A Zinc‐Dependent Alcohol Dehydrogenase (ADH) from Thauera aromatica, Reducing Cyclic α‐ and β‐Diketones

Abstract

Zinc-dependent alcohol dehydrogenases (ADHs) are valuable biocatalystsf or the synthesis of chiral hydroxy compoundss uch as a-hydroxy ketones andd iols, both valuable precursors for the synthesis of various pharmaceuticals.H owever, while highly active on aliphatic or phenyl-substituted diketones,m ost well characterized ADHs show no significant activity on cyclic a-a nd b-diketones.T herefore, this study aimed at the detection of anovel ADHcapable to reduce these special targets.Itinvolved arational screeningo fb iochemical pathways for enzymes with structurally related natural substrates. Thes od etected 6-hydroxycyclohex-1-ene-1-carbonyl-CoA dehydrogenase (ThaADH) from Thauera aromatica was cloned, expressedi nEscherichiac oli and purified by affinity chromatography.T he characterization revealed as ubstrate specificity with highest activitieso nc yclic a-a nd b-diketones including 1,2-cyclohexanedionea nd 1,3-cyclopentanedione. Structural reasonsf or this extraordinary substrate spectrumw ere investigated with ah omology model created via Swiss Model server. Although the quality of the model may be improved, it suggestst hat ab ulky aromaticr esidue,t hat plays ac rucial role in the definition of the substrate binding pockets of most ADHs,i sr eplaced by ag lycine residue in ThaADH. We propose that this structural difference leads to the formation of one large binding pocket insteado ft wo smallero nes and consequently to ap reference for cyclic diketones over linear bulky substrates.T hus,w eh ave achieved both provision of an ovelb iocatalyst with high potential in chiral synthesis,a nd ap ossible explanation for the measured differencest ok nown ADHs.T he described structural motif might be usedf or identification of further enzymeswith ar elated substrate scope.

Advanced Insights into Catalytic and Structural Features of the Zinc‐Dependent Alcohol Dehydrogenase from Thauera aromatica

“…In accordance with our previous findings, we found a sharp pH-optimum for the reductive activity of ThaADH, [19] but were able to refine the absolute value to pH 6.5. The highest oxidative activity was obtained at a more alkaline pH of 9.0 (Figure S1).…”

“…In our previous study, we determined the molecular weight of the recombinant, Strep-tagged monomer subunit of ThaADH as about 40 kDa. [19] Here, size exclusion chromatography (SEC) showed the enzyme in solution exclusively as a dimer with a molecular weight of 89 kDa (Figure S3), which is in good agreement with the description of the dimeric structure of the native enzyme with a molecular mass of 78000 � 10000 kDa. [27] On the other hand, blue native PAGE documented tetrameric and higher oligomeric states (Figure S5).…”

“…Compared to other published ADHs, the observed activities and stabilities of ThaADH under synthetically relevant conditions are rather promising. As expected, activity [19] and stability towards temperature behave inversely. However, ThaADH shows t 1/2 in the range of 67 d (at 6 °C) to 31 h (at 50 °C) (Figure 1A), which is considerably higher than those of…”

“…Based on our previous observations on the reaction scope of ThaADH, [19] we conducted a detailed examination of the oxidative (Figure 4) as well as reductive (Figure 5) activities.…”

“…In accordance with our previous findings, ThaADH selectively mono-reduced 1,2-cyclohexanedione to the corresponding α-hydroxy ketone 2-hydroxycyclohexanone (Scheme 1A); diol formation was not observed. [19] The preferred reaction product was (S)-2-hydroxycyclohexanone with an ee of 75 % (Table 2). With the chiral substrates 2-bromo-and 2-chlorocyclohexanone, the (S)-configured compounds were clearly preferred (Scheme 1B).…”