![]() ![]() Thus, CtADH prefers to accommodate a bulky phenyl substituent in the outer substrate binding tunnel as indicated in Figure 3c,d, but can essentially place it alternatively as well, namely at the inner region of the tunnel occupied by the n-propyl chain in Figure 3c,d. 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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