Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis.
Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.
Kim
Sung-Jin
SJ
Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340, USA.
Kim
Mi-Ran
MR
Bedgar
Diana L
DL
Moinuddin
Syed G A
SG
Cardenas
Claudia L
CL
Davin
Laurence B
LB
Kang
ChulHee
C
Lewis
Norman G
NG
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D008958:Models, Molecular; D008969:Molecular Sequence Data; D005810:Multigene Family; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C520967:CAD5 protein, Arabidopsis; C018656:cinnamyl alcohol dehydrogenase