Quinohaemoprotein alcohol dehydrogenase apoenzyme from Pseudomonas testosteroni.
Cell-free extracts of Pseudomonas testosteroni, grown on alcohols, contain quinoprotein alcohol dehydrogenase apoenzyme, as was demonstrated by the detection of dye-linked alcohol dehydrogenase activity after the addition of PQQ (pyrroloquinoline quinone). The apoenzyme was purified to homogeneity, and the holoenzyme was characterized. Primary alcohols (except methanol), secondary alcohols and aldehydes were substrates, and a broad range of dyes functioned as artificial electron acceptor. Optimal activity was observed at pH 7.7, and the presence of Ca2+ in the assay appeared to be essential for activity. The apoenzyme was found to be a monomer (Mr 67,000 +/- 5000), with an absorption spectrum similar to that of oxidized cytochrome c. After reconstitution to the holoenzyme by the addition of PQQ, addition of substrate changed the absorption spectrum to that of reduced cytochrome c, indicating that the haem c group participated in the enzymic mechanism. The enzyme contained one haem c group, and full reconstitution was achieved with 1 mol of PQQ/mol. In view of the aberrant properties, it is proposed to distinguish the enzyme from the common quinoprotein alcohol dehydrogenases by using the name 'quinohaemoprotein alcohol dehydrogenase'. Incorporation of PQQ into the growth medium resulted in a significant shortening of lag time and increase in growth rate. Therefore PQQ appears to be a vitamin for this organism during growth on alcohols, reconstituting the apoenzyme to a functional holoenzyme.
D000429:Alcohol Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D001051:Apoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D004790:Enzyme Induction; D007700:Kinetics; D011549:Pseudomonas / Q000201:enzymology*; D013053:Spectrophotometry; D013379:Substrate Specificity
D001051:Apoenzymes; D001059:Apoproteins; D000429:Alcohol Oxidoreductases; C019641:alcohol dehydrogenase (acceptor); D002118:Calcium