Patricia J. Harvey

Veratryl alcohol as a mediator and the role of radical cations in lignin biodegradation by Phanerochaete chrysosporium

The oxidative capacity of the ligninase from Phanerochaete chrysosporium toward monomethoxylated and dimethoxylated aromatic compounds was investigated. Phenylacetic acid derivatives were shown to be decarboxylated by the ligninase, via C‐C bond cleavage. Dimethoxylated substrates were much more readily oxidised by the ligninase than were monomethoxylated derivatives, but oxidation of the latter could be stimulated by catalytic amounts of 1,4‐dimethoxybenzene or veratryl alcohol. A mechanism based upon the ability of radical cations to function as one‐electron oxidants is described. The role of redox mediators in lignin degradation, and the biological significance of veratryl alcohol as a secondary metabolite of P. chrysosporium are discussed.

Phytoremediation of polyaromatic hydrocarbons, anilines and phenols.

Phytoremediation technologies based on the combined action of plants and the microbial communities that they support within the rhizosphere hold promise in the remediation of land and waterways contaminated with hydrocarbons but they have not yet been adopted in large-scale remediation strategies. In this review plant and microbial degradative capacities, viewed as a continuum, have been dissected in order to identify where bottlenecks and limitations exist. Phenols, anilines and polyaromatic hydrocarbons (PAHs) were selected as the target classes of molecule for consideration, in part because of their common patterns of distribution, but also because of the urgent need to develop techniques to overcome their toxicity to human health.Depending on the chemical and physical properties of the pollutant, the emerging picture suggests that plants will draw pollutants including PAHs into the plant rhizosphere to varying extents via the transpiration stream. Mycorrhizosphere-bacteria and -fungi may play a crucial role in establishing plants in degraded ecosystems. Within the rhizosphere, microbial degradative activities prevail in order to extract energy and carbon skeletons from the pollutants for microbial cell growth. There has been little systematic analysis of the changing dynamics of pollutant degradation within the rhizosphere; however, the importance of plants in supplying oxygen and nutrients to the rhizosphere via fine roots, and of the beneficial effect of microorganisms on plant root growth is stressed.In addition to their role in supporting rhizospheric degradative activities, plants may possess a limited capacity to transport some of the more mobile pollutants into roots and shoots via fine roots. In those situations where uptake does occur (i.e. only limited microbial activity in the rhizosphere) there is good evidence that the pollutant may be metabolised. However, plant uptake is frequently associated with the inhibition of plant growth and an increasing tendency to oxidant stress. Pollutant tolerance seems to correlate with the ability to deposit large quantities of pollutant metabolites in the ‘bound’ residue fraction of plant cell walls compared to the vacuole. In this regard, particular attention is paid to the activities of peroxidases, laccases, cytochromes P450, glucosyltransferases and ABC transporters. However, despite the seemingly large diversity of these proteins, direct proof of their participation in the metabolism of industrial aromatic pollutants is surprisingly scarce and little is known about their control in the overall metabolic scheme. Little is known about the bioavailability of bound metabolites; however, there may be a need to prevent their movement into wildlife food chains. In this regard, the application to harvested plants of composting techniques based on the degradative capacity of white-rot fungi merits attention.

On the mechanism of enzymatic lignin breakdown

The recent discovery of an extracellular enzyme from Phanerochaete chrysosporium capable of degrading lignin model compounds has countered the view that reactive diffusible oxygen species are responsible for lignin biodegradation. In this paper we propose a mechanism which accounts for both the results obtained in enzymatic lignin degradation studies and in studies involving active oxygen species. The quintessence of the mechanism is initial one‐electron oxidation of the lignin model compounds or of a specific lignin subunit followed by subsequent breakdown reactions via radical cation intermediates. The implication of this type of mechanism on the oxidative biodegradation of the natural lignin polymer is discussed.

Single‐electron transfer processes and the reaction mechanism of enzymic degradation of lignin

Lignin degradation Single‐electron transfer Cα‐Cβ bond cleavage Phanerochaete chrysosporium Peroxidase compound I

On the mechanism of oxidation of non-phenolic lignin model compounds by the laccase-ABTS couple

On the Mechanism of Oxidation of Non-Phenolic Lignin Model Compounds by the Laccase-ABTS Couple By Andreas Muheim, Armin Fiechter, Patricia J. Harvey and Hans E. Schoemaker 1 Eidgen ssische Technische Hochschule Z rich, Institut f r Biotechnologie, ETH-H nggerberg, CH-8093 Z rich, Switzerland 2 Imperial College of Science,Technology and Medicine, Department of Biology, Prince Consort Road, London SW7 2BB, United Kingdom 3 DSM Research, Bio-organic Chemistry Section, P.O. Box 18, NL-6160 MD Geleen,The Netherlands

Oxidation of phenolic compounds by ligninase.

The kinetics of oxidation of phenolic compounds by ligninase was investigated in the presence and absence of dimethoxylated compounds (veratryl alcohol and 3,4-dimethoxyphenyl acetic acid). In all cases, the phenolic compounds were found to be preferentially oxidised compared to the dimethoxylated compounds. Veratryl alcohol but not 3,4-dimethoxyphenyl acetic acid enhanced their oxidation, but only when present in at least 200-fold molar excess compared with the phenolic compounds. Ligninase was inactivated in the course of oxidation of the phenolic compounds. Inactivation was associated with the accumulation of compound III, formed by reaction of compound II with H2O2. Inactivation was reversed with additions of more enzyme but not with additions of veratryl alcohol. Evidence of inactivation was also obtained during the course of veratryl alcohol oxidation, but the extent was much less, supporting the concept of a substrate-modified compound II intermediate able to promote reaction with reductant over reaction with H2O2. A model to describe the mechanism by which ligninase catalyses net depolymerisation of lignin as opposed to further polymerisation is presented. It involves spatial separation between ligninase, lignin and phenolic lignin breakdown products and invokes the concepts of radical cations as mediators between enzyme and lignin as well as of radical cation charge transfer reactions through the structure of lignin.

Prospects for the phytoremediation of organic pollutants in Europe.

Reference LBE-ARTICLE-2002-007View record in Web of Science Record created on 2005-03-02, modified on 2016-08-08

Isolation and characterization of the storage protein of yam tubers (Dioscorea rotundata)

Abstract The major proteins of the yam tuber, which were identified as storage proteins by virtue of their abundance ( ca 85 % of the total protein content), amino acid composition (high in amide content) and cellular location within the tuber, were isolated by ion-exchange chromatography and characterized using, in particular, polyacrylamide gel techniques. They consist principally of subunits of one size, apparent MW 31 000, and N -terminal amino acid glutamine/glutamic acid, of which there are a number of charge isomers; these usually contain one intra-chain disulphide bond. The subunits associated into polymers depending on the protein concentration, pH value and ionic strength of the milieu and, therefore, a value for the MW of the native protein(s) is not given. The storage proteins are not glycoproteins. They are intracellularly located as protein ’aggregates‘ within cellular protein vacuoles, and also within the cytoplasm.

Pre-steady-state kinetic study on the formation of compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2.10(5) M-1.s-1) for the formation of Compound I was independent of pH (pH 3.0-6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5-10 s at pH 3.1. The rate of this reaction was not affected by the H2O2 concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9.10(4) M-1.s-1 to 6.8.10(6) M-1.s-1 when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0.10(3) M-1.s-1 at pH 6.0 to 1.0.10(5) M-1.s-1 at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.

The role of peroxidases, radical cations and oxygen in the degradation of lignin

Ligninase is an extracellular peroxidase produced by several species of white-rot fungi. It is able to oxidize methoxylated substrates to radical cation intermediates that can undergo C—H or C—C bond cleavage, thereby providing the basis for the oxidation of veratryl alcohol or degradation of lignin model compounds respectively. In some cases, the radical cation intermediate can act as an oxidant, accepting an electron from a suitable donor. It can thus function as a mediator, causing oxidation in a polymer not immediately accessible to the enzyme. This could be important in the degradation of natural lignocellulose substrates. However, the removal of a single electron by a mediator would leave a radical in the polymer. We propose that oxygen will bind to this radical to generate active oxygen species. This provides a potential mechanism for the auto-oxidation of lignin at a distance from the enzyme. A scheme is presented to account for the observation that ligninase can open the ring of veratryl alcohol.