Determination of r-3-hydroxyacyl-acp-coa transferase (phag) structure
| dc.contributor.author | Arsad, Hasni | |
| dc.date.accessioned | 2015-07-30T00:33:23Z | |
| dc.date.available | 2015-07-30T00:33:23Z | |
| dc.date.issued | 2010 | |
| dc.description.abstract | R-3-hydroxyacyl-ACP-CoA transferase (PhaG) catalyzes the conversion of (R)-3- hydroxyacyl-ACP to (R)-3-hydroxyacyl-CoA derivatives, which serve as the ultimate precursor for polyhydroxyalkanoate (PHA) polymerization from unrelated substrates. PHA is a family of bioplastic that has a good potential to replace fossilbased thermoplastics because it is biodegradable. The transferase enzyme PhaG of a locally isolated strain, Pseudomonas sp. USM 4-55, was recently doned(GeneBank accession number EU305558). Currently there is no known 3D structure with high I similarity to PhaG. In order to over express, the phaG gene was cloned into expression vector pQE-30 and it was successfully overexpressed by induction with 0.5 mM IPTG in the host Escherichia coli strain SG 13009. The PhaG was purified by metal affinity chromatography followed by gel filtration chromatography to a single band under Coomassie Blue SDS-PAGE analysis. The purified protein was confirmed as PhaG by de novo peptide sequencing. The pI of PhaGp sp USM 4-55 is 7.45, as determined by IEF. Protein crystallization screening was then performed by applying micro batch and hanging drop method. However, none of the crystallization screening kit used were able to produce PhaG protein crystal. A 3-D model of PhaG was predicted using threading method. The 2-hydroxy-6-oxo-6-phenylhexa-2,4- dienote hydrolase enzyme from Rhodococcus sp. strain RHAI (pdb id: lC4X) structure was used as the template. The Modeller9v4 program was used to model the PhaGp . sp USM 4-55. The RMS value of the PhaG model 132, and the template J C4X is O.75A. Interestingly, binding site prediction using SiteMap on the proposed model, showed a binding pocket that can accomodate the ligand, R-3-hydroxyoctanoate- CoA by using Glide. Consequently, the reaction mechanism of PhaG is proposed based on the docking analysis and the mechanism of malonyl CoA-ACP transacylase (FabD). | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/881 | |
| dc.language.iso | en | en_US |
| dc.title | Determination of r-3-hydroxyacyl-acp-coa transferase (phag) structure | en_US |
| dc.type | Thesis | en_US |
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