Formaldehyde Dehydrogenase Activity Research Articles

Oxidative and assimilative enzyme activities in continuous cultures of the obligate methylotrophMethylobacillus flagellatum

In methanol-limited continuous cultures of the obligate methylotrophic bacterium Methylobacillus flagellatum grown at rates from 0.05 to 0.63 h-1, and also in an oxyturbidostat culture of M. flagellatum growing at the rate of 0.73 h-1, levels of methanol dehydrogenase, enzymes of formaldehyde oxidation (both linear and cyclic) and assimilation (RuMP cycle), a number of intermediary metabolism and TCA cycle enzymes and also 'dye-linked' formaldehyde dehydrogenase were determined. It was shown that the activities of dissimilatory enzymes, with the exception of 'dye-linked' formaldehyde dehydrogenase, decreased with increasing growth rate. Activities of assimilative enzymes and activities of the TCA cycle enzymes detected as well as the 'dye-linked' formaldehyde dehydrogenase activity, increased with increasing growth rate. A periplasmic location was shown for the latter enzyme and a role in formaldehyde detoxification was proposed.

Demonstration of formaldehyde dehydrogenase activity in formaldehyde-resistant Enterobacteriaceae

Clinical isolates of different Enterobacteriaceae strains and genetically modified variants which were resistant to the disinfectant formaldehyde were investigated. In cell-free extracts of all formaldehyde-resistant strains a glutathione-dependent formaldehyde dehydrogenase activity was demonstrated. In contrast cell extracts from formaldehyde sensitive strains did not show any formaldehyde dehydrogenase activity. The enzymatic degradation of formaldehyde seems to play an important role in formaldehyde resistance.

Demonstration of formaldehyde dehydrogenase activity in formaldehyde-resistant Enterobacteriaceae

Clinical isolates of different Enterobacteriaceae strains and genetically modified variants which were resistant to the disinfectant formaldehyde were investigated. In cell-free extracts of all formaldehyde-resistant strains a glutathione-dependent formaldehyde dehydrogenase activity was demonstrated. In contrast cell extracts from formaldehyde sensitive strains did not show any formaldehyde dehydrogenase activity. The enzymatic degradation of formaldehyde seems to play an important role in formaldehyde resistance.

Monomorphism of Formaldehyde Dehydrogenase in Different Populations

Blood samples from Koreans, Chinese, Hungarians and Germans were analyzed by isoelectric focusing on polyacrylamide gels and stained for formaldehyde dehydrogenase (FDH) activity. Three activity bands (one major and two minor) were observed in all blood samples studied. No distinct intra- and interpopulation differences were observed in the intensity of the three bands. Human autopsy liver samples also showed a similar three-activity band profile. An additional cathodic band was detected in a single case of autopsy liver extract from a Chinese subject. An apparent identity of FDH with the class III alcohol dehydrogenase was confirmed.

Formaldehyde as a carbon and energy source in yeast growth

Summary Formaldehyde can simultaneously be utilized by yeasts during growth on another heterotrophic substrate (e.g. glucose, xylose, methanol). From transient state growth experiments it became evident with Hansenula polymorpha that up to a molar mixing ratio of formaldehyde to glucose of 3 the former seems to play the role of an energy donor for glucose carbon incorporation. This also holds for the non-methylotrophic yeast Candida utilis. At higher mixing ratios formaldehyde is used as a carbon and energy source besides glucose in the case of H. polymorpha. Formaldehyde utilization in the mixture is limited by the capacity of formate dehydrogenase. In contract, with C. utilis formaldehyde dehydrogenase activity limits consumption of this substrate in the mixture. The results with this yeast suggest that formaldehyde is not used significantly as a carbon source.

Histochemical localization of formaldehyde dehydrogenase in the rat

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or tenmicrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD +, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowman's and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose ( M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560–567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.

Energetics of mixotrophic and autotrophic C1-metabolism by Thiobacillus acidophilus

Although the facultatively autotrophic acidophile Thiobacillus acidophilus is unable to grow on formate and formaldehyde in batch cultures, cells from glucose-limited chemostat cultures exhibited substrate-dependent oxygen uptake with these C1-compounds. Oxidation of formate and formaldehyde was uncoupler-sensitive, suggesting that active transport was involved in the metabolism of these compounds. Formate- and formaldehyde-dependent oxygen uptake was strongly inhibited at substrate concentrations above 150 and 400 μM, respectively. However, autotrophic formate-limited chemostat cultures were obtained by carefully increasing the formate to glucose ratio in the reservoir medium of mixotrophic chemostat cultures. The molar growth yield on formate (Y=2.5 g ·mol-1 at a dilution rate of 0.05 h-1) and RuBPCase activities in cell-free extracts suggested that T. acidophilus employs the Calvin cycle for carbon assimilation during growth on formate. T. acidophilus was unable to utilize the C1-compounds methanol and methylamine. Formate-dependent oxygen uptake was expressed constitutively under a variety of growth conditions. Cell-free extracts contained both dye-linked and NAD-dependent formate dehydrogenase activities. NAD-dependent oxidation of formaldehyde required reduced glutathione. In addition, cell-free extracts contained a dye-linked formaldehyde dehydrogenase activity. Mixotrophic growth yields were higher than the sum of the heterotrophic and autotrophic yields. A quantitative analysis of the mixotrophic growth studies revealed that formaldehyde was a more effective energy source than formate.

Biochemical characterization of mutants of the facultatively ribulose monophosphate type methylotroph Acetobacter methanolicus MB 58 defective in C1 metabolism

Summary 18 methanol negative mutants of Acetobacter methanolicus MB 58 induced by MNNG or UV light were tested for enzymes of the linear and the cyclic dissimilatory sequence of formaldehyde as well as of phosphofructokinase. Most of the mutants are pleiotrophic ones. The highest proportion of defects was found in C1 regulated methanol dehydrogenase and in constitutive formaldehyde dehydrogenase activity. The cytochrome c deficiency was always combined with that of the methanol dehydrogenase. Formate dehydrogenase was only absent in one pleiotrophic mutant. In none of the heterotrophically grown mutants any enzyme of the dissimilatory RuMP cycle as well as phosphofructokinase were defective. Hexulose-6-phosphate isomerase could not be further induced by methanol in 3 methanol oxidation positive mutants indicating the existance of isoenzymes. No enzyme lesion was detected in 2 mutants which should be defective in other assimilatory enzymes.

High-performance liquid chromatographic determination of formate as benzimidazole in biological samples

Formate was determined as benzimidazole by high-performance liquid chromatography after reaction with o-phenylenediamine at 130°C for 2 h in 1 M perchloric acid. The useful concentration range was 1.6–40 μmol/l and the determination limit was 20 pmol. The recoveries from rat liver homogenate and human urine were 90.3±2.9 and 89.4±2.5%, respectively. Using this method, the activity of formaldehyde dehydrogenase in biological samples could be measured, and also the formate concentration in the liver and urine of rats to which methanol had been administered.

Isolation, complementation and partial characterization of mutants of the methanol autotroph Xanthobacter H4-14 defective in methanol dissimilation.

Seven mutants of Xanthobacter H4-14, unable to grow on methanol but capable of growth on formate, were isolated and complemented with a chromosomal clone bank constructed in the broad-host-range cosmid pVK100. One mutant could not be complemented but the others fell into four distinct complementation groups that involved three different recombinant clones. All of the complementing regions were separated by at least 10 kbp. The five complementation classes had different phenotypic characteristics and were defective in different aspects of methanol and formaldehyde oxidation. Class I mutants were defective in methanol oxidation, class II mutants were impaired in formaldehyde oxidation, class III mutants appeared to be defective in a regulatory element involving the methanol oxidation system, and class IV mutants appeared to be defective in a regulatory element involving formaldehyde oxidation. Class V mutants exhibited a methanol-sensitive phenotype, which was correlated with an imbalance between methanol and formaldehyde dehydrogenase activities. Analysis of this class suggested it was defective in a repressor that regulated methanol dissimilation functions.

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.

Inhibition of CO 2 production from aminopyrine or methanol by cyanamide or crotonaldehyde and the role of mitochondrial aldehyde dehydrogenase in formaldehyde oxidation

Previous results have shown that cyanamide or crotonaldehyde are effective inhibitors of the oxidation of formaldehyde by the low- K m mitochondrial aldehyde dehydrogenase, but do not affect the activity of the glutathione-dependent formaldehyde dehydrogenase. These compounds were used to evaluate the enzyme pathways responsible for the oxidation of formaldehyde generated during the metabolism of aminopyrine or methanol by isolated hepatocytes. Both cyanamide and crotonaldehyde inhibited the production of 14CO 2 from 14C-labeled aminopyrine by 30–40%. These agents caused an accumulation of formaldehyde which was identical to the loss in CO 2 production, indicating that the inhbition of CO 2 production reflected an inhibition of formaldehyde oxidation. The oxidation of methanol was stimulated by the addition of glyoxylic acid, which increases the rate of H 2O 2 generation. Crotonaldehyde inhibited CO 2 production from methanol, but caused a corresponding increase in formaldehyde accumulation. The partial sensitivity of CO 2 production to inhibition by cyanamide or crotonaldehyde suggests that both the mitochondrial aldehyde dehydrogenase and formaldehyde dehydrogenase contribute towards the metabolism of formaldehyde which is generated from mixed-function oxidase activity or from methanol, just as both enzyme systems contribute towards the metabolism of exogenously added formaldehyde.

Induction and selection of formaldehyde-based resistance inPseudomonas aeruginosa

A formaldehyde resistant (R) phenotype ofPseudomonas aeruginosa was isolated from a formaldehydesensitive (S) parent by sequential treatment with 1,3,5-tris-(ethyl)hexahydro-s-triazine (ET). The resistance of the (R) strain to treatment with ET was approximately 3-fold higher than the parental (S) strain. Two modes of resistance to ET, and simultaneous resistance to formaldehyde, are demonstrated: (1) transient or induced resistance is expressed during shor-term exposure to ET, and this resistance is gradually lost during subsequent growth in the absence of ET, and (2) resistance that results from a stable phenotypic change in the (S) strain following sequential treatment with ET ((R) strain phenotype). The observed activities of three forms of the formaldehyde oxidizing enzyme, formaldehyde dehydrogenase, are strongly correlated with the relative response of the (S) and (R) strains to treatment with ET. The observed resistance of the (R) strain appears to be due to high levels of an NAD+-linked, glutathione-dependent form of formaldehyde dehydrogenase as well as a dye-linked formaldehyde dehydrogenase. The transient or induced response of the (R) strain involves an increase in activity of the dye-linked formaldehyde dehydrogenase. The induced response of the (S) strain and an ATCC strain ofP. aeruginosa, however, is correlated with the two forms of the NAD+-linked enzyme (glutathione-dependent (EC 1.2.1.1) and independent (EC 1.2.1.46)) with no contribution from the dye-linked enzyme.

The formaldehyde dehydrogenase of Rhodococcus erythropolis, a trimeric enzyme requiring a cofactor and active with alcohols.

During growth on compounds containing methyl groups a formaldehyde dehydrogenase is induced in the gram-positive bacteria Rhodococcus erythropolis. This formaldehyde dehydrogenase has been purified to homogeneity using affinity chromatography and permeation chromatography. The isoelectric point of the enzyme was 4.7. The molar mass of the native enzyme was determined as 130 000 g/mol. Sodium dodecyl sulfate gel electrophoresis yielded a single subunit with a molar mass of 44000 g/mol. These results, together with cross-linking experiments which yielded monomer, dimer, and trimer bands, are consistent with a trimeric subunit structure of the formaldehyde dehydrogenase. A heat-stable cofactor of low molar mass was required for activity with formaldehyde as substrate. This cofactor was found to be oxidizable, but active only in its reduced form. Preparative electrofocusing revealed that the cofactor is a weak acid with a pK of about 6.5. The enzyme was active with the homologous series of the primary alcohols, ethanol up to octanol, without requiring the presence of the cofactor. A mutant without formaldehyde dehydrogenase activity was not impaired in its growth with ethanol as substrate. It is suggested that the alcohols mimic the true substrate of the formaldehyde dehydrogenase, which could be a hydroxymethyl derivative of the cofactor, resulting from the addition of formaldehyde.

An unusual formaldehyde oxidizing system inRhodococcus erythropolisgrown on compounds containing methyl groups

Abstract During utilization of compounds containing methyl groups, the non-methylotrophic bacteria Rhodococcus erythropolis oxidized the methyl groups entirely to carbon dioxide. This oxidation was linked to the presence of an NAD-dependent formaldehyde dehydrogenase activity which was lost on dialysis. The activity could be restored by the addition of boiled extract but not by adding the known cofactors glutathione or tetrahydrofolate. A further dehydrogenase activity with formaldehyde as substrate was found in ethanolgrown cells. This activity could be differentiated from that in methyl group metabolizing cells.

Inhibition of the oxidation of acetaldehyde and formaldehyde by hepatocytes and mitochondria by crotonaldehyde

Crotonaldehyde was oxidized by disrupted rat liver mitochondrial fractions or by intact mitochondria at rates that were only 10 to 15% that of acetaldehyde. Although a poor substrate for oxidation, crotonaldehyde is an effective inhibitor of the oxidation of acetaldehyde by mitochondrial aldehyde dehydrogenase, by intact mitochondria, and by isolated hepatocytes. Inhibition by crotonaldehyde was competitive with respect to acetaldehyde, and the K i for crotonaldehyde was about 5 to 20 μ m. Crotonaldehyde had no effect on the oxidation of glutamate or succinate. Very low levels of acetaldehyde were detected during the metabolism of ethanol. Crotonaldehyde increased the accumulation of acetaldehyde more than 10-fold, indicating that crotonaldehyde, besides inhibiting the oxidation of added acetaldehyde, also inhibited the oxidation of acetaldehyde generated by the metabolism of ethanol. Formaldehyde was a substrate for the low- K m mitochondrial aldehyde dehydrogenase, as well as for a cytosolic, glutathione-dependent formaldehyde dehydrogenase. Crotonaldehyde was a potent inhibitor of mitochondrial oxidation of formaldehyde, but had no effect on the activity of formaldehyde dehydrogenase. In hepatocytes, crotonaldehyde produced about 30 to 40% inhibition of formaldehyde oxidation, which was similar to the inhibition produced by cyanamide. This suggested that part of the formaldehyde oxidation occurred via the mitochondrial aldehyde dehydrogenase, and part via formaldehyde dehydrogenase. The fact that inhibition by crotonaldehyde is competitive may be of value since other commonly used inhibitors of aldehyde dehydrogenase are irreversible inhibitors of the enzyme.

Oxidation of formaldehyde and acetaldehyde by NAD +-dependent dehydrogenases in rat nasal mucosal homogenates

Homogenates of respiratory and olfactory tissue from the rat nasal cavity were examined for their capacity to catalyze the NAD +-dependent oxidation of formaldehyde (in the presence and absence of glutathione) and of acetaldehyde. Both aldehydes were oxidized efficiently by nasal mucosal homogenates, and formaldehyde dehydrogenase (FDH) and aldehyde dehydrogenase (AldDH) were tentatively identified in both tissue samples. At least two isozymes of AldDH, differing with respect to their apparent K m and V max values with acetaldehyde as substrate, were found in the nasal mucosa, one of which may catalyze the oxidation of both formaldehyde and acetaldehyde. The specific activity of FDH in the olfactory mucosa was twice that in the respiratory mucosa, whereas the specific activity of the higher K m isozyme of AldDH was five to eight times greater in respiratory than in olfactory tissue. The specific activity of the lower K m isozyme of AldDH was similar in respiratory and olfactory homogenates. Repeated exposures of rats to formaldehyde (15 ppm, 6 hr/day, 10 days) or to acetaldehyde (1500 ppm, 6 hr/day, 5 days) did not substantially affect the specific activities of FDG and AldDH in nasal mucosal homogenates. Glutathione is a cofactor for FDH; the concentration of nonprotein sulfhydryls in respiratory mucosal homogenates was approximately 2.8 μmoles/g and was not changed significantly by repeated exposures to formaldehyde (15 ppm, 6 hr/day, 9 days). These data indicate that the rat nasal mucosa, which is the major target site for both aldehydes in inhalation toxicity studies, can metabolize both formaldehyde and acetyldehyde, and that the specific activities of formaldehyde and aldehyde dehydrogenase in homogenates of the nasal mucosa are essentially unchanged following repeated exposures to toxic concentrations of either compound.

Burden and biochemical effects of extended tetrahydrofuran vapour inhalation of three concentration levels.

Adult male rats exposed to tetrahydrofuran vapour at 8.2 (200 p.p.m.), 41 (1,000 p.p.m.) or 82 mumol/l (2,000 p.p.m.) for 2 to 18 weeks, five days a week, 6 hrs daily, showed dose-dependent brain and perirenal fat solvent burden linearly correlated to each other. After two weeks of exposure, the body burden of tetrahydrofuran seems to decrease. This might have been caused by increased oxidative metabolism as enhanced 7-ethoxycoumarin O-deethylase activity was detected in liver and kidneys in the 2nd week and onwards. The exposure also caused inhibition of alcohol and formaldehyde dehydrogenase activities in liver at the highest dose. Biochemical effects in the cerebellum were not detected while gluteal muscle specimens showed increased succinate dehydrogenase activity in a dose-related manner. This points to effects on the energy metabolism. Muscle acetylcholine esterase activity was also increased showing possible effects on the myoneural junctions.

More Than One Amine Oxidase is Involved in the Metabolism by Yeasts of Primary Amines Supplied as Nitrogen Source

Twenty-seven yeast species were tested for growth on methylamine and n-butylamine as sole nitrogen sources. Five species (including Saccharomyces cerevisiae) failed to grow on either amine, and a further five grew only on n-butylamine. The remainder grew on both amines. Amine oxidase activity was detected in extracts of all the strains which could grow on amines, but was generally absent from cells grown on ammonia or nitrate. From measurements in cell-free extracts of oxidase activity for a number of different amines, it is concluded that most such strains have at least two amine oxidases of different substrate specificity, and that the substrate specificity also varies between different yeast species. Based on these observations, four different groups of yeasts could be distinguished. Formaldehyde dehydrogenase activity was elevated in cells grown on amines or nitrate compared with those grown on ammonia, and activity was generally higher in cells grown on methylamine than in those grown on n-butylamine or nitrate.

Purification and characterization of [formula omitted] hydrolase from a methanol-utilizing yeast, Kloeckera sp. No. 2201

S- Formylglutathione hydrolase (EC 3.1.2.12), a glutathione thiol esterase, was purified from a methanol-utilizing yeast, Kloeckera sp. No. 2201, to homogeneity as judged by polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was determined to be 58 000 by gel filtration. The enzyme appeared to be composed of two identical subunits ( M r = 31 000 ). The apparent K m for S- formylglutathione was 0.077 mM. The optimum temperature was 50°C and the optimum pH was 6.4–6.6. The enzyme was inhibited by several types of sulfhydryl reagents. The purified enzyme preparation contained no activity of formaldehyde dehydrogenase or of formate dehydrogenase. It is thought that three enzymes, formaldehyde dehydrogenase, S- formylglutathione hydrolase and formate dehydrogenase, participate in the oxidation of formalhyde to CO 2 in Kloeckera sp. No. 2201.