Abstract

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescence 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. fluorescence ProDH were estimated to be 35 mM and 116 micromol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30 degrees 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. fluorescence ProDH.

Authors

Mohammadi HS, Omidinia E

Institution

Biochemistry Dept., Pasteur Institute of Iran, Tehran, Iran 13164. skandar@pasteur.ac.ir

Source

Prikladnaia biokhimiia i mikrobiologiia 48:2 pg 191-8

MeSH

Amino Acid Sequence
Bacterial Proteins
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Flavin-Adenine Dinucleotide
Hydrogen-Ion Concentration
Kinetics
Models, Molecular
Molecular Sequence Data
Molecular Weight
Open Reading Frames
Plasmids
Proline Oxidase
Pseudomonas fluorescens
Recombinant Fusion Proteins
Sequence Homology, Amino Acid
Structural Homology, Protein
Temperature

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

22586912