Aryl-alcohol Dehydrogenase Research Articles

Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme.

A cDNA clone encoding a ligninolytic aryl-alcohol dehydrogenase (AAD; EC 1.1.1.91) from the white-rot basidiomycete fungus Phanerochaete chrysosporium was isolated and characterized. The nucleotide sequence obtained reveals an open reading frame encoding a protein of 385 amino acids. Substantial homology (49.3% identity and 67.3% similarity, respectively) was observed between AAD and an open reading frame sequence present on chromosome III of Saccharomyces cerevisiae. A Southern blot analysis showed the presence of multiple AAD gene-related sequences in P. chrysosporium and in other white-rot fungi including Bjerkandera adusta and Fomes lignosus. Northern blot analyses are in line with the view that the levels and appearance of AAD mRNA correlate with the level and appearance of AAD activity and that, under conditions of nitrogen limitation, the AAD mRNA levels are higher than in carbon limited cultures. This is consistent with the regulation of the enzyme by carbon or nitrogen limitation being at the level of transcription. Moreover, the appearance of AAD-specific transcripts correlates with the appearance of lignin peroxidase-specific transcripts in the same cultures. This co-appearance is in line with the proposed synergistic interaction of the two enzymes in lignin biodegradation, which suggests a similar regulation. The AAD encoding cDNA was expressed in Escherichia coli to yield high levels of active enzyme, and the recombinant enzyme was purified by using metal chelate affinity chromatography.

Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H 2 O 2 Production by Pleurotus eryngii

The existence of a redox cycle leading to the production of hydrogen peroxide (H(2)O(2)) in the white rot fungus Pleurotus eryngii has been confirmed by incubations of 10-day-old mycelium with veratryl (3,4-dimethoxybenzyl) and anisyl (4-methoxybenzyl) compounds (alcohols, aldehydes, and acids). Veratraldehyde and anisaldehyde were reduced by aryl-alcohol dehydrogenase to their corresponding alcohols, which were oxidized by aryl-alcohol oxidase, producing H(2)O(2). Veratric and anisic acids were incorporated into the cycle after their reduction, which was catalyzed by aryl-aldehyde dehydrogenase. With the use of different initial concentrations of either veratryl alcohol, veratraldehyde, or veratric acid (0.5 to 4.0 mM), around 94% of veratraldehyde and 3% of veratryl alcohol (compared with initial concentrations) and trace amounts of veratric acid were found when equilibrium between reductive and oxidative activities had been reached, regardless of the initial compound used. At concentrations higher than 1 mM, veratric acid was not transformed, and at 1.0 mM, it produced a negative effect on the activities of aryl-alcohol oxidase and both dehydrogenases. H(2)O(2) levels were proportional to the initial concentrations of veratryl compounds (around 0.5%), and an equilibrium between aryl-alcohol oxidase and an unknown H(2)O(2)-reducing system kept these levels steady. On the other hand, the concomitant production of the three above-mentioned enzymes during the active growth phase of the fungus was demonstrated. Finally, the possibility that anisaldehyde is the metabolite produced by P. eryngii for the maintenance of this redox cycle is discussed.

Anisaldehyde production and aryl-alcohol oxidase and dehydrogenase activities in ligninolytic fungi of the genus Pleurotus.

A variety of simple aromatic compounds were identified in liquid cultures of the basidiomycetes Pleurotus cornucopiae, P. eryngii, P. floridanus, P. pulmonarius, P. ostreatus, and P. sajor-caju by using gas chromatography-mass spectrometry. Such compounds were detected in fungal cultures on lignin- and straw-containing media, but it was found that they were also produced in the absence of aromatic precursors. Anisylic and hydroxybenzylic compounds (such as alcohols, aldehydes, and acids) were identified, p-anisaldehyde being the most characteristic extracellular metabolite synthesized by these ligninolytic fungi. Small amounts of 3-chloro-p-anisaldehyde were also detected in several species. It is postulated that the balance between the more-or-less-oxidized aromatic compounds can be explained in terms of the activity of fungal enzymes, including aryl-alcohol oxidase and dehydrogenase. The former enzyme shows high affinity for p-anisyl alcohol, which is oxidized to p-anisaldehyde with production of H2O2. The aryl-alcohol dehydrogenase was detected only in the mycelium, where it reduces aromatic aldehydes in the presence of NADPH. Both enzymes could be involved in the redox cycling of these aromatic compounds, providing H2O2 to ligninolytic peroxidases.

Purification and properties of an aryl‐alcohol dehydrogenase from the white‐rot fungus Phanerochaete chrysosporium

An intracellular aryl-alcohol dehydrogenase (previously referred to as aryl-aldehyde reductase) was purified from the white-rot fungus Phanerochaete chrysosporium. The enzyme reduced veratraldehyde to veratryl alcohol using NADPH as a cofactor. Other aromatic benzaldehydes were also reduced, but not aromatic ketones. Methoxy-substituted rings were better substrates than hydroxylated ones. The enzyme was also able to reduce a dimeric aldehyde (4-benzyloxy-3-methoxybenzaldehyde). The highest reduction rate was measured when 3,5-dimethoxybenzaldehyde was used as a substrate. On SDS/PAGE the purified enzyme showed one major band with a molecular mass of 47 kDa, whereas gel filtration suggested a molecular mass of 280 kDa. Polyclonal antibodies raised against the gel purified 47-kDa protein were able to immunoprecipitate the aryl-alcohol dehydrogenase indicating that its activity possibly resides entirely in this protein fragment. The pI of the enzyme was 5.2 and it was most active at pH 6.1. The aryl-alcohol dehydrogenase was partially inhibited by typical oxidoreductase inhibitors.

Aldehyde Reductase from Crithidia fasciculata: Purification and Characterization

Crithidia fasciculata, a pterin requiring protozoa, contained a NADPH-dependent dihydro-6-formylpterin reducing enzyme. The aldehyde reductase was purified and characterized. This enzyme was soluble and constitutive, and had the ability to catalyze the interconversion between aldehydes and alcohols. The optimal pH was 7.0 for the reductase activity and 9.0 for the dehydrogenase activity. The enzyme had a broad substrate specificity and reduced aromatic and aliphatic aldehydes such as dihydro-6-formylpterin, benzaldehyde, pyridine-3-aldehyde, butyraldehyde, DL-glyceraldehyde, and acetaldehyde. The enzyme was inhibited by sulfhydryl reagents and heavy metals, but not inhibited by chelating reagents. Its molecular weight was determined to be 66,000 and 72,000 by gel filtration and sedimentation equilibrium analysis, respectively. The value of 39,000 was obtained by sodium dodecyl sulfate gel electrophoresis indicating a dimeric structure. The aldehyde reductase of Crithidia may be classified as NADP+-dependent aryl-alcohol dehydrogenase [EC 1.1.1.91].

Metablites of [formula omitted] acid V. Isolation and properties of [formula omitted] alcohol dehydrogenase

An aryl alcohol dehydrogenase (aryl alcohol: NADP +oxidoreductase. EC 1.1.1.91), that catalyzes the interconversion of benzyl alcohol and benzaldehyde as well as the oxidation of p- aminobenzyl alcohol has been isolated from Mycobacterium tuberculosis cells (American Type Culture Collection No. 607) grown in the presence of p- aminobenzoic acid. The partially purified enzyme shows a major peak with a minor contaminating component upon ultracentrifugation. The enzyme shows a broad substrate specificity and is sensitive to the buffer composition. The enzyme is inhibited by p- chloromercuribenzoate and 1,10-o- phenanthroline . The apparent K m values and the calculation of the approximate equilibrium constant are also presented.