular weight PHAs under nutrient- limited conditions in the presence of a carbon source. PHA synthases are representative of enzymes involved in polymerizations in which a soluble substrate is transformed into an insoluble inclusion during the polymerization process. The initiation, elongation, and termination phases of this non-template driven polymerization process are not well understood. This thesis is focused on the initiation and the elongation phases leading to granule formation. For the first time, we have observed intermediate species in the in vitro reaction containing a mutant Class III synthase, D302A-PhaCPhaEAv, with its natural substrate (R)-3-hydroxybutyryl-CoA (HB-CoA). Analysis of reaction products by SDS-PAGE gel, Westerns with PHA and PhaCPhaEAv antibodies, and autoradiography showed different migratory properties of the mutant synthase after its reaction with substrate at various substrate to enzyme ratios (S/E). These results indicate that PhaCAv has been modified with hydroxybutyrate oligomers ((HB)n). The site of labeling was established to be C149, by trypsin digestion of the (HB)n modified synthase (n=3-10 at S/E = 5), reverse-phase HPLC separation of peptides, and mass spectrometry analysis. Similar intermediates have also been detected with the wild-type (wt) PhaCPhaEAv, and shown to be chemically competent. Thus, the mechanism of initiation of this synthase is through self- priming. Kinetic analysis of the reaction of HB-CoA with the wt synthase at S/E ratios of 70,000 was mechanistically informative.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemistry.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Tian, Jiamin, 1974-

Advisor dc:contributor.advisor

• JoAnne Stubbe.

• Chemistry.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/30240

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/30240