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.
Goffner
D
D
Signaux et Messages Cellulaires chez les Végétaux-UMR CNRS-UPS 5546, Université Paul Sabatier, Toulouse, France.
Van Doorsselaere
J
J
Yahiaoui
N
N
Samaj
J
J
Grima-Pettenati
J
J
Boudet
A M
AM
D015096:3-Hydroxysteroid Dehydrogenases / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005052:Eucalyptus / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D015870:Gene Expression; D017343:Genes, Plant; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D005810:Multigene Family; D010802:Phylogeny; D018547:Plant Stems / Q000201:enzymology; D010946:Plants, Medicinal; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018744:DNA, Plant; D011994:Recombinant Proteins; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; C018656:cinnamyl alcohol dehydrogenase