A NOVEL AROMATIC ALCOHOL-DEHYDROGENASE IN HIGHER-PLANTS - MOLECULAR-CLONING AND EXPRESSION

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.195) catalyses the convers ion of p-hydroxy-cinnamaldehydes to the corresponding alcohols and is considered a key enzyme in lignin biosynthesis. In a previous study, a n atypical form of CAD (CAD I) was identified in Eucalyptus gunnii [12 ]. We report here the molecular cloning and characterization of the co rresponding cDNA, CAD 1-5, which encodes this novel aromatic alcohol d ehydrogenase. The identity of CAD 1-5 was unambiguously confirmed by s equence comparison of the cDNA with peptide sequences derived from pur ified 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 an d coniferaldehyde, but they do not accept sinapaldehyde. Moreover, rec ombinant CAD 1 can also utilize a wide range of aromatic substrates in cluding unsubstituted and substituted benzaldehydes. The open reading frame of CAD 1-5 encodes a protein with a calculated molecular mass of 35790 Da and an isoelectric point of 8.1. Although sequence compariso ns with proteins in databases revealed significant similarities with d ihydroflavonol-4-reductases (DFR; EC 1.1.1.219) from a wide range of p lant species, the most striking similarity was found with cinnamoyl-Co A reductase (CCR; EC 1.2.1.44), the enzyme which directly precedes CAD in the lignin biosynthetic pathway. RNA blot analysis and immunolocal ization experiments indicated that CAD 1 is expressed in both lignifie d and unlignified tissues/cells. Based on the catalytic activity of CA D 1 in vitro and its localization in planta, CAD 1 may function as an 'alternative' enzyme in the Lignin biosynthetic pathway. However, addi tional roles in phenolic metabolism are not excluded.