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.

Aldehyde dehydrogenase from Pseudomonas testosteroni was purified to homogeneity. The enzyme has a pH optimum of 8.2, uses a wide range of aldehydes as substrates and cationic dyes (Wurster's blue, phenazine methosulphate and thionine), but not anionic dyes (ferricyanide and 2.6-dichloroindophenol), NAD(P)+ or 02, as electron acceptors. Haem c and pyrroloquinoline quinone appeared to be absent but the common cofactors of molybdenum hydroxylases were present. Xanthine was not a substrate and allopurinol was not an inhibitor. Alcohols were inhibitors only when turnover of the enzyme occurred in aldehyde conversion. The enzyme has a relative molecular mass of 186000, consists of two subunits of equal size ( M , 92000), and 1 enzyme molecule contains 1 FAD, 1 molybdopterin cofactor, 4 Fe and 4 S. It is a novel type of NAD(P)+-independent aldehyde dehydrogenase since its catalytic and physicochemical properties are quite different from those reported for already known aldehyde-converting enzymes like haemoprotein aldehyde dehydrogenase (EC 1.2.99.3), quinoprotein alcohol dehydrogenases (EC 1.1.99.8) and molybdenum hydroxylases.Aldehydes are toxic compounds, omnipresent in the natural environment mainly as a result of combustion processes. They are also intermediates in the biological degradation of alkanes, alcohols, and several xenobiotics. Both facts probably explain the general occurrence and the variety of microbial aldehyde-oxidizing activities. One of these is dyelinked aldehyde dehydrogenase activity, frequently encountered in cell extracts of alkane-grown or alcohol-grown bacteria. Since purification of this activity from methanotrophic bacteria resulted in a haem-c-containing protein [I], dyelinked aldehyde dehydrogenase has been classified as a haemoprotein. It should be realized, however, that it is far from clear whether haem c is the only prosthetic group and whether all dye-linked aldehyde dehydrogenases are haemoproteins. Thus, for the enzyme from acetic acid bacteria it has been stated that it is quite probably a quinoprotein containing pyrroloquinoline quinone (PQQ) [2]. Although no unequivocal evidence for the presence of PQQ was given and the quantity of it in the enzyme has not been reported, the cofactor seems at least suited to catalyze such a reaction since several quinoprotein alcohol dehydrogenases are also able to oxidize aldehydes very efficiently [3].

Cell-free extracts of Pseudomonas aeruginosa strains, grown on ethanol, showed dye-linked alcohol dehydrogenase activities. The enzyme responsible for this activity was purified to homogeneity. It appeared to contain two molecules of pyrroloquinoline quinone per enzyme molecule. In many respects, it resembled other quinoprotein alcohol dehydrogenases (EC 1.1.99.8), having a substrate specificity intermediate between that of methanol dehydrogenases and ethanol dehydrogenases in this group. On the other hand, it also showed dissimilarities: the enzyme was found to be a monomer (Mr 101 000), to need only one molecule of the suicide substrate cyclopropanol to become fully inactivated, and to have a different aromatic amino acid composition.

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Hexose oxidase from the red seaweed, Chondrus crispus was purified to homogeneity. The enzyme appeared to be encapsulated in particles obtained after mechanical disintegration of the fronds. Liberation of the enzyme in soluble form required either waiting for the spontaneous development of a suitable microbial flora in the suspension, or treatment with a mixture of proteases (pronase). As deduced from (SDS/)PAGE, the enzyme has a molecular mass of 87 kDa and probably consists of subunits of 36 kDa and 25 kDa. The low isoelectric point of 2.8 and the presence of 25% (by mass) sugars indicate that the enzyme is a strongly acidic glycoprotein. The absorption spectrum of isolated enzyme minus that of the substrate-reduced enzyme, and the EPR spectrum of the free radical observed in the reduced enzyme revealed the presence of a flavin. This cofactor is probably covalently bound since flavins were not released upon denaturation of the enzyme by heat or acid treatment. Taking free FAD as a reference compound, the enzyme contains 1 mol flavin/mol enzyme. EPR spectroscopy of the purified preparation showed the presence of Cu'+. However, since the amount was substoichiometric, substrate addition did not affect the signal, and the addition of chelator or Cu'+ did not affect the activity, the presence of this metal ion seems adventitious. It is concluded that the large discrepancies between the presently and the previously reported Keywords: hexose oxidase ; Chondrus crispus; red seaweed ; covalently bound flavin.Similarly to glucose oxidase, hexose oxidase (D-hexose : 0, oxidoreductase) oxidizes glucose at its C1 position to gluconolactone (which, depending on pH, may hydrolyze to gluconic acid) with formation of hydrogen peroxide. However, as indicated by the names, hexose oxidase has a much broader substrate specificity, oxidizing a wide range of aldose sugars. Moreover, substantial differences exist in enzyme characteristics. Glucose oxidase is a fungal enzyme that contains FAD. Hexose oxidase has been found in the red seaweeds Chondrus crispus [l], Iridophycusflaccidum [2] and Euthora cristata [l], and such an enzyme may occur in the fruit-juice sacs of young oranges [3j. The enzyme has been purified from C. crispus and the following characteristics were reported by the authors [l]. The molecular mass of the enzyme was 130 kDa, no flavins or other cofactors were present, except Cu2 I , which was present in the exorbitantly high ratio of 12 mol/mol enzyme, the enzyme was a glycoprotein (70% carbohydrate), and was inhibited by the chelators diethyldithiocarbamate and, to a lesser extent, KCN, azide and hydroxylamine. Since the unusual cofactor composition and the potential of hexose oxidase for biotechnological applications aroused our interest, we attempted to characterize the enzyme further. MATERIALS AND METHODSMaterials. C. crispus was collected at the intertidal zone of the Oosterschelde estuary. The collected weed was washed with tap water, dried in air at 40"C, grounded in a blendor (Culatti Mikro-Schlagmuhle) to ...