Characterization of a Saccharomyces cerevisiae NADP(H)-dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family.

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.     

Multifunctionality of liver alcohol dehydrogenase. Studies of aldehyde dehydrogenase activity

Kinetic studies of the liver alcohol dehydrogenase catalyzed dehydrogenation of aldehydes were carried out over a wide range of octanal concentrations. The effect of specific inhibitors of liver alcohol dehydrogenase on aldehyde dehydrogenase activity was examined. The results were consistent with a steady-state random mechanism with the formation of the ternary E · NADH octanal complex at low temperatures. This ternary complex becomes inconspicuous at high temperatures. The aldehyde dehydrogenase activity was found to associate with all ethanol-active isozymes. The dual dehydrogenase reactions are catalyzed by the same molecule, presumably in the region of the same domain. However, the two activities respond differently to structural changes.     

The involvement of alcohol dehydrogenase and aldehyde dehydrogenase in alcohol/aldehyde metabolism in Drosophila melanogaster

In this study we have examined the roles of alcohol dehydrogenase, aldehyde oxidase, and aldehyde dehydrogenase in the adaptation of Drosophila melanogaster to alcohol environments. Fifteen strains were characterized for genetic variation at the above loci by protein electrophoresis. Levels of in vitro enzyme activity were also determined. The strains examined showed considerable variation in enzyme activity for all three gene-enzyme systems. Each enzyme was also characterized for coenzyme requirements, effect of inhibitors, subcellular location, and tissue specific expression. A subset of the strains was chosen to assess the physiological role of each gene-enzyme system in alcohol and aldehyde metabolism. These strains were characterized for both the ability to utilize alcohols and aldehydes as carbon sources as well as the capacity to detoxify such substrates. The results of the above analyses demonstrate the importance of both alcohol dehydrogenase and aldehyde dehydrogenase in the in vivo metabolism of alcohols and aldehydes.     

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.     

Polymorphism of aldehyde dehydrogenase and alcohol sensitivity

The metabolism of acetaldehyde has received considerable attention in the past years owing to its acute and chronic toxic effects in humans. Aldehyde dehydrogenase (ALDH) catalyzes the oxidation of acetaldehyde in liver and other organs. Two major isozymes of hepatic ALDH (ALDH I or E2 and ALDH II or E1), which differ in their structural and functional properties, have been characterized in humans. The ALDH I with a low Km for acetaldehyde is predominantly of mitochondrial origin and ALDH II which has a relatively higher Km is of cytosolic origin. An inherited deficiency of ALDH I isozyme has been found among Japanese and Chinese which is primarily responsible for producing acute alcohol sensitivity symptoms (flushing response) after drinking mild doses of alcohol. Biochemical, immunochemical and molecular genetics data indicate a structural mutation in the ALDH I isozyme gene responsible for the loss in catalytic activity. Population genetic studies indicate a wide prevalence of this ALDH polymorphism among individuals of Mongoloid race. Flushing response to alcohol shows familial resemblances and preliminary family data from Japan, China and Korea hint to an autosomal codominant inheritance for ALDH I isozyme deficiency. The ALDH polymorphism is apparently responsible for the low incidence of alcoholism in Japanese, Chinese and Koreans. Alcohol-induced sensitivity due to ALDH isozyme deficiency may act as an inhibitory factor against excessive alcohol drinking thereby imparting a protection against alcoholism.     

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2′5′ ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K_m = 12 micromolar) and coniferaldehyde (K_m = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.     

Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase

The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red-brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and paper-making value of forest trees while the similarity with brown-midrib mutants suggests a route to more digestible forage crops.     

Unusual NADPH conformation in the crystal structure of a cinnamyl alcohol dehydrogenase from Helicobacter pylori in complex with NADP(H) and substrate docking analysis

Cinnamyl alcohol dehydrogenase is a zinc- and NADPH-dependent dehydrogenase catalyzing the reversible conversion of p-hydroxycinnamaldehydes to their corresponding hydroxycinnamyl alcohols. A CAD homolog from Helicobacter pylori (HpCAD) possesses broad substrate specificities like the plant CADs and additionally a dismutation activity converting benzaldehyde to benzyl alcohol and benzoic acid. We have determined the crystal structure of HpCAD complexed with NADP(H) at 2.18 Å resolution to get a better understanding of this class of CAD outside of plants. The structure of HpCAD is highly homologous to the sinapyl alcohol dehydrogenase and the plant CAD with well-conserved residues involved in catalysis and zinc binding. However, the NADP(H) binding mode of the HpCAD has been found to be significantly different from those of plant CADs.Structured summary of protein interactionsHpCAD and HpCAD bind by x-ray crystallography (View interaction)     

Characterization of rat cornea aldehyde dehydrogenase

Aldehyde dehydrogenase has been purified from rat cornea in a single step. The enzyme is a class 3 aldehyde dehydrogenase. Cornea aldehyde dehydrogenase is a 100-kDa dimer composed of 51-kDa subunits, prefers NADP+ as coenzyme, and preferentially oxidizes benzaldehyde-like aromatic aldehydes as well as medium chain length (4-9 carbons) aliphatic aldehydes. The substrate and coenzyme specificity, immunochemical properties, effect of disulfiram, pH profile, and isoelectric point of cornea aldehyde dehydrogenase are identical to those of tumor-associated aldehyde dehydrogenase, the prototype class 3 enzyme. The substrate and coenzyme preferences are consistent with a role for cornea aldehyde dehydrogenase in the oxidation of a variety of aldehydes generated by lipid metabolism, including lipid peroxidation.     

An enzymatic model for rat liver alcohol dehydrogenase in the presence of low-Km aldehyde dehydrogenase

Four isoenzymes of aldehyde dehydrogenase were partially purified from rat liver mitochondria by hydroxylapatite chromatography and gel filtration. While three forms display low affinity for acetaldehyde, the fourth is active at extremely low aldehyde concentrations (Km less than or equal to 2 microM) and allows the oxidation of the acetaldehyde formed by catalysis of alcohol dehydrogenase at pH 7.4. Different models of alcohol dehydrogenase have been examined by analysis of progress curves of ethanol oxidation obtained in the presence of low-km aldehyde dehydrogenase. According to the only acceptable model, when the acetaldehyde concentration is kept low by the action of aldehyde dehydrogenase, NADH no longer binds to alcohol dehydrogenase, but acetaldehyde still competes with ethanol for the active site of the enzyme. The seven kinetic parameters of the two enzymes (four for alcohol dehydrogenase and three for aldehyde dehydrogenase) and the equilibrium constant of the reaction catalyzed by alcohol dehydrogenase have been determined by applying a new fitting procedure here described.     

Intralobular distribution of rat liver aldehyde dehydrogenase and alcohol dehydrogenase

1. The activity of liver microsomal high Km-ALDH and mitochondrial low Km-ALDH, which may be primarily responsible for the oxidation of acetaldehyde after ethanol administration was found to be predominantly distributed in the centrilobular area. 2. The activities of other ALDH isozymes in mitochondrial and soluble fractions were evenly distributed in periportal and perivenous regions. 3. The activity of ADH which is involved in production of acetaldehyde was predominantly located in the periportal area. 4. From these results it seems unlikely that a concentration of acetaldehyde after ethanol ingestion is higher in perivenous hepatocytes than in periportal ones. Additional data would be needed to understand fully the mechanism by which ethanol induces predominantly centrilobular liver injury.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Genotypes of alcohol dehydrogenase and aldehyde dehydrogenase loci in Japanese alcohol flushers and nonflushers

Summary A much higher incidence of alcohol flushing among Orientals in comparison to Caucasians, i.e., >50% vs 5%–10%, has been attributed to racial differences in alcohol-metabolizing enzymes. A large majority of Orientals are atypical in alcohol dehydrogenase-2 locus (ADH ₂ ), and their livers exhibit significantly higher ADH activity than the livers of most Caucasians. Approximately 50% of Orientals lack the mitochondrial aldehyde dehydrogenase (ALDH₂) activity, and elimination of acetaldehyde might be disturbed. We determined by means of hybridization of genomic DNA samples with allele specific oligonucleotide probes, genotypes of the ADH ₂ and ALDH ₂ loci in Japanese alcohol flushers and nonflushers. We found that all individuals with homozygous atypical ALDH ₂ ² /ALDH ₂ ² and most of those with heterozygous atypical ALDH ₁ ² /ALDH ₂ ² were alcohol flushers, while all subjects with homozygous usual ALDH ₁ ² /ALDH ₁ ² were nonflushers. Frequency of the atypical ADH ₂ ² was found to be higher in alcohol flushers than in nonflushers, but the statistical significance was not established in the sample size examined.     

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase fromEucalyptus xylem

Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions ofEucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl andp-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42–43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.Abbreviations CAD cinnamyl alcohol dehydrogenase - DTT dithiothreitol - NCS N chlorosuccinimide - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by the European Economic Community project AGRE 0021 (OPLIGE) in the scope of the ECLAIR PROGRAMME. The authors whis to thank Drs. L. Davin and N. Lewis (Washington State University) for kindly providing synthesized substrates, Dr. Annie Boudet for excellent technical assistance, and Dr. M. Campbell for fruitful discussions (Université Paul Sabatier, Toulouse, France). We would also like to thank Dr. M. M. Cordonnier-Pratt and Dr. L. Pratt (University of Georgia, Athens, USA) for helpful advice and antibody production.