MhyADH: Biocatalytic and Structural Characterisation

The enzyme MhyADH (Michael Hydratase/Alcohol Dehydrogenase)[1] [2] is a recently discovered hydratase/alcohol dehydrogenase that has its origin in the cyclohexanol degrading bacterium Alicycliphilus denitrificans DSMZ 14773 (isolated from anaerobic sewage sludge). The hydratase activity of MhyADH is catalysing the enantioselective addition of water to alpha, beta- unsaturated carbonyl compounds (natural reaction: 2-cyclohexenone to 3-hydroxycyclohexanone), a reaction that is not easily done with chemical methods. Only a few examples using non-enzymatic methods are reported so far.[3] [4] [5] Furthermore the ADH activity is catalysing subsequently the conversion of 3-hydroxycyclohexanone to 1,3-cyclohexanedione. MhyADH consists of three subunits each one of different size and each one harbouring a different co-factor, namely molybdopterin (large subunit), FAD (medium subunit) and [2Fe-2S] cluster (small subunit). [1] [2] In the project submitted herein, MhyADH should be investigated in detail from a biocatalytical and structural point of view. This includes the testing of the substrate spectrum and the structural elucidation of the enzyme to learn more about the mechanism behind the catalysed Michael-addition reaction. Furthermore attempts to express the enzyme in a heterologous fashion using for example E. coli as a host system will be done. The work is planned to be done at the Delft University of Technology under the supervision of Dr. Ulf Hanefeld (Biocatalysis and Organic Chemistry group). [1] Jin J, Straathof AJJ, Pinske MWH, Hanefeld U: Purification, characterization, and cloning of a bifunctional molybdoenzyme with hydratase and alcohol dehydrogenase activity. Appl. Microbiol. Biorechnol 2011, 86:1831- 1840. [2] Jin J, Osakam PC, Karmee SK, Straathof AJJ, Hanefeld U: MhyADH catalysed Michael addition of water and in situ oxidation. Chem. Commun. 2010, 46:8588-8590. [3] Boersma AJ, Coquiere D, Geerdink D, Rosati F, Feringa BL, Roelfes G: Catalytic enantioselective syn hydration of enones in water using a DNA-based catalyst. Nat. Chem. 2010, 2:911-995. [4] Feng X, Yun J: Catalytic enantioselective boron conjugate addition to cyclic carbonyl compounds: a new approach to cyclic beta-hydroxy carbonyls. Chem. Commun. 2009:6577-6579. [5] Hartmann E, Vyasa DJ, Oestreich M: Enantioselective formal hydration of a,b-unsaturated acceptors: asymmetric conjugate addition of silicon and boron nucleophiles. Chem. Commun. 2011, 47:7917-7932

The initial goal of the project was the characterisation of an enzyme (Michael Hydratase Alcohol Dehydrogenase) for the use in organic synthesis for the production of pharmaceutical building blocks (for example secondary alcohols). An earlier published work reported that the described enzyme has a bifunctional activity, meaning it is able to catalyse both a hydration (addition of water to double bonds) and dehydrogenation reaction. However, the focus of the project was set on the hydration reaction, since water would serve as a substrate, which allows a very effective synthetic strategy. After initial problems reproducing the earlier published work on the enzymatic activity, it was observed that the same reaction could be catalysed by simple amino acids. It was shown that the earlier reported enzymatic activity might not be catalysed by the enzyme but rather by amino acids. This observation was investigated further and started as a new project as an alternative to the biocatalytic version. Under optimised conditions the reaction catalysed by amino acids is a very simple route for the synthesis of secondary alcohols. The beauty of the system is its simplicity. Since water serves as a substrate, only an acceptor substrate, aqueous buffer and L-lysine are necessary for the reaction to proceed. During the study all natural amino acids were tested as potential catalysts. L-Lysine turned out to be the best candidate. Amino acids are very cheap and environmentally benign catalysts. They are produced in large scale from natural sources and are easily disposable. The developed reaction system for the hydration of double bonds using amino acids as cheap and environmentally friendly catalysts represents a simple alternative to conventional systems. The advantage of the system is given by its simplicity and eco friendliness.