E. Azoulay

Alkane oxidation in Candida tropicalis

Abstract Reexamination of NAD+ reduction by cell-free extracts of Candida tropicalis in the presence of n-decane provides conclusive evidence that this reaction is due to the contamination of some decane samples by an impurity, and does not correspond to a dehydrogenation of the alkane itself. The actual pathway of alkane oxidation by C. tropicalis is a microsomal and inducible hydroxylase system. This system involving cytochrome P450 and specific for NADPH is similar to the mammalian hydroxylase system. The mitochondrial fragments of C. tropicalis contain an ATP-dependent transhydrogenase, the presence of which explains the fact that crude particle preparations, but not purified microsomes, can substitute NADH or NAD+ for NADPH in alkane hydroxylation. The same transhydrogenase is also responsible for the previously reported observation that ATP increases NAD+ reduction by these enzymic preparations in the presence of impure decane. In the active microsomal particles, the hydroxylase system is associated with alcohol and aldehyde dehydrogenases, which are also present in mitochondria and which are partly solubilized in the course of some procedures of cell extraction. The induction of these dehydrogenases and of the hydroxylase system is studied.

Déshydrogénation d'hydrocarbures paraffiniques par les suspensions non-proliférantes et les extraits de Pseudomonas aeruginosa

Abstract 1. 1. Under anaerobic conditions and in the presence of paraffinic hydrocarbons (C 7 to C 10 ), resting-cells of Pseudomonas aeruginosa (Sol 20) grown on these substrates reduce pyocyanin, a natural pigment produced by the organism concerned. 2. 2. A n -heptane dehydrogenase has been extracted from the cells and its properties have been studies. The enyzme utilizes either diphosphopyridine nucleotide or pyocyanin as hydrogen acceptor, with similar affinities. Triphosphopyridine nucleotide is approximately eight times less efficient. 3. 3. The cell-free extracts contain a reduced diphosphopyridine nucleotide oxidase, which is specifically inhibited by mercapto-ethanol, and have dehydrogenase activities for 1- n -heptanol and heptylic aldehyde. 4. 4. The extracts consume O 2 in the presence of n -heptane and their respiratory activity is consistently increased by the addition of either diphosphopyridine nucleotide or pyocyanin.

Isolement et caracterisation des enzymes responsables de l'oxydation des hydrocarbures

Abstract 1. 1. The enzymic system responsible for the transformation of n -heptane to heptanol-1 was isolated by fractionated centrifugation from cell-free extracts from bacteria grown on n -heptane. 2. 2. This hydrocarbon oxidase system, specific for paraffinic hydrocarbons requires the presence of Fe 2+ and O 2 . The activity is consistently increased by the addition of NAD. 3. 3. The presence of the reversible hydrocarbon dehydrogenase and olefin reductase activities, specific for NAD in these same systems shows that the oxidation of hydrocarbon to primary alcohol is effected according to two different steps.

Extraction and properties of alcohol dehydrogenase from Pseudomonas aeruginosa.

Abstract 1. 1. An aldehyde dehydrogenase (aldehyde:NAD(P) oxidoreductase) from a strain of Pseudomonas aeruginosa (Sol 20) grown with paraffin hydrocarbons as sole carbon source was obtained and partially purified from cell-free extracts. 2. 2. The aldehyde dehydrogenase was specific for aliphatic aldehydes and required the presence of Fe2+ or Ca2+, and a flavin such as FAD or riboflavin. 3. 3. The action of compounds having −SH groups established that CoA and mercaptoethanol were activators of the enzyme. 4. 4. The aldehyde dehydrogenase was only found in preparations from bacteria grown on higher alcohols, aliphatic acids or hydrocarbons. This confirms that the aldehyde is an intermediate in the metabolic degradation of hydrocarbons by bacteria.

Étude des mutants chlorate-résistants chez Escherichia coli K12 I. Reconstitution in vitro de l'activité nitrate-réductase particulaire chez Eschrichia coli K12

Abstract Study of chlorate-resistant mutants in Escherichia coli K 12. I. Restoration in vitro of nitrate reductase activity in particular Using chlorate as a selective agent, we have isolated two genetically distinct types of chlorate-resistant mutants in Escherichia coli K 12: chl A 15 and chl B 24 . Both of these types are defective in several enzymatic activities specific to anaerobic respiration, and in particular, in nitrate reductase (NADH: nitrate oxidoreductase, EC 1.6.6.1) and hydrogen lyase (formate:cytochrome b 1 oxidorecductase. EC 1.2.2.1). By mixing the cell-free extracts of these two mutants under certain precise conditions, we achieve a restoration of the nitrate reductase activity lost by each of the two organisms. When this complementation is performed with high-speed supernatants of the extracts (170 000 × g for 90 min), particles having nitrate reductase activity are formed de novo . The behavior of these particles on sucrose gradients is the same as that of particles having nitrate reductase activity isolated from the wild strain. We describe the conditions for this type of complementation, the nature of the reaction, and the biochemical characteristics of the synthesized particles. The restoration of nitrate reductase activity by complementation occurs only in the absence of O 2 , in a narrow range of temperature and pH (32°, pH 7–7.6), and at certain protein concentrations. The kinetics of complementation were studied by following the increase of nitrate reductase activity. The reaction is complete after 2 h of incubation. The enzymatic activity thus reconstituted is equal to 19 units/mg of total protein and represents a tenth of the wild-type activity. In the course of complementation 15–20%, of the total soluble protein becomes particulate. This includes the cytochrome b 1 which was present in the soluble state in the mutant extracts. From these results, we present an hypothesis as to the nature and role of the chl B gene product.

Participation du cytochrome P 450 dans l'oxydation des alcanes chez Candida tropicalis

Candida tropicalis strain 101 possesses a hydroxylase system when grown on tetradecane as the carbon source which is active towards hydrocarbons and fatty acids. This system including cytochrome P 450 and NADPH‐cytochrome c reductase has been localized exclusively in the microsomal fraction.

Alcool- et aldéhyde-déshydrogénases particulaires de Candida tropicalis cultivé sur hydrocarbures

Cell-free extract of a strain of Candida tropicalis cultivated on hydrocarbon catalyse in the presence of NAD+ the oxidation of higher alcohols to the corresponding fatty acids; two enzymes, an alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) and an aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3), have been studied. Assay of these enzymes (340-nm spectrometry) fails with substrates barely soluble in water. The assay is therefore carried out with emulsions prepared with Tween-80 or with deoxycholate. Alternatively the substrates were solubilized with dimethyl formamide. Km values obtained with 1-decanol using the modifications described were 31 mM, 7 mM and 0.7 mM, respectively. Kinetics properties and particularly the variation of the Km and νmax as a function of chain length of the substrates are reported. Alkane and the corresponding alcohol and aldehyde are inducers of these enzymes.

Mise en évidence de deux systèmes de transport du phosphate chez Candida tropicalis

Summary Candida tropicalis has two phosphate transport systems, one of which is constitutive and has a low affinity for its substrate (Kmapp 1.2.10−3 M). The other one characterized by a high affinity for H2PO4− (Kmapp = 4.5.10−6 M) appears only under phosphate starvation conditions. The regulation of the latter would act on the one hand on the synthesis of binding proteins for Pi (repression-derepression) and on the other on the activation or inactivation of the carriers.

Isolation and study of the enzymes involved in the metabolism of hydrocarbons by Candida tropicalis

SummaryIn cell free extracts prepared from protoplasts of n-tetradecane grown cells of Candida tropicalis we have found an ATP and NAD+ dependent alkane-dehydrogenase, an alcohol-dehydrogenase, an aldehyde-dehydrogenase and acyl-CoA synthetases. The study of these enzymes and their regulation allows us to propose a scheme of degradation of n-decane.

Characterization of the membrane‐bound nitrate reductase activity of aerobically grown chlorate‐sensitive mutants of escherichia coli K12

Wild-type strains of Escherichia coli can grow in the presence of chlorate aerobically but under anaerobic conditions growth is inhibited. This observation has been explained on the assumption that under anaerobic conditions, chlorate, an analogue of nitrate, induces nitrate reductase (EC 1.7.99.4) and is converted by the enzyme to the toxic compound chlorite with the result that cell growth ceases: aerobic growth in the presence of chlorate is allowed since under these conditions nitrate reductase activity is repressed [ 11. Recently mutants have been isolated which show a chlorate-sensitive phenotype when grown under aerobic conditions in the presence of a fermentable carbon source [2] and a detailed biochemical characterization of one of these mutants, strain 72, has indicated that the primary genetic lesion occurs in the biosynthetic pathway for ubiquinone8 [3]. In addition, it was noticed that strain 72, when grown aerobically in the presence of nitrate, produced significantly higher activities of reduced benzylviologen-dependent nitrate reductase than an equivalent culture of the parent strain similarly grown, however this activity represented only 2-10% of that found in cultures of either strain 72 or the wild-type grown anaerobically in the presence ofnitrate [2,3]. The nitrate reductase activity found in aerobically-grown strain 72 was destroyed