Characterization of <i>Arabidopsis thaliana</i> cinnamyl alcohol dehydrogenases, and phenylpropenal allylic double bond reductases, and their homologues
Cinnamyl alcohol dehydrogenases (CAD) are members of a NADPH-dependent oxidoreductase gene family involved in the last step of monolignol biosynthesis in the phenylpropanoid metabolic pathway. Monolignols can be converted into lignins and lignans, with the former having structural roles in cell wall biosynthesis, and the latter, as plant defense substances, in vascular plants, respectively. To establish whether there were phenylpropanoid metabolic networks present in Arabidopsis thaliana involving CAD's, the amino acid sequence of 17 putative genes annotated as CAD homologues in the TAIR database were first compared in silico to that of two bona fide CAD genes from tobacco and loblolly pine. In this way, only 9 (AtCAD1–9) of the 17 were correctly annotated as being putative CAD's. These nine were next individually heterologously expressed and characterized in terms of their kinetic properties. AtCAD4 and 5 had the highest overall catalytic activities, whereas AtCAD2–3 and 7–8 were moderately active, and AtCAD1, 6 and 9 had no CAD activity when assayed with various p-hydroxycinnamyl aldehydes in vitro. In particular, there was no substrate specific preference with AtCAD6, 7 and 8, with these being of the highest homology to that of an aspen sinapyl alcohol dehydrogenase (SAD) claimed by others to be sinapyl aldehyde specific. No strict substrate specificity has been established for any members of the CAD family.
Next, to identify gene expression patterns in Arabidopsis, histochemical localization was performed using all AtCAD promoter-GUS reporter fusions. Based on the above in vitro enzyme characterization data, the putative AtCADs were conveniently classified into three groups, AtCAD4 and 5 (catalytically most active), AtCAD2, 3, 7 and 8 (lower activity), and AtCAD1, 6 and 9 (no detectable activity). In the study of gene expression of all nine genes throughout growth/development, AtCAD4 and 5 were found to be strongly expressed in lignifying (vascular) tissues, whereas AtCAD7 and 8 were only moderately expressed in the lignifying tissues, and AtCAD2 and 3 were not. AtCAD1, 6 and 9, although of unknown biochemical function, were also expressed in lignifying tissues. Thus, through the enzyme kinetic characterization and histochemical localization, AtCAD4 (At3g19450) and 5 (At4g34230) were identified as the main enzymes of the Arabidopsis CAD metabolic network.
In a second objective, a phenylpropenal double-bond reductase (PPDBR), which is a NADPH-dependent reductase, was isolated from Pinus taeda cell suspension cultures, and which can reduce the C7–C 8 double bond of various substrates, e.g., coniferyl and dehydrodiconiferyl aldehydes. Its encoding gene was obtained by peptide sequencing approach and cDNA cloning. Recombinant PPDBR readily reduced both the 7–8/7'–8' double bonds of coniferyl/dehydrodiconiferyl aldehydes into their dihydro-forms, but not the corresponding coniferyl/dehydrodiconiferyl alcohols. Therefore, formation of dihydro-derivatives, for example, in 8–5' linked lignan biosynthesis, can occur via a three-step process; phenylpropenol oxidation, phenylpropenal double bond reduction (PPDBR), and phenylpropanal reduction to the corresponding alcohol.
An Arabidopsis homolog of P. taeda PPDBR, alkenal double-bond reductase (AtDBR1; At5g16970) was cloned, with the recombinant protein purified and characterized. AtDBR1 also catalyzed the reduction of the 7–8 double bond of both p-coumaryl and coniferyl aldehydes in vitro, as well as being able to reduce 4-hydroxy-(2E)-nonenal, which is a toxic substance in mammalian cells. An X-ray structural determination of AtDBR1 identified the protein as a dimer with two domains and a catalytic site in-between. Tyr-260 is proposed to have an important role in overall catalysis.