Abstract
The Michael hydratase – alcohol dehydrogenase (MhyADH) from Alicycliphilus denitrificans was previously identified as a bi-functional enzyme performing a hydration of α,β-unsaturated ketones and subsequent oxidation of the formed alcohols. The investigations of the bi-functionality were based on a spectrophotometric assay and an activity staining in a native gel of the dehydrogenase. New insights in the recently discovered organocatalytic Michael addition of water led to the conclusion that the previously performed experiments to identify MhyADH as a bi-functional enzyme and their results need to be reconsidered and the reliability of the methodology used needs to be critically evaluated.
Highlights
The Michael addition of water to α,β-unsaturated ketones is a very interesting but rarely investigated reaction in organic synthesis and only a few procedures are described (Riedel and Krekeler 1972; Mahajan et al 2006; Wang et al 2006; Boersma et al 2010a; Boersma et al 2010b)
Staining of 3-hydroxycyclohexanone dehydrogenase activity in native gel The results from previous experiments indicated that Michael hydratase – alcohol dehydrogenase (MhyADH) is a bi-functional enzyme that possesses next to its hydratase activity a dehydrogenase activity
TADH has a homotetrameric structure with a molecular weight of around 149 kDa (Höllrigl et al 2008) MhyADH was described as a heterotrimeric enzyme comprised of three subunits with an approximate molecular weight of 20, 30, and 90 kDa, respectively (Jin et al 2011)
Summary
The Michael addition of water to α,β-unsaturated ketones is a very interesting but rarely investigated reaction in organic synthesis and only a few procedures are described (Riedel and Krekeler 1972; Mahajan et al 2006; Wang et al 2006; Boersma et al 2010a; Boersma et al 2010b). In contrast to the low number of chemical methods, nature is capable of performing this reaction and many examples have been described (Jin and Hanefeld 2011) The beauty of this reaction is obvious: Using water as a nucleophile and solvent would allow a green route to hydroxy ketones. One drawback of the enzymes described is their narrow substrate scope caused by their involvement in (primary) metabolic pathways where specificity is highly important. For their optimal use in biocatalysis, this ability is not desired and new hydratases with a broader substrate spectrum are required. Even though no extensive study on the substrate acceptance is reported and the hydratase involved in this
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