Purification of formaldehyde and formate dehydrogenases from pea seeds by affinity chromatography and S-formylglutathione as the intermediate of formaldehyde metabolism

Formaldehyde dehydrogenase (EC 1.2.1.1) and formate dehydrogenase (EC 1.2.1.2) have been isolated in pure form from pea seeds by a rapid procedure which employs column chromatographies on 5′-AMP-Sepharose, Sephacryl S-200, and DE32 cellulose. The apparent molecular weights of formaldehyde and formate dehydrogenases are, respectively, 82,300 and 80,300 by gel chromatography, and they both consist of two similar subunits. The isoelectric point of formaldehyde dehydrogenase is 5.8 and that of formate dehydrogenase is 6.2. The purified formate dehydrogenase gave three corresponding protein and activity bands in electrophoresis and isoelectric focusing on polyacrylamide gel whereas formaldehyde dehydrogenase gave only one band. Formaldehyde dehydrogenase catalyzes the formation of S-formylglutathione from formaldehyde, and glutathione. Formate dehydrogenase can, besides formate, also use S-formylglutathione and two other formate esters as substrates. S-Formylglutathione has a lower K_m value (0.45 mm) than formate (2.1 mm) but the maximum velocity of S-formylglutathione is only 5.5% of that of formate. Pea extracts also contain a highly active S-formylglutathione hydrolase which has been separated from glyoxalase II (EC 3.1.2.6) and partially purified. S-Formylglutathione hydrolase is apparently needed between formaldehyde and formate dehydrogenases in the metabolism of formaldehyde in pea seeds, in contrast to what was recently reported for Hansenula polymorpha, a yeast grown on methanol.     

Formaldehyde production with heat-treated cells of methanol yeast

Chemostat-grown cells of a methanol yeast, Candida boidini S2 AOU-1, were investigated for formaldehyde production. The productivity and catalytic stability were improved by preincubation of the cells at 37°C for 24 h in 0.1 M potassium phosphate buffer (pH 7.5) containing 50 mM NaN₃. These cells produced 1150 mM formaldehyde after a 10-h reaction.     

Characterization of crystalline formate dehydrogenase from Candida methanolica

The crystalline formate dehydrogenase from Candida methanolica, which showed the highest specific activity (7.52 U/mg) so far reported, was characterized in detail. The enzyme is a dimer composed of identical subunits, each containing one SH group related to the catalytic activity. The molecular mass of the enzyme is about 82-86 kDa. The Km values were found to be 3.0 mM for formate and 0.11 mM for NAD+. Even if the enzyme was incubated at pH 6.5-9.5 or at 55 degrees C, the activity remained at 100%. Hg2+, Ni2+, NaCN, NaN3 and p-chloromercuribenzoate strongly inhibited the enzyme activity, while the enzyme showed relatively high resistance to various chelating agents. The amino acid composition and some other physicochemical properties of the enzyme were studied. Immunological studies revealed that formate dehydrogenases of methanol-utilizing yeasts immunologically more or less resemble each other, but differ from those of methanol-utilizing bacteria. Furthermore, yeast formate dehydrogenases can be immunologically classified into three types: (a) the Candida type, (b) the Torulopis/Hansenula/Pichia type and (c) the formaldehyde-resistant yeast type. For simple and large-scale preparation of the enzyme for practical use, treatment of cells of C. methanolica with the commercial cationic detergent, 'Benzalkonium' cation, is useful: the total and specific activities of the enzyme are 1.17-fold and 3.10-fold higher than those of the crude cell-free extract, respectively.     

The Structure of an Arabinogalactan Isolated from Bamboo Shoot

It was found that azide bound to alcohol oxidase non-covalently and caused a color change from yellow to red. Alcohol oxidase was purified with a high yield from methanol-limited chemostat-grown cells of Candida boidinii S2 as an enzyme-azide complex by a simple procedure. That is, a cell-free extract was prepared from cells treated with a cationic detergent, Cation M2, and alcohol oxidase amounted to more than 80% of the soluble protein. Inactivation of the enzyme by H₂O₂ and aldehydes was decreased in the complex. The results were discussed as to the production of various aldehydes with the yeast cells.     

Dehydrogenases of the pentose phosphate pathway in rat liver peroxisomes

Subcellular distribution of pentose-phosphate cycle enzymes in rat liver was investigated, using differential and isopycnic centrifugation. The activities of the NADP+-dependent dehydrogenases of the pentose-phosphate pathway (glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase) were detected in the purified peroxisomal fraction as well as in the cytosol. Both dehydrogenases were localized in the peroxisomal matrix. Chronic administration of the hypolipidemic drug clofibrate (ethyl-alpha-p-chlorophenoxyisobutyrate) caused a 1.5-2.5-fold increase in the amount of glucose-6-phosphate and phosphogluconate dehydrogenases in the purified peroxisomes. Clofibrate decreased the phosphogluconate dehydrogenase, but did not alter glucose-6-phosphate dehydrogenase activity in the cytosolic fraction. The results obtained indicate that the enzymes of the non-oxidative segment of the pentose cycle (transketolase, transaldolase, triosephosphate isomerase and glucose-phosphate isomerase) are present only in a soluble form in the cytosol, but not in the peroxisomes or other particles, and that ionogenic interaction of the enzymes with the mitochondrial and other membranes takes place during homogenization of the tissue in 0.25 M sucrose. Similar to catalase, glucose-6-phosphate dehydrogenase and phosphogluconate dehydrogenase are present in the intact peroxisomes in a latent form. The enzymes have Km values for their substrates in the millimolar range (0.2 mM for glucose-6-phosphate and 0.10-0.12 mM for 6-phosphogluconate). NADP+, but not NAD+, serves as a coenzyme for both enzymes. Glucose-6-phosphate dehydrogenase was inhibited by palmitoyl-CoA, and to a lesser extent by NADPH. Peroxisomal glucose-6-phosphate and phosphogluconate dehydrogenases have molecular mass of 280 kDa and 96 kDa, respectively. The putative functional role of pentose-phosphate cycle dehydrogenases in rat liver peroxisomes is discussed.     

Thiols in formaldehyde dissimilation and detoxification

Glutathione is not a universal coenzyme for formaldehyde oxidation. MySH (mycothiol, 1-O-(2'-[N-acetyl-L-cysteinyl]amido-2'-deoxy-alpha-D-glucopyranosyl)-D-m yo-inositol) is GSH's counterpart as coenzyme in formaldehyde dehydrogenase from certain gram-positive bacteria. However, formaldehyde dissimilation and detoxification not only proceed via thiol-dependent but also via thiol-independent dehydrogenases. The distinct structures and enzymatic properties of MySH-dependent and GSH-dependent formaldehyde dehydrogenases could provide clues for development of selective drugs against pathogenic Mycobacteria. It is to be expected that other new types of thiol-dependent formaldehyde dehydrogenases will be discovered in the future. Indications exist that the product of thiol-dependent formaldehyde oxidation, the thiol formate ester, is not only hydrolytically converted into thiol and formate but can also be oxidatively converted in some cases by a molybdoprotein aldehyde dehydrogenase into the corresponding carbonate ester, decomposing spontaneously into CO2 and the thiol.     

Peroxisomes of alkane-utilizing yeasts metabolic functions and practical aspects

One of the most striking features of alkane-grown yeast cells is conspicuous appearance of peroxisomes in harmony with a high level of catalase. This unique phenomenon was first demonstrated in the authors′ laboratory, and the metabolic functions of peroxisomes in yeasts utilizing alkanes has been estabilished with intact peroxisomes isolated by density gradient centrifugation. The organelles participate in the degradation of fatty acids derived from alkanes to C²-units and the synthesis of gluconeogenic intermediates from C²-units. The abundant appearance of peroxisomes in alkane-utilizing cells has allowed successful production of several useful enzymes including catalase, D-amino acid oxidase, uricase, acyl-CoA oxidase etc. Yeast cells will be an excellent system for investigation the functions and development of peroxisomes because biogenesis of the organelles is induced only by transferring the cells into alkane medium from glucose or ethanol medium.     

Oxidation of C1 compounds by Pseudomonas sp. MS

Pseudomonas sp. MS is capable of growth on a number of compounds containing only C₁ groups. They include trimethylsulphonium salts, methylamine, dimethylamine and trimethylamine. Although formaldehyde and formate will not support growth they are rapidly oxidized by intact cells. Methanol neither supports growth nor is oxidized. A particulate fraction of the cell oxidizes methylamine to carbon dioxide in the absence of any external electron acceptor. Formaldehyde and formate are more slowly oxidized to carbon dioxide by the particulate fraction, although they do not appear to be free intermediates in the oxidation of methylamine. Soluble NAD-linked formaldehyde dehydrogenase and formate dehydrogenase are also present. The particulate methylamine oxidase is induced by growth on methylamine, dimethylamine and trimethylamine, whereas the soluble formaldehyde dehydrogenase and formate dehydrogenase are induced by trimethylsulphonium nitrate as well as the aforementioned amines.     

Regulation of protein synthesis in methylotrophic yeasts: Repression of methanol dissimilating enzymes by nitrogen limitation

The influence of nitrogen limitation on the regulation of the methanol oxidizing enzymes alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two methylotrophic yeastsHansenula polymorpha andKloeckera sp. 2201 was studied in continuous culture. When shifted from carbon-limited growth conditions (with a mixture of glucose and methanol as carbon sources) to a nitrogen-limited environment both cultures were found to go through a transition phase where neither enhanced residual concentrations of the nitrogen source nor of one of the two carbon sources could be detected in the supernatant. As soon as nitrogen became a limiting substrate an immediate reorganisation of the cell composition was initiated: protein content of the cells dropped to approximately 40% of its initial value, glycogen was synthesized and the enzyme composition of the cells was changed. The peroxisomal enzymes alcohol oxidase and catalase in both organisms and the two dehydrogenases for formaldehyde and formate in cells ofKloeckera sp. 2201 were subject to degradation (catabolite inactivation). The measured rates of inactivation indicated that in cells ofH. polymorpha this process might be limited to peroxisomes, whereas inKloeckera sp. 2201 the degradation was found to affect peroxisomal as well as cytoplasmic enzymes. In contrast to methanol dissimilating enzymes the net rate of synthesis of hexokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was not affected by this process but those enzymes were synthesized with increased rates.     

Formaldehyde incorporation by a new methylotroph (L3).

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Formaldehyde incorporation by a new methylotroph (L3)

A number of bacterial strains have been isolated and investigated in our search for a promising organism in the production of single-cell protein from methanol. Strain L3 among these isolates was identified as an obligate methylotroph which grew only on methanol and formaldehyde as the sole sources of carbon and energy. The organism also grew well in batch and chemostat mixed-substrate cultures containing methanol, formaldehyde, and formate. Although formate was not utilized as a sole carbon and energy source, it was readily taken up and oxidized by either formaldehyde- or methanol-grown cells. The organism incorporated carbon by means of the ribulose monophosphate pathway when growing on either methanol, formaldehyde, or various mixtures of C1 compounds. Its C1-oxidation enzymes included phenazine methosulfate-linked methanol and formaldehyde dehydrogenase and a nicotinamide adenine dinucleotide-linked formate dehydrogenase. Identical inhibition by formaldehyde of the first two dehydrogenases suggested that they are actually the same enzyme. The organism had a rapid growth rate, a high cell yield in the chemostat, a high protein content, and a favorable amino acid distribution for use as a source of single-cell protein. Of special interest was the ability of the organism to utilize formaldehyde via the ribulose monophosphate cycle.     

Respiratory resistance of methylotrophic bacteria to formate and cyanide

Whole cells and cell-free preparations of the methylotrophic bacteria, Pseudomonas sp. AM 1 and Achromobacter parvulus, can oxidize formate at tis concentration in the reaction medium up to 1 M. The respiration of whole cells is registered at a concentration of formate greater than 10(-2) M, while that of cell-free extracts at a formate concentration greater than 5 X 10(-5) M. This seems to be due to the presence of a permeability barrier in cells for formate. The oxidation of reduced TMPD and exogenous cytochrome c by the membrane preparations of the two bacteria is inhibited by formate and cyanide; Ki50% = 2.5 X 10(-2) and 10(-6) M, respectively. The oxidation of NADH by the membrane preparations of the bacteria is not inhibited by 1 M formate and 5 X 10(-4) M cyanide but is inhibited by formaldehyde with Ki50% = 3 X 10(-2) M. Formaldehyde has no effect on the oxidation of reduced TMPD and cytochrome c at concentrations greater than 2 X 10(-1) M. These data indicate that respiration of the studied methylotrophic bacteria in the presence of high formate concentrations should be attributed in the presence of a branched electron transport chain in them; one branch of the chain is resistant to formate and cyanide, but is sensitive to formaldehyde.     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

Steady-state kinetics of formaldehyde dehydrogenase and formate dehydrogenase from a methanol-utilizing yeast, Candida boidinii.

Initial velocity studies and product inhibition studies were conducted for the forward and reverse reactions of formaldehyde dehydrogenase (formaldehyde: NAD oxidoreductase, EC 1.2.1.1) isolated from a methanol-utilizing yeast Candida boidinii. The data were consistent with an ordered Bi-Bi mechanism for this reaction in which NAD+ is bound first to the enzyme and NADH released last. Kinetic studies indicated that the nucleoside phosphates ATP, ADP and AMP are competitive inhibitors with respect to NAD and noncompetitive inhibitors with respect to S-hydroxymethylglutathione. The inhibitions of the enzyme activity by ATP and ADP are greater at pH 6.0 and 6.5 than at neutral or alkaline pH values. The kinetic studies of formate dehydrogenase (formate:NAD oxidoreductase, EC 1.2.1.2) from the methanol grown C. boidinii suggested also an ordered Bi-Bi mechanism with NAD being the first substrate and NADH the last product. Formate dehydrogenase the last enzyme of the dissimilatory pathway of the methanol metabolism is also inhibited by adenosine phosphates. Since the intracellular concentrations of NADH and ATP are in the range of the Ki values for formaldehyde dehydrogenase and formate dehydrogenase the activities of these main enzymes of the dissimilatory pathway of methanol metabolism in this yeast may be regulated by these compounds.     

Topographical localization of peroxisomal acyl-CoA ligases: differential localization of palmitoyl-CoA and lignoceroyl-CoA ligases

We found that peroxisomal lignoceroyl-CoA ligase, like palmitoyl-CoA ligase, is present in the peroxisomal membrane whereas the peroxisomal beta-oxidation enzyme system is localized in the matrix. To further define the role of peroxisomal acyl-CoA ligases (membrane component) in providing acyl-CoA for peroxisomal beta-oxidation, we examined the transverse topographical localization of enzymatic sites of palmitoyl-CoA and lignoceroyl-CoA ligases in the peroxisomal membranes. The disruption of peroxisomes by various techniques resulted in the release of a "latent" pool of lignoceroyl-CoA ligase activity while palmitoyl-CoA ligase activity remained the same. Proteolytic enzyme treatment inhibited palmitoyl-CoA ligase activity in intact peroxisomes but had no effect on lignoceroyl-CoA ligase activity. Lignoceroyl-CoA ligase activity was inhibited only if peroxisomes were disrupted with detergent before trypsin treatment. Antibodies to palmitoyl-CoA ligase and to peroxisomal membrane proteins (PMP) inhibited palmitoyl-CoA ligase in intact peroxisomes, and no pool of "latent" activity appeared when antibody-treated peroxisomes were disrupted with detergent. On the other hand, disruption of PMP antibody-treated peroxisomes with detergent resulted in the appearance of a "latent" pool of lignoceroyl-CoA ligase activity. These results demonstrate that the enzymatic site of palmitoyl-CoA ligase is on the cytoplasmic surface whereas that for lignoceroyl-CoA ligase is on the luminal surface of peroxisomal membranes. This implies that palmitoyl-CoA is synthesized on the cytoplasmic surface and is then transferred to the matrix through the peroxisomal membrane for beta-oxidation in the matrix.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological responses of a methylotrophic bacterium after sudden shifts from C-limited chemostat to C-excess batch growth conditions

The response of steady-state continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady-state cultures was low ( D = 0.046 h⁻¹), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol l⁻¹ to 280 mmol l⁻¹, uncoupled growth occurred and the molar cell yield decreased. Conversely, steady-state cultures at high growth rate ( D = 0.2 h⁻¹) showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1⁻¹). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1⁻¹ Slow-growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast-growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate.
It is concluded that slow-growing cultures are more stable than fast-growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow-growing and fast-growing cultures.     

Alcohol Dehydrogenase from Methylobacterium organophilum

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10⁻⁵ M for methanol and 8.2 × 10⁻⁵ M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.     

Effect of increasing methanol concentrations on physiology and cytology ofCandida boidinii

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology ofCandida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rateD 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S ₀ 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentrationS ₀>K _I (K ₁ equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.