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

Abstract

The asymmetric reduction of ketones to chiral hydroxyl compounds by alcohol dehydrogenases (ADHs) is an established strategy for the provision of valuable precursors for fine chemicals and pharmaceutics. However, most ADHs favor linear aliphatic and aromatic carbonyl compounds, and suitable biocatalysts with preference for cyclic ketones and diketones are still scarce. Among the few candidates, the alcohol dehydrogenase from Thauera aromatica (ThaADH) stands out with a high activity for the reduction of the cyclic α-diketone 1,2-cyclohexanedione to the corresponding α-hydroxy ketone. This study elucidates catalytic and structural features of the enzyme. ThaADH showed a remarkable thermal and pH stability as well as stability in the presence of polar solvents. A thorough description of the substrate scope combined with the resolution and description of the crystal structure, demonstrated a strong preference of ThaADH for cyclic α-substituted cyclohexanones, and indicated structural determinants responsible for the unique substrate acceptance.

Extended Scope and Understanding of Zinc‐Dependent Alcohol Dehydrogenases for Reduction of Cyclic α‐Diketones

“…In contrast, RhjADH did not accept this substrate, but reduced aliphatic 2,3‐pentanedione, which is a typical substrate of CPCR2, with good activity (1.1 U/mL). As our former studies indicated that good acceptance of cyclic and aliphatic α‐substituted ketones is almost mutually exclusive, [5a,b] and the observed activity was in accordance with the lower sequential similarity of RhjADH with ThaADH, we assumed that RhjADH was not able to convert cyclic diketones effectively. Consequently, we excluded this enzyme from further study.…”

“…Detailed examination of the reducing activities of purified CibADH, DecADH and ThpADH, respectively, revealed an overall very similar substrate scope of all three enzymes (Figure 1), which also strongly correlated with the substrate acceptance of ThaADH [5b] . Methylated cyclohexanones (2‐, 3‐ and 4‐methylcyclohexanone), cyclic diketones whose carbonyl groups were not in the α ‐position (1,3‐ and 1,4‐cyclohexanedione) or compounds with aromatic substituents, such as acetophenone and benzaldehyde, were not converted.…”

“…Only a few ADHs are capable to generate sterically demanding cyclic hydroxyketones with α‐ or β‐substitutions [2c,4a–f] . However, through a rational screening of metabolic pathways we succeeded in identifying a NADH‐dependent ADH of the medium chain dehydrogenase/reductase (MDR) superfamily that reduces 1,2‐cyclohexanedione to ( S )‐α‐hydroxycyclohexanone with outstanding activity [5a,b] . In this, the enzyme from Thauera aromatica (ThaADH) differs significantly from other known representatives of the MDR‐ADH superfamily.…”

“…In this, the enzyme from Thauera aromatica (ThaADH) differs significantly from other known representatives of the MDR‐ADH superfamily. The special substrate acceptance goes along with a very specific design of the enzyme's active site [5b] . Most apparent is the formation of a single large binding pocket instead of the usual division into a large and a small binding pocket, [6a,b] due to the replacement of a bulky aromatic amino acid residue [5a,b] …”

“…The second carbonyl function could only be substituted by halogens (Br, Cl, F); methyl substitutions were not accepted. Cyclic diketones in which the carbonyl functions were not in the α‐position and/or which had slightly smaller or larger rings were hardly converted [5b] …”