Active site alanine preceding catalytic cysteine determines unique substrate specificity in bacterial CoA-acylating prenal dehydrogenase.

In detoxification and fermentation processes, acylating dehydrogenases catalyze the reversible oxidation of aldehydes to their corresponding acyl-CoA esters. Here, we characterize an enzyme from Aquincola tertiaricarbonis L108 responsible for prenal (3-methyl-2-butenal) to 3-methylcrotonyl-CoA oxidation. Enzyme kinetics demonstrate a preference for C5 substrates not yet observed in aldehyde dehydrogenases. Compared to acetaldehyde and acetyl-CoA, conversion of valeraldehyde and valeryl-CoA is > 100- and 8-fold more efficient, respectively. Enzyme variants with A254I, A254P, and A254G mutations indicate that active site Ala preceding the catalytic C255 is crucial for this unique specificity. These results shed new light on evolutionary adaptation of aldehyde dehydrogenases toward xenobiotics and structure-guided design of highly specific enzymes for production of biofuels, such as linear or iso-branched butanols and pentanols.