The angle of a side-chain decides regio- and enantioselectivity in Alcohol Dehydrogenase A

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for asymmetric synthesis of organic compounds.1 This enzyme is capable of catalyzing enantio- and regioselectivity of phenyl-substitute a-hydroxy ketones (acyloins), which are precursors for the synthesis of a range of biologically active compounds.1,2 ADH-A catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared to the 2-hydroxylated derivatives (R)-phenyl-1,2-ethanediol. ADH-A is highly selective towards secondary-alcohols and displays very low activities with corresponding primary-alcohol derivatives.2,3 Apparently, when this selectivity was tested with substrate contained two secondary-alcohols, we analyzed the catalytic efficiency and the regioselectivity towards (1R,2S)-2.2 The conclusions were yielded that ADH-A is a comparably inefficient catalyst for oxidation of vicinal diols, but displays regioselectivity, oxidizing primarily the benzylic carbon of this substrate.2 Please click Additional Files below to see the full abstract

Characterization and Directed Evolution of an Alcohol Dehydrogenase : A Study Towards Understanding of Three Central Aspects of Substrate Selectivity

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme. Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme. This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides

Facile Synthesis of 2-Hydroxyacetophenone from Racemic Styrene Oxide Catalyzed by Engineered Enzymes

We describe a system that allows for biocatalyzed in vivo synthesis of α-hydroxy ketones from racemic epoxide starting material by in vivo co-expression of native and engineered epoxide hydrolase and alcohol dehydrogenases. The constructed expression system exploits the host cell metabolism for supply and regeneration of precious nicotinamide dinucleotide coenzyme. Racemic styrene oxide added to growth medium passively enters the cells and is hydrolyzed into (1R)-phenylethane-1,2-diol, which is subsequently oxidized to the acyloin 2-hydroxyacetophenone. Produced 2-hydroxyacetophenone escapes the cells via passive diffusion into the growth medium. Thus, co-expression of potato epoxide hydrolase and engineered alcohol dehydrogenase variants can be employed for robust and facile production of 2-hydroxyacetophenone from racemic styrene oxide

The Crystal Structure of Tyrosinase from <i>Verrucomicrobium spinosum</i> Reveals It to Be an Atypical Bacterial Tyrosinase

Tyrosinases belong to the type-III copper enzyme family, which is involved in melanin production in a wide range of organisms. Despite similar overall characteristics and functions, their structures, activities, substrate specificities and regulation vary. The tyrosinase from the bacterium Verrucomicrobium spinosum (vsTyr) is produced as a pre-pro-enzyme in which a C-terminal extension serves as an inactivation domain. It does not require a caddie protein for copper ion incorporation, which makes it similar to eukaryotic tyrosinases. To gain an understanding of the catalytic machinery and regulation of vsTyr activity, we determined the structure of the catalytically active “core domain” of vsTyr by X-ray crystallography. The analysis showed that vsTyr is an atypical bacterial tyrosinase not only because it is independent of a caddie protein but also because it shows the highest structural (and sequence) similarity to plant-derived members of the type-III copper enzyme family and is more closely related to fungal tyrosinases regarding active site features. By modelling the structure of the pre-pro-enzyme using AlphaFold, we observed that Phe453, located in the C-terminal extension, is appropriately positioned to function as a “gatekeeper” residue. Our findings raise questions concerning the evolutionary origin of vsTyr

Relaxation of Nonproductive Binding and Increased Rate of Coenzyme Release in an Alcohol Dehydrogenase Increases Turnover With a Non-Preferred Alcohol Enantiomer

Alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber DSM 44541 is a promising biocatalyst for redox transformations of arylsubstituted sec-alcohols and ketones. The enzyme is stereoselective in the oxidation of 1-phenylethanol with a 300-fold preference for the (S)-enantiomer. The low catalytic efficiency with (R)-1-phenylethanol has been attributed to nonproductive binding of this substrate at the active site. Aiming to modify the enantioselectivity, to rather favor the (R)-alcohol, and also test the possible involvement of nonproductive substrate binding as a mechanism in substrate discrimination, we performed directed laboratory evolution of ADH-A. Three targeted sites that contribute to the active-site cavity were exposed to saturation mutagenesis in a stepwise manner and the generated variants were selected for improved catalytic activity with (R)-1-phenylethanol. After three subsequent rounds of mutagenesis, selection and structure-function analysis of isolated ADH-A variants, we conclude: (1) W295 has a key role as a structural determinant in the discrimination between (R)- and (S)-1-phenylethanol and a W295A substitution fundamentally changes the stereoselectivity of the protein. One observable effect is a faster rate of NADH release, which changes the rate-limiting step of the catalytic cycle from coenzyme release to hydride transfer. (2) The obtained change in enantiopreference, from the (S)- to the (R)-alcohol, can be partly explained by a shift in the nonproductive substrate binding modes

Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (S)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (R)-phenylethane-1,2-diol. The enzyme is also highly selective for sec-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1R,2S)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (S)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher kcat value with 1-phenylpropane-(1R,2S)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1R,2S)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites. Keywords: alcohol dehydrogenase; alcohol oxidation; biocatalysis; crystal structure; directed evolution; enzyme engineering; molecular dynamics simulations; stereoselectivit

Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1‑Phenylethane-1,2-diol and Its Corresponding Acyloin

Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants

Stereo- and Regioselectivity in Catalyzed Transformation of a 1,2-Disubstituted Vicinal Diol and the Corresponding Diketone by Wild Type and Laboratory Evolved Alcohol Dehydrogenases

ADH-A from Rhodococcus ruber DSM 44541 catalyzes the oxidation of (<i>S</i>)-1-phenylethanol 3000-fold more efficiently as compared with the 2-hydroxylated derivative (<i>R</i>)-phenylethane-1,2-diol. The enzyme is also highly selective for <i>sec</i>-alcohols with comparably low activities with the corresponding primary alcohols. When challenged with a substrate containing two secondary alcohols, such as 1-phenylpropane-(1<i>R</i>,2<i>S</i>)-diol, ADH-A favors the oxidation of the benzylic carbon of this alcohol. The catalytic efficiency, however, is modest in comparison to the activity with (<i>S</i>)-1-phenylethanol. To investigate the structural requirements for improved oxidation of vicinal diols, we conducted iterative saturation mutagenesis combined with activity screening. A first-generation variant, B1 (Y54G, L119Y) displays a 2-fold higher <i>k</i>_cat value with 1-phenylpropane-(1<i>R</i>,2<i>S</i>)-diol and a shift in the cooperative behavior in alcohol binding, from negative in the wild type, to positive in B1, suggesting a shift from a less active enzyme form (T) in the wild type to a more active form (R) in the B1 variant. Also, the regiopreference changed to favor oxidation of C-2. A second-generation variant, B1F4 (F43T, Y54G, L119Y, F282W), shows further improvement in the turnover and regioselectivity in oxidation of 1-phenylpropane-(1<i>R</i>,2<i>S</i>)-diol. The crystal structures of the B1 and B1F4 variants describe the structural alterations to the active site, the most significant of which is a repositioning of a Tyr side-chain located distal to the coenzyme and the catalytic zinc ion. The links between the changes in structures and stereoselectivities are rationalized by molecular dynamics simulations of substrate binding at the respective active sites