title: Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4.
abstract: The cinnamyl alcohol dehydrogenase (CAD) multigene family in planta encodes proteins catalyzing the reductions of various phenylpropenyl aldehyde derivatives in a substrate versatile manner, and whose metabolic products are the precursors of structural lignins, health-related lignans, and various other metabolites. In Arabidopsis thaliana, the two isoforms, AtCAD5 and AtCAD4, are the catalytically most active being viewed as mainly involved in the formation of guaiacyl/syringyl lignins. In this study, we determined the crystal structures of AtCAD5 in the apo-form and as a binary complex with NADP+, respectively, and modeled that of AtCAD4. Both AtCAD5 and AtCAD4 are dimers with two zinc ions per subunit and belong to the Zn-dependent medium chain dehydrogenase/reductase (MDR) superfamily, on the basis of their overall 2-domain structures and distribution of secondary structural elements. The catalytic Zn2+ ions in both enzymes are tetrahedrally coordinated, but differ from those in horse liver alcohol dehydrogenase since the carboxyl side-chain of Glu70 is ligated to Zn2+ instead of water. Using AtCAD5, site-directed mutagenesis of Glu70 to alanine resulted in loss of catalytic activity, thereby indicating that perturbation of the Zn2+ coordination was sufficient to abolish catalytic activity. The substrate-binding pockets of both AtCAD5 and AtCAD4 were also examined, and found to be significantly different and smaller compared to that of a putative aspen sinapyl alcohol dehydrogenase (SAD) and a putative yeast CAD. While the physiological roles of the aspen SAD and the yeast CAD are uncertain, they nevertheless have a high similarity in the overall 3D structures to AtCAD5 and 4. With the bona fide CAD's from various species, nine out of the twelve residues which constitute the proposed substrate-binding pocket were, however, conserved. This is provisionally considered as indicative of a characteristic fingerprint for the CAD family.
authors.affiliation: School of Molecular Biosciences, Washington State University, Pullman, 99164-4660, USA.
authors.forename: Buhyun
authors.initials: B
authors.lastname: Youn
authors.forename: Roy
authors.initials: R
authors.lastname: Camacho
authors.forename: Syed G A
authors.initials: SG
authors.lastname: Moinuddin
authors.forename: Choonseok
authors.initials: C
authors.lastname: Lee
authors.forename: Laurence B
authors.initials: LB
authors.lastname: Davin
authors.forename: Norman G
authors.initials: NG
authors.lastname: Lewis
authors.forename: Chulhee
authors.initials: C
authors.lastname: Kang
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry*; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019281:Dimerization; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009249:NADP / Q000737:chemistry; D020033:Protein Isoforms; D015032:Zinc / Q000737:chemistry
chemical_list: D029681:Arabidopsis Proteins; D020033:Protein Isoforms; D009249:NADP; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C520968:CAD4 protein, Arabidopsis; C520967:CAD5 protein, Arabidopsis; C018656:cinnamyl alcohol dehydrogenase; D015032:Zinc