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

Abstract

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. Methoxysubstituted 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 SDSjPAGE 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.Lignin, the structural component of woody plants, is a highly irregular, three-dimensional biopolymer composed of oxygenated phenylpropane units.White-rot fungi are the most effective lignin degraders in nature [l]. Since the discovery of lignin peroxidases in P. chrysosporium, tremendous progress has been made in understanding the mechanism of lignin biodegradation by fungi. Presently most of the oxidative reactions observed in lignin biodegradation can be rationalized by one-electron oxidations of phenols [e.g. by laccase or Mn(I1)-dependent peroxidase to yield phenoxy-radicals] and of non-phenolic compounds by lignin peroxidase to yield radical-cation intermediates [2]. Spontaneous chemical reactions triggered by the radicals formed appear to lead to the observed degradative reactions like Ca-Cfl bond cleavage, demethoxylation, aromatic ring cleavage etc. [3].However, when isolated lignins are reacted with laccases or peroxidases in vitro polymerization occurs [4]. We have thus suggested that in addition to oxidative reactions at least two further phenomena are needed for lignin catabolism by whiterot fungi to take place. Firstly, peroxidase-induced polymerization and peroxidase-induced depolymerization results in a dynamic system where the equilibrium may be shifted towards degradation by fungal uptake of smaller fragments. Secondly, the importance of reductive reactions in lignin degradation has been re-emphasized [5]. Veratryl alcohol (3,4-dimethoxybenzyl alcohol) is a secondary metabolite of P. chrysosporium [6] and appears to have an important role in lignin biodegradation itself [7]. Veratryl alcohol is also metabolized by the fungus and thus serves as the most simple model compound for lignin degradation. The oxidation and metabolism of this compound and of its methyl ether, shows several key reactions for lignin degradation. The first discovery was the lignin peroxidase-mediated aromaticring opening [8]. Secondly, the involvemen...

Novel Scheme for Biosynthesis of Aryl Metabolites from l -Phenylalanine in the Fungus Bjerkandera adusta

“…AAO activity was quantified by measuring the oxidation of veratryl alcohol to veratraldehyde (22). AAD activity was quantified by measuring the oxidation of NADPH during reduction of veratraldehyde, as previously described (23). AADD activity was measured by using veratric acid as the substrate (9).…”

“…Guillén and Evans (9) have suggested that intracellular dehydrogenases, such as aryl-aldehyde dehydrogenase (AADD), reduce aromatic acids to aldehydes and that aryl-alcohol dehydrogenase (AAD) reduces aromatic aldehydes to alcohols in Pleurotus eryngii. An intracellular AAD was purified from the fungus P. chrysosporium (23). This enzyme reduced veratraldehyde and other aryl aldehydes to veratryl alcohol and corresponding aryl alcohols by using NADPH as a cofactor.…”

Polyacrylamide Gel Electrophoresis an Important Tool for the Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms

“…In the case of NADP + -dependent trans-dihydrodiol dehydrogenases, almost all investigations have been done in mammalian tissues (Carbone et al, 2008;Chang et al, 2009;Chen et al, 2008) but only a few reports about these important enzymes have been done in fungi (Bezalel et al, 1997;Hammel, 1995;Sutherland et al, 1993) particularly in Phanerochaete chrisosporium (Bogan & Lamar, 1996;Muheim et al, 1991). At date, there is no any report about the detection of dihydrodiol dehydrogenase activities by means of electrophoretic zymograms in any organism.…”

Chiral alcohol production by NADH‐dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH

“…Among the ketones tested, the e.e. values of the produced alcohols (19)(20)(21)(22)(23)25) were > > 99%, except those of ECHB (24) and isopropyl 4-bromo-3-hydroxybutanoate (26), which were 98 and 98.4%, respectively.…”