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

Abstract

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.

Metabolic engineering of Methylobacterium extorquens AM1 for the production of butadiene precursor

“…Trace concentration of crotyl diphosphate was detected, and 0.51 μg/mL of crotonol (7.0 μM) was produced at 4 h. This result indicated that low activity of ADHE2 was one major bottleneck resulting in the lack of reduction of crotonyl-CoA into crotyl alcohol. Just recently, Becher et al characterized a novel CoA-acylating aldehyde dehydrogenase responsible for prenal (3-methyl-2-butenal) to 3-methylcrotonyl-CoA oxidation [48], which could possibly improve the activity for the first step of reduction of crotyl-CoA to crotonaldehyde. Future direction will be focused on improving catalytic efficiency of crotyl-CoA into crotyl alcohol to realize the production of butadiene precursor from methanol.…”

“…This was likely because of low activity of ADHE2 towards crotonyl-CoA reduction. In the future, it should be possible to address this issue including protein design and manipulating the flux through the EMC pathway [14, 48, 49]. Although further enzymes and strain optimization are required to make this system industrially relevant, this novel work is the first example for biosynthesis of butadiene precursors.…”

Actinobacterial Degradation of 2-Hydroxyisobutyric Acid Proceeds via Acetone and Formyl-CoA by Employing a Thiamine-Dependent Lyase Reaction

“…In DSM 45062 cultures, consumption of substrates and formation of metabolites were routinely monitored by HPLC (Shimadzu Corporation) equipped with a Hi-Plex H column (300 mm × 7.7 mm; Agilent Technologies) and refractive index detector (Becher et al, 2018). In addition, acetone was quantified by headspace GC (Agilent Technologies) employing an Optima Delta-3 column (60 m × 0.32 mm × 0.25 µm; Macherey-Nagel) and flame ionization detection (Becher et al, 2018).…”

“…In DSM 45062 cultures, consumption of substrates and formation of metabolites were routinely monitored by HPLC (Shimadzu Corporation) equipped with a Hi-Plex H column (300 mm × 7.7 mm; Agilent Technologies) and refractive index detector (Becher et al, 2018). In addition, acetone was quantified by headspace GC (Agilent Technologies) employing an Optima Delta-3 column (60 m × 0.32 mm × 0.25 µm; Macherey-Nagel) and flame ionization detection (Becher et al, 2018). Formation of ketones in bacterial cultures and in the lyase assays was verified by a GC system (Agilent Technologies) equipped with an Optima Delta-3 column (30 m × 0.25 mm × 0.25 µm; Macherey-Nagel) and mass spectrometer (Becher et al, 2018).…”

“…In addition, acetone was quantified by headspace GC (Agilent Technologies) employing an Optima Delta-3 column (60 m × 0.32 mm × 0.25 µm; Macherey-Nagel) and flame ionization detection (Becher et al, 2018). Formation of ketones in bacterial cultures and in the lyase assays was verified by a GC system (Agilent Technologies) equipped with an Optima Delta-3 column (30 m × 0.25 mm × 0.25 µm; Macherey-Nagel) and mass spectrometer (Becher et al, 2018). The resulting mass spectra were compared with spectra from pure standard chemicals as well as with most-probable matches by the National Institute of Standards and Technology library database.…”