title: Purification and characterization of a zinc-dependent cinnamyl alcohol dehydrogenase from Leucaena leucocephala, a tree legume.
abstract: A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13 % and specific activity of 812. 5 nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38 kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75 μM(-1) s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH 8.8 and 40 °C, respectively. The enzyme activity was enhanced in the presence of 2.0 mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0 mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.
authors.affiliation: Department of Biochemistry, University of Lucknow, Lucknow, 226007, India.
authors.forename: Brijesh
authors.initials: B
authors.lastname: Pandey
authors.forename: Veda P
authors.initials: VP
authors.lastname: Pandey
authors.forename: A K
authors.initials: AK
authors.lastname: Shasany
authors.forename: U N
authors.initials: UN
authors.lastname: Dwivedi
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D007887:Fabaceae / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D015032:Zinc / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase; D015032:Zinc