R-1-phenyl ethanol, however, is oxidized under CtADH catalysis as well, albeit with a significantly lower specific activity ( Table 2). This orientation, in which the phenyl ring is embedded in a hydrophobic environment within the outer part of the substrate-binding tunnel formed by Val141, Leu148, Ala184/185 and Trp191 ( Figure 3a), would lead to the product S-1-phenyl hexanol and is thus consistent with the preference of CtADH for the (S)-enantiomer of 1-phenyl ethanol, a CtADH substrate in oxidation direction ( Table 2). In Figure 3b, this is illustrated with the R-specific alcohol dehydrogenase from Lactobacillus brevis (LbRADH) which accordingly contains a much shorter C-terminal segment ( Figure 3a).įurther, similar to what had been done for SyADH, we placed n-pentyl phenyl ketone such that its re-plane faces NADPH. In this way, the helix α9 stabilizes the dimeric architecture ( Figure 3b) simultaneously, however, it blocks the surface used by the majority of SDR enzymes to coordinate a second dimer and thus to establish a homotetramer with D2 point symmetry. This helix sticks out from the main domain since it has a special double role at the level of the quaternary structure: as typical for SDR enzymes, the large helices α4/α5 and α6 function as a dimerization module and form a four-helix bundle with a second CtADH monomer ( Figure 3b) yet, in contrast to most SDR enzymes (but similar to SyADH), the particular helix α9 crosses over to the second subunit and forms a helical pair with its counterpart within the dimer. Like SyADH, CtADH contains a non-canonical helix α9 in its C-terminal segment ( Figure 3a). CtADH/NADPH/substrate complexes modelled on the basis of crystal structures of CtADH and its closest homologue suggested preliminary hints to rationalize the enzyme’s substrate preferences Biotransformations with selected ketones-performed with a coupled regeneration system for the co-substrate NADPH-resulted in conversions of more than 99% with all tested substrates and with excellent enantioselectivity for the corresponding S-alcohol products. These studies revealed a broad pH profile and an extended substrate spectrum with the highest activity for compounds containing halogens as substituents and a moderate activity for bulky–bulky ketones. A novel alcohol dehydrogenase from Comamonas testosteroni (CtADH) was identified in silico, recombinantly expressed and purified, enzymatically and biochemically investigated as well as structurally characterized. Due to their high regio- and stereospecificity, they are key components in a wide range of industrial applications. Alcohol dehydrogenases catalyse the conversion of a large variety of ketone substrates to the corresponding chiral products.
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