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Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling

Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization... The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K m and V max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Applied Biochemistry and Microbiology Springer Journals

Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling

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References (1)

Publisher
Springer Journals
Copyright
Copyright © 2012 by Pleiades Publishing, Ltd.
Subject
Life Sciences; Microbiology; Medical Microbiology; Biochemistry, general
ISSN
0003-6838
eISSN
1608-3024
DOI
10.1134/S0003683812020081
Publisher site
See Article on Publisher Site

Abstract

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K m and V max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH.

Journal

Applied Biochemistry and Microbiology – Springer Journals

Published: Feb 29, 2012

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