Eucalyptus gunnii CCR and CAD2 promoters are active in lignifying cells during primary and secondary xylem formation in Arabidopsis thaliana.

Cell-specific expression patterns of the Eucalyptus gunnii cinnamoyl coenzymeA reductase (EgCCR) and cinnamyl alcohol dehydrogenase (EgCAD2) promoters were analyzed by promoter-GUS histochemistry in the primary and secondary xylem tissues from floral stems and roots of Arabidopsis thaliana. Expression patterns indicated that the EgCCR and EgCAD2 genes were expressed in a coordinated manner in primary and secondary xylem tissues of the Arabidopsis floral stem and root. Both genes were expressed in all lignifying cells (vessel elements, xylem fibers and paratracheal parenchyma cells) of xylem tissues. The capacity for long-term monolignol production appeared to be related to the cell-specific developmental processes and biological roles of different cell types. Our results suggested that lignification of short-lived vessel elements was achieved by a two-step process involving (i) monolignol production by vessel elements prior to vessel programmed cell death and (ii) subsequent monolignol production by vessel-associated living paratracheal parenchyma cells following vessel element cell death. EgCCR and EgCAD2 gene expression patterns suggested that the process of xylem cell lignification was similar in both primary and secondary xylem tissues in Arabidopsis floral stems and roots.

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: Identification of New Sites of Promoter Activity in Transgenic Poplar.

Stem sections from poplar that were stably transformed with a eucalypt cinnamyl alcohol dehydrogenase promoter-[beta]-glucuronidase construct were prepared by using either a technique routinely used in herbaceous species or a technique designed to take into account the particular anatomy of woody plants. Although both preparation techniques confirmed the pattern of expression previously observed (C. Feuillet, V. Lauvergeat, C. Deswarte, G. Pilate, A. Boudet and J. Grima-Pettenati [1995] Plant Mol Biol 27: 651-657), the latter technique also allowed the detection of other sites of promoter activity not revealed by the first technique. In situ hybridization confirmed the expression pattern obtained with the second sample preparation technique.

Kinetic and inhibition studies of cinnamoyl-CoA reductase 1 from Arabidopsis thaliana.

Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignin monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. AtCCR1, one of the two distinct isoforms isolated from Arabidopsis thaliana, was shown to be involved in lignin biosynthesis during development. Here, we report on the purification of the recombinant AtCCR1 protein expressed in Escherichia coli and the subsequent determination of its kinetic properties (K(m) and k(cat)/K(m) values) towards its main substrates i.e. feruloyl-CoA, sinapoyl-CoA, and p-coumaroyl-CoA esters. In addition, the potential inhibitory effect of five substrate-like analogs possessing an N-acetylcysteamine thioester group was tested on CCR activity using either feruloyl-CoA or sinapoyl-CoA as substrates. The K(i) values were in the range of 4.4-502 microM and the type of inhibition was found to be either uncompetitive or noncompetitive. Interestingly, for compounds 3 and 5, the type of inhibition was found to be different depending on the substrate used to monitor the enzyme activity. The best inhibitors were those possessing the feruloyl (compound 3) and sinapoyl (compound 5) aromatic moiety (4.1 and 7.1 microM) while the enzyme activity was monitored using the corresponding substrates.

Biochemistry and molecular biology of lignification.

Lignins, which result from the dehydrogenative polymerization of cinnamyl alcohols, are complex heteropolymers deposited in the walls of specific cells of higher plants. Lignins have probably been associated to land colonization by plants but several aspects concerning their biosynthesis, structure and function are still only partially understood. This review focuses on the modern physicochemical methods of structural analysis of lignins, and on the new approaches of molecular biology and genetic engineering applied to lignification. The principles, advantages and limitations of three important analytical tools for studying lignin structure are presented. They include carbon 13 nuclear magnetic resonance, analytical pyrolysis and thioacidolysis. The use of these methods is illustrated by several examples concerning the characterization of grass lignins,'lignin-like'materials in protection barriers of plants and lignins produced by cell suspension cultures. Our present limited knowledge of the spatio temporal deposition of lignins during cell wall differentiation including the nature of the wall components associated to lignin deposition and of the cross-links between the different wall polymers is briefly reviewed. Emphasis is placed on the phenylpropanoid pathway enzymes and their corresponding genes which are described in relation to their potential roles in the quantitative and qualitative control of lignification. Recent findings concerning the promoter sequence elements responsible for the vascular expression of some of these genes are presented. A section is devoted to the enzymes specifically involved in the synthesis of monolignols: cinnamoyl CoA reductase and cinnamyl alcohol dehydrogenase. The recent characterization of the corresponding cDNAs/genes offers new possibilities for a better understanding of the regulation of lignification. Finally, at the level of the synthesis, the potential involvement of peroxidases and laccases in the polymerization of monolignols is critically discussed. In addition to previously characterized naturally occurring lignin mutants, induced lignin mutants have been obtained during the last years through genetic engineering. Some examples include plants transformed by O-methyltransferase and cinnamyl alcohol dehydrogenase antisense constructs which exhibit modified lignins. Such strategies offer promising perspectives in gaining a better understanding of lignin metabolism and functions and represent a realistic way to improve plant biomass. Contents Summary 203 I. Introduction 204 II. Main structural features of lignins 205 III. Lignification and cell wall differentiation: spatio-temporal deposition of lignins and inter-relations with other wall components 213 IV. Enzymes and genes involved in the biosynthesis and polymerization of monolignols 216 V. Lignin mutants as a way to improve plant biomass and to explore lignin biochemistry and metabolism 226 VI. Concluding remarks 229 Acknowledgements 230 References 230.

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.

Two cinnamoyl-CoA reductase (CCR) genes from Arabidopsis thaliana are differentially expressed during development and in response to infection with pathogenic bacteria.

Cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) catalyses the conversion of cinnamoyl-CoAs into their corresponding cinnamaldehydes, i.e. the first step of the phenylpropanoid pathway specifically dedicated to the monolignol biosynthetic branch. In previous work, we described the isolation and characterisation of the first cDNA encoding CCR in Eucalyptus (Lacombe, E., Hawkins, S., Van Dorsselaere, J., Piquemal, J., Goffner, D., Poeydomenge, O., Boudet, A.M., Grima-Pettenati, J., 1997. Cinnamoyl CoA reductase, the first committed enzyme of the lignin branch biosynthetic pathway: cloning, expression and phylogenetic relationships. Plant Journal 11, 429--441) and shown the role of this enzyme in controlling the carbon flux into lignins (Piquemal, J., Lapierre, C., Myton, K., O'Connell, A., Schuch, W., Grima-Pettenati, J., Boudet, A.M., 1998. Down-regulation of cinnamoyl-CoA reductase induces significant changes of lignin profiles in transgenic tobacco plants. Plant Journal 13, 71--83). Here, we report the characterisation of two functionally and structurally distinct cDNA clones, AtCCR1 and AtCCR2 (81.6% protein sequence identity) in Arabidopsis thaliana. The two recombinant proteins expressed in Escherichia coli are able to use the three cinnamoyl-CoAs tested but with different levels of efficiency. AtCCR1 is five times more efficient with feruloyl-CoA and sinapoyl-CoA than AtCCR2. In addition, the two genes are differentially expressed during development and in response to infection. AtCCR1 is preferentially expressed in tissues undergoing lignification. In contrast, AtCCR2, which is poorly expressed during development, is strongly and transiently induced during the incompatible interaction with Xanthomonas campestris pv. campestris leading to a hypersensitive response. Altogether, these data suggest that AtCCR1 is involved in constitutive lignification whereas AtCCR2 is involved in the biosynthesis of phenolics whose accumulation may lead to resistance.

Characterization of a bean (Phaseolus vulgaris L.) malic-enzyme gene.

We have isolated a genomic clone encoding a plant NADP(+)-dependent malic enzyme (NADP-ME). This clone, isolated from bean (Phaseolus vulgaris L.), covers the entire gene (exons, introns) and 5'-flanking regions. DNA sequencing defines 20 exons spanning approximately 4.5 kb, which range over 48-235 bp in size. All 19 introns are fairly small (79-391). The first intron resides in the 5'-untranslated leader sequence. Introns 10, 11 and 16 are located at positions identical to a rat malic-enzyme gene. In the promoter region, a TATA box (TATATATA) is easily recognized 41 bp upstream of a single transcription-initiation site. Two potential cis-acting elements with homology to elements from plant genes, activated by UV light and fungal elicitors, were identified at positions -153 and -312, respectively. Southern-blot analysis suggests a single gene copy, but also other distantly related genes, in the bean genome. The deduced NADP-ME protein of 589 amino acids exhibits features consistent with a cytoplasmic location. We describe the organization of the NADP-ME protein into functional domains located on separate exons. The evolution of malic-enzyme genes coding for isoforms in different cellular compartments of plants and animals is discussed.

Proteolytic activities and ribulose 1,5 bisphosphate carboxlyase degradation in leaves of soybean (Glycine max L. Merril) with different nitrogen status.

Changes in soluble proteins and Rubisco (E.C.4.1.1.39) contents were examined in leaves of nitrogen-deprived and nitrogen-sufficient soybeans. Rubisco content was very responsive to nitrogen stress, and this protein appeared to be the largest source of mobilizable nitrogen in the senescent leaf. Loss of soluble proteins and Rubisco was associated with a decrease in the activities of several proteolytic enzymes measured using artificial substrates: carboxypeptidase, aminopeptidase and haemoglobinase.The in vitro activity of enzyme(s) which can degrade Rubisco was investigated using endogenous Rubisco and in vitro radiolabelled Rubisco as substrates. Highest endopeptidic cleavage of endogenous Rubisco occured at pH 4; the enzyme responsible for this breakdown appeared to be a sulfhydryl-dependent proteinase. In contrast, [(14)C] Rubisco was attacked preferentially at pH 9, by a peptide hydrolase sensitive to EDTA. No increase in Rubisco-degrading activities was detected in nitrogen-deficient soybean leaves compared to control plant leaves.

Molecular cloning and expression of a Eucalyptus gunnii cDNA clone encoding cinnamyl alcohol dehydrogenase.

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.

Tissue- and cell-specific expression of a cinnamyl alcohol dehydrogenase promoter in transgenic poplar plants.

Cinnamyl alcohol dehydrogenase (CAD) which catalyses the synthesis of the cinnamyl alcohols, the immediate precursors of lignins, from the corresponding cinnamaldehydes is considered to be a highly specific marker for lignification. We have isolated and characterized a CAD genomic clone from eucalyptus, a woody species of economic importance. The full-length promoter (EuCAD, 2.5 kb) and a series of 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene. These constructs were tested in a homologous transient expression system of eucalyptus protoplasts which enabled the identification of several regions involved in transcriptional control. In order to study the spatial and developmental regulation of the CAD gene, the chimeric gene fusion (EuCAD-GUS) was then transferred via Agrobacterium tumefaciens-mediated transformation into poplar, an easily transformable woody angiosperm. Quantitative fluorometric assays conducted on eight independent in vitro transformants showed that GUS activity was highest in roots followed thereafter by stems and leaves. Histochemical staining for GUS activity on both in vitro primary transformants and more mature greenhouse-grown plants indicated a specific expression in the vascular tissues of stems, roots, petioles and leaves. At the onset of xylem differentiation, GUS activity was detected in parenchyma cells differentiating between the xylem-conducting elements. After secondary growth has occurred, GUS activity was localized in xylem ray cells and parenchyma cells surrounding the lignified phloem and sclerenchyma fibers. This first characterization of a woody angiosperm CAD promoter provides functional evidence for the role of CAD in lignification and suggests that parenchyma cells expressing CAD may provide lignin precursors to the adjacent lignified elements (vessels and fibres).

Immunolocalization of cinnamyl alcohol dehydrogenase 2 (CAD 2) indicates a good correlation with cell-specific activity of CAD 2 promoter in transgenic poplar shoots.

Cinnamyl alcohol dehydrogenase 2 (CAD 2) localization and the cell-specific activity of the eucalyptus CAD 2 promoter were investigated by CAD 2 immunogold localization and promoter beta-glucuronidase (GUS) histochemistry in apical and mature parts of stable transformed poplar (Populus tremula x P. alba) stems. Both CAD 2 protein and GUS activity were found to be confined in the same types of cells in the shoot apices, particularly in the determined meristematic cells in leaf axils and shell zones, procambium and developing tracheids. Within mature stems, CAD 2 and GUS were also identified in cambium and in fully or partially lignified cells derived from it (young xylem, developing phloem fibres, chambered parenchyma cells around phloem). Additionally, GUS activity was found in the scale leaves of apical shoot buds and in the roots (namely in the procambium, cambium, phellogen, young xylem, pericycle) of transformed plants. By employing immunogold cytochemistry, CAD 2 was shown to be localized in the cytoplasm within cambial, ray and young xylem cells in stems, the gold particles being randomly attached to endoplasmic reticulum and Golgi-derived vesicles. These results support a crucial role for CAD 2 in lignification and indicate a new role for this enzyme in branching events within the shoot apex and during lateral root formation.

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.

Cloning and characterization of two maize cDNAs encoding cinnamoyl-CoA reductase (CCR) and differential expression of the corresponding genes.

Cinnamoyl-CoA Reductase (CCR, EC 1.2.1.44) catalyses the first step of the lignin pathway. Two full-length cDNAs identified by sequence analysis as CCR-encoding cDNAs were isolated from a maize root cDNA library. These two cDNAs designated ZmCCR1 and ZmCCR2 exhibit 73% sequence conservation at the nucleotide level for their coding regions and are relatively divergent at their 5'- and 3'-untranslated regions. They both contain a common signature which is thought to be involved in the catalytic site of CCR. Northern blot analysis indicated that ZmCCR2 was expressed at very low levels in roots whereas ZmCCR1 was widely expressed in different organs. The high level of ZmCCR1 gene expression along the stalk suggests that the corresponding enzyme is probably involved in constitutive lignification.

Purification and characterization of cinnamyl alcohol dehydrogenase from tobacco stems.

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. In this paper, we report the purification of CAD to homogeneity from tobacco (Nicotiana tabacum) stems. The enzyme is low in abundance, comprising approximately 0.05% of total soluble cell protein. A simple and efficient purification procedure for CAD was developed. It employs three chromatography steps, including two affinity matrices, Blue Sepharose and 2'5' ADP-Sepharose. The purified enzyme has a specific cofactor requirement for NADP and has high affinity for coniferyl alcohol (K(m) = 12 micromolar) and coniferaldehyde (K(m) = 0.3 micromolar). Two different sized polypeptide subunits of 42.5 and 44 kilodaltons were identified and separated by reverse-phase HPLC. Peptide mapping and amino acid composition analysis of the polypeptides showed that they are closely related, although not identical.

Characterization of cis-elements required for vascular expression of the cinnamoyl CoA reductase gene and for protein-DNA complex formation.

Cinnamoyl-CoA reductase (CCR) catalyses the first specific step in the biosynthesis of monolignols, the monomeric units of lignins. We examined the developmental regulation of the Eucalyptus gunnii EgCCR promoter by analysing the expression of EgCCR-GUS fusions in tobacco. EgCCR promoter activity was strongest in lignified organs (stems and roots) consistent with the EgCCR mRNA level in these organs. Histochemical analysis showed expression in vascular tissues (cambium, young differentiating xylem, ray cells, internal and external phloem) of stems and roots in agreement with in situ hybridization data. Promoter deletion analysis and gain-of-function experiments identified the sequences between positions -119 and -77 as necessary and sufficient for expression in vascular tissues of stems. Electrophoretic mobility-shift assays showed that this region is specifically recognized by nuclear proteins present in tobacco stems, giving rise to two retarded complexes, LMC1 and LMC2. Using overlapping EgCCR fragments and mutated oligonucleotides as competitors in gel-shift assays, two DNA-protein interaction sites were mapped. Finally, the role of protein-protein interactions in the formation of the LMC1 and LMC2 complexes was investigated using the detergent sodium deoxycholate, and protein fractionation onto a heparin Sepharose column.

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.

Purification and characterization of isoforms of cinnamyl alcohol dehydrogenase from Eucalyptus xylem.

Two distinct isoforms of cinnamyl alcohol dehydrogenase, CAD 1 and CAD 2, have been purified to homogeneity from xylem-enriched fractions of Eucalyptus gunii Hook and partially characterized. They differ greatly in terms of both physical and biochemical properties, and can be separated by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The native molecular weight of of CAD 1 is 38 kDa as determined by gel-filtration chromatography on Superose 6, and this isoform is likely to be a monomer since it yields a polypeptide of 35 kDa upon sodium dodecyl sulfatepolyacrylamide gel electrophoresis. It has a low substrate affinity for coniferyl and p-coumaryl alcohols and their corresponding aldehydes. No activity with sinapyl aldehyde and alcohol was detected. The more abundant isoform is CAD 2, which has a native molecular weight of 83 kDa and is a dinier composed of two subunits of slightly different molecular weights (42-43 kDa). These subunits show identical peptide patterns after digestion with N-chlorosuccinimide. The isoform, CAD 2, has a high substrate affinity for all the substrates tested. The two isoforms are immunologically distinct as polyclonal antibodies raised against CAD 2 do not cross-react with CAD 1. The characterization of two forms of CAD exhibiting such marked differences indicates their involvement in specific pathways of monolignol utilisation.

Rational inhibitor design, synthesis and NMR spectroscopic study by transferred nuclear overhauser spectroscopy of novel inhibitors of cinnamyl alcohol dehydrogenase, a critical enzyme in lignification.

Cinnamyl alcohol dehydrogenase is one of the enzymes controlling the first two committed steps of lignification. Using a 3-dimensional similarity model of this enzyme, a series of novel phosphonates (1-5) was designed as potential inhibitors. Phosphonates 1-5 were synthesized in good yield by reaction of the corresponding cinnamaldehydes with tetraethylmethylene diphosphonate. Monophosphonic acids 6 and 7 were obtained by basic hydrolysis of the corresponding phosphonates while phosphonamidate 8 was synthesized by reacting benzylamine with the iminium salt intermediate of the monophosphonic acid. Using recombinant cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) the inhibitory activity of these compounds was evaluated and compared with that of the carbonyl analogues. Inhibition kinetic studies showed compounds 2 and 3 to be mixed type linear inhibitors while compound 4 was uncompetitive. 1H NMR studies of inhibitor 2, for which Ki and Ki' were 20 and 86 microM, respectively, in the presence of CAD based on selective line-broadening showed an increased interaction of the 3-OMe group of the aromatic ring of the inhibitor with the active site of the CAD. A transferred nuclear overhauser effect spectroscopy (TRNOESY) experiment for inhibitor 2 with CAD was used to determine the conformation of this compound bound to CAD. These results were found to be consistent with the 3-dimensional structural model of the enzyme.

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)