Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

This review discusses recent achievements in the field of cofactor regeneration for the nicotinamide cofactors NADH and NADPH. The examples discussed include alcohol dehydrogenases, formate dehydrogenase, glucose dehydrogenase and a hydrogenase. For the reaction either one-phase systems or two-phase systems in combination with an organic solvent are discussed. For the enantioselective reduction of 2-octanone to (R)-2-octanol it could be shown that enzyme coupled NADPH regeneration with glucose dehydrogenase and glucose results in shorter reaction times and higher yields when compared to the substrate coupled regeneration with 2-propanol.

ADH: alcohol dehydrogenase; LDH: Lactose dehydrogenase; GDH: Glucose dehydrogenase; FDH: Formate dehydrogenase; LB-ADH: alcohol dehydrogenase from Lactobacillus brevis; HL-ADH: alcohol dehydrogenase from horse liver; TB-ADH: alcohol dehydrogenase from Thermoanaerobicum brockii; PS-GDH: Glucose dehydrogenase from Pseudomonas species; [BMIM][PF₆]: Butyl-methyl-imidazoliumhexafluorophosphate     

Selective recruitment of <Emphasis Type="Italic">Adh</Emphasis> genes for distinct enzymatic functions in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.     

The effect of insertion of the maize transposable elementMutator is dependent on genetic background

A secondary mutant, derived from an allele of maize alcohol dehydrogenase 1 (Adh1) carrying a Mutator transposable element (Mu1) in its first intron, was reported to exhibit a threefold decrease in ADH enzymatic activity and steady-state RNA levels compared to the original mutant. The original mutant,Adh1-S3034 (abbreviatedS3034), was previously characterized at the molecular level. The derivative, abbreviatedS3034b, has now been cloned; at the DNA sequence level the insertion and surroundingAdh1 sequences are indistinguishable fromS3034. Furthermore, in our lines there is no difference in relative ADH activities between products of the two putative alleles. A comparison of gene expression in heterozygotes obtained by crossing to different tester lines reveals a correlation between the measured decrease in levels of ADH polypeptide produced by the mutant allele and the background in which it is measured; this effect is distinct from any background-related variation in the expression of the progenitor allele. It does not appear to be attributable to alternative patterns of DNA modification. It appears to reflect a background-associated difference in the level of normalAdh1-RNA produced. Thus the previously reported distinction betweenS3034 andS3034b may be due to differences in the extent to which the mutant allele and a given genetic background interact to produce functionalAdh1-RNA.This research was supported by United States Public Health Service Grant GM38616 and United States Department of Agriculture Grant 87-CRCR-1-2500 to J.S. D.O. was supported by an NIH predoctoral training grant to the Department of Genetics.     

Adh4, an alcohol dehydrogenase controls alcohol formation within bacterial microcompartments in the acetogenic bacterium Acetobacterium woodii

Acetobacterium woodii utilizes the Wood-Ljungdahl pathway for reductive synthesis of acetate from carbon dioxide. However, A. woodii can also perform non-acetogenic growth on 1,2-propanediol (1,2-PD) where instead of acetate, equal amounts of propionate and propanol are produced as metabolic end products. Metabolism of 1,2-PD occurs via encapsulated metabolic enzymes within large proteinaceous bodies called bacterial microcompartments. While the genome of A. woodii harbours 11 genes encoding putative alcohol dehydrogenases, the BMC-encapsulated propanol-generating alcohol dehydrogenase remains unidentified. Here, we show that Adh4 of A. woodii is the alcohol dehydrogenase required for propanol/ethanol formation within these microcompartments. It catalyses the NADH-dependent reduction of propionaldehyde or acetaldehyde to propanol or ethanol and primarily functions to recycle NADH within the BMC. Removal of adh4 gene from the A. woodii genome resulted in slow growth on 1,2-PD and the mutant displayed reduced propanol and enhanced propionate formation as a metabolic end product. In sum, the data suggest that Adh4 is responsible for propanol formation within the BMC and is involved in redox balancing in the acetogen, A. woodii.     

Molecular analysis of a somaclonal mutant of maize alcohol dehydrogenase

Summary Plants regenerated from tissue cultures of maize were screened for variants of ADH1 and ADH2. Root extracts of 645 primary regenerant plants were tested, and one stable mutant of Adh1 was detected. The mutant gene (Adh1-Usv) produces a functional enzyme with a slower electrophoretic mobility than that of the progenitor Adh1-S allele, and is stably transmitted to progeny. The mutant was not present among four other plants derived from the same immature embryo, and therefore arose as a consequence of the culture procedure. The gene of Adh1-Usv was cloned and sequenced. A single base change in exon 6 was the only alteration found in the gene sequence. This would translate in the polypeptide sequence to a valine residue substituting for a glutamic acid residue, resulting in the loss of a negative charge and the production of a protein with slower electrophoretic mobility.Abbreviations kb kilobase pairs - ADH alcohol dehydrogenase     

Three alcohol dehydrogenase genes in wild and cultivated barley: Characterization of the products of variant alleles

Barley (Hordeum vulgare) and its wild progenitor (H. spontaneum) have three loci for alcohol dehydrogenase (EC 1.1.1.1; ADH). The Adh1 locus is constitutively expressed in seed tissues, whereas expression of the loci Adh2 and Adh3 requires anaerobic induction. The Adh3 gene is well expressed in aleurone and embryo tissues kept under N₂ for 2–3 days. Using N₂-treated embryos, a diverse collection of H. spontaneum was screened in starch gels for electrophoretic variants at the Adh3 locus. Four variants were found: two were conventional mobility variants (Adh3 S, Adh3 V); one was a null variant (Adh3 n); and the fourth (Adh3 I) variant lacked active homodimers and showed reduced heterodimer activity. The ³⁵S-labeled monomers induced under N₂ in the lines homozygous for Adh1, Adh2, or Adh3 variants were immunoprecipitated with antiserum raised against maize ADH. Fluorography after separation by SDS-PAGE and by urea-isoelectric focusing indicated that the Adh3 n allele was CRM- and that the Adh3 I gene product was smaller than normal. The Adh1 and Adh3 variants showed independent segregation.     

Mutant alleles that are altered in quantitative,organ-specific behavior

Three new mutant alleles of maize alcohol dehydrogenase-1 (Adh 1) were recovered following allyl alcohol selection of pollen. Each is altered in quantitative, organ-specific, regulatory properties. All mutant sites act in cis to the structural gene component. One mutant arose spontaneously, one followed indirectly from irradiation with high Z accelerated particles, and one was induced by an autonomous mutator system. Each mutant is assessed in three organs by utilizing ADH allozyme ratios that were quantified at the level of ADH enzyme activity and either [³H]-Leu incorporation into newly synthesized ADH 1 subunits or direct protein determinations. One mutation simultaneously raises Adh 1 expression in one organ and lowers it in another, another affects expression in one organ only, and another is extremely underexpressed in all organs but is unstable. This unstable allele has generated derivative mutant alleles that have less or zero ADH expression. We do not yet know whether or not coding sequences are involved in these mutants. We conclude that information for organ specificity and quantitative behavior resides near or within Adh 1 coding sequences.     

Impaired retinol utilization in Adh4 alcohol dehydrogenase mutant mice

Adh4, a member of the mouse alcohol dehydrogenase (ADH) gene family, encodes an enzyme that functions in vitro as a retinol dehydrogenase in the conversion of retinol to retinoic acid, an important developmental signaling molecule. To explore the role of Adh4 in retinoid signaling in vivo, gene targeting was used to create a null mutation at the Adh4 locus. Homozygous Adh4 mutant mice were viable and fertile and demonstrated no obvious defects when maintained on a standard mouse diet. However, when subjected to vitamin A deficiency during gestation, Adh4 mutant mice demonstrated a higher number of stillbirths than did wild‐type mice. The proportion of liveborn second generation vitamin A‐deficient newborn mice was only 15% for Adh4 mutant mice but 49% for wild‐type mice. After retinol administration to vitamin A‐deficient dams in order to rescue embryonic development, Adh4 mutant mice demonstrated a higher resorption rate at stage E12.5 (69%), compared with wild‐type mice (30%). The relative ability of Adh4 mutant and wild‐type mice to metabolize retinol to retinoic acid was measured after administration of a 100‐mg/kg dose of retinol. Whereas kidney retinoic acid levels were below the level of detection in all vehicle‐treated mice (<1 pmol/g), retinol treatment resulted in very high kidney retinoic acid levels in wild‐type mice (273 pmol/g) but 8‐fold lower levels in Adh4 mutant mice (32 pmol/g), indicating a defect in metabolism of retinol to retinoic acid. These findings demonstrate that another retinol dehydrogenase can compensate for a lack of Adh4 when vitamin A is sufficient, but that Adh4 helps optimize retinol utilization under conditions of both retinol deficiency and excess. Dev. Genet. 25:1–10, 1999. © 1999 Wiley‐Liss, Inc.     

Phenotype Dependence of the Combining Ability in Mutant Sugar Beet

Repetitive gametic selection for a higher frequency of the Adh1-Ssemilethal mutant allele of the alcohol dehydrogenase (ADH) gene yielded viable homozygotes Adh1-SS. The plants varied in phenotype from weak mutant to nearly normal (restored). Phenotypically different plants were individually tested for combining ability. This parameter was high in plants with the mutant phenotype and tended to decrease, rather than further increase, in plants with a restored normal phenotype. The results are discussed in terms of viability restoration mechanisms in homozygotes for semilethal mutant alleles.     

Structural and biochemical characterisation of a NAD+-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni²⁺-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni²⁺, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD⁺/NADH, whereas no activity has been observed with NADP⁺/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn²⁺. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni²⁺-dependent activity towards 1,3-propanediol.     

Catalytic properties of alcohol dehydrogenase isozymes specified by duplicate genes in the diploid plant Stephanomeria exigua

The alcohol dehydrogenase (ADH) isozymes induced in flooded roots of the diploid plant Stephanomeria exigua are specified by tightly linked genes comprising a complex locus, Adh1. Individuals homozygous for a complex with two active genes which specify electrophoretically different subunits have three ADH-I isozymes, two intragenic homodimers and an intergenic heterodimer. Individual isozymes were partially purified from plants homozygous for several different Adh1 complexes and apparent K _m values for acetaldehyde, ethanol, NAD, and NADH and responses to temperature, pH, and two different alcohols were determined. The two homodimeric enzymes specified by a particular Adh1 complex generally differed in one or more of the properties studied, and in three of four cases, intergenic heterodimers differed significantly from intermediacy, often having lower K _m values than either homodimer. None of the isozymes studied could be considered greatly divergent or defective. Constraints on evolution of duplicate genes which form intergenic heterodimers are considered.     

Characterization of mutation-induced changes in the maize (Zea mays L.) ADH1-1S1108 alcohol dehydrogenase

The homodimeric alcohol dehydrogenase gene product of maize (Zea mays L.)Adh1-1S1108 mutation was purified and compared with the parentalAdh1-1S enzyme. The mutant alcohol dehydrogenase activity had pH optima and substrate specificity similar to those of the parental enzyme, but exhibited somewhat increased and decreasedK _mvalues for acetaldehyde and NADH, respectively. The mutant enzyme was also markedly less stable than the enzyme from parental tissues to temperatures as low as 50°C. Sequence analysis of a polymerase chain reaction (PCR)-generated cDNA clone revealed a G-to-C mutation at position 406 and a C-to-T mutation at position 974. These would result in residue 103 of each protein subunit being changed from an alanine to a proline and residue 292 being changed from an alanine to a valine. Whether one or both of these changes in primary sequence is responsible for the altered substrate affinities and stability is not yet understood.     

Biochemical genetics of alcohol dehydrogenase isozymes in the gray short-tailed opossum (Monodelphis domestica)

Polyacrylamide gel-isoelectric focusing (PAGE-IEF) methods were used to examine the multiplicity, tissue distribution, and biochemical genetics of alcohol dehydrogenase (ADH) isozymes among gray short-tailed opossums (Monodelphis domestica). Seven ADH isozymes were resolved and distinguished on the basis of their isoelectric points, tissue distributions, and substrate and inhibitor specificities. ADH1 and ADH2 exhibited Class I properties and were observed in liver (and intestine) extracts. ADH3, ADH4, and ADH5 showed “high-K _m ” (possibly Class IV) properties, with ADH3 and ADH4 exhibiting high activity in cornea, ear, stomach, and esophagus extracts. ADH6 and ADH7 exhibited Class III properties, including activities as formaldehyde dehydrogenases, with each showing different tissue distribution characteristics; ADH6 was widely distributed, and ADH7 was restricted to prostate extracts. An additional form of formaldehyde dehydrogenase (FDH) was observed, which was inactive with hexenol and ethanol as substrates. Isoelectric point variants were observed for ADH3 (three forms) and for ADH4 (two forms), and the inheritance of ADH3 was studied in 15 families ofM. domestica. The data were consistent with codominant inheritance of two alleles (ADH3*A andADH3*B) at a single autosomal locus (designatedADH3) and with a model involving a dimeric ADH isozyme: ADH3 (γ₂ isozyme, forming three dimers designated γ ₂ ¹ , γ¹ γ², and γ ₂ ² in heterozygous individuals).     

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.

Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O₂ deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O₂ levels were reduced below 5%, and were most intense at 0% O₂.

In vivo³⁵S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O₂, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O₂, the synthesis of all three ADH peptides increased markedly.

Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O₂ deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O₂, and about 100-fold after 1 day at 0% O₂.

We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O₂, that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

     

Purification and characterization of an alcohol dehydrogenase from Lithospermum erythrorhizon cell cultures

An NAD-dependent alcohol dehydrogenase has been purified to apparent homogeneity from cell suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), using protamine sulphate and ammonium sulphate precipitation and chromatography on DEAE-Sephacel, Superdex 200, hydroxyapatite and HiTrap blue. The enzyme is a homodimer with a M_r of ca. 77,000. Each subunit with a M_r of 40,000 contains two zinc atoms. Its isoelectric point was found at pH 5.0. The best alcohol substrate of the enzyme is ethanol. The pH optimum for ethanol oxidation is at pH 8.7 and for acetaldehyde reduction at pH 4.6. The Michaelis constants for ethanol and NAD are 2.49 and 0.05 (pH 8.7), and for acetaldehyde and NADH 2.2 and 0.078 mM (pH 4.6), respectively. Partial amino acid sequences of the purified enzyme showed high homology to alcohol dehydrogenases from other plants.Abbreviations ADH alcohol dehydrogenase - DTT dithiothreitol - PMSF dephenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - IAA indole-3-acetic acid - TFA trifluoroacetic acid     

Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of Saccharomyces cerevisiae

The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated NDH2 has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.     

Ethanol-resistant mutants of Nicotiana plumbaginifolia are deficient in the expression of pollen and seed alcohol dehydrogenase activity

Summary Six independent mutant lines ofNicotiana plumbaginifolia resistant to ethanol, designated E3, E8, E101, E112, E144 and E251, were isolated as germinating seedlings on selective medium. In all cases, resistance to ethanol was conferred by a single recessive nuclear mutation at the same locus. Mutant seeds and pollen lacked detectable ADH activity, with the exception of E251 where a residual activity was detected. An antiserum directed againstArabidopsis thaliana ADH detected an ADH-related polypeptide of 44 kDa present in wild-type seeds and, to a lesser extent, in the seeds of the leaky mutant E251. No ADH-related polypeptide could be detected in seeds of the other mutants. However, all of them had a nearly normal level of ADH mRNA except one which did not synthesize any mRNA. These results suggest that these ethanol-resistant mutants are impaired in one of the structural genes coding for alcohol dehydrogenase. The corresponding locus has been designatedAdh1.Abbreviations ADH alcohol dehydrogenase - EMS ethyl methane-sulfonate - MTT dimethyl thiazol tetrazolium - NAD nicotinamide adenine dinucleotide - NBT nitro blue tetrazolium - p-cells protoplast-derived cells - PMS phenazine methosulfate - SDS sodium dodecyl sulfate     

Mutations of the Adh1 gene in maize following infection with barley stripe mosaic virus

Summary Mutations at the Adh1 locus in maize were selected from plants infected with barley stripe mosaic virus (BSMV). Pollen from the infected inbred line 1s2p, which is homozygous for Adh1-S (abbreviated S), Adh2-P, c and r was treated with allyl alcohol and applied to silks of a tester stock homozygous for Adh1-F, Adh2-N, C and R. From these pollinations 356 kernels arose on the F₁ ears. Of these eight showed no activity of the S allele in scutellar samples while two exhibited low levels. Five of the putative mutant kernels germinated and two of these contained the contamination markers Adh2-P, c and r. The newly arisen mutations were designated S5446 and S5453. S5453 exhibited an abnormally low level of ADH activity in the F₁ scutellum. In the F₂ generation the mutant reverted at a high frequency with only about 5% of the S5453 alleles expressing low levels. DNA blotting and hybridization analyses showed no alterations in the restriction patterns of S5453 when compared to the progenitor S allele. S5446 which exhibited no ADH activity in the F₁ scutellum is unstable in the pollen; reversion frequencies approaching 10^-2 were observed in samples from some plants. Restriction digestion patterns of DNA from this mutant revealed the presence of a 3.3 kb insertion at Adh. The insert does not appear to contain sequences homologous to the BSMV genome but rigorous analyses remain to be carried out. It is hypothesized that BSMV infection may mobilize endogenous but dormant transposable elements in maize.     

Genetic determination of the developmental program for maize scutellar alcohol dehydrogenase: Involvement of a recessive,trans-acting,temporal-regulatory gene

The developmental program of alcohol dehydrogenase (ADH) activity in the scutellum of maize strain R6-67 is different from that of W64A. The level of scutellar ADH activity in R6-67 remains relatively high during the course of early sporophytic development as compared to the commonly observed pattern. In the typical inbred strain W64A, the activity of ADH declines substantially during that period. The variance values from the crosses between R6-67 and W64A reveal that the trait is under genetic control. Detailed genetic analysis suggests that a single gene is responsible for the altered developmental program of ADH activity in R6-67. This gene meets the criteria for temporal regulatory genes and is different from Adh2, the structural gene which codes the ADH-2 isozyme. We have designated this gene as Adr1 (alcohol dehydrogenase regulator, #1). Adr1 is unlinked to Adh2. There is no de novo synthesis of ADH in the scutellum during germination, and the difference in the activity level reflects the difference in the amount of enzyme protein as demonstrated by density labeling and rocket immunoelectrophoresis. Thus, it appears that Adr1 may regulate the degradation of ADH.