ABSTRACT
Previous studies have suggested that an association exists between the proline dehydrogenase gene (PRODH) and increased schizophrenia risk. We examined the prevalence of the PRODH 757C/T (Arg185Trp), 1766A/G (Gly521Arg), and 1852G/A (intronic mutation) polymorphisms in 175 patients with schizophrenia and 185 control subjects. All subjects were of Iranian ancestry. The PRODH 757TT, 1852AA, and 1766GG genotypes were associated with an increased risk of schizophrenia (odds ratio = 1.38, 95% confidence interval: 0.88-2.16, P = 0.001, P = 0.001, respectively). The activity alleles in the PRODH genotype combinations were associated with an increased risk of schizophrenia (haplotype analysis, TAG genotype P = 0.007). Our findings support a major role for the PRODH 757TT, 1766GG, and 1852AA genotypes alone and in combination for schizophrenia susceptibility.
Subject(s)
Alleles , Polymorphism, Single Nucleotide , Proline Oxidase/genetics , Schizophrenia/genetics , Adolescent , Adult , Age Factors , Case-Control Studies , Educational Status , Female , Gene Expression , Gene Frequency , Genetic Predisposition to Disease , Haplotypes , Humans , Introns , Male , Middle Aged , Odds Ratio , Risk , Schizophrenia/physiopathologyABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Proline Oxidase/metabolism , Pseudomonas fluorescens/enzymology , Recombinant Fusion Proteins/metabolism , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Flavin-Adenine Dinucleotide/metabolism , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Sequence Data , Molecular Weight , Open Reading Frames/genetics , Plasmids , Proline Oxidase/genetics , Proline Oxidase/isolation & purification , Pseudomonas fluorescens/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Sequence Homology, Amino Acid , Structural Homology, Protein , TemperatureABSTRACT
BACKGROUND AND OBJECTIVES: Proline dehydrogenase (ProDH; 1.5.99.8) plays an important role in specific determination of plasma proline level in biosensor and diagnostic kits. The goal of this research was to isolate and characterize ProDH enzyme from Iranian soil microorganisms. MATERIALS AND METHODS: Screening of L-proline degradative enzymes from soil samples was carried out employing enrichment culture techniques. The isolate was characterized by biochemical reactions and specific PCR amplification. The target ProDH was purified and the effects of pH and temperature on the activity and stability were also tested. RESULTS: Among the 250 isolates recovered from 40 soil samples, only one strain characterized as Pseudomonas entomophila displayed the highest enzyme activity toward L-proline (350 U/l) than others. The enzyme of interest was identified as a ProDH and had K(m) value of 32 mM for L-proline. ProDH exhibited its best activity at temperature range of 25 to 35°C and its highest activity was achieved at 30°C. It was almost stable at temperatures between 25-30°C for 2 hours. The optimum pH activity of ProDH reaction was 8.5 and its activity was stable in pH range of 8.0-9.0 upto 24 hours. The enzyme was purified with a yield of 8.5% and a purification factor of 37.7. The molecular mass of the purified ProDH was about 40 kDa, and determined to be a monomeric protein. CONCLUSION: This is the first report concerning the ProDH production by a P. entomophila bacterium isolated from soil sample.
