Alcohol dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecane and hexadecanol metabolism.

Multiple alcohol dehydrogenases (ADH) were demonstrated in Acinetobacter sp. strain HO1-N. ADH-A and ADH-B were distinguished on the basis of electrophoretic mobility, pyridine nucleotide cofactor requirement, and substrate specificity. ADH-A is a soluble, NAD-linked, inducible ethanol dehydrogenase (EDH) exhibiting an apparent Km for ethanol of 512 microM and a Vmax of 138 nmol/min. An ethanol-negative mutant (Eth1) was isolated which contained 6.5% of wild-type EDH activity and was deficient in ADH-A. Eth1 exhibited normal growth on hexadecane and hexadecanol. A second ethanol-negative mutant (Eth3) was acetaldehyde dehydrogenase (ALDH) deficient, having 12.5% of wild-type ALDH activity. Eth3 had threefold-higher EDH activity than the wild-type strain. ALDH is a soluble, NAD-linked, ethanol-inducible enzyme which exhibited an apparent Km for acetaldehyde of 50 microM and a Vmax of 183 nmol/min. Eth3 exhibited normal growth on hexadecane, hexadecanol, and fatty aldehyde. ADH-B is a soluble, constitutive, NADP-linked ADH which was active with medium-chain-length alcohols. Hexadecanol dehydrogenase (HDH), a soluble and membrane-bound, NAD-linked ADH, was induced 5- to 11-fold by growth on hexadecane or hexadecanol. HDH exhibited apparent Kms for hexadecanol of 1.6 and 2.8 microM in crude extracts derived from hexadecane- and hexadecanol-grown cells, respectively. HDH was distinct from ADH-A and ADH-B, since HDH and ADH-A were not coinduced; Eth1 had wild-type levels of HDH; and HDH requires NAD, while ADH-B requires NADP. NAD- and NADP-independent HDH activity was not detected in the soluble or membrane fraction of extracts derived from hexadecane- or hexadecanol-grown cells. NAD-linked HDH appears to possess a functional role in hexadecane and hexadecanol dissimilation.     

Inhibition of glucose metabolism by n-hexadecane in Cladosporium (Amorphotheca) resinae.

When Cladosporium resinae is provided with n-hexadecane and glucose, n-hexadecane is used preferentially. Studies using [14C]glucose indicated that n-hexadecane did not inhibit glucose uptake but did retard oxidation of glucose to CO2 and assimilation of glucose carbon into trichloroacetic acid-insoluble material. Glucose could be recovered quantitatively from hydrocarbon-grown cells that had been transferred to glucose. Four enzymes that may be involved in glucose metabolism, hexokinase, glucose-6-phosphate dehydrogenase, glucose-phosphate isomerase, and succinate dehydrogenase, were not detected in cells grown on hexadecane but were present in cells grown on glucose. Addition of hexadecane to extracts of glucose-grown cells resulted in immediate loss of activity for each of the four enzymes, but two other enzymes did not directly involved in glucose metabolism, adenosine triphosphatase and alanine-ketoacid aminotransferase, were not inhibited by hexadecane in vitro. Cells grown on hexadecane and transferred to glucose metabolize intracellular hexadecane; after 1 day, activity of hexokinase, glucose-6-phosphate dehydrogenase, glucosephosphate isomerase, and succinate dehydrogenase could be detected and 22% of the intracellular hydrocarbon had been metabolized. Hexadecane-grown cells transferred to glucose plus cycloheximide showed the same level of activity of all the four enzymes as cells transferred to glucose alone. Thus, intracellular n-hexadecane or a metabolite of hexadecane can inthesis of those enzymes is not inhibited.     

Fatty aldehyde dehydrogenases in Acinetobacter sp. strain HO1-N: role in hexadecanol metabolism

The role of fatty aldehyde dehydrogenases (FALDHs) in hexadecane and hexadecanol metabolism was studied in Acinetobacter sp. strain HO1-N. Two distinct FALDHs were demonstrated in Acinetobacter sp. strain HO1-N: a membrane-bound, NADP-dependent FALDH activity induced 5-, 15-, and 9-fold by growth on hexadecanol, dodecyl aldehyde, and hexadecane, respectively, and a constitutive, NAD-dependent, membrane-localized FALDH. The NADP-dependent FALDH exhibited apparent Km and Vmax values for decyl aldehyde of 5.0, 13.0, 18.0, and 18.3 microM and 537.0, 500.0, 25.0, and 38.0 nmol/min in hexadecane-, hexadecanol-, ethanol-, palmitate-grown cells, respectively. FALDH isozymes ald-a, ald-b, and ald-c were demonstrated by gel electrophoresis in extracts of hexadecane- and hexadecanol-grown cells. ald-a, ald-b, and ald-d were present in dodecyl aldehyde-grown cells, while palmitate-grown control cells contained ald-b and ald-d. Dodecyl aldehyde-negative mutants were isolated and grouped into two phenotypic classes based on growth: class 1 mutants were hexadecane and hexadecanol negative and class 2 mutants were hexadecane and hexadecanol positive. Specific activity of NADP-dependent FALDH in Ald21 (class 1 mutant) was 85% lower than that of wild-type FALDH, while the specific activity of Ald24 (class 2 mutant) was 55% greater than that of wild-type FALDH. Ald21R, a dodecyl aldehyde-positive revertant able to grow on hexadecane, hexadecanol, and dodecyl aldehyde, exhibited a 100% increase in the specific activity of the NADP-dependent FALDH. The oxidation of [3H]hexadecane byAld21 yielded the accumulation of 61% more fatty aldehyde than the wild type, while Ald24 accumulated 27% more fatty aldehyde, 95% more fatty alcohol, and 65% more wax ester than the wild type. This study provides genetic and physiological evidence for the role of fatty aldehyde as an essential metabolic intermediate and NADP-dependent FALDH as a key enzyme in the dissimilation of hexadecane, hexadecanol, and dodecyl aldehyde in Acinetobactor sp. strain HO1-N.     

Cytosolic alcohol dehydrogenases from yeast Torulopsis candida]

A comparative study of cell cytosol alcohol dehydrogenase (ADH) from yeast Torulopsis candida IBFM-Y-127 grown on glucose and hexadecane which were the only source of carbon, was made. In both cases ADH had a pH optimum within the range of 7.0--10.0, when various normal primary alcohols (C2--C16) were used. The enzyme was active only in the presence of NAD, which cannot be substituted by NADP. The total activity of ADH decreased approximately 8-fold when the length of hydrocarbon radicals was changed from C2 up to C16. When the cells were grown on hexadecane, only ethyl, n-buthyl, n-amyl and n-hexyl alcohols were active as substrates. The dehydration rate of each alcohol was far lower than that for the cytosol of glucose-grown cells. In the latter case the enzyme activity also decreased with an increase in the alcohol radical from C2 to C6. In all cases studied methyl alcohol and cyclic (cinnamyl alcohol--C8) alcohol were not dehydrated at all. Disc-electrophoresis in polyacrylamide gel, involving gel colouration for the assay of enzyme activity showed that glucose--grown cell cytosol contained three forms of ADH. One of those forms was highly active when short--chain normal primary alcohols were used; this form may be probably regarded as "classical" ADH (EC 1.1.1.1). The two other forms caused intensive dehydration of long-chain alcohols (the best substrates were C7--C10 alcohols for one form and C10--C14 for the others). The two forms of ADH are probably isoenzymes of octanol dehydrogenase (EC 1.1.1.73). Cytosol of cells grown on n-alcane, had a reduced number of ADH forms. The data obtained are discussed in terms of the regulatory role of carbon and energy source (glucose or hexadecane) in the redistribution of alcohol dehydrogenases between structural components of cells (mitochondria) and cytosol.     

Studies on methanol — oxidizing yeast

Oxidation of methanol, formaldehyde and formic acid was studied in cells and cell-free extract of the yeast Candida boidinii No. 11Bh. Methanol oxidase, an enzyme oxidizing methanol to formaldehyde, was formed inducibly after the addition of methanol to yeast cells. The oxidation of methanol by cell-free extract was dependent on the presence of oxygen and independent of any addition of nicotine-amide nucleotides. Temperature optimum for the oxidation of methanol to formaldehyde was 35 degrees C, pH optimum was 8.5. The Km for methanol was 0.8mM. The cell-free extract exhibited a broad substrate specificity towards primary alcohols (C1--C6). The activity of methanol oxidase was not inhibited by 1mM KCN, EDTA or monoiodoacetic acid. The strongest inhibitory action was exerted by p-chloromercuribenzoate. Both the cells and the cell-free extract contained catalase which participated in the oxidation of methanol to formaldehyde; the enzyme was constitutively formed by the yeast. The pH optimum for the degradation of H2O2 was in the same range as the optimum for methanol oxidation, viz. at 8.5. Catalase was more resistant to high pH than methanol oxidase. The cell-free extract contained also GSH-dependent NAD-formaldehyde dehydrogenase with Km = 0.29mM and NAD-formate dehydrogenase with Km = 55mM.     

Oxidation of fatty alcohols to acids in the caecum of a gourami (Trichogaster cosby).

Oxidation of fatty alcohols to acids in gourami caeca was investigated by measuring the reduction of NAD+ and the formation of labeled hexadecanoic acid from [1(-14)C]hexadecanol. Virtually all dehydrogenase activity is in the microsomal fraction. Maximal activity is obtained with NAD+ as cofactor whereas with NADP+ 60% of that activity is obtained. The enzyme is rather specific for long chain alcohols and 2 NADH are formed for each molecule of hexadecanol oxidized to acid. It is stabilized by mercaptoethanol, and completely inhibited by p-chloromercuribenzoate. The activity is optimal at pH 9.5. At higher pH, small amounts of aldehyde are found. The first reaction in the sequence, fatty alcohol leads to aldehyde leads to acid seems to occur under the more physiological condition at a much slower rate than the second reaction so that free aldehyde is not detected. Addition of palmitic acid indicated an uncompetitive product inhibition. The oxidation of alcohol to acid is reversible only to a very minor extent even in the presence of NADPH, CoA, ATP and Mg2+. Location, activity and properties of the enzyme are in agreement with the earlier observation from dietary experiments that in the gourami fatty alcohols of wax esters are oxidized to acids in the course of absorption.     

Membrane-bound respiratory of Spirillum itersonii.

The membrane-bound respiratory system of the gram-negative bacterium Spirillum itersonii was investigated. It contains cytochromes b (558), c (550), and o (558) and beta-dihydro-nicotinamide adenine dinucleotide (NADH) and succinate oxidase activities under all growth conditions. It is also capable of producing D-lactate and alpha-glycerophosphate dehydrogenases when grown with lactate or glycerol as sole carbon source. Membrane-bound malate dehydrogenase was not detectable under any conditions, although there is high activity of soluble nicotinamide adenine dinucleotide: malate dehydrogenase. When grown with oxygen as the sole terminal electron acceptor, approximately 60% of the total b-type cytochrome is present as cytochrome o, whereas only 40% is present as cytochrome o in cells grown with nitrate in the presence of oxygen. Both NADH and succinate oxidase are inhibited by azide, cyanide, antimycin A, and 2-n-heptyl-4-hydroxyquinoline-N-oxidase at low concentrations. The ability of these inhibitors to completely inhibit oxidase activity at low concentrations and their effects upon the aerobic steady-state reduction levels of b- and c-type cytochromes as well as the aerobic steady-state reduction levels obtained with NADH, succinate, and ascorbate-dichlorophenolindophenol suggest that presence of an unbranched respiratory chain in S. itersonii with the order ubiquinone leads to b leads to c leads to c leads to oxygen.     

A Medium for the Isolation of Capsular Bacteria from Kefir Grains

Secondary alcohols (C₃ to C₁₀) were oxidized to the corresponding methylketones by resting mycelia of Scedosporium sp. A-4 grown on propane, but 3-pentanol and 3-hexanol were not oxidized. The oxidation of 2-propanol to acetone was inhibited by pyrazole, potassium cyanide, sodium azide and Hg^{2 +}. Alcohol dehydrogenase activity was found in the cell-free soluble fraction and this activity requires a cofactor, specifically NAD⁺. The oxidation of both 1-propanol and 2-propanol may be catalyzed by the same alcohol dehydrogenase.     

New mutants resistant to glucose repression affected in the regulation of the NADH reoxidation

Spontaneous mutants resistant to vanadate, arsenate or thiophosphate were isolated from a haploid strain of Saccharomyces cerevisiae. These three anions have an inhibitory effect on some mitochondrial functions and at the level of glyceraldehyde 3-phosphate dehydrogenase, a glycolysis enzyme. All the selected mutants had the same phenotype: they were deficient in alcohol dehydrogenase I, the terminal enzyme of the glycolysis, and possessed a high content of cytochrome c oxidase, the terminal enzyme of the respiratory chain. Moreover, cytochrome c oxidase biosynthesis had become insensitive to the catabolite repression, while the biosynthesis of the other enzymes sensitive to this phenomenon were always inhibited by glucose. Metabolic effects of this pleiotropic mutation manifested themselves in the following ways. 1. Growth rate and final cell mass were enhanced, compared to the wild type, when cells were grown on glucose or on glycerol, but not on lactate or ethanol. 2. Growth under anaerobiosis was nil and mutants did not ferment. 3. Mitochondrial respiration of the mutant strains was identical to the wild type with succinate or 2-oxo-glutarate as substrate, and weak with ethanol. But with added NADH, respiration rate of the mutants was higher than that of the wild type and partially insensitive to antimycin, even when cells were grown in repression conditions. It is postulated that in mutants strains, NADH produced at the level of glyceraldehyde 3-phosphate dehydrogenase, failing to be reoxidized via alcohol dehydrogenase, could be reoxidized with a high turnover owing to the enhancement of the amount of cytochrome c oxidase. Since NADH reoxidation is partially insensitive to antimycin, a secondary pathway going from external NADH dehydrogenase to cytochrome c oxidase is suggested.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

Inducible long chain alcohol oxidase from alkane-grown Candida tropicalis

Summary The oxidation of primary aliphatic alcohols by microsomal membrane fractions of alkane grown Candida tropicalis was shown to be due to the action of an inducible alcohol oxidase with a wide substrate specificity towards aliphatic alcohols. Stoichiometric studies showed that NADH production, in the presence of fatty alcohols, was due to the activity of an inducible fatty aldehyde dehydrogenase. The oxidase activity could be measured directly by hydrogen peroxide production via a peroxidase and a chromogenic redox indicator.     

Peroxisomal Localization of Enzymes Related to Fatty Acid β-Oxidation in an n-Alkane-grown Yeast,Candida tropicalis

In Candida tropicalis cells grown on n-alkanes (C₁₀-C₁₃), the levels of the activities of the enzymes related to fatty acid β—oxidation—acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase—were found to be higher than those in cells grown on glucose, indicating that these enzymes were induced by alkanes. The enzymes were first confirmed to be localized only in peroxisomes, while none of these enzymes nor acyl-CoA dehydrogenase, which is known to participate in the initial step of mitochondrial β-oxidation in mammalian cells, were detected in yeast mitochondria under the conditions employed.

The significance of the peroxisomal β-oxidation system in the metabolism of alkanes by the yeast was also discussed.     

Mitochondrial regulation of caspase activation by cytochrome oxidase and tetramethylphenylenediamine via cytosolic cytochrome c redox state

Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential.     

A physiological study of formate dehydrogenase,formate oxidase and hydrogenlyase fromEscherichia coli K-12

Escherichia coli was grown under various culture conditions. Variations in the levels of formate dehydrogenase which reacts with methylene blue (MB) or phenazine methosulfate (PMS) (N enzyme), formate dehydrogenase which reacts with benzyl viologen (BV) (H enzyme), formate oxidase and hydrogenlyase were analyzed. It was observed that formate dehydrogenase N and formate oxidase were induced by nitrate and repressed by oxygen. Synthesis of formate dehydrogenase H and hydrogenlyase was induced by formate and repressed by nitrate and oxygen. Selenite was required for the biosynthesis of formate dehydrogenase H and hydrogenlyase. Activity of both formate oxidase and hydrogenlyase was inhibited by azide and KCN but not by N-heptyl hydroxyquinoline-N-oxide (HOQNO); on the other hand, formate oxidase was extremely sensitive to HOQNO. Data were obtained which suggest that cytochromes are not involved in hydrogen formation from formate. Part of this work was carried out when the senior author was visiting Research Biologist in the Laboratory of Dr. J. A. de Mosss at the University of California, San Diego. Thanks are given to Dr. De Moss for his hospitality and advise and to Dr. Warren Butler of the University of California, San Diego for making available his spectrophotometer to carry out cytochrome analyses. Most of this work was sustained by a grant from the Research Corporation, Brown Hazen Fund and the financial help of the C.O.F.A.A. from the Instituto Politécnico Nacional.     

Metabolism of toluene and xylenes by Pseudomonas (putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.

Pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, TOL. These compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the TOL plasmid. The specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three respective substrates are independent of the carbon source used for growth, suggesting that a single set of nonspecific enzymes is responsible for the dissimilation of the breakdown products of toluene and m- and p-xylene. Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are coincidently and possible coordinately induced by toluene and the xylenes, and by the corresponding alcohols and aldehydes. They are not induced in cells grown on m-toluate but catechol 2,3-oxygenase can be induced by m-xylene.     

Alcohol oxidase of Aspergillus flavipes grown on hexadecanol

Abstract: The activity of alcohol oxidase in Aspergillus flavipes was induced by growth on hexadecanol, though highest activities were obtained using a mixture of hexadecanol and olive oil. The enzyme showed a wide range of substrate specificity towards aliphatic primary alcohols from C₈ to C₁₈. The preferred substrate was decanol. The enzyme had an optimum pH of 9.5. It also used cis -unsaturated alcohols better than the trans -isomers. ω-Hydroxy fatty acids and α,ω-diols were not attacked.     

Sulfite oxidation by iron-grown cells of Thiobacillus ferrooxidans at pH 3 possibly involves free radicals, iron, and cytochrome oxidase

Thiobacillus ferrooxidans cells grown on ferrous iron oxidized sulfite to sulfate at pH 3, possibly by a free radical mechanism involving iron and cytochrome oxidase. A purely chemical system with low concentrations of Fe3+ simulated the T. ferrooxidans system. Metal chelators, ethylenediamine tetraacetic acid (EDTA), 4,5-dihydroxy-1-3-benzene disulfonic acid (Tiron), o-phenanthroline, and 2,2'-dipyridyl, inhibited both sulfite oxidation systems, but the T. ferrooxidans system was inhibited only after the initial brief oxygen consumption. EDTA and Tiron, strong chelators of Fe3+, inhibited the oxidation at lower concentrations than o-phenanthroline and 2,2'-dipyridyl, strong chelators of Fe2+. Inhibition of Fe3+-catalyzed sulfite oxidation by EDTA and Tiron was instant, but the inhibition by o-phenanthroline and dipyridyl was briefly delayed, presumably for the reduction of Fe3+ to Fe2+. Mannitol, a free radical scavenger, inhibited both systems to the same extent. Cyanide and azide inhibited only the T. ferrooxidans system, suggesting a role of cytochrome oxidase. It is proposed that sulfite is oxidized by a free radical mechanism initiated by Fe3+ on the cell surface of T. ferrooxidans. Cytochrome oxidase is possibly involved in the regeneration of Fe3+ from Fe2+ by the normal Fe2+-oxidizing system of T. ferrooxidans.     

Derepression of FAD Pyrophosphorylase and Flavin Changes during Growth of Kloeckera sp. No. 2201 on Methanol

A methanol-utilizing yeast Kloeckera sp. No. 2201, when grown with methanol as a sole carbon and energy source, accumulated about three times much flavin as those grown with glucose, ethanol, or glycerol. A high proportion of the total flavin was FAD in methanol-grown cells. A remarkable derepression of FAD pyrophosphorylase accompanied by an inducible formation of an FAD-dependent alcohol oxidase which catalyzes oxidation of methanol, the first step in the oxidation sequence, was observed during growth of the yeast on methanol. Significant elevations of riboflavin synthetase and flavokinase were also found. Formate, as well as methanol, effectively induced both FAD pyrophosphorylase and methanol-oxidizing enzymes (alcohol oxidase, formaldehyde dehydrogenase, formate dehydrogenase, and catalase). Observations with other methanol-utilizing yeasts also gave essentially same results. These results led to the conclusion that cellular flavin level might be under control with level of flavoprotein physiologically required.