Aromatic Alcohol Dehydrogenase Research Articles

Effect of Allelic Variants of Aromatic Alcohol Dehydrogenase CADim on Micromorphological and Chemical Tissue Indices in the Spring Bread Wheat Triticum aestivum L.

Effect of Allelic Variants of Aromatic Alcohol Dehydrogenase CADim on Micromorphological and Chemical Tissue Indices in the Spring Bread Wheat Triticum aestivum L.

Chromosomal localization of aromatic alcohol dehydrogenase fast-migrating isoenzyme Aadh1F (CAD1-F) gene in Triticum aestivum L. bread wheat

Differences in isoenzyme pattern of aromatic alcohol dehydrogenase, NADP-AADH or CAD, were found in the Triticum aestivum L. winter bread wheat cultivars by the method of electrophoresis in the starch gel. A standard three-component spectrum is present in the cv. Zitnica (former Yugoslavia); additional fact-migrating isoenzymes appear in the cv. Novosibirskaya 9 (Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Russia). The presence of fast-migrating CAD isoenzymes is designated as FF phenotype; their absence, as 00 phenotype. Hybridological analysis was carried out; the excess of “null” genotypes was found in F2 progenies. Hybridization with nulli-tetrasomic lines of the chromosomes of the fifth homeologous group was conducted for the gene localization. The segregation analysis demonstrated the most probable localization of the CAD1-F gene in the chromosome 5A. The plants with FF and 00 genotypes differed in a number of chemical and anatomical traits, as well as in grain productivity. The results obtained are discussed in connection with the function of this enzyme in the wheat plant tissues.

Inheritance and phenotype expression of functional and null alleles of aromatic alcohol dehydrogenase (CAD) in diploid wheats

Functional F and null 0 alleles of the CAD1 (Aadh1) gene, which controls the biosynthesis of aromatic alcohol dehydrogenase, were studied in hybrids of the diploid wheat T. monococcum L. and Triticum sinskajae A.Filat. et Kurk. The gene CAD1 is located in chromosome 5A and is linked with the awnless gene awnS (La) with a recombination frequency of about 32%. Plants with genotypes FF, F0, and 00 were significantly different in the height and mechanical strength of the stalk (culm). The elastic limit of the culm tissues of plants FF was considerably higher than in 00 plants. F0 heterozygotes had intermediate values. The thickness of the wall of the sclerenchyma was thinner in plants with genotype 00. The chemical structure of lignin of plants with the functional CAD allele contained units of a phloroglucinol series missing in the mutant plants. The CAD genotypes had no effect on the relative content of cellulose and lignin in stalks ofdiploid wheat and insignificantly influenced the ratio of H :G : S units in the lignin structure, as well as some components of extractives.

The order of the bs, Skdh, and Aadh1 genes in chromosome 5R of rye Secale cereale L.

Segregation analysis was performed in the progenies obtained in analyzing crosses of hybrids of spring and winter accessions of rye Secale cereale L. and wild S. montanum subsp. anatolicum (Grossh.) Tzvel. (syn. S. strictum (J. Presl) J. Presl). The test genes controlled the brittle stem (bs), the allelic variants of aromatic alcohol dehydrogenase (Aadh 1) and shikimate dehydrogenase (Skdh), and the growth habit (Vrn 1). A linkage was observed in the inheritance of the brittle stem and the aromatic alcohol dehydrogenase and shikimate dehydrogenase alloenzymes. The order of genes was established to be bs-Skdh-Aadh 1, and the genetic distances were estimated to be bs-(9.0%)-Skdh, bs-(10.8%)-Aadh 1, and Skdh-(5.3%)-Aadh 1. The recombination coefficient between the Skdh and Aadh 1 genes varied from 2.2 to 18.2%, averaging 5.3%. The growth habit was inherited independently of the bs-Skdh-Aadh 1 linkage group.

Alcohol Dehydrogenases as Tools for the Preparation of Enantiopure Metabolites of Drugs with Methyl Alkyl Ketone Moiety

Three dehydrogenases – (R)-alcohol dehydrogenase from L. kefir, (S)-aromatic alcohol dehydrogenase from T. sp. and (S)-alcohol dehydrogenase from T. brockii – were tested for the preparation of enantiopure hydroxyl metabolites of pentoxifylline (PTX), propentofylline (PPT) and denbufylline (DBF). These metabolites have an important pharmacological significance. The experimental conditions were optimized for biocatalytic reactions. LKADH produced the chiral secondary alcohols: (R)-OHPTX, (R)-OHPPT and (R)-OHDBF, in an anti- Prelog’s rule configuration. In contrast, TBADH and SAADH also generated chiral secondary alcohols, but according to Prelog’s rule, giving (S)-OHPTX, (S)-OHPPT and (S)-OHDBF respectively. All the ADHs tested were characterized by a high enantioselectivity (ees of 99–100%), but the yield of bioconversion was only satisfactory for the reactions performed using LKADH, being in the 96–98% range for PPT and PTX respectively.

A novel NADPH-dependent aldehyde reductase gene from Vigna radiata confers resistance to the grapevine fungal toxin eutypine.

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzyl aldehyde, is a toxin produced by Eutypa lata, the causal agent of eutypa dieback of grapevines. It has previously been demonstrated that tolerance of some cultivars to this disease was correlated with their capacity to convert eutypine to the corresponding alcohol, eutypinol, which lacks phytotoxicity. We have thus purified to homogeneity a protein from Vigna radiata that exhibited eutypine-reducing activity and have isolated the corresponding cDNA. This encodes an NADPH-dependent reductase of 36 kDa that we have named Vigna radiata eutypine-reducing enzyme (VR-ERE), based on the capacity of a recombinant form of the protein to reduce eutypine into eutypinol. The strongest homologies (86.8%) of VR-ERE at the amino acid level were found with CPRD14, a drought-inducible gene of unknown function, isolated from Vigna unguiculata and with an aromatic alcohol dehydrogenase (71.7%) from Eucalyptus gunnii. Biochemical characterization of VR-ERE revealed that a variety of compounds containing an aldehyde group can act as substrates. However, the highest affinity was observed with 3-substituted benzaldehydes. Expression of a VR-ERE transgene in Vitis vinifera cells cultured in vitro conferred resistance to the toxin. This discovery opens up new biotechnological approaches for the generation of grapevines resistant to eutypa dieback.

A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

A novel type of pathogen defense-related cinnamyl alcohol dehydrogenase.

We describe an aromatic alcohol dehydrogenase with properties indicating a novel type of function in the defense response of plants to pathogens. To obtain the enzyme free of contamination with possible isoforms, a parsley (Petroselinum crispum) cDNA comprising the entire coding region of the elicitor-responsive gene, ELI3, was expressed in Escherichia coli. In accord with large amino acid sequence similarities with established cinnamyl and benzyl alcohol dehydrogenases from other plants, the enzyme efficiently reduced various cinnamyl and benzyl aldehydes using NADPH as a co-substrate. Highest substrate affinities were observed for cinnamaldehyde, 4-coumaraldehyde and coniferaldehyde, whereas sinapaldehyde, one of the most efficient substrates of several previously analyzed cinnamyl alcohol dehydrogenases and a characteristic precursor molecule of angiosperm lignin, was not converted. A single form of ELI3 mRNA was strongly and rapidly induced in fungal elicitor-treated parsley cells. These results, together with earlier findings that the ELI3 gene is strongly activated both in elicitor-treated parsley cells and at fungal infection sites in parsley leaves, but not in lignifying tissue, suggest a specific role of this enzyme in pathogen defense-related phenylpropanoid metabolism.

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homolgous to a range of dehydrogenases including alcohol dehydrogenases, l-threonine-3-dehydrogenase, d-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dhydrogenase to provide the template, with additional input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands wasdesigned based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.

Use of Isozymes to Characterize Triticum aestivum — Aegilops markgrafii Addition Lines

Summary Sixteen leaf and three seed isozymes were tested for their ability to identify alien chromosomes in Triticum aestivum — Aegilops markgrafii amphiploid and six descending disomic addition lines. Four leaf isozymes (NADP-dependent aromatic alcohol dehydrogenase, aspartate aminotransferase, esterase, leucine aminopeptidase) and three seed enzymes (alcohol dehydrogenase, (s-amylase, esterase) from Ae. markgrafii were found to be informative in differentiating both the amphiploid and five of the six different addition lines from wheat. Leaf peroxidase, an eighth informative marker among the 19 isozyme systems tested was only in the amphiploid present, none of the six addition lines showed the Aegilops-specific band.

Polyethylene glycol dehydrogenating activity demonstrated by dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila M402.

Ethylene glycols and polyethylene glycols inhibited the growth of a photosynthetic bacterium, Rhodopseudomonas acidophila, strain M402 under aerobic-dark and anaerobic-light culture conditions. However, polyethylene glycol dehydrogenating activity was demonstrated with phenazine methosulfate (PMS) as an electron acceptor in parallel with the PMS-linked aromatic alcohol dehydrogenase activity. Addition of ethylene glycols or polyethylene glycols to the culture neither induced aromatic alcohol dehydrogenase, nor accelerated polyethylene glycol dehydrogenating activity. Purified PMS-linked aromatic alcohol dehydrogenase had high affinities toward diethylene glycol and polyethylene glycols. This indicated that the dye-linked aromatic alcohol dehydrogenase is capable of oxidizing these xenobiotic synthetic polymer alcohols, polyethylene glycols. There is no evidence on the existence of a specific polyethylene glycol dehydrogenase in this bacterium.

Purification and properties of dye-linked aldehyde dehydrogenase in Rhodopseudomonas acidophila M402.

Rhodopseudomonas acidophila M402 grown under aerobic-dark condition shows two bands of dye-linked aldehyde dehydrogenase activity by polyacrylamide gel electrophoresis. The slower-migrating band coincided with the dye-linked aromatic alcohol dehydrogenase that was active on aromatic and aliphatic alcohols and aldehydes. The faster-migrating band was a new dye-linked aldehyde dehydrogenase. The latter enzyme was purified 125-fold by ultracentrifugation and column chromatographies on DEAE-cellulose, Bio-Gel HTP, and Sepharose CL-6B. The enzyme has a relative molecular mass of 70,000 dal tons, and a subunit size of 35,000 dalton indicates the dehydrogenase has a dimeric structure. The isoelectric point was pH 4.74. NAD+ and NADP+ do not act as electron acceptors. The enzyme was active specifically on straight chain aldehydes (C3–C10) rather than on benzaldehyde and its substitutes. Aromatic and aliphatic alcohols were inert for this enzyme. The Michaelis constant (m\i) were 1.6, 0.6, 0.9, 3.6, 0.5, 0.3, 0.1,...

The NAD‐dependent alcohol dehydrogenase (ADH) of hansenula polymorpha cbs 4732 – an electrophoretic study (II)

AbstractUsing gradient polyacrylamide gel electrophoresis (gradient between 10–20%) we were able to show up to six clearly distinguishable ADH‐bands in Hansenula polymorpha CBS 4732. In contrast to commercial ADH from Saccharomyces cerevisiae the ADH‐forms of Hansenula polymorpha CBS 4732 have a much broader substrate specificity. Furthermore, the occurence of secondary alcohol dehydrogenases and of an aromatic alcohol dehydrogenase could be demonstrated.

Isoenzymes and their Importance for Breeding Autopolyploids

AbstractIsoenzyme analyses have been carried out in polyploid rye. Taking into consideration zymograms at the diploid level, conclusions can be drawn concerning the genetics of band positions and band intensity. This is rather clear with the aspartate aminotransferase and the NAD‐specific aromatic alcohol dehydrogenase. For both isoenzymes one gene locus with two alleles is responsible in the zones analyzed. In both cases a superdominance is assumed. Thereby nulliplex‐, simplex‐, duplex‐, triplex‐ and quadru‐plex types can be distinguished. For the banding pattern with the leucine aminopeptidase two gene loci with two alleles each are responsible. With allelic interaction codominance is supposed. On this base it is possible to differentiate between homozygosity and heterozygosity. Difficulties in autopolyploid breeding can be overcome by means of genetically‐analyzed isoenzymes as markers. This is demonstrated by examples.

Isoenzyme Variation in the Barley Genus Hordeum L. I. Alcohol Dehydrogenase and Superoxide Dismutase

Summary Electropboretic evidence is presented on the existence of three genetically independent isoenzymes, i.e. heterozymes of aliphatic alcohol dehydrogenase (ADH). One (ADH-A) is constitutive in the seed and young seedling, whereas two (ADH-B and ADH-D) are induced by anaerobiosis and by germination in the presence of 2,4-D. All three are dimeric, NAD-dependent, and capable of forming intergenic hybrid isoenzymes between each other. Considerable differences in the ability to induce ADH-B and ADH-D were found to exist between some species, Hordeum bulbosum and H. murinum s.l. representing the extreme cases. Aromatic alcohol dehydrogenase (AADH) exhibits its own system of heterozymes different from those of aliphatic alcohol dehydrogenase. The following heterozymes of AADH were described in the barley species: a dimerie NADP-dependent AADH-A; a monomeric NADP-dependent AADH-B; a slow-moving coon zyme-unspecific AADH-C, dominating in the root tissue; a dimeric NAD-dependent AADH-E; a leaf-specific, NADP-dependent AADH-F; and AADH-R, a fast-moving, rootspecific and NADP-dependent heterozyme. The degree of evolutionary clectrophoretic variation of the heterozymes studied proved to be quite limited in most species and within the whole barley genus, the barley species sharing many common electromorphs with the species of the Triticinae subtribe (wheat and rye). -Most of the clectrophoretic variation is inherent in two allogamous perennials, H. bulbosum and H. brevisubulatum s. l, whereas self-pollinating species proved essentially monomorphic or revealed rare additional allozymes. The diploid H. marinum s. str. and the tetraploid H. geniculatum, a morphologically very similar couple of species, can distinctly be distinguished from each other and from the remainder species by clectrophoretic and isoelectric phenotypes of SOD-A, an evolutionarily conservative chloroplastic heterozyme of superoxide dismutase.

NAD-dependent aromatic alcohol dehydrogenase in wheats (Triticum L.) and goatgrasses (Aegilops L.): evolutionary genetics

Evolutionary electrophoretic variation of a NAD-specific aromatic alcohol dehydrogenase, AADH-E, in wheat and goatgrass species is described and discussed in comparison with a NAD-specific alcohol dehydrogenase (ADH-A) and a NADP-dependent AADH-B studied previously. Cultivated tetraploid emmer wheats (T. turgidum s. l.) and hexaploid bread wheats (T. aestivum s. l.) are all fixed for a heterozygous triplet, E(0.58)/E(0.64). The slowest isoenzyme, E(0.58), is controlled by a homoeoallelic gene on the chromosome arm 6AL of T. aestivum cv. 'Chinese Spring' and is inherent in all diploid wheats, T. monococcum s. Str., T. boeoticum s. l. and T. urartu. The fastest isoenzyme, E(0.64), is presumably controlled by the B- and D-genome homoeoalleles of the bread wheat and is the commonest alloenzyme of diploid goat-grasses, including Ae. speltaides and Ae. tauschii. The tetraploid T. timopheevii s. str. has a particular heterozygous triplet E(0.56)/E(0.71), whereas the hexaploid T. zhukovskyi exhibited polymorphism with electromorphs characteristic of T. timopheevii and T. monococcum. Wild tetraploid wheats, T. dicoccoides and T. araraticum, showed partially homologous intraspecific variation of AADH-E with heterozygous triplets E(0.58)/E(0.64) (the commonest), E(0.58)/E(0.71), E(0.45)/E(0.58), E(0.48)/E(0.58) and E(0.56)/E(0.58) recorded. Polyploid goatgrasses of the D-genome group, excepting Ae. cylindrica, are fixed for the common triplet E(0.58)/E(0.64). Ae. cylindrica and polyploid goatgrasses of the C(u)-genome group, excepting Ae. kotschyi, are homozygous for E(0.64). Ae. kotschyi is exceptional, showing fixed heterozygosity for both AADH-E and ADH-A with unique triplets E(0.56)/E(0.64) and A(0.49)/A(0.56).

Isoenzymes of aromatic alcohol dehydrogenase in rye and triticale

One minor and two major genetically independent isoenzymes (heterozymes) of NADP-dependent aromatic alcohol dehydrogenase (labelled AADH-M, -A, and -B in the decreasing order of their anodaj mobility), one major heterozyme of NAD-specific AADH (labelled AADH-E), and one major coenzyme unspecific heterozyme (AADH-C) have been specified in rye seedlings by means of Polyacrylamide gel electrophoresis. Intraspecific and interspecific alloenzymic variation of the AADH heterozymes in the rye genus Secale and in some related species ( Triticale , wheat, wheatgrass) is described. Dimeric structure of the NADP-dependent AADH-A and of the NAD-dependent AADH-E and monomeric structure of the NADP-dependent AADH-B is supposed from the electrophoretic data.

Isoenzyme als biochemische markerfaktoren für roggenchromosomen

Summary The alcohol dehydrogenase, amylase and esterase zymotypes in endosperm and the aspartate aminotransferase, endopeptidase, esterase, glucosidase, leucine aminopeptidase, NAD- and NADP-dependent aromatic alcohol dehydrogenase, peroxidase and acid phosphatase zymotypes in leaves of Triticum aestivum cv. “Holdfast”, Secale cereale cv. “King II”, 8x-triticale “Holdfast-King II” and in 17 disomic or ditelosomic “King II” chromosome addition lines to “Holdfast” were determined by disc electrophoresis and isoelectric focusing, respectively. The results confirm informations on the localization of structural genes involved in the production of several isoenzymes on specific rye chromosomes or chromosome arms. Additionally the genetic control of multiple forms of glucosidase, leaf-esterase, NAD- and NADP-dependent aromatic alcohol dehydrogenase by three rye chromosomes is determined. The analysis of isoenzyme markers demonstrated chromosome changes in 5 of 17 addition lines. The modifications of rye chromosomes are less surprising than the occurrence of telosomic wheat chromosomes. These findings explain the necessity of a permanent control in maintaining and propagating such plant material. The analysis of isoenzymes can be used as a valuable tool for this control.

Nachweis von multiplen Formen der Alkoholdehydrogenase in Blattmaterial von Triticum aestivum L. “Carola”

Summary Multiple forms of the NAD-dependent aromatic alcohol dehydrogenase were analysed in leaves of monosomic plant offsprings of the wheat cultivar “Carola”. By referring by the leucine aminopeptidase zymotypes we succeeded in distinguishing for one chromosome each of the group 6 nulli-, mono- or disomic plants. Disc-electrophoretic fractionation of the NAD-AADH showed zymograms with maximum four bands. Three are considered as homo- and/or heterodimeric enzyme forms (NAD-AADH- 1: ββ, δδ, βδ, NAD-AADH-2: αδ, αδ, NAD-AADH-3: αα). The protein subunits α, β and δ are coded by one structural gene, on each chromosomes 6A, 6B and 6D.

NADP-dependent aromatic alcohol dehydrogenase in polyploid wheats and their diploid relatives. On the origin and phylogeny of polyploid wheats

The three major isoenzymes of the NADP-dependent aromatic alcohol dehydrogenase (ADH-B), distinguished in polyploid wheats by means of polyacrylamide gel electrophoresis, are shown to be coded by homoeoalleles of the locus Adh-2 on short arms of chromosomes of the fifth homoeologous group. Essentially codominant expression of the Adh-2 homoeolleles of composite genomes was observed in young seedlings of hexaploid wheats (T. aestivum s.l.) and tetraploid wheats of the emmer group (T. turgidum s.l.), whereas only the isoenzyme characteristic of the A genome is present in the seedlings of the timopheevii-group tetraploids (T. timopheevii s.str. and T. araraticum).The slowest-moving B(3) isoenzyme of polyploid wheats, coded by the homoeoallele of the B genome, is characteristic of the diploid species Aegilops speltoides S.l., including both its awned and awnless forms, but was not encountered in Ae. bicornis, Ae. sharonensis and Ae. longissima. The last two diploids, as well as Ae. tauschii, Ae. caudata, Triticum monococcum s.str., T. boeoticum s.l. (incl. T. thaoudar) and T. urartu all shared a common isoenzyme coinciding electrophoretically with the band B(2) controlled by the A and D genome homoeoalleles in polyploid wheats. Ae. bicomis is characterized by the slowest isoenzyme, B(4), not found in wheats and in the other diploid Aegilops species studied.Two electrophoretic variants of ADH-B, B(1) and B(2), considered to be alloenzymes of the A genome homoeoallele, were observed in T. dicoccoides, T. dicoccon, T. turgidum. s.str. and T. spelta, whereas B(2) was characteristic of T. timopheevii s.l. and only B(1) was found in the remaining taxa of polyploid wheats. The isoenzyme B(1), not encountered among diploid species, is considered to be a mutational derivative which arose on the tetraploid level from its more ancestral form B(2) characteristic of diploid wheats.The implication of the ADH-B isoenzyme data to the problems of wheat phylogeny and gene evolution is discussed.