title: Expression, Purification and Characterization of the Proline Dehydrogenase Domain of PutA from Pseudomonas putida POS-F84.
abstract: The objective of the present work was to express a truncated form of Pseudomonas putida PutA that shows proline dehydrogenase (ProDH) activity. The putA gene encoding ProDH enzyme was cloned into pET23a vector and expressed in Escherichia coli strain BL-21 (DE3) plysS. The recombinant P. putida enzyme was biochemically characterized and its three dimensional structure was also predicted. ProDH encoding sequence showed an open reading frame of 1,035-bp encoding a 345 amino acid residues polypeptide chain. Purified His-tagged enzyme gave a single band with a molecular mass of 40 kDa on SDS-PAGE. The molecular mass of the isolated enzyme was found to be about 40 kDa by gel filtration. This suggested that the enzyme of interest consists of one subunit. The K m and V max values of recombinant P. putida ProDH were estimated to be 31 mM and 132 μmol/min, respectively. The optimum pH and temperature for the catalytic activity of the enzyme was about pH 8.5 and 30 °C. The modeling analysis of the three dimensional structure elucidated that Ser-165, Lys-195 and Ala-252 were key residues for the ProDH activity. This study provides data on the cloning, sequencing and recombinant expression of PutA ProDH domain from P. putida POS-F84.
authors.affiliation: Department of Biochemistry, Pasteur Institute of Iran, Pasteur Street, Tehran, Iran.
authors.forename: Eskandar
authors.initials: E
authors.lastname: Omidinia
authors.affiliation: Department of Marine Biology, Bandar Abbas Branch, Islamic Azad University, Bandar Abbas, Islamic Republic of Iran.
authors.forename: Rahman
authors.initials: R
authors.lastname: Mahdizadehdehosta
authors.affiliation: Department of Biochemistry, Pasteur Institute of Iran, Pasteur Street, Tehran, Iran.
authors.forename: Hamid Shahbaz
authors.initials: HS
authors.lastname: Mohammadi
keywords: Characterization; Expression; Proline dehydrogenase (ProDH); Pseudomonas putida