Characterization of active and inactive forms of the phenol hydroxylase stimulatory protein DmpM

The stimulatory protein DmpM of phenol hydroxylase from methylphenol-degrad ing Pseudomonas sp. strain CF600 has been found to exist in two forms. DmpM purified from the native strain was mostly active in stimulating phenol hy droxylase activity, whereas an inactive form accumulated in a recombinant s train. Both forms exhibited a molecular mass of 10 361.3 +/- 1.3 Da by elec trospray mass spectrometry, but nondenaturing gel filtration showed molecul ar masses of 31 600 Da for the inactive form and 11 500 Da for the active f orm. 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 combi nation of circular dichroism and fluorescence spectroscopies, activity assa ys, and native and urea gel electrophoresis were used to further characteri ze reactivation with urea. These results showed that dissociation of the di meric form of DmpM precedes denaturation at low protein concentrations and results in activation. The same concentration of urea that effects dissocia tion also converts the monomeric form to a different conformation.