Purification and partial characterization of the hydroxylase component of the methanesulfonic acid mono-oxygenase from methylosulfonomonas methylovora strain M2.
The reductase enzyme and the hydroxylase enzyme of the three-component methanesulfonic acid mono-oxygenase (MSAMO) from Methylosulfonomonas methylovora were purified. Purification of the reductase from M. methylovora using a range of chromatographic techniques was accompanied by complete loss of activity. Expression of the reductase as a glutathionine S-transferase fusion protein in Escherichia coli cells was successful as judged from the size of the polypeptide band obtained on induction with isopropyl thio-beta-D-galactoside. Subsequent affinity purification of the fusion protein, however, led to a protein extract containing only glutathionine S-transferase protein, indicating that the fusion protein was unstable in vitro. The hydroxylase component of the MSAMO was purified from M. methylovora to near electrophoretic homogeneity using Q-Sepharose, hydroxyapatite and Mono Q chromatography. SDS/PAGE of the purified hydroxylase showed a single band at approximately 43.7 kDa for the alpha-subunit and a double band at approximately 23 kDa for the beta-subunit. MS scans obtained with matrix-assisted laser desorption/ionization and electrospray ionization showed single peaks for both subunits, with a mass of 48 145.4 Da for alpha, 20 479.1 Da for beta, and 68 624.5 for the alphabeta-monomer. Gel filtration revealed a mass of 209 kDa, suggesting an alpha3beta3 structure for the native enzyme. Purified hydroxylase enzyme exhibited absorbance maxima at 330 nm, 460 nm and 570 nm, indicating the presence of iron-sulfur centres. The protein preparations contained 1 mol sulfide and 3-4 mol iron per mol alphabeta-monomer. Chromium, cobalt, copper, lead, nickel, molybdenum, tungsten and vanadium were not found. Flavins were also absent. Antibodies raised against the native hydroxylase enzyme cross-reacted with cell-free extract from M. methylovora cells grown with methanesulfonate, but not with extract from cells grown with methanol, confirming that MSAMO was specifically induced during growth on methanesulfonate.
Reichenbecher
W
W
Department of Biological Sciences, University of Warwick, Coventry, UK.
Murrell
J C
JC
D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D008698:Mesylates / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D008698:Mesylates; D011993:Recombinant Fusion Proteins; C045880:methanesulfonic acid; D007544:Isopropyl Thiogalactoside; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C104883:methanesulfonic acid monooxygenase; D005982:Glutathione Transferase