Project Number: 1935-41000-067-01
Project Type: Reimbursable

Start Date: Aug 01, 2003
End Date: Sep 14, 2007

Objective:
Determine the 3-dimensional structures of bacterial poly(hydroxyalkanoate) synthase (PHAS) enzymes and their catalytic domains. Molecular cloning of the corresponding genes, and subsequent high-level expression and large-scale purification of the enzymes and their subfragments will be carried out. The gene products will be used to grow crystals for subsequent X-ray diffraction studies to determine their structures.

Approach:
The genes coding for Class I, II and III poly(hydroxyalkanoate) synthases (PHAS) will be cloned by PCR from Ralstonia eutropha, Pseudomonas aeruginosa, and Allochromatium venosum, respectively. Subgenomic sequences coding for the alpha/beta-hydrolase folds of these enzymes will also be PCR-cloned. The cloned DNAs will be expressed as affinity-tagged fusion polypeptides in Escherichia coli. After large-scale isolation of the gene products, the affinity tags will be removed by using appropriate protease. Crystallization conditions for the isolated PHASes and their subfragments will be screened using the sparse matrix sampling approach and subsequently refined. Using the protein crystals, X-ray diffraction data will be collected and analyzed for model building. Refinement of the structures will finally be carried out using XPLOR method.