Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM.
The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrading Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hydroxylase activity, whereas an inactive form accumulated in a recombinant strain. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by electrospray mass spectrometry, but nondenaturing gel filtration showed molecular masses of 31 600 Da for the inactive form and 11 500 Da for the active form. Cross-linking and sedimentation velocity results were consistent with the inactive form being a dimer. Partial thermal or chemical denaturation, or treatment with trifluoroethanol, readily activated dimeric DmpM. A combination of circular dichroism and fluorescence spectroscopies, activity assays, and native and urea gel electrophoresis were used to further characterize reactivation with urea. These results showed that dissociation of the dimeric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissociation also converts the monomeric form to a different conformation.
Cadieux
E
E
Department of Chemistry and Biochemistry, Concordia University, Montreal, Quebec, Canada.
Powlowski
J
J
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006358:Hot Temperature; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D017510:Protein Folding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014270:Trifluoroethanol / Q000494:pharmacology; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; C121254:DmpM protein, Pseudomonas; D020033:Protein Isoforms; D011994:Recombinant Proteins; D015534:Trans-Activators; D014270:Trifluoroethanol; D014508:Urea; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase; D005990:Glycerol