title: Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana.
abstract: Alcohol dehydrogenases (ADH) catalyze the interconversion between alcohols and aldehydes with the reduction of nicotinamide adenine dinucleotide (NAD(+)) to NADH. In this study, for the first time we report an over-expression and purification strategy for the Arabidosis thaliana ADH (AtADH), and characterize its enzymatic properties. AtADH was expressed in an Escherichia coli system, the polyhistidine-tag was removed after the recombinant AtADH protein was purified by metal chelating affinity chromatography. Activity assays demonstrated that AtADH has distinct enzymatic properties when compared with many well-known ADHs. It held peak activity at pH 10.5 and showed broad substrate selectivity for primary and secondary alcohols. The kinetic Km parameters for both ethanol and coenzyme were in the order of mM. This relative low affinity may reflect the need of the plant to maintain a supply of NAD(+) in nature. Different from yeast ADH, AtADH showed almost the same activity for short straight chain alcohols and reduced activity for secondary alcohols. This broad spectrum in alcohol selection and the observed higher catalytic activity (high Vmax (EtOH)) may result from the requirement of the single enzyme to accommodate many substrates.
authors.affiliation: The Nurturing Station for the State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin'an, Zhejiang 311300, China.
authors.forename: Fangfang
authors.initials: F
authors.lastname: Cheng
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Yan
authors.initials: Y
authors.lastname: An
authors.forename: Jianqin
authors.initials: J
authors.lastname: Huang
authors.forename: Yingwu
authors.initials: Y
authors.lastname: Xu
mesh_terms: D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D029681:Arabidopsis Proteins; D009243:NAD; D000426:Alcohol Dehydrogenase
keywords: Alcohol dehydrogenases; Arabidopsis thaliana; Enzymatic activity; Protein purification