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United States Department of Agriculture

Agricultural Research Service

Title: Sequence analysis, structure prediction and functional validation of phaC1/phaC2 genes of Pseudomonas sp. LDC-25 and its importance in polyhydroxyalkanoate accumulation

Authors
item Sujatha, Kabilan - LADY DOAK COLLEGE
item Mahalakshmi, Ayyasamy - LADY DOAK COLLEGE
item SOLAIMAN, DANIEL
item Shenbagarathai, Rajaiah - LADY DOAK COLLEGE

Submitted to: Journal of Biomolecular Structure and Dynamics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 15, 2009
Publication Date: June 1, 2009
Citation: Sujatha, K., Mahalakshmi, A., Solaiman, D., Shenbagarathai, R. 2009. Sequence analysis, structure prediction and functional validation of phaC1/phaC2 genes of Pseudomonas sp. LDC-25 and its importance in polyhydroxyalkanoate accumulation. Journal of Biomolecular Structure and Dynamics. 26(6):771-780.

Interpretive Summary: Polyhydroxyalkanoates (PHAs) are a family of bioplastic materials produced by bacteria. Many microbes can use agricultural products and coproducts to synthesize the biodegradable PHAs. The key enzymes used by bacteria to produce PHAs are coded by genes labeled as phaC1 and phaC2. An in-depth understanding of the structure of these genes and their corresponding gene-products (i.e., PhaC1 and PhaC2 enzymes) is needed to modify them by genetic and protein engineering approaches to achieve a high-level PHA production or to obtain novel PHAs. We report in this study the cloning and characterization of phaC1 and phaC2 genes from two strains of newly isolated Pseudomonas bacteria. We applied a computer program to successfully construct the 3-dimensional structure of PhaC1 and PhaC2 by emulation to an enzyme (called carboxyesterase) whose structure had been experimentally determined. It is anticipated that the results of this study will be useful for the selective modification of the building blocks (i.e., amino acids) of PhaC1 or PhaC2 to improve PHA production-yields or properties.

Technical Abstract: Polyhydroxyalkanoates (PHAs) are attractive biomaterials in both conventional medical devices and tissue engineering. PHA synthase is responsible for catalyzing the formation of polyhydroxyalkanoates (PHA), but its structural information is limited. Hence, the focus of this study is to predict 3D model for phaC1 and phaC2 genes of field-soil strain Pseudomonas sp. LDC-25 and to validate the functional properties through in vitro studies. The phaC1/phaC2 genes were amplified, cloned and sequenced. The sequence analysis showed larger-than-90% homology to pha loci and presence of alpha/beta hydrolase fold, but phaC2 loci of LDC-25 exhibits variation in the conserved residue (Ser is replaced by Ala). The threading approach demonstrated that carboxylesterase (d1tqha) can be used as the modeling template. The predicted models showed the presence of conserved residues at, 122 (G), 205 (S) and 236 (S). In vitro studies also supported that PHA accumulation ability was less in Pseudomonas sp. LDC-25 compared to Pseudomonas sp. LDC-5. FT-IR spectrum showed PHA specific peaks at 1735.62 cm-1. Results of this study would help to detect the functional domains of the protein in order to elucidate their structure/function characteristics with special emphasis on invariant conserved residues.

Last Modified: 8/27/2014
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