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AIR2-CT93-1661
Structure, Function and Industrial Applications of Plant Laccases and Peroxidases |
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Proposal No: | AIR2-CT93-1661 |
| Date Prepared: | September 1999, October 1996 | |
| Source: | Final report December 1998 |
Summary
Objectives This project concerned phenol oxidases (laccases) and peroxidases, mainly from higher plants. These two groups of enzymes oxidise aromatic compounds, using oxygen and hydrogen peroxide respectively as the electron acceptor. The electron donors are diverse aromatic substances such as the phenolic precursors of lignin synthesis in wood or certain coloured pollutants. The work thus had both basic and commercial significance. Oxidases and peroxidases are distinct groups of enzymes; it is therefore a challenge to define their distinctive biological roles. In particular, at the start of the project there was controversy as to which group is the more important in lignification.
Activities The enzymes used in this work came from several complementary sources. Native laccases were isolated from abundant natural sources (poplar and the fungus Rigidoporus lignosus) and novel native peroxidases from poplar xylem, Arabidopsis cell cultures and zucchini. Later, the genes for numerous plant laccases (poplar and tobacco) and peroxidases (poplar and Arabidopsis), were identified and the encoded proteins expressed in heterologous systems (poplar laccase and some of the Arabidopsis peroxidases in Escherichia coli, other Arabidopsis peroxidases and poplar xylem peroxidases in baculovirus-transformed insect cells). This provided sufficient quantities of enzyme for thorough chemical, biochemical and molecular biological characterisation.
To enable an evaluation of the potential of the new peroxidases for enzyme-linked immunoassay kits, peroxidases and Rubella antigen E1 were expressed insect cell lines and the products tested in reference immunoassays.
Arabidopsis peroxidases were characterised by X-ray crystallography at unprecedented resolution, the metallochemistry and glycobiology of the laccases were described in depth, and numerous Arabidopsis peroxidases were shown to differ in catalytic kinetics. No significant differences between the various laccases and peroxidases could be found in substrate preference or reaction products formed. Novel phenolic components of the plant cell wall (potential substrates for peroxidases and laccases) were discovered and characterised chemically, and it was shown that wall phenolics can be extensively cross-linked by peroxidases and/or laccases within the protoplast, prior to secretion into the cell wall. A novel interaction of peroxidase, ascorbate and hydroxyl radicals was described, potentially a new chapter in understanding plant cell growth and wall-turnover in vivo.
To assess the biological roles and to characterise the expression of the enzymes in Planta, transformant plants over- and under-expressing several of the enzymes (laccase-modified poplar and tobacco; peroxidase-modified poplar) were produced. An important observation in this work was that none of the laccases could be shown to be essential for lignification: this finding shifts emphasis to peroxidases as the more likely catalysts of lignification. This view was backed up by in vitro enzyme assays, which showed that plant laccases have very low specific activities and would thus be unlikely to occur in xylem in sufficient amounts to account for observed rates of lignin biosynthesis.
Introduction
Peroxidases and laccases are enzymes that oxidise a wide range of aromatic compounds, using hydrogen peroxide and oxygen, respectively, as the electron acceptor. These enzymes occur in plants and fungi, where they presumably serve different physiological roles. The electron donor substrates for these enzymes are diverse aromatic substances including:
The enzymes are therefore not only important in plant and fungal physiology but also possess considerable industrial potential. Typical reactions catalysed by these two groups of enzymes can result in pairs of aromatic molecules becoming cross-linked to give a diversity of products. The laccases contain copper and the peroxidases contain iron. For this and many other reasons, they are quite distinct groups of enzymes, although the aromatic end-products of their action are not necessarily different. It is therefore an important challenge, and the basic aim of the present collaborative project, to define their respective structures, enzymological properties, expression, biological roles, and applicability in industry. The eight principal, inter-related aims of the present project were as indicated in the next section.
Results
To purify certain laccases and peroxidases from abundant natural sources Two coniferyl alcohol oxidising enzymes were identified in crude cell wall extracts of poplar xylem. One was purified to homogeneity and its physico-chemical properties were determined. A purification protocol was developed that enabled separation and purification of the anionic peroxidases present in poplar xylem. The subsequent protein characterisation revealed that two of the isoenzymes could be correlated with lignifying tissues.
To identify the genes for several plant laccases and peroxidases The corresponding cDNA clones for both proteins were obtained from a poplar xylem library along with three other laccase cDNAs obtained by heterologous screening with an Acer laccase cDNA. All five genes contain the expected features of laccases including a hydrophobic signal peptide, potential N-glycosylation sites, blue copper oxidase signature, etc. As determined by Northern analysis and RT-PCR, all five are most highly expressed in stem tissue.
Screening of a tobacco stem cDNA library with a heterologous sycamore cDNA probe has led to the isolation of three laccase-encoding cDNA clones. They were characterised by sequencing and shown to be different. One of them corresponds to a full-length cDNA sequence that was subsequently used to prepare different gene constructs in order to under- or over-express the corresponding gene in transgenic tobacco plants. The corresponding cDNA and genomic clones were isolated for the lignin-correlated peroxidase isoenzyme PXP 3-4. Surprisingly, all xylem peroxidase clones isolated contained a putative vacuolar targeting signal. Because homologues that were closely related to PXP 3-4 were also isolated, an epitope tagging strategy was developed for in situ localisation of PXP 3-4 expression only.
To produce trangformant plants over- or under-expressing the cloned laccases and peroxidases Antisense constructs were made with four of the five poplar laccase genes and used in poplar transformation experiments. Transgenic poplars expressing low levels of laccase transcripts were obtained for three of the four antisense populations.
To produce high levels of specific laccases and peroxidases by expression of the cloned genes in heterologous systems
Work was concentrated on the cloning and E. coli expression of the two extracellular neutral ATP N and anionic ATP A2 peroxidases. The former had never been seen as a protein and therefore its properties were unknown, whereas the horseradish homologue of the latter, HRP A2, was well-characterised and desirable due to a greater turnover stability, but had never been cloned. ATP A2 was isolated and cloned from cell suspension culture and shown to be well-transcribed from day 4 to 57 in root tissue. Its expression in a number of different vector - E. coli hosts, as well as the yield of active peroxidase folded from inclusion body protein, has been very low until recently. Fourteen out of the twenty-three Arabidopsis peroxidase clones that were expressed in baculovirus yielded an active enzyme. Some of these enzymes could be purified by various chromatography methods. The catalytic profile of these recombinant peroxidases was characterised. Expression products of El in E. coli could never be demonstrated. Expression in insect cells in the presence of tunicamycin resulted in a mixture of unglycosylated and glycosylated El proteins, unsuitable for use in EIA. Finally, the El protein was expressed in human cells, with immunological properties resembling those of the natural El protein. As availability of larger quantities of novel peroxidases was a problem, production of A. t. peroxidase was scaled-up in insect cells in batch sizes up to 300 ml.
To characterise the transformed plants phenotypically and in terms of their phenolic chemistry Laccase antisense constructs in poplar expressing low levels of laccase transcripts were obtained for three of the four antisense populations. No significant differences were observed in either lignin content or composition in antisense plants as compared to controls. However, in one line, lac3as, a three-fold increase in soluble phenolic compounds was observed, The nature of this compound remains to be elucidated. Some difficulties appeared in the development of the tools required for screening of transgenic tobacco plants with altered laccase expression: lack of specificity of the antibodies prepared for Western-blot analysis and impossibility to optimise the conditions for testing the laccase activity of crude plant extracts. So, as no means were available for an analysis at the protein level, the characterisation of the transgenic plants was undertaken at the RNA level by Northern-blot analysis. This task was not achieved at the end of the AIR2 contract and it is still too early to draw conclusions concerning the involvement of laccase(s) in lignin biosynthesis in tobacco plants. Some preliminary results were obtained concerning the expression of the cloned laccase gene, indicating that it is preferentially expressed in stems and roots of tobacco plantlets older than four weeks. Another important result was obtained from structural characterisation of N-glycans of a laccase purified from the culture medium of suspension-cultured sycamore cells. It was shown that the sycamore laccase contains N-glycans harbouring the Lewis a-containing antigen that was for the first time described in plants during this study. As Lewis a-containing N-glycans are widely distributed in plants and are expressed at the cell surface, a potential function in cell/cell function is suggested for this oligosaccharide structure.
To investigate the function of peroxidase PXP 3-4, constructs were made with the PXP 3-4 cDNA in either sense or antisense orientation. These constructs were transformed into poplar and over-expressing and under-expressing lines were identified. Sense plants showed up to 150-fold higher total peroxidase activity in the xylem. No obvious phenotypes were observed. The analysis of these transgenic plants is currently ongoing. These plants will be very useful for the isolation of large amounts of peroxidase; +50 % of extracted proteins from the xylem corresponds to the recombinant peroxidase, corresponding to 100 mg/kg wood. To investigate the function of the putative C-terminal targeting peptide, transgenic plants expressing PXP 3-4 without this peptide have been generated. Northern analysis showed specific expression patterns of Arabidopsis peroxidases in roots, stems, leaves and flowers. These observations were verified by a cDNA array procedure. Roots contained the highest level of isoperoxidases, while leaves contained the lowest. Only four isoperoxidases could be detected in flowers and stems. Finally, the expression of an anionic pectin-binding peroxidase was visualised in zucchini seedlings by in situ hybridisation. Its expression was found mainly in dermal tissues and in differentiating xylem. Taken together, the data on biochemical properties and genes expression strongly support specificities in the role and function of individual isoperoxidases. A direct relation with lignification however remains to be elucidated. Mutants deficient in specific peroxidases are being collected and analysed for their impact on this important biological process.
To characterise the available enzymes in terms of protein chemistry The slight differences in the EPR magnetic parameters observed for the five laccases studied, suggested no changes on coordinating residues, but only little strains caused by non-co-ordinating residues that can be indirectly responsible for subtle geometrical distortions of the metal sites. Changing the pH of the solution, these magnetic parameters were not significantly altered, hence, the geometry of the metal sites was not affected by the acidity of the medium. All the enzymes were found to belong to the high-redox laccase group; however the redox potentials are strictly dependent on the pH of the solution - in particular, they decrease as the pH increases. Differences in Eº values can be ascribed to minor structural changes in the co-ordination environment of the type-I copper site owing to factors such as solvent accessibility, internal hydrogen bonding and dielectric anisotropy, mediated by the protein environment. These effects may change the relative energies of the metal oxidised and reduced states, directly influencing the redox potential value of the metal sites and hence the oxidising capabilities of the proteins. Although the E' values may give indications on the kinetic behaviour of the enzymes, other factors mediating the molecular recognition processes can also play a role in determining their catalytic properties. The ability of Pox B laccase to oxidise non-phenolic substrates (veratryl alcohol) varied with mediator: substrate ratio. In particular, it increased with increases in the mediator concentration. The efficiency of the two ABTS and HBT mediators is different, HBT being more active.
Arabidopsis peroxidase ATP A2 was characterised by absorption spectroscopy and x-ray crystallography and the structure solved to 1.45 angstrom resolution, the best so far for a plant peroxidase. Expression and folding of ATP N follow a pattern applicable to the majority of plant peroxidases. The electronic absorption spectrum showed a HRP C like peroxidase with a five-co-ordinated haem iron. The crystal structure of this recombinant Arabidopsis peroxidase has been solved to 2 angstrom resolution. The enzymatic properties and the stability were analysed. Optimal reaction with the substrate ATBS was seen at pH 5. The specific activity of ATP N was similar to that of HRP C at pH 5 but six-fold higher at pH 7. ATP N only showed stability from pH 5 to 8.5. Hence the stability of ATP N is very low compared with HRP C.
To characterise the available enzymes in terms of the reactions catalysed by them All enzymes studied had the same substrate specificity. Laccases had a much lower specific activity than peroxidases, generally needing 100 times the amount of protein to catalyse comparable reaction rates. Basic iso-peroxidases appeared to be far more active than the acidic isozymes. The major product formed by coniferyl alcohol was isolated and characterised as dehydrodiconiferyl alcohol. Owing to the small product yields of most substrates it has proven impossible to determine their identity.
There were some differences between the 14 recombinant Arabidopsis peroxidases, purified from insect cells, observed with extensin or scopoletin as substrates. However, all the peroxidases were able to oxidise the common hydrogen donors such as o-phenylene diamine, guaiacol, ABTS or ferulic acid, although with some preferences and at a specific pH. Calcium modulated the activity of some of the peroxidases. The level of activity of one of them was closely dependent on the Ca2+ concentration present in the medium in which it was pre-incubated. Certain peroxidases were able to produce significant level of hydrogen peroxide when cysteine was used as an electron donor. The various recombinant peroxidases exhibited also quite different binding properties. Some were strongly hydrophobic or were able to bind heparin. Four of them had an affinity for the Ca-pectate structure, a property also shown by native isoperoxidases which could be important for the regulation of their activity in vivo. These pectin-binding peroxidases have cationic amino acid located in the same position.
A series of phenolic compounds was isolated by alkali treatment from maize cell walls. Preliminary NMR analysis of compound 'F'suggests an 8-8-linked dimer between ferulic acid and another, as yet unidentified, phenol.
Studies of the feruloyl-polysaccharide ester-linked conjugates present in graminaceous cell walls, by techniques avoiding the use of alkali, revealed the universal occurrence of a novel structural unit, 2-0-beta-D-xylopyranosyl-(5-0-feruloyl)-L-arabinose (XFA), which is attached to the backbone of xylans. This structural unit was found in the cell walls of all twenty-one species of grass and cereal examined and of one palm. Several more complex structures were partially characterised, and shown to be based on XFA but with additional 0-xylose residues (1,3)-linked to the xylose residue of XFA. In addition, evidence was obtained that these larger structures are endogenously partially 0-acetylated. The biological reasons for the complexity are thus unclear, and could potentially determine the mode of oxidative coupling to which these feruloyl groups are subject in the presence of laccases and peroxidases. Scission of plant cell wall polysaccharides in vivo has generally been assumed to be enzymic. However, in the presence of L-ascorbate, such polysaccharides are shown to undergo non-enzymic scission under physiologically relevant conditions. Scission of xyloglucan by 1mM ascorbate had a pH optimum of 4.5, and the maximum scission rate was reached after a 10-25-min delay. Catalase prevented the scission, whereas added hydrogen peroxide increased the scission rate and shortened the delay. Ascorbate caused detectable xyloglucan scission above about five micromolar. Dehydroascorbate was much less effective. Added Cu2+ also increased the rate of ascorbate-induced scission; EDTA was inhibitory. The rate of scission in the absence of added metals appeared to be attributable to the traces of Cu present in the xyloglucan. Ascorbate-induced scission of xyloglucan was inhibited by radical scavengers: their effectiveness was proportional to their rate-constants for reaction with hydroxyl radicals. It is proposed that ascorbate non-enzymically reduces oxygen to hydrogen peroxide and Cu2+ to Cu+, and that hydrogen peroxide and copper react to form hydroxyl ions that causes oxidative scission of polysaccharide chains.
Time course studies of the fate of [14C] cinnamic acid, fed to maize suspension cell cultures, showed the appearance, within 2 minutes, of a dimer phenolic product. From previous work it is known that this is too early for ferulate esters to have reached the cell wall and therefore be intra-protoplasmically. The major product does not co-chromatograph with standard 8-5 or 5-5 linked dimers. The precise identity of this dimeric product has not been determined.
To evaluate the potential of the individual specific enzymes in industrial applications The bleaching activities obtained with the plant laccases were very low compared to the microbial laccases despite that they were dosed nearly ten times higher. However, the plant laccases did show significant bleaching at high pH with some mediators, especially Rhus laccase and Poplar laccase with phenoxazine derivatives. Our assumption that plant laccases may be somewhat more alkaline than most microbial laccases was supported by these results. However, the very poor specific activity of these plant laccases - probably due to low oxidation potentials - will probably be prohibitive for most industrial applications.
Two reference ElAs requiring an antibody-peroxidase or a hapten-peroxidase conjugate were developed. For a third system, an E1-peptide and the full E1 protein had to be expressed in heterologous systems. To assess the possibilities of conjugating proteins with peroxidases different from HRP and A. t. peroxidases, a recombinant Copriitus ctitereus peroxidase (rCIP) was used, but procedures were not successful, whereas a new approach to conjugate a hapten like T4 to HRP proved successful
Discussion
Transformants with very high expression levels of specific isoperoxidases have been obtained. These plants can now be used as raw material for industrial applications. Plants regulated in lignin-correlated peroxidase activity potentially have modified lignin amount or composition, which could be of economical or environmental benefit for the paper and pulp industry.
Plant peroxidases are important enzymes, both for the physiology of plants and for various industrial uses. This work has allowed the synthesis of many recombinant peroxidases, which are now available for various uses. It has also given evidence for the diversity of catalytic and binding properties of the different isoperoxidases. A technique was initiated to study the expression pattern of a large number of peroxidases in a single step. This will be useful to study the involvement of peroxidases in various developmental and stress responses. The present work has contributed significantly to the elucidation of the potential for biotechnology applications of plant peroxidase genes. Genes have been cloned from Arabidopsis and recombinant proteins have been produced in E. coli and folded into active peroxidases (Patent Applicationt PA 1998 01154). A great deal of experience concerning the expression of proteins in insect cells and especially in mammalian cells was gained. More specifically, false-positive interference in EIA with insect cell expressed antigens could be assigned to the presence of insect cell protein contaminants in the recombinant antigen combined with the presence of human antibodies directed against insect cell pro teins.
The major impact of the tobacco laccase work is not related to lignin biosynthesis but a spin-off of relevance to plant glycobiology. Indeed, the discovery of Lewis a epitopes in plant N-glycans has illustrated that N-glycan biosynthesis and maturation are at least as complex in plants as in animals. Following on from these results, several laboratories are now studying potential functions of these plant Lewis a N-glycans and are attempting purification and cloning of the glycosyltransferases responsible of their biosynthesis.
The present project provides the first report of a plant laccase purified and characterised from a lignifying tissue in angiosperms. Until now, plant laccase research had focused either on the biochemical or on the molecular characterisation of laccases, rather than the integrated biochemical and molecular approach, adopted here, as a first step towards understanding the physiological role of individual laccases.
The laccase metallo-enzyme work achieved the following goals:
In this perspective, the use of lignolytic enzymes in the pulp industry or the wastewater treatment seems to be very promising.
High-resolution peroxidase structures have been solved at unprecedented resolution by X-ray crystallography for the well-characterised stable anionic A2 type and for the very reactive but less stable neutral N type.
The project has shown that, while different peroxidases may have different a rates of reaction, they all have very gimilar substrate specificity and produce essentially the same product profiles. Thus, it was concluded that observed differences between plant tissues in the range of phenolic coupling products formed are most likely to be due to different contents of 'dirigent' proteins or to differences in the molecular environment of the phenolic substrates rather than to different peroxidase/laccase isoenzyme profiles.
The work on novel maize (and other graminaceous) cell wall phenolic esters has considerably extended our knowledge of the chemical structures of the potential substrates of plant peroxidases and laccases.
The finding that oxidation of feruloyl estersi is an intra-protoplasmic event has a profound consequences for our understanding of the biosynthesis of the primary cell wall, and the biological roles of intra- and extra-protoplasmic isoperoxidases. As a result of this discovery, far greater emphasis will in the future have to be placed on a the intra-protoplasmic enzymes, which in the past have tended to be ignored by scientists interested in plant cell walls.
9 8. To evaluate the potential of the individual specific enzymes in industrial a applications. Although only three plant laccases were investigated, it seems to be a general tendency that these laccases show a better performance in dye bleaching at alkaline pH than do microbial laccases. The very poor specific activity of the plant laccases is a major drawback. However, the knowledge obtained about the various laccases with respect to structure-function relations may help in future development of ideal enzymes for various applications.
A major outcome of the work on novel peroxidases for enzyme-linked immunoassays (ElAs) can be summarised in terms of benefits and identification of pitfalls, with development in some cases of proper solutions to avoid those pitfalls. The major benefit for the diagnostic industry concerns the positive outcome of the feasibility study. It has been indicated that the concept of generating recombinant tracer enzymes, peptide-recombinant tracer enzyme conjugates and recombinant antigen-HRP conjugates may become valuable tools in EIA development. The feasibility of recombinant protein-enzyme conjugates has been clearly demonstrated. All these findings together will enable the production of recombinant proteins with excellent immunological properties and will improve the reproducibility from batch-to-batch, with major benefits for correct immune diagnostic methods.
Exploitation
The project resulted in four patents. Several laccases were produced in sufficient quantities to be evaluated by Novo Nordisk as potential agents for decolorising waste dyes: the plant laccases had the industrially relevant advantage of being more alkali-tolerant than the fungal laccases, but their specific activity turned out to be too low for commercial exploitation. The consortium has also published a large number of original scientific papers and conference contributions.
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