Coniferyl Alcohol Oxidase Research Articles

Coniferyl Alcohol Oxidase Activity in the Cell Wall of Callisthephus sinensis

The oxidation of coniferyl alcohol by cell walls isolated from lignifying hypocotyls of Callistephus sinensis was studied in the absence and in the presence of H 2 O 2 . The results showed that the oxidation of coniferyl alcohol by cell walls in the absence of H 2 O 2 (coniferyl alcohol oxidase activity) was approximately 3% of that occurring in the presence of H 2 O 2 . The in vivo significance of this cell wall-located coniferyl alcohol oxidase activity was studied in C. sinensis hypocotyls by histochemical tests, which revealed that coniferyl alcohol oxidase was located in the H 2 O 2 -producing lignifying xylem cells. The results obtained from these histochemical probes strongly suggest that coniferyl alcohol oxidase activity of C. sinensis cell walls is physiologically irrelevant in tissues that accumulate H 2 O 2 , such as the lignifying xylem, where the peroxidatic activities favorably compete with the oxidase activities during the polymerization of cinnamyl alcohols to lignins.

Coniferyl alcohol oxidase activity of a cell-wall-located class III peroxidase

Coniferyl alcohol oxidase activity was determined in cell walls from hypocotyls of the following species belonging to the family Asteraceae: Calendula officinalis, Callistephus sinensis, Cosmos bipinnanthus, Helianthus annuus, Helianthus debilis and Zinnia elegans. In all the cases studied, coniferyl alcohol oxidase activity was partially located ionically-bound to cell walls and resided in a basic peroxidase, the activity of which was stimulated by H 2 O 2 . This enzymatic activity was insensitive to freezing and was inactivated by high H 2 O 2 concentrations, as tested both in vitro and in situ by using purified cell wall fractions. The peroxidase with coniferyl alcohol oxidase activity was purified from Z. elegans hypocotyls until apparent homogeneity, as checked by SDS-PAGE. It showed a visible spectrum typical of a haem-containing high-spin ferric secretory (class III) plant peroxidase. Coniferyl alcohol oxidase activity of this basic peroxidase constitutes about 0.25% of the activity shown in the presence of H 2 O 2 . The significance of the coniferyl alcohol oxidase activity in vivo was studied in Z. elegans hypocotyls by means of histochemical tests, which revealed that it was located in the H 2 O 2 -producing lignifying xylem cells. The results obtained from the histochemical probes suggest that the coniferyl alcohol oxidase activity of this basic peroxidase is physiologically irrelevant in tissues that accumulate H 2 O 2 , as is the case of the lignifying xylem, where the peroxidase activity of the enzyme favorably competes with the oxidase activity of the enzyme.

Coniferyl alcohol oxidase operates through a bound free-radical intermediate

Oxidase activity was enriched in extracts of the developing, lignifying xylem of Sitka spruce, Leyland cypress, Lawson cypress and Wych elm obtained by a procedure that selects cell-wall-associated glycoproteins. All of the xylem extracts were able to oxidise the monolignol, coniferyl alcohol, and had a greater affinity for coniferyl alcohol than ABTS. The oxidases were strongly inhibited by Cu-chelators and had inhibition profiles broadly similar to catechol oxidase-type polyphenol oxidases. Analysis of the oxidation products of coniferyl alcohol generated in the presence of the spin trap POBN, by electron paramagnetic resonance spectroscopy, showed that a spin adduct of parameters a n = 15.65–15.74 G and a h = 2.73–2.78 G had been generated which can be most readily assigned to a POBN adduct of the β-carbon-centred free radical of coniferyl alcohol. Electron paramagnetic resonance spectra of the extracts from the four species contained signals representative of Cu 2+ and a free radical species of g value ∼2.0036. The intensity of the free radical signal was diminished by addition of coniferyl alcohol but was restored upon aeration. The Cu 2+ signals were similarly altered by this treatment, but diminution was less marked. Cycles of depletion then recovery of the free radical signal could be obtained by addition of aliquots of coniferyl alcohol follwed by aeration which strongly suggests that the free radical is directly involved in the oxidation mechanism. We propose that coniferyl alcohol oxidase operates via a bound free radical which is re-oxidised via a charge relay mechanism involving bound Cu 2+ ions and molecular oxygen.

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl2 extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees.

Purification of coniferyl alcohol oxidase from lignifying xylem of sitka spruce using immobilised metal affinity chromatography

Summary Extracts of lignifying xylem of Sitka spruce, obtained by a combined extraction/affinity method that selects for cell-wall-associated glycoproteins, were enriched in oxidase and peroxidase activity but peroxidase activity was approximately 80 times more abundant than oxidase activity. Previous attempts to purify the oxidase using ion-exchange, hydrophobic interaction and gel filtration chromatographic methods have been unsuccessful and it has proved particularly difficult to separate completely oxidase activity from peroxidase activity. All of the oxidase activity in the xylem cell wall extracts bound to an affinity matrix loaded with immobilised copper (Cu2+) ions and was fractionated into two main peaks by eluting the bound proteins with a gradient of histidine. The second oxidase peak, which represents a subset of oxidase that has higher affinity for the bound metal ions, contained a third of the total oxidase activity at a 2-fold increase in specific activity. In addition, this fraction had greatly reduced although still detectable peroxidase activity. Oxidase activity was split into a bound and an unbound fraction when extracts were applied to a matrix of immobilised cobalt (Co2+) ions. In this case, a larger proportion of the applied oxidase activity than peroxidase activity bound to the Co2+-loaded matrix and the specific activity was higher than that in the unbound fraction. This suggested that this bound fraction of the oxidase population had a higher affinity for metal ions and could therefore be isolated from peroxidase. When applied to a Zn2+-loaded matrix, the oxidase activity also split into bound and unbound portions but effectively all of the peroxidase activity was eluted in the unbound fraction yielding a bound fraction enriched in oxidase activity with negligible peroxidase contamination. The nature of this «high affinity fraction» of oxidase and the heterogeneity in the oxidase population is discussed. Gel permeation on Superose-6 partially resolved oxidase from the residual peroxidase activity in the Zn-bound fraction and gave an apparent Mr of 62 kDa for the oxidase activity. However, an examination of the protein profile of fractions enriched in oxidase activity by SDS-PAGE strongly suggests that a protein band of apparent Mr 80 kDa is responsible for the oxidase activity. This apparent disparity is discussed. In summary, immobilised metal affinity chromatography on Zn-loaded matrix provides a rapid method to separate oxidase from peroxidase activities enabling studies of the substrate specificity and the physical properties of the oxidase to be carried out.

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce ( Picea sitchensis )

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce ( Picea sitchensis )

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl2. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent Mr approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent Mr approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed.

Extraction and Partial Purification of Cell-Wall-Associated Coniferyl Alcohol Oxidase from Developing Xylem of Sitka Spruce

Article Extraction and Partial Purification of Cell-Wall-Associated Coniferyl Alcohol Oxidase from Developing Xylem of Sitka Spruce was published on January 1, 1996 in the journal Holzforschung (volume 50, issue 6).

Coniferyl alcohol oxidase ? a catechol oxidase?

The physico-chemical properties of coniferyl alcohol oxidase (CAO), a copper containing glycoprotein spatiotemporally associated with lignification in conifers, is reported here. By electron paramagnetic resonance spectroscopy, only type 3 copper was indicated in CAO. CAO oxidizes several laccase substrates; however, it is not a blue-copper protein and monoclonal antibodies against both native and deglycosylated CAO did not recognize any of several laccases. The N-terminal sequence of CAO, H2N-X E L A Y S P P Y X P S, was non-homologous with known enzymes. Transparent copper, tetrameric structure, aminoacid composition, phenylhydrazine and tropolone inhibition, and SDS enhancement of CAO activity indicate that CAO is an o-diphenol oxidase.

A laccase‐like phenoloxidase is correlated with lignin biosynthesis in Zinnia elegans stem tissues

SummaryStem tissues from different internodes of 4–6 week‐old Zinnia elegans cv. Envy plants were sectioned and stained with chromogenic substrates previously used in studies of laccases (p‐diphenol:O2 oxidoreductases) isolated from tree tissues. The pattern of color development found when stem sections were stained in the presence and absence of H2O2 suggested that p‐diphenol:O2 oxidoreductase activity was tightly correlated spatially and temporally with the lignification of secondary cell walls in developing primary xylem. The correlation between this laccase‐like phenoloxidase activity and lignification appeared tighter than that between lignification and peroxidases stained using the same substrates. Zymogram analysis of the phenoloxidase activities catalyzed by enzymes that were not boiled prior to separation by SDS—PAGE suggested that a single enzyme was predominantly responsible for the laccase‐like phenoloxidase activity in Zinnia stems. Some of this enzyme was released from cell wall residue by washing with high ionic strength buffer; however, substantial amounts of the enzyme could only be recovered after treatment of the residue with cell wall‐degrading enzymes. This phenoloxidase appears to share significant characteristics with the coniferyl alcohol oxidase isolated from developing secondary xylem in pines, which suggests that such enzymes may be widespread in vascular plants.

Electrophoretic analysis of coniferyl alcohol oxidase and related laccases.

Gradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI approximately 9 pine "laccase" recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2-[N-morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell-wall-bound glycoprotein (38% glycan), M(r) 107,500, pI 7.6, pH and temperature optima 6.3 and 30 degrees C, respectively. By graphite-furnace atomic-absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI approximately 9 pine "laccase" following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate-deglycosylated CAO.

Cell wall-bound coniferyl alcohol oxidase associated with lignification in conifers

An enzyme utilizing O2 to oxidize coniferyl alcohol into a lignin-like product is described. This enzyme has been found firmly bound to cell walls in developing xylem of Pinus strobus, Abies balsamea, Larix laricina, Picea rubens and Pinus banksiana. In each of these conifers, oxidase activity arises in synchrony with initiation of lignification and disappears with termination of lignification. By combined gas chromatography-mass spectrometry, the enzyme was found to oxidize coniferyl alcohol into dehydrodiconiferyl alcohol and pinoresinol, and by UV microscopy it was determined that under aerobic in vitro conditions the enzyme converts coniferyl alcohol into hydrophobic globules similar to guaiacyl lignin. By isoelectric focusing, several oxidase isoenzymes were resolved; all appear to be glycoproteins.

Evidence for coniferyl-alcohol oxidase promotion of lignification in developing xylem of conifers.

Conference Article| May 01 1992 Evidence for coniferyl-alcohol oxidase promotion of lignification in developing xylem of conifers Rodney A Savidge; Rodney A Savidge 1Dept Forest Resources, Univ New Brunswick, Fredericton, NB, E3B 6C2, Canada Search for other works by this author on: This Site PubMed Google Scholar Preethi V Randeniya Preethi V Randeniya 1Dept Forest Resources, Univ New Brunswick, Fredericton, NB, E3B 6C2, Canada Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1992) 20 (2): 229S. https://doi.org/10.1042/bst020229s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Rodney A Savidge, Preethi V Randeniya; Evidence for coniferyl-alcohol oxidase promotion of lignification in developing xylem of conifers. Biochem Soc Trans 1 May 1992; 20 (2): 229S. doi: https://doi.org/10.1042/bst020229s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1992 Biochemical Society1992 Article PDF first page preview Close Modal You do not currently have access to this content.