Purification And Enzymatic Characterization Of Alcohol Dehydrogenase From Arabidopsis Thalina

Alcohol dehydrogenase is the key enzyme of the primary short-chain alcohol metabolism in many organisms.The classic alcohol dehydrogenase is a Zn-binding enzyme that acts as a dimer and relies on an NAD(P)+ co-factor to interconvert ethanol and acetaldehyde. The plant ADHs generally contain three functional domains, which commonly are ADH N domain of GroES structure, NAD(P)(+)-binding protein by Rossmann fold and zinc binding site, these functional domains would be considered much conservative in all plant species. In order to explore the structure of arabidopsis thaliana ethanol dehydrogenase and its biochemical characteristics, arabidopsis ADH was constructed as a fusion protein and expressed in prokaryotic cells.AtADH expression vectors were constructed with pET-HTT vector and Rosetta expression stain. Through screening of different expression conditions, an optimal expression strategy by inducing at 20 ℃ for 14 hours with 0.1% IPTG was achieved. The soluble protein were received, then induced and purified amounts of pure protein(>90%).Infinite M200 Multimode Reader(TECAN) was used to record the absorbance at 340 nm in a 96-well plate. The results suggest that AtADH held peak activity at p H 10.5 and showed broad substrate selectivity for primary and secondary alcohols. the Michaelis constant Km(NAD) was measured to be 1.65±0.42 mM and the maximum velocity value Vmax(NAD) was 7.9±2.6 U/mg。According to sequence comparison and molecular dynamics results, we found the catalytic domain is connected to coenzyme DB through linkers 176 SCG and 321 FFGNYKP. The glycine residue may facilitate the relative movement between the two domains and contribute to coenzyme binding and catalytic activity. Through comparing the wild type and mutant ADH protein enzyme activity, the results show no significant change.