The biosynthesis and assembly of methanol dehydrogenase in bacterium W3A1.
Bacterium W3A1, a restricted facultative methylotroph, produces a periplasmic methanol dehydrogenase composed of two identical subunits of Mr = 57,300, and two noncovalently bound methoxatin prosthetic groups. A precursor form of Mr = 1,500 larger than the mature subunit was identified among the products of an in vitro translation of total RNA isolated from bacterium W3A1. The precursor form of the protein could not be detected in cells during in vivo pulse-labeling studies, suggesting that the processing of this precursor occurs entirely co-translationally. Whereas the holoenzyme was detectable only as a dimer, removal of the prosthetic group yielded an apoenzyme that could be detected as either a dimeric or monomeric species. After readdition of the purified prosthetic group to the apoenzyme, only the dimeric form of the protein, bearing the cofactor and exhibiting an absorption spectrum similar to that of the holoenzyme, was detected. Neither the mature apoprotein nor the holoenzyme demonstrated any affinity for phospholipid membranes, as assayed by their inability to bind to liposomes. Taken together, these data suggest a scheme of co-translational processing and export of the apoprotein subunits, followed by assembly of the subunits and prosthetic groups in the periplasmic space to form the mature holoenzyme. The suitability of bacterium W3A1, and other methylotrophic bacteria, for use in studies of protein biosynthesis and export, is also discussed.
D000429:Alcohol Oxidoreductases / Q000096:biosynthesis*; D001419:Bacteria / Q000201:enzymology*; D001965:Bromides / Q000378:metabolism; D002850:Chromatography, Gel; D003429:Cross Reactions; D004792:Enzyme Precursors / Q000032:analysis; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D017680:Potassium Compounds; D013053:Spectrophotometry
D001965:Bromides; D004792:Enzyme Precursors; D046911:Macromolecular Substances; D008563:Membrane Lipids; D010743:Phospholipids; D017680:Potassium Compounds; D008715:Methionine; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); C039004:potassium bromide; D011188:Potassium