NAD(P)⁺‐independent aldehyde dehydrogenase from Pseudomonas testosteroni

Abstract

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].

Formylmethanofuran dehydrogenases from methanogenic Archaea Substrate specificity, EPR properties and reversible inactivation by cyanide of the molybdenum or tungsten iron‐sulfur proteins

“…Rather, it suggests that the formyl group is dehydrogenated while still bound to the primary amino group of methanofuran yielding N-carboxymethanofuran as product (reaction b) which should break down non-enzymically to CO, and methanofuran (Ewing et al, 1980) : 0 R Reaction (b) indicates that formylmethanofuran dehydrogenase belongs to the group of molybdenum enzymes that catalyze an insertion of an oxygen atom derived from H,O into a C-H bond (Pilato and Stiefel, 1993). Enzymes belonging to this group are xanthine dehydrogenases and xanthine oxidases (Bray, 1988;Wootton et al, 1991), molybdenum-containing formate dehydrogenases (Adams and Mortenson, 1985;Barber et al, 1986;Friedebold and Bowien, 1993), formate-ester dehydrogenase (van Ophem et al, 1992), aldehyde oxidase (Branzoli and Massey, 1974), aldehyde dehydrogenase (Poels et al, 1987), aldehyde oxidoreductase (White et al, 1993), nicotine dehydrogenase (Freudenberg et al, 1988), nicotinate dehydrogenase and 6-hydroxynicotinate dehydrogenase (Nagel and Andreesen, 1990), isonicotinate dehydrogenase and 2-hydroxyisonicotinate dehydrogenase (Kretzer and Andreesen, 1991), quinoline oxidoreductase (Hettrich et al, 1991), quinoline-4-carboxylic acid oxidoreductase (Bauer and Lingens, 19921, quinaldine oxidoreductase (de Beyer and Lingens, 1993), quinaldic acid 4-oxidoreductase (Fetzner and Lingens, 1993), picolinate dehydrogenase (Siegmund et al, 1990), 2-furoyl-coenzyme A dehydrogenase , and pyrimidine oxidase and pyridoxal oxidase (Burgmayer and Stiefel, 1985). Interestingly, one of these enzymes, milk xanthine oxidase, can even catalyze the dehydrogenation of formamide to carbamic acid (Morpeth et al, 1984) which is a reaction also catalyzed by formylrnethanofuran dehydrogenase.…”

Xanthine dehydrogenase and 2-furoyl-coenzyme A dehydrogenase from Pseudomonas putida Fu1: two molybdenum-containing dehydrogenases of novel structural composition

“…Heterocyclic aromatic compounds are usually converted by molybdoenzymes such as xanthine dehydrogenase-oxidase (4,9,53) and the closely related aldehyde dehydrogenase-oxidase (39,40), which represent the prototypes of this class of enzymes (9, 25), for both enzymes convert a broad spectrum of heterocyclic compounds (25), in contrast to nicotine dehydrogenase (13), nicotinate dehydrogenase (11; Nagel, Ph.D. thesis, 1989), and quinoline dehydrogenase (M. Blaschke, Diplom thesis, University of Gottingen, Gottingen, Federal Republic of Germany, 1990). They all contain molybdenum, flavin, and nonheme iron-sulfur as redoxactive centers, and all are composed of three subunits after SDS-polyacrylamide gel electrophoresis or proteolytic treatment with molecular weights of about 90,000, 42,000, and 20,000, exhibiting mostly a subunit structure of L2M2S2 (Table 2).…”

Quinaldine 4‐Oxidase from Arthrobacter sp. Rü61a, a Versatile Procaryotic Molybdenum‐Containing Hydroxylase Active towards N‐Containing Heterocyclic Compounds and Aromatic Aldehydes

“…Quinaldine 4-oxidase and I1 OR generally are more active with aromatic than with aliphatic aldehydes. Based on the data published, this feature apparently does not apply for the procaryotic aldehyde dehydrogenases, since they all convert aliphatic aldehydes far better than quinaldine 4-oxidase and IlOR do Poels et al, 1987: van Ophem et al, 1992White et al, 1991;Strobe1 et al, 1992;Turner et al, 1987). However, eucaryotic aldehyde oxidase, the molybdenumcontaining enzyme well characterized with respect to Nheterocyclic and aldehyde substrate specificity, proved to be similar in so far that it converts N-containing heterocyclic compounds (e.g.…”

“…Xanthine was not a substrate. NAD(P)' or 0, were not used as electron acceptors (Poels et al, 1987). Actually, none of the bacterial aldehyde oxidoreductases described uses 0, as electron acceptor.…”