A novel aromatic alcohol dehydrogenase in higher plants: molecular cloning and expression.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the conversion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, an atypical form of CAD (CAD 1) was identified in Eucalyptus gunnii [12]. We report here the molecular cloning and characterization of the corresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol dehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by sequence comparison of the cDNA with peptide sequences derived from purified CAD 1 protein and by functional expression of CAD 1 recombinant protein in Escherichia coli. Both native and recombinant CAD 1 exhibit high affinity towards lignin precursors including 4-coumaraldehyde and coniferaldehyde, but they do not accept sinapaldehyde. Moreover, recombinant CAD 1 can also utilize a wide range of aromatic substrates including unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35,790 Da and an isoelectric point of 8.1. Although sequence comparisons with proteins in databases revealed significant similarities with dihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of plant species, the most striking similarity was found with cinnamoyl-CoA reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocalization experiments indicated that CAD 1 is expressed in both lignified and unlignified tissues/cells. Based on the catalytic activity of CAD 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the lignin biosynthetic pathway. However, additional roles in phenolic metabolism are not excluded.

Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships.

Cinnamoyl CoA:NADP oxidoreductase (CCR, EC 1.2.1.44) catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, i.e. the first specific step in the synthesis of the lignin monomers. The cloning of a cDNA encoding CCR in Eucalyptus gunnii (EUCCR) is reported here. The identity of the EUCCR cDNA was demonstrated by comparison with peptide sequence data from purified CCR and functional expression of the recombinant enzyme in Escherichia coli. Sequence analysis revealed remarkable homologies with dihydroflavonol-4-reductase (DFR), the first enzyme of the anthocyanin biosynthetic pathway. Moreover, significant similarities were found with mammalian 3 beta-hydroxysteroid dehydrogenase and bacterial UDP-galactose-4-epimerase, suggesting that CCR shared a common ancestor with these enzymes and can therefore be considered as a new member of the mammalian 3 beta-hydroxysteroid dehydrogenase/ plant dihydroflavonol reductase superfamily. In Eucalyptus gunnii, CCR is encoded by one gene containing four introns whose positions are similar to those of introns I, II, III and V in DFR genes from dicots. In agreement with the involvement of CCR in lignification, the CCR transcript was shown to be expressed in lignified organs, i.e. root and stem tissues, and was localized mainly in young differentiating xylem. On the other hand, its abundance in Eucalyptus leaves suggests that monolignols may be precursors of end products other than lignins. This first characterization of a gene corresponding to CCR opens new possibilities to genetically engineer plants with lower lignin content. This is particularly important for woody plants such as Eucalyptus which are used for pulp making.

Site-directed mutagenesis of a serine residue in cinnamyl alcohol dehydrogenase, a plant NADPH-dependent dehydrogenase, affects the specificity for the coenzyme.

Using recombinant cinnamyl alcohol dehydrogenase isoform 2 (CAD2, EC 1.1.1.195), an NADPH-dependent aromatic alcohol dehydrogenase involved in lignification in vascular plants, we have investigated the detailed steady-state kinetic mechanism of CAD2 and the role of a serine residue in determining the cofactor specificity of CAD2. Site-directed mutagenesis (S212D) and overexpression of the WT and mutant S212D forms of CAD2 in Escherichia coli, followed by kinetic studies on the purified WT and mutant proteins, confirmed the involvement of S212D in recognizing the phosphate group of NADPH and provided information on the structural requirements for NADPH specificity. From substrate kinetic patterns and product inhibition studies both WT and S212D mutant forms of CAD2 have been shown to follow rapid equilibrium random bireactant kinetics with the value of the interaction factor (alpha) for WT (0.25) being significantly less than that for S212D CAD2 (0.45). The changes in binding energy arising from the mutation on the binding of the 2'-phosphate site of the coenzyme were assessed. A marked degree of physical interaction was detected between the enzymatic binding sites of the coniferyl alcohol substrate and the 2'-phosphate binding region, which are quite distant in the three-dimensional structure. The inhibition by 2',5'-ADP and 5'-AMP was found to be weak for both WT and S212D CAD2. Strong substrate inhibition was detected for CAD2, and its implications for plant physiological studies were assessed.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction of specific carbohydrates into Eucalyptus gunnii cells increases their freezing tolerance.

The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long-term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.

Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from Phaseolus vulgaris.

Cinnamyl alcohol dehydrogenase (CAD) catalyses the reduction of hydroxycinnamaldehydes (p-coumaryl, coniferyl, sinapyl) to the corresponding alcohols which are the monomeric precursors of lignins. We have demonstrated the occurrence of two isoforms of CAD (CAD1 and CAD2) in bean which differ in terms of subunit Mr, specific activity, substrate affinity and antigenicity. The most abundant polypeptide in bean pods, organs with very limited lignification, is a low affinity CAD isoform (CAD1). This enzyme which is distinct from a benzyl alcohol dehydrogenase with broad substrate specificity, was purified to apparent homogeneity and partial amino acid sequencing was carried out using internal peptides obtained by trypsin cleavage.

A molecular model for cinnamyl alcohol dehydrogenase, a plant aromatic alcohol dehydrogenase involved in lignification.

The plant aromatic alcohol dehydrogenase, cinnamyl alcohol dehydrogenase (CAD2 from Eucalyptus) was found by sequence analysis of its cloned gene to be homologous to a range of dehydrogenases including alcohol dehydrogenases, L-threonine-3-dehydrogenase, D-xylose reductase and sorbitol dehydrogenase. A homology model of CAD2 was built using the X-ray crystallographic coordinates of horse-liver alcohol dehydrogenase to provide the template, with additional modelling input from other analogous regions of structure from similar enzymes where necessary. The structural model thus produced rationalised the Zn-binding properties of CAD2, indicated the possession of a Rossmann fold (GXGXXG motif), and explained the class A stereospecificity (pro-R hydrogen removal from substrate alcohol) and aromatic substrate specificity of the enzyme. A range of potential ligands was designed based on the homology model and tested as inhibitors of CAD2 and horse liver alcohol dehydrogenase.

Cinnamyl-alcohol dehydrogenase, a molecular marker specific for lignin synthesis: cDNA cloning and mRNA induction by fungal elicitor.

Cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) catalyzes the final step in a branch of phenylpropanoid synthesis specific for production of lignin monomers. We have isolated a full-length cDNA clone encoding CAD, as a molecular marker specific for lignification, by immunoscreening a lambda gt11 library containing cDNAs complementary to mRNA from elicitor-treated cell cultures of bean (Phaseolus vulgaris L.). The clone comprises a single long open reading frame of 1767 base pairs, 31 base pairs of 5' leader, and 152 base pairs of 3' untranslated sequence. The deduced 65-kDa CAD polypeptide has several features that are strongly conserved in alcohol dehydrogenases. Addition of fungal elicitor to cell cultures stimulates CAD transcription, which leads to a remarkably rapid, but transient, accumulation of CAD mRNA, with no detectable lag and maximal levels after 1.5 hr. Southern blot analysis of bean genomic DNA indicates that elicitor-induced CAD is encoded by a single gene. The regulatory significance of the rapid activation of this CAD gene and the possible existence of a second, divergent CAD gene involved in lignification during xylogenesis are discussed.