Microbody of methanol-grown yeasts. Localization of catalase and flavin-dependent alcohol oxidase in the isolated microbody.

Profuse appearance of microbodies was observed in the cells of methanol-utilizing yeasts in connection with the enhanced catalase activity. These microbodies were isolated successfully by means of sucrose gradient centrifugation from the methanol-grown cells of Kloeckera sp. no. 2201. Localization of a flavin-dependent alcohol oxidase as well as characteristic microbody enzymes (catalase and D-amino acid oxidase) were ascertained in the isolated microbodies, whereas formaldehyde and formate dehydrogenases were detected in the cytoplasmic region. Localization of catalase in the isolated microbody was also demonstrated by the cytochemical technique with 3,3'-diaminobenzidine.     

NAD-linked formate dehydrogenase from methanol-grown Pichia pastoris NRRL-Y-7556

An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 mm) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 mm for NAD and 16 mm for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts.     

Initial microbial adhesion is a determinant for the strength of biofilm adhesion

Abstract A considerable amount of methylformate accumulated in the culture medium of methanol-grown methylotrophic yeasts. Methylformate is considered as an intermediate in a novel formaldehyde oxidation pathway. Through investigations with Pichia methanolica , methylformate formation was found to be catalysed by a new type of alcohol dehydrogenase, which was named methylformate synthase. When cells were grown on a relatively high concentration of methanol or exposed to a high concentration of formaldehyde, formation of methylformate was enhanced and the level of methylformate synthase in the cells increased. How methylformate synthase is involved in formaldehyde oxidation and formaldehyde detoxification is discussed.     

Structural aspects of the dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila.

1. A dye-linked alcohol dehydrogenase was purified 60-fold from extracts of Rhodopseudomonas acidophila 10050 grown aerobically on ethanol. 2. The properties of this enzyme were identical with those of the alcohol dehydrogenase synthesized by this organism during growth on methanol anaerobically in the light, and they are judged to be the same enzyme. 3. The enzyme gave a single protein band, coincident with alcohol dehydrogenase activity, during electrophoresis on polyacrylamide gel. 4. The amino acid composition, ioselectric point, u.v. and visible absorption spectra of the enzyme were determined and compared with those of other similar enzymes. 5. The presence of 0.7--1.0 g-atom of non-haem, acidlabile iron/mol of enzyme was shown by atomic absorption spectrophotometry and colorimetric assay. The iron could not be dissociated from the enzyme by dialysis against chelating agents. 6. E.p.r. spectroscopy of the enzyme did not indicate any redox function for the iron during alcohol dehydrogenation, but showed a signal at g = 2.00 consistent with the presence of a protein-bound organic free radical. 8. Antisera were raised against alcohol (methanol) dehydrogenases purified from Rhodopseudomonas acidophila, Paracoccus denitrificans and Methylophilus methylotrophus. 9. The antiserum to the Rhodopseudomonas acidophila enzyme cross-reacted with neither of the two other antisera, nor with crude extracts of methanol-grown Hyphomicrobium X and Pseudomonas AM1, thus emphasizing its singular biochemical properties.     

Extended superfamily of short alcohol-polyol-sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25% sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcohol-polyol-glucose dehydrogenases.     

Alcohol Dehydrogenase Activities of Wine Yeasts in Relation to Higher Alcohol Formation

Alcohol dehydrogenase activities were examined in cell-free extracts of 10 representative wine yeast strains having various productivities of higher alcohols (fusel oil). The amount of fusel alcohols (n-propanol, isobutanol, active pentanol, and isopentanol) produced by the different yeasts and the specific alcohol dehydrogenase activities with the corresponding alcohols as substrates were found to be significantly related. No such relationship was found for ethanol. The amounts of higher alcohols formed during vinification could be predicted from the specific activities of the alcohol dehydrogenases with high accuracy. The results suggest a close relationship between the control of the activities of alcohol dehydrogenase and the formation of fusel oil alcohols. Also, new procedures for the prediction of higher alcohol formation during alcoholic beverage fermentation are suggested.     

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.     

Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.     

Microbial production of methyl ketones. Purification and properties of a secondary alcohol dehydrogenase from yeast.

Cell-free extracts derived from yeasts Candida utilis ATCC 26387, Hansenula polymorpha ATCC 26012, Pichia sp. NRRL-Y-11328 Torulopsis sp. strain A1 and Kloeckera sp. strain A2 catalyzed an NAD+-dependent oxidation of secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol) to the corresponding methyl ketones (acetone, 2-butanone, 2-pentanone, 2-hexanone). We have purified a NAD+-specific secondary alcohol dehydrogenase from methanol-grown yeast, Pichia sp. The purified enzyme is homogenous as judged by polyacrylamide gel electrophoresis. The purified enzyme catalyzed the oxidation of secondary alcohols to the corresponding methyl ketones in the presence of NAD+ as an electron acceptor. Primary alcohols were not oxidized by the purified enzyme. The optimum pH for oxidation of secondary alcohols by the purified enzyme is 8.0. The molecular weight of the purified enzyme as determined by gel filtration is 98 000 and subunit size as determined by sodium dodecyl sulfate gel electrophoresis is 48 000. The activity of the purified secondary alcohol dehydrogenase was inhibited by sulfhydryl inhibitors and metal-binding agents.     

Development of Microbodies in the yeast Kloeckera growing on methanol.

A number of microbodies appear regularly in methanol-grown yeast cells, but rarely in ethanol- or glucose-grown cells. When one of representative methanol-utilizing yeasts, Kloeckera sp.no. 2201 (also known as Candida bodinii), was cultured on glucose and then transferred into a methanol medium, microbodies of small size could be observed in 2-h old cells. The number of microbodies per sectioned cell reached five to six after 4 h of cultivation. Though the number of microbodies did not change during prolonged cultivation, their size became larger with the passage of cultivation time. The activities of catalase and alcohol oxidase were confirmed in the particulate fractions throughout the cultivation period, whereas the activities of formaldehyde dehydrogenase and formate dehydrogenase were not detected in the particles. The activity of isocitrate lyase was detected in the particulate fractions only at the early growth phase.     

NAD- and co-substrate (GSH or factor)-dependent formaldehyde dehydrogenases from methylotrophic microorganisms act as a class III alcohol dehydrogenase

Abstract The methylotrophic yeasts, Hansenula polymorpha and Candida boidinii , and the methylotrophic Gram-negative bacteria, Paracoccus denitrificans and Thiobacillus versutus (but not Methylophaga marina ), contain NAD/GSH-dependent formaldehyde dehydrogenase when grown on C₁-compounds. The enzymes appeared to be similar to each other and to the mammalian counterparts with respect to substrate specificity, including the ability to act as an alcohol dehydrogenase class III. The Gram-positive bacteria, Amycolatopsis methanolica and Rhodococcus erythropolis , possess NAD/Factor-dependent formaldehyde dehydrogenase when grown on C₁-compounds or on C₁-unit-containing substrates, respectively. These enzymes also exhibit alcohol dehydrogenase class III activity. Thus, like the mammalian ones, methylotrophic formaldehyde dehydrogenases show dual substrate specificity, suggesting that this is an inherent property of the enzyme.     

The primary structure of alcohol dehydrogenase from Drosophila lebanonensis. Extensive variation within insect 'short-chain' alcohol dehydrogenase lacking zinc

Insect alcohol dehydrogenase is highly different from the well-known yeast and mammalian alcohol dehydrogenases. The enzyme from Drosophila lebanonensis has now been characterized by protein analysis and was found to have a 254-residue protein chain with an acetyl-blocked N-terminal Met. Comparisons with the structures of the enzyme from other species allows judgement of the extent of variability within the insect alcohol dehydrogenases. They have diverged to a considerable extent; two forms analyzed at the protein level differ at 18% of all residues, and all the known Drosophila alcohol dehydrogenase structures reveal differences at 72 positions. Some deviations, against a background similarity, in the extent of changes are noted among the parts corresponding to different exons. The structural variation within Drosophila is about as large as the one for the mammalian zinc-containing alcohol dehydrogenase. Consequently, the results illustrate Drosophila relationships and establish great variations also for group of alcohol dehydrogenases lacking zinc.     

Sequence of the gene for a NAD(P)-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) from a marine methanotroph Methylobacter marinus A45

Abstract A fragment of Methylobacter marinus A45 DNA has been cloned and sequenced, and an open reading frame has been identified that could code for a 46-kDa polypeptide. Comparison of the deduced amino acid sequence of the polypeptide against the protein data bank has revealed strong similarity with a number of alcohol dehydrogenases, with highest similarity towards class III alcohol dehydrogenases, which recently have been shown to be identical to glutathione-dependent formaldehyde dehydrogenases. We were unable to measure appreciable levels of NAD(P)-dependent formaldehyde dehydrogenases or alcohol dehydrogenase activities using aldehydes or primary or secondary alcohols in cell-free extracts from batch cultures of M. marinus A45. However, formaldehyde dehydrogenases activity was detected on zymograms. Our data suggest that, although NAD(P)-linked formaldehyde dehydrogenase or alcohol dehydrogenase activities are undetectable in cell-free extracts of most methylotrophs employing the ribulose monophosphate pathway for formaldehyde assimilation and dissimilation, the gene encoding formaldehyde dehydrogenase is present in M. marinus A45 and may be present in more of these organisms as well.     

Alcohol and aldehyde dehydrogenase from Saccharomyces cerevisiae: specific activity and influence on the production of acetic acid, ethanol and higher alcohols in the first 48 h of fermentation of grape must

The changes in the specific activity of alcohol dehydrogenase (ADH-I and ADH-II) and aldehyde dehydrogenases [AIDH-NADP+ and AIDH-NAD(P)+] from Saccharomyces cerevisiae during the first 48 h of fermentation of grape must were investigated. The biosynthesis of ADH-I and AIDH-NADP+ took place basically during the adaptation of the yeasts to the must (first 4 h), while that of ADH-II occurred immediately after exponential growth (after 12 h). From the products produced by the yeast, only the specific rate of production of ethanol was found to be directly related to the specific activity of ADH-I.     

Factors affecting the stability of methanol dehydrogenase from Paracoccus denitrificans

Methanol dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methanol-grown cells. The enzyme was composed of subunits of M(r) 67,000 and 12,000, and non-covalently bound pyrroloquinoline quinone. It exhibited a pH optimum at pH values of 9.0 and above. It was not stable at pH greater than 9.0, but exhibited little loss of activity after prolonged incubation at pH values as low as 4.5. Methyl dehydrogenase was relatively stable to thermal denaturation. The thermal stability was enhanced by the presence of Ca2+ and diminished by the presence of EDTA. These data suggest a structural role for Ca2+ in this enzyme, similar to what has been observed with quinoprotein glucose and ethanol dehydrogenases.     

Alcohol Dehydrogenases: Identification and Names for Gene Families

Plant gene products that have been described as `alcohol dehydrogenases' are surveyed and related to their CPGN nomenclature. Most are Zn-dependent medium chain dehydrogenases, including `classical' alcohol dehydrogenase (Adh1), glutathione-dependent formaldehyde dehydrogenase (Fdh1), cinnamyl alcohol dehydrogenase (Cad2), and benzyl alcohol dehydrogenase (Bad1). Plant gene products belonging to the short-chain dehydrogenase class should not be called alcohol dehydrogenases unless such activity is shown.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

Thermostable NAD-linked secondary alcohol dehydrogenase from propane-grown Pseudomonas fluorescens NRRL B-1244.

NAD-linked alcohol dehydrogenase activity was detected in cell-free crude extracts from various propane-grown bacteria. Two NAD-linked alcohol dehydrogenases, one which preferred primary alcohols (alcohol dehydrogenase I) and another which preferred secondary alcohols (alcohol dehydrogenase II), were found in propane-grown Pseudomonas fluorescens NRRL B-1244 and were separated from each other by DEAE-cellulose column chromatography. The properties of alcohol dehydrogenase I resembled those of well-known primary alcohol dehydrogenases. Alcohol dehydrogenase II was purified 46-fold; it was homogeneous as judged by acrylamide gel electrophoresis. The molecular weight of this secondary alcohol dehydrogenase is 144,500; it consisted of four subunits per molecule of enzyme protein. It oxidized secondary alcohols, notably, 2-propanol, 2-butanol, and 2-pentanol. Primary alcohols and diols were also oxidized, but at a lower rate. Alcohols with more than six carbon atoms were not oxidized. The pH and temperature optima for secondary alcohol dehydrogenase activity were 8 to 9 and 60 to 70 degrees C, respectively. The activation energy calculated from an Arrhenius plot was 8.2 kcal (ca. 34 kJ). The Km values at 25 degrees C, pH 7.0, were 8.2 X 10(-6) M for NAD and 8.5 X 10(-5) M for 2-propanol. The secondary alcohol dehydrogenase activity was inhibited by strong thiol reagents and strong metal-chelating agents such as 4-hydroxymercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 5-nitro-8-hydroxyquinoline, and 1,10-phenanthroline. The enzyme oxidized the stereoisomers of 2-butanol at an equal rate. Alcohol dehydrogenase II had good thermal stability and the ability to catalyze reactions at high temperature (85 degrees C). It appears to have properties distinct from those of previously described primary and secondary alcohol dehydrogenases.     

The detection of sorbitol dehydrogenase in the cytoplasm of liver cells in birds and its relation to alcohol dehydrogenase and glucose-6-phosphate dehydrogenase

Comparative studies have been made in the specific activity of sorbitol dehydrogenase, glucose-6-phosphate and alcohol dehydrogenases in the cytoplasm from the liver of wild and domestic ducks, hen and pheasant. High activity of all the three enzymes was found in ducks indicating the effective sorbitol (polyol) metabolism of glucose. The activity of glucose-6-phosphate dehydrogenase is an order lower as compared with the activity of sorbitol and alcohol dehydrogenases in the cytoplasm of hen liver. The same relationship was found for the activity of sorbitol dehydrogenase in the cytoplasm of pheasant liver.     

Alcohol-oxidizing enzymes from various organisms.

1. Enzymes that catalyse the oxidation of aliphatic alcohols to aldehydes are reviewed. 2. Special attention is given to phenazine methosulphate-linked alcohol dehydrogenases from bacteria and to flavin-containing alcohol oxidases from yeasts, moulds and higher plants. 3. Some properties of the microsomal ethanol-oxidative system of rat liver are discussed.