ABSTRACT
The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm. The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCR-based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene-specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
Subject(s)
Genes, Plant , Lignin/metabolism , Magnoliopsida/genetics , Monophenol Monooxygenase/genetics , Oxidoreductases/genetics , Acrolein/analogs & derivatives , Acrolein/metabolism , Amino Acid Sequence , Cell Wall/enzymology , Cloning, Molecular , Copper , Evolution, Molecular , Glycoproteins/genetics , Glycoproteins/metabolism , Laccase , Magnoliopsida/enzymology , Metalloproteins/genetics , Metalloproteins/metabolism , Molecular Sequence Data , Monophenol Monooxygenase/metabolism , Multigene Family , Oxidoreductases/metabolism , Phenols/metabolism , Plant Stems/enzymology , Sequence Homology, Amino Acid , Substrate Specificity , Trees/enzymology , Trees/geneticsABSTRACT
Cinnamoyl-coenzyme A:NADP oxidoreductase (CCR, EC 1.2.1.44), the entry-point enzyme into the monolignol biosynthetic pathway, was purified to apparent electrophoretic homogeneity from differentiating xylem of Eucalyptus gunnii Hook. The purified protein is a monomer of 38 kD and has an isoelectric point of 7. Although Eucalyptus gunnii CCR has approximately equal affinities for all possible substrates (p-coumaroyl-coenzyme A, feruloyl-coenzyme A, and sinapoyl-coenzyme A), it is approximately three times more effective at converting feruloyl-coenzyme A than the other substrates. To gain a better understanding of the catalytic regulation of Eucalyptus CCR, a variety of compounds were tested to determine their effect on CCR activity. CCR activity is inhibited by NADP and coenzyme A. Effectors that bind lysine and cysteine residues also inhibit CCR activity. As a prerequisite to the study of the regulation of CCR at the molecular level, polyclonal antibodies were obtained.
ABSTRACT
Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamyl aldehydes (sinapyl, paracoumaryl, coniferyl aldehydes) to the corresponding alcohols which are the direct monomeric precursors of lignins. Recently, we have purified from Eucalyptus gunnii two isoforms of CAD (CAD1 and CAD2), distinct in their biochemical and functional properties. In this paper, we report the cloning of a CAD cDNA (pEuCAD2) isolated by screening a lambda gt11 library generated from cell suspension culture of Eucalyptus gunnii, using a tobacco CAD cDNA as a probe. This full-length clone (1392 bp) encodes a protein of 356 amino acids which corresponds to the subunit molecular weight of the CAD2 isoform. Sequence analysis revealed that CAD2 is very well conserved among species (78% homology with CAD from tobacco, a herbaceous angiosperm, and 81% with the partial sequence from a gymnosperm, loblolly pine). The identity of this clone was unambiguously demonstrated (1) by comparison with peptide sequence data from purified CAD2 and (2) by functional expression of the recombinant enzyme in Escherichia coli. Recombinant CAD showed the same properties as the natural isoform CAD2, in terms of electrophoretic mobility, polypeptide structure, substrate specificity and antigenicity. The CAD2 transcript is equally abundant in stems and leaves and at the limit of detection in roots. At the tissue level the CAD2 gene is highly expressed in xylem and virtually undetectable in phloem.
