Structural and biochemical characterisation of a NAD+-dependent alcohol dehydrogenase from Oenococcus oeni as a new model molecule for industrial biotechnology applications

Journal
Applied microbiology and biotechnology  
Volume
97
Issue
20
Start Page
8963
End Page
8975
Citation
Applied Microbiology and Biotechnology 20 (97): 8963-8975 (2013)
Publisher DOI
10.1007/s00253-013-4725-0
Scopus ID
2-s2.0-84885369980
Publisher
Springer
Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni2+-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni2+, activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD +/NADH, whereas no activity has been observed with NADP +/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn2+. The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni2+-dependent activity towards 1,3-propanediol. © 2013 Springer-Verlag Berlin Heidelberg.
Subjects
1,3-propanediol
Alcohol dehydrogenase
Dimer
NAD(H)-dependent
Oenococcus oeni
Zinc
DDC Class
570: Biowissenschaften, Biologie