A Gelatin Film Method for Improved Histochemical Localization of Dehydrogenases in Plant Cells

Glucose-6-phosphate and 6-phosphogluconate dehydrogenases diffused from frozen sections of Vicia faba embryos during histochemical incubation. In the liquid incubation medium, the dehydrogenases catalysed the oxidation of substrate and reduction of NADP. NADPH₂ thus formed could lead to artifactual deposition of formazan in frozen sections. The addition of 20% polyvinyl alcohol to the incubation medium was found unsatisfactory in preventing this loss which appeared to be overcome by incorporating the reaction mixture into a gelatin film. Equal volumes of 10% gelatin solution in 0.05 M phosphate buffer at pH 7.8, and the enzyme reaction medium containing twice the normal concentration of substrate (0.014 M), of 0.007 M pyridine nucleotide, of 0.02 M KCN and of 0.0024 M NBT in the buffer, were mixed and layered onto polyethylene, and allowed to set in the dark at room temperature for 30-60 min. The solidified medium and its support were cut into strips and layed onto unfixed, frozen sections of plant tissues which were incubated at 20 C. Evidence is presented to support the supposition that the enzymes are retained in the sections during the reaction.     

Cyclopropanol in the exploration of bacterial alcohol oxidation

Abstract Cyclopropanol selectively inhibits bacterial alcohol oxidation proceeding via NAD-independent, quinoprotein alcohol dehydrogenases. Thus, for instance, alcohol oxidation by Pseudomonas aeruginosa , grown on ethanol, was inhibited for about 50% by cyclopropanol treatment. Accordingly, cell-free extracts of untreated cells had nearly equal activities of quinoprotein and NAD-dependent alcohol dehydrogenases, whereas only the latter enzyme activity was found in cell-free extracts of cyclopropanol-treated cells. Upon incubation of Hyphomicrobium X with cyclopropanol, oxidation of alcohols was blocked while formaldehyde oxidation was not. Therefore, methanol dehydrogenase in this organism is not specifically involved in formaldehyde oxidation. The examples show that cyclopropanol-derived substrates are potential tools in revealing the physiological role of bacterial alcohol dehydrogenases.     

SCOPE AND LIMITATIONS OF THE USE OF NICOTINOPROTEIN ALCOHOL DEHYDROGENASE FOR THE COENZYME-FREE PRODUCTION OF ENANTIOPURE FINE-CHEMICALS

Nicotinoprotein alcohol dehydrogenases are enzymes that contain non-dissociable NAD(P)(H) in the active site. The suitability of a nicotinoprotein alcohol dehydrogenase as coenzyme-independent alternative to classic alcohol dehydrogenases for enantioselective synthetic applications was studied. To this end the NADH-containing nicotinoprotein, np-ADH, from Rhodococcus erythropolis DSM 1069 was used as a model enzyme in different types of conversion: asymmetric synthesis, kinetic resolution and racemization. The enzyme was found to catalyze the asymmetric reduction of ketones using cheap reductants, such as ethanol, with high stereoselectivity, but the reaction was too slow to obtain good yields. Kinetic resolutions of racemic alcohols failed due to dismutation of the aldehyde that was used as cosubstrate. Racemization of a secondary alcohol via the corresponding ketone could not be achieved, which was due to an unidentified side reaction. This evaluation shows that, for developing biotransformations of industrial interest using nicotinoprotein alcohol dehydrogenases, the attention should be focused on enzymes with a higher reactivity towards prochiral ketones and secondary alcohols.     

Alcohol dehydrogenase (ADH) in yeasts. II. NAD+-and NADP+-dependent alcohol dehydrogenases in Saccharomycopsis lipolytica.

In Sm. lipolytica one NAD+-dependent and three NADP+-dependent alcohol dehydrogenases are detectable by polyacrylamide gelelectrophoresis. The NAD+-dependent ADH (ADH I), with a molecular weight of 240,000 daltons, reacts more intensively with long-chain alcohols (octanol) than with short-chain alcohols (methanol, ethanol). The ADH I is not or only minimally subject to glucose repression. Besides the ADH I band no additional inducible NAD+-dependent ADH band is gel-electrophoretically detectable during growth of yeast cells in medium containing ethanol or paraffin. The ADH I band is very probably formed by two ADH enzymes with the same electrophoretic mobility. The NADP+-dependent alcohol dehydrogenases (ADH II--IV) react with methanol, ethanol and octanol with different intensity. In polyacrylamide gradients two bands of NADP+-dependent ADH are detectable: one with a molecular weight of 70,000 daltons and the other with 120,000 daltons. The occurrence of the three NADP+-dependent alcohol dehydrogenases is regulated by the carbon source of the medium. Sm. lipolytica shows a high tolerance against allylalcohol. Resistant mutants can be isolated only at concentrations of 1 M allylalcohol in the medium. All isolates of allylalcohol-resistant mutants show identical growth in medium containing ethanol as the wild type strain.     

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.     

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.     

A new NADH-dependent,zinc containing alcohol dehydrogenase from Bacillus thuringiensis serovar israelensis involved in oxidations of short to medium chain primary alcohols

The escalation in genome sequencing has presented a mass of potentially useful new alcohol dehydrogenases (ADHs) in the form of putative open reading frame (ORF). To take advantage of such available resources, PCR primers based on the genome sequence of Bacillus thuringiensis serovar israelensis were used to clone a gene encoding a hypothetical alcohol dehydrogenase (named as BtADH). Activity studies of the translation product revealed that the alcohol dehydrogenases catalyse the inter-conversion of aliphatic aldehydes and corresponding primary alcohol with chain length of two to ten carbons. The required co-factor for such inter-conversion was found to be NAD(H). The ADH gene was engineered for heterologous expression in Escherichia coli, and the enzyme was produced in a soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined.The findings lead us to propose that BtADH represents a novel primary–secondary alcohol dehydrogenase that acts on primary alcohols of medium chain lengths and simple ketones. Besides, BtADH shares high sequence similarity with well known ADHs from thermophilic origins. Such biochemical characterisation of BtADH provides valuable information for the study of sequence–function relationship including source of thermal stability, cofactor and substrate preferences.     

Differences between alcohol dehydrogenases. Structural properties and evolutionary aspects.

Comparisons of the primary structures of yeast and horse liver alcohol dehydrogenases reveal that the enzymes are homologous but distantly related. The overall positional identity is 25% between common regions, and several deletions/insertions occur in either enzyme, the longest apparently corresponding to 21 residues, showing that the different subunit sizes are largely explained by internal differences. Variabilities in the structural similarities can be coupled with functional requirements but not directly with whole domains in the previously known tertiary structure of the horse protein. The two most similar regions of the enzymes affect active-site segments and the two most dissimilar regions seem to affect a loop structure without known function, and a segment participating in subunit interactions. The dissimilarities may probably be correlated with changes in zinc-binding properties and quaternary structures. The extra region corresponding to the large internal chain-length difference shows an apparent coincidence in sequence to a following segment of the horse enzyme, and additional elements of internal coincidences, or superficial similarities with other dehydrogenases, are noticed. These characteristics are not fully distinguishable from chance distributions but in view of the extensive species variations in alcohol dehydrogenases some evolutionary considerations may not be excluded, in which case a model relating all regions of these and associated enzymes to a common ancestor is shown to be compatible with all known observations.     

Histochemical localization of primary and secondary alcohol dehydrogenases in whole-body, freeze-dried sections of mice

Summary Whole-body sagittal sections of frozen, C57BL/6J, adult, male mice were used for the localization of primary and secondary alcohol dehydrogenases in most tissues of the body. The reduction of Nitro BT with NAD⁺ as coenzyme, as described originally by Hardonk (1965), was utilized for the generation of coloured final reaction deposits. Ethanol was used as a substrate for primary alcohol dehydrogenase; 2-propanol, -methylbenzyl alcohol and 2-butanol were used as substrates for secondary alcohol dehydrogenase. Liver and bronchial epithelium showed the highest activities for both enzymes; oesophageal and upper gastric epithelium showed a high activity of primary alcohol dehydrogenase. Pyrazole, indazole and imidazole inhibited primary, but not secondary, alcohol dehydrogenase. Dimethylsulphoxide and menthol slightly inhibited both enzymes. Oleic acid, sulphydryl agents,p-chloromercuribenzoate, and copper sulphate also inhibited both enzymes. Slight inhibition of secondary dehydrogenase was observed on co-administration of several alcohols.As expected,N-nitrosonornicotine did not function as a substrate for alcohol dehydrogenases. When this compound was present in the histochemical incubation media, no activity was seen at any of the usual sites of these enzymes. The distribution of the alcohol dehydrogenase activities found in this study correlates with the distribution of radioactivity in oesophagus, bronchi and liver after administration of [¹⁴C]nitrosonornicotine. This suggests that the alcohol dehydrogenases may be involved in the metabolism of hydroxylated nitrosonornicotine, a metabolite of the most abundant known carcinogen in cigarette smoke.     

Purification, cloning, and overexpression of an alcohol dehydrogenase from Nocardia globerula reducing aliphatic ketones and bulky ketoesters

For the huge amount of chiral chemicals and precursors that can potentially be produced by biocatalysis, there is a tremendous need of enzymes with new substrate spectra, higher enantioselectivity, and increased activity. In this paper, a highly active alcohol dehydrogenase is presented isolated from Nocardia globerula that shows a unique substrate spectrum toward different prochiral aliphatic ketones and bulky ketoesters as well as thioesters. For example, the enzyme reduced ethyl 4-chloro-3-oxo butanoate with an ee >99% to (S)-4-chloro-3-hydroxy butanoate. Very interesting is also the fact that 3-oxobutanoic acid tert-butylthioester is reduced with 49.4% of the maximal activity while the corresponding tert-butyloxyester is not reduced at all. Furthermore, it has to be mentioned that acetophenone, a standard substrate for many known alcohol dehydrogenases, is not reduced by this enzyme. The enzyme was purified from wild-type N. globerula cells, and the corresponding 915-bp-long gene was determined, cloned, expressed in Escherichia coli, and applied in biotransformations. The N. globerula alcohol dehydrogenase is a tetramer of about 135 kDa in size as determined from gel filtration. Its sequence is related to several hypothetical 3-hydroxyacyl-CoA dehydrogenases whose sequences were derived by whole-genome sequencing from bacterial sources as well as known mammalian 3-hydroxyacyl-CoA dehydrogenases and ß-hydroxyacyl-CoA dehydrogenases from different clostridiae.     

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.     

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.     

Cloning, sequence analysis, and expression in Escherichia coli of the gene encoding phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10

The gene encoding phenylacetaldehyde reductase (PAR), a useful biocatalyst for producing chiral alcohols, was cloned from the genomic DNA of the styrene-assimilating Corynebacterium sp. strain ST-10. The gene contained an opening reading frame consisting of 1,158 nucleotides corresponding to 385 amino acid residues. The subunit molecular weight was calculated to be 40,299, which was in agreement with that determined by polyacrylamide gel electrophoresis. The enzyme was sufficiently expressed in recombinant Escherichia coli cells for practical use and purified to homogeneity by three-column chromatography steps. The predicted amino acid sequence displayed only 20–29% identity with zinc-containing, NAD⁺-dependent, long-chain alcohol dehydrogenases. Nevertheless, the probable NAD⁺- and zinc-binding sites are conserved although one of the three catalytic zinc-binding residues of the zinc-containing, long-chain alcohol dehydrogenases was substituted by Asp in PAR. The protein contains 7.6 mol zinc/mol tetramer. Therefore, the enzyme was considered as a new member of zinc-containing, long-chain alcohol dehydrogenases with a particular and broad substrate specificity. Received: 5 March 1999 / Received last revision: 10 May 1999 / Accepted: 16 May 1999     

Ancestral gene fusion in cellobiose dehydrogenases reflects a specific evolution of GMC oxidoreductases in fungi

Cellobiose dehydrogenases (CDHs) are extracellular hemoflavoenzymes that are thought to be involved in the degradation of two of the most abundant biopolymers in the biosphere, cellulose and lignin. To date, these enzymes, consisting of a cytochrome domain and a flavin domain, have been detected and sequenced exclusively in the kingdom of fungi. Independent phylogenetic analyses of two distinct domains of CDH genes reveal that they evolved in parallel as fused genes. Whereas the cytochrome domains are unique sequence motifs, the flavin domains clearly belong to the glucose-methanol-choline (GMC) oxidoreductase family--an evolution line of widespread flavoproteins extending from the Archae to higher eukaryotes. The most probable unrooted phylogenetic tree obtained from our analysis of 52 selected GMC members reveals five principal evolutionary branches: cellobiose dehydrogenase, cholesterol oxidase (COX), hydroxynitrile lyase, alcohol oxidase (AOX)/glucose oxidase (GOX)/choline dehydrogenase, and a branch of dehydrogenases with various specificities containing also an Archaeon open reading frame (ORF). Cellobiose dehydrogenases cluster with cholesterol oxidases and the clade of various specificities, whereas hydroxynitrile lyases are closely related to glucose oxidases, alcohol oxidases, and choline dehydrogenases. The results indicate that the evolutionary line from a primordial GMC flavoprotein to extant cellobiose dehydrogenases was augmented after an early acquisition of the cytochrome domain to form two distinct branches for basidiomycetes and ascomycetes. One ascomycetous evolutionary line of CDHs has acquired a carbohydrate-binding module (CBM) of type 1, the sequence of which is similar to that of corresponding domains in several glycosidases. This is the first attempt towards a comprehensive phylogenetic analysis of cellobiose dehydrogenases.     

The possible occurrence of zinc in ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279

The activity of polyhydric alcohol dehydrogenases in Mycobacterium sp. 279 was studied under limitation of zinc in the growth medium. It was found that the activity of ribitol and sorbitol dehydrogenases were markedly lowered and that of D-arabinitol dehydrogenase remained unchanged in the Zn2+-deficient cells. Other ions tested i.e., Co2+, Cu2+, Ni2+ and Mn2+ failed to substitute Zn2+ ions in their effect on the enzyme activities. The Zn2+-responsive enzymes were sensitive to the chelating agents (1,10-phenanthroline, 2,2'-dipyridyl), whereas D-arabinitol dehydrogenase was insensitive. The results indicate possible existence of a zinc component in the ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279.     

On the specificity of a histochemical method for localizing dihydrofolate dehydrogenase

Synopsis The enzymic reduction of folate in the histochemical method described previously by the authors for localizing dihydrofolate dehydrogenase was followed spectrophotometrically by determining the decrease in the folate concentration in the medium during the incubation. In addition the different histochemical localizations of dihydrofolate dehydrogenase and NADH₂ dehydrogenase were compared in smears of normal and leukaemic human blood.     

A quantitative study of the validity of the histochemical demonstration for pyridine nucleotide-linked dehydrogenases

Summary A quantitative histochemical and biochemical study has been made of the loss of pyridine nucleotide-linked dehydrogenases from frozen histological sections of rat liver. Glucose-6-phosphate, 6-phosphogluconate and lactate dehydrogenases were lost rapidly from the sections during incubation in the histochemical medium, but -OH-butyrate dehydrogenase was lost at a much slower rate. It was shown that a dehydrogenase reaction can occur in a section lacking that particular dehydrogenase if the section is incubated in the presence of another containing the dehydrogenase. The validity of the tetrazolium reaction for demonstrating pyridine-nucleotide-linked dehydrogenases is considered in the light of these results.     

The effect of functionally bound vitamins and their coenzyme forms on the activity of 2-oxoacid dehydrogenases in mouse organs

Ever-growing doses (beginning from the therapeutic up to 10-fold doses of a complex of five functionally bound vitamins (B1, FMN, nicotinamide, pantothenate, lipoic acid) being administered to F1 mice (CBA x Black) induced a constant and considerable rise of the pyruvate- and 2-oxoglutarate dehydrogenase (PDG and OGDG) activity in the mouse organs. In the in vitro experiments the addition of the corresponding coenzymes or their mixture (in the optimal concentrations) to the incubation medium containing mitochondria of the mouse liver led to a greater activation of these dehydrogenases in the group of animals which were preliminary injected the above complex of vitamins.     

Localization and cytophotometric analysis of cathepsin B activity in unfixed and undecalcified cryostat sections of whole rat knee joints

Cathepsin B activity is demonstrated histochemically with a post-coupling method using Z-Arg-Arg-4-methoxy-2-naphthylamide as substrate and Fast Blue BB as coupling reagent in unfixed and undecalcified cryostat sections of whole rat knee joints. Sections were attached to transparent tape to keep the integrity of the tissue intact, such attachment being essential for precise precipitation of the final reaction product at sites of enzyme activity. Also essential was inclusion of polyvinyl alcohol in the enzyme incubation medium. High cathepsin B activity was found in osteoclasts, chondrocytes, fibroblasts, synovial cells, and bone marrow cells in knee joints after induction of arthritis. The final reaction product was precipitated as fine cytoplasmic granules probably corresponding to lysosomes. The reaction was specific because addition to the incubation medium of selective inhibitors of cathepsin B-like activity completely blocked the activity. The amount of final reaction product in synovium and in bone marrow cells was analyzed cytophotometrically. Specific formation of final reaction product was linear with incubation time up to 60 min at 37 degrees C and with section thickness up to 12 microns. Variation of the substrate concentration in the incubation medium revealed a KM value of 1.86 +/- 0.36 mM in synovial cells and 2.48 +/- 0.51 mM in bone marrow cells and Vmax values (expressed as mean integrated absorbance) of 1.18 +/- 0.10 in synovial cells and 1.02 +/- 0.11 in bone marrow cells. Both KM and Vmax values were significantly different in synovial cells and bone marrow cells (p less than 0.01) which could be owing to the presence of different isoenzymes in these tissues. We conclude that the described post-coupling method is sufficient to yield precise localization and that the method is valid for quantitative purposes.     

Simian liver alcohol dehydrogenase: isolation and characterization of isoenzymes from Macaca mulatta

Like human liver alcohol dehydrogenase, that of Macaca mulatta can be purified and separated into anodic and cathodic pyrazole-insensitive and cathodic pyrazole-sensitive enzyme forms. Their inhibition by 4-methylpyrazole and their substrate specificities are analogous to those observed for the corresponding isoenzymes of human liver. However, on the basis of data available so far, the physiochemical and compositional characteristics, i.e., molecular weight, zinc content, and dimeric structure, of all simian alcohol dehydrogenase forms are virtually identical with those of other mammalian alcohol dehydrogenases studied up to now. Zinc is essential for their enzymatic function, as demonstrated by inhibition with chelating agents.