Michael hydratase alcohol dehydrogenase or just alcohol dehydrogenase?

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.

Enantioselective Michael Addition of Water

“…When using 3 a as substrate under aerobic conditions (Table 1 , control 1), no conversion to 2 a was detected, indicating that no oxidation occurs. In previous studies 2b , [ 14 ] we were able to show that a chemically catalyzed addition reaction occurs when 2-cyclohexenone ( 1 h ) is used as a substrate. Therefore, any undesired background reaction needed to be ruled out.…”

“…Since whole cells were used in this transformation, the hydratase activity needed to be critically evaluated. [ 11 ]–[ 14 ] Instead of a direct addition of water, the conversion of 1 a to 2 a could also occur via a two-step approach (Scheme Scheme 1 ). Indeed, the enantioselective hydroxylation of a range of THF and THP derivatives was reported for R. rhodochrous strains.…”

Alicycliphilus : current knowledge and potential for bioremediation of xenobiotics

“…Nevertheless, in a more detailed analysis only the ADH activity was confirmed, therefore the hydration of the 2‐cyclohexenone double bond seems to take place spontaneously (Resch et al . ).…”

“…The subsequent purification, characterization and cloning of the 2-cyclohexenone hydratase suggested it was a bi-functional Michael hydratase/alcohol dehydrogenase enzyme (Jin et al 2011). Nevertheless, in a more detailed analysis only the ADH activity was confirmed, therefore the hydration of the 2-cyclohexenone double bond seems to take place spontaneously (Resch et al 2014).…”

The selective addition of water

“…This was due to a coupled assay used, indicating that great care has to be taken when identifying these enzyme activities. 128,129 To summarise, the addition of water is a demanding task. Chemically only the most simple molecules with a limited number of functional groups can be converted reasonably well.…”