Veratryl alcohol oxidation

These findings led to the proposition that the veratryl alcohol is degraded via the quinone intermediates (Figure 5) to CO2 through a series of transformations involving lignin peroxidase, perhydroxy radicals and the NADP-dependent aryl alcohol oxidoreductase. Veratraldehyde, the major product of lignin peroxidase catalyzed veratryl alcohol oxidation, is rapidly reduced back to veratryl alcohol it is the further metabolism of the side products of the oxidative process, viz. the quinones and lactones, that drives the overall transformation towards completion (34). 

In Figure 1 the isolated reaction products of the veratryl alcohol oxidation are depicted. 

Oxygen uptake during substrate oxidation was measured with a Clark oxygen electrode (Rank Brothers, Cambridge, U.K.) at room temperature with 1 mM substrate in 0.25 mM sodium tartrate buffer, pH 3.0 (3 mL). Rates are expressed relative to veratryl alcohol oxidation. 

As already mentioned, in some enzymes radicals generated on surface tryptophan and tyrosine radicals by electron transfer to the ferryl species are involved in abstraction of electrons from substrates [33-36, 40]. Mutation of Trpl71 on the surface of P. chrysosporium LiP to a phenylalanine or serine completely suppresses the veratryl alcohol oxidizing activity of the enzyme [40]. A similar depression in the oxidation of veratryl alcohol occurs on mutation of Trpl64 in the versatile peroxidase from P. eryngii [34, 35]. 

Lignin peroxidase Phanerochaete 3.0 Veratryl alcohol oxidation [10-12]... 

D Annibale and his colleagues [99] performed experiments on VA oxidation using MnP from Lentinus edodes. They demonstrated two different types of reactions occurring in the presence and absence of GSH. When GSH was not included in the reaction mixture of MnP, Mn +, VA, H2O2 and chelator, aromatic ring cleavage, side chain oxidization and dimerization of VA were detected. (The main metabolite was y-muconolactone (58%), and three dimerization products were also observed [31] 2.3 and 1.8%, respectively). However, with the addition of GSH, veratraldehyde was the only metabolite formed. Veratryl alcohol oxidation was 32 and 14% in nonthiol and thiol mediated reactions, respectively. Under both conditions, oxidation depended strictly on the presence of both Mn + and H2O2. 

A D Annibale, C Crestini, E De Mattia, GG Sermanni. Veratryl alcohol oxidation by manganese-dependent peroxidase from Lentinus edodes. J Biotechnol 48 231-239, 1996. 

The addition of 0.5 equivalent of a two-electron reducing agent like veratryl alcohol generates LiP-Cpd II (with a Soret band at 420 nm), which corresponds to the iron(IV) 0x0 without oxidation of the porphyrin ligand. Site-directed mutations have demonstrated a key role for Trp-171 in veratryl alcohol oxidation, this amino acid being probably the location of an intermediate radical cation in the oxidation cascade. 

Figure 3. Peroxidase activity in nutrient nitrogen deficient cultures of P. chrysosporium. Manganese dependent (open square) and lignin (closed diamond) peroxidase activities were determined spectro-photometrically by following guaiacol and veratryl alcohol oxidation, respectively.

Another iron porphyrin complex with 5,10,15,20-tetrakis(2, 6 -dichloro-3 -sulfonatophenyl)porphyrin was applied in ionic liquids and oxidized veratryl alcohol (3,4-dimethoxybenzyl alcohol) with hydrogen peroxide in yields up to 83% to the aldehyde as the major product [145]. In addition, TEMPO was incorporated via... 

VP (E.C. 1.11.1.46) was first described in liquid cultures of Pleurotus eryngii growing on peptone as nitrogen source [82, 83] and Bjerkandera sp. [84], VP is a heme containing structural hybrid between MnP and LiP, as it is able to oxidize Mn2+, veratryl alcohol, simple amines, phenolic, nonphenolic and high molecular... 

Although compound I formation is not influenced by pH, reactions of compounds I and II are significantly affected by pH. These reactions are acid-catalyzed 16,17). The rate constant for the oxidation of veratryl alcohol or fenocyanide by lignin peroxidase compound I is 10 times greater at pH 3.5 than at pH 6.0. The enhancement in rate is of the same magnitude for compound II reacting with veratryl alcohol. Therefore, the observed pH dependency for Vmax in catalysis is due to the pH-dependent reactions between the compounds I and n and the reducing substrates. 

Activity Assays. The standard activity assay mixture of 3 ml contained about 0.1 U/ml lignin peroxidase, 0.4 mM veratryl alcohol (Fluka, purum >97%) and 0.1M sodium tartrate, pH 3.0. The reaction was started by adding 15 fil of 54 mM H2O2 to make a final concentration of 0.28 mM in the reaction. The production of veratraldehyde was followed by recording the change of absorbance for 12 seconds at 310 nm in a cuvette which was thermostated to 37°C. The reaction was started 24 seconds before the recording. One unit of lignin peroxidase is defined as the amount of enzyme required to oxidize one imol of veratryl alcohol to veratraldehyde in one minute. 

LiP catalyzes the oxidation of a low-molecular-weight redox mediator, veratryl alcohol, which in mrn mediates one-electron oxidation of lignin to generate aryl cation radicals [100]. The radicals facilitate a wide variety of reactions such as carbon-carbon cleavage, hydroxylation, demethylation, and so on. Dezotti et al. [101] reported enzymatic removal of color from extraction stage effluents using lignin and horseradish peroxidases immobilized on an activated silica gel support. 

In our studies on the lignin peroxidase catalyzed oxidation of veratraldehyde and veratric acid no degradation was observed under the reaction conditions used. However, ligninolytic cultures of P. chrysosporium degraded veratraldehyde and veratric acid via reduction to veratryl alcohol... 

Reductase Activity Measurement. Reductase activity was measured by the reduction of 200 /iM veratraldehyde to veratryl alcohol in the presence of 250 /tM NADPH. The reaction was carried out in 20 raM Tris buffer pH 7.4 (optimal pH = 6) with 20% glycerol and 1 mM EDTA. The decrease in absorbance of NADPH at 365 nm was measured. NADPH was slowly oxidized in those mixtures which were not purified by ion exchange even in the absence of veratraldehyde. This unspecific reaction was considered in the calculation of the reductase activity. One unit of reductase reduced 1 /imol min-1 of veratraldehyde to veratryl alcohol at room temperature (25°C). 

It has now been found that the quinones 13, 14 and 15 from the aerobic oxidation of veratryl alcohol by lignin peroxidase were also reduced by fungal mycelium to yield the corresponding hydroquinones. For quinone 14 this reduction had already been reported by Buswell et al. (17) in a study of vanillic acid metabolism. 

Metabolism of Monomeric Lignin Models. Leisola and coworkers (31,37) have reported on the products of the lignin peroxidase catalyzed oxidation of veratryl alcohol. Veratraldehyde was the major product (> 70% yield), together with a number of minor products, the quinones 13 and 14 and the ringopened lactones 16 and 17. In addition, Shimada et al. (38,39) showed that the 6-lactone 18 was also formed. Recently we obtained evidence that the ortho-quinone 15 and the 6-lactone 19 were also products of the lignin peroxidase catalyzed oxidation of veratryl alcohol (Schmidt ef al, Biochemistry, in press). Mechanisms for the formation of those compounds... 

In this scheme veratryl alcohol was viewed to be metabolized by the combined action of oxidative systems (the lignin peroxidase and possibly other active oxygen species) and reductive conversions (aldehyde and quinone reductions). A possible route via veratric acid was discounted because both veratraldehyde and veratric acid were not substrates for the lignin peroxidase under the condition studied. However, both veratraldehyde and veratric acid were rapidly and quantitatively reduced by ligninolytic cultures of P. chrysosporium (21). 

VAO activity was measured with a mixture of 1 mM veratryl alcohol, 250 mM sodium tartrate buffer, pH 5.0 and enzyme. Oxidation to vera-... 

Lignin peroxidase activity, (i.e., peroxide-dependent oxidation of veratryl alcohol at pH 3) was not detected over the 30 days tested, while laccase appeared at day 7. Culture medium from day 7 onwards could also oxidize veratryl alcohol to aldehyde with concomitant conversion of oxygen to hydrogen peroxide. This activity, which was optimal at pH 5.0, was named veratryl alcohol oxidase (VAO). The extracellular oxidative enzyme activities (laccase and veratryl alcohol oxidase) could be separated by ion-exchange chromatography (Figure 2). Further chromatography of the coincident laccase and veratryl alcohol oxidase (peak 2), as described elsewhere (25) resulted in the separation of two veratryl alcohol oxidases from the laccase. 

Enzyme Properties. The two isolated veratryl alcohol oxidases had very similar properties (Table I). The difference in isoelectric points might be accounted for by aspartate content all other amino acid contents except glycine were the same within experimental error (5%). The specific activities (veratryl alcohol as substrate) were significantly different, but both enzymes contained a flavin prosthetic group (25) and converted one molecule of oxygen to one molecule of hydrogen peroxide during alcohol oxidation. 

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