title: Proline dehydrogenase from Pseudomonas fluorescence: gene cloning, purification, characterization and homology modeling.
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.affiliation: Biochemistry Dept., Pasteur Institute of Iran, Tehran, Iran 13164. skandar@pasteur.ac.ir
authors.forename: H Shahbaz
authors.initials: HS
authors.lastname: Mohammadi
authors.forename: E
authors.initials: E
authors.lastname: Omidinia
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010957:Plasmids; D011394:Proline Oxidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D040681:Structural Homology, Protein; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D011394:Proline Oxidase