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

• Published in: Applied microbiology and biotechnology Vol. 97; no. 20; pp. 8963 - 8975
• Main Authors: Elleuche, Skander, Fodor, Krisztian, Klippel, Barbara, von der Heyde, Amélie, Wilmanns, Matthias, Antranikian, Garabed
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer Berlin Heidelberg 01.10.2013
• Subjects: Alcohol Dehydrogenase - chemistry; Alcohol Dehydrogenase - genetics; Alcohol Dehydrogenase - isolation & purification; Alcohol Dehydrogenase - metabolism; Aldehydes - metabolism; Amino Acid Sequence; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Dimerization; Enzyme Stability; Kinetics; Life Sciences; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Molecular Sequence Data; NAD - metabolism; NADP - metabolism; Nickel - metabolism; Oenococcus - enzymology; Oenococcus - genetics; Propylene Glycols - metabolism; Sequence Alignment; Substrate Specificity; Dimer; Alcohol dehydrogenase; 1,3-propanediol; NAD(H)-dependent; Zinc
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-4725-0

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 Ni 2+ -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 Ni 2+ , 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 Zn 2+ . The present study demonstrates that O . oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni 2+ -dependent activity towards 1,3-propanediol.