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

Abstract

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.

A Genomewide Analysis of the Cinnamyl Alcohol Dehydrogenase Family in Sorghum [Sorghum bicolor(L.) Moench] IdentifiesSbCAD2as theBrown midrib6Gene

“…The last step in monolignol biosynthesis is the reduction of cinnamyl aldehyde precursors (reviewed by Humphreys and Chapple 2002), catalyzed by the enzyme cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.95; Mansell et al 1974). Two enzymes with cinnamyl alcohol dehydrogenase activity were identified in Eucalyptus gunnii, and named EgCAD1 and EgCAD2 (Goffner et al 1992). The enzymes are structurally unrelated and share no sequence similarity.…”

Characterization of three cinnamyl alcohol dehydrogenases from Carthamus tinctorius

“…CtCAD2, in contrast, showed high similarity to PtreCAD (79.8% identity) (Li et al 2001) and AtCAD5 (74.8% identity) (Youn et al 2006). The CtCADs showed low amino acid sequence identities to EgCAD1-type enzymes (8.6-10.5%), which are close to cinnamoyl CoA reductases (CCRs) (Goffner et al 1992).…”

“…Individual angiosperm species, in contrast, have a number of CAD isoforms. These isoforms can show different or comparable characteristics (Goffner et al 1992;Kim et al 2004;Li et al 2001;Mansell et al 1976;Pillonel et al 1992;Wyrambik and Grisebach 1975). Wyrambik and Grisebach (1975) undertook one of the earliest studies of different plant CAD isoforms.…”

“…Eucalyptus gunnii was also reported to have several CAD isoforms. Goffner et al (1992) reported the isolation of two structurally distinct CAD isoforms from E. gunnii. One E. gunnii CAD (EgCAD1) was only capable of catalyzing coniferaldehyde reduction, whereas the second CAD (EgCAD2) reduced a wider range of substrates: coniferaldehyde, sinapaldehyde, and p-coumaraldehyde.…”

“…One E. gunnii CAD (EgCAD1) was only capable of catalyzing coniferaldehyde reduction, whereas the second CAD (EgCAD2) reduced a wider range of substrates: coniferaldehyde, sinapaldehyde, and p-coumaraldehyde. Interestingly, a cDNA encoding an EgCAD1-type enzyme showed higher similarity to CCRs (cinnamoyl CoA reductases) than to typical plant CADs (Goffner et al 1992).…”