Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-diol and Its Corresponding Acyloin (original) (raw)
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Journal of Molecular Catalysis B-enzymatic, 2002
A NAD-dependent secondary alcohol dehydrogenase (SAD) has been extracted from cells of the sterol-degrading bacterium, Rhodococcus sp. GK1 (CIP 105335). The dehydrogenase was partially purified by means of ammonium sulfate fractionation (60% saturation) and filtration on a Sepharose CL-6B column. The obtained enzyme sample was active with aliphatic secondary alcohols, such as 2-hexanol, and as reductase with aliphatic monoketones and diketones, such as 2-hexanone and 2,3-hexanedione. A hydrophobic environment was required for catalysis: methyl on one side and either methyl, ethyl, propyl, butyl, pentyl or hexyl on the other side of the function being transformed. The K m value for NAD or NADH with, respectively 2-propanol or acetone was around 1.60×10 −4 M at pH 7.0 and 30 • C. The enzyme affinity (1/K m ) for the examined 2-alcohols and 2-ketones (three to eight C atoms) increased with increasing the chain length. Its activity with 2-octanone was somewhat higher than that with 3-octanone, reflecting a better enzyme affinity for a function positioned at C-2. The K m values for the 2-alcohols (pH 7.0, 30 • C) ranged from 6.0 × 10 −2 M for 2-propanol to 1.8 × 10 −3 M for 2-octanol. Reciprocally, the K m values for the 2-ketones ranged from 6.5 × 10 −2 M for acetone to 2.1 × 10 −3 M for 2-octanone. With 2-hexanol as the substrate, the optimal temperature was around 55 • C and the activation energy of the system was 9.49 kcal/mol. The SAD was specific for the (S)-(+)-stereoisomers of 2-butanol.
2013
The NAD + regeneration system present in E. coli cells was exploited for the oxidation and deracemisation of secondary alcohols with the over-expressed alcohol dehydrogenase from Rhodococcus ruber DSM 44541 (E. coli/ADH-A). Thus, various racemic alcohols were selectively oxidised employing lyophilised or resting E. coli/ADH-A cells, without requiring external cofactor or co-substrate. Simple addition of these substrates to the E. coli/ADH-A cells in buffer afforded the corresponding ketones and the remaining enantioenriched (R)-alcohols. This methodology was applied for the desymmetrisation of a meso-diol, and for the synthesis of the highly valuable raspberry ketone. Moreover, a biocatalytic concurrent process was developed with resting cells of E. coli/ADH-A, ADH from Lactobacillus brevis (LBADH) and glucose dehydrogenase (GDH), for the deracemisation of various secondary alcohols, producing the desired enantiopure alcohols in up to >99% ee starting from the racemic mixture. The reaction time of deracemisation for 1phenylethanol was estimated to be less than 30 min. The stereoinversion of (S)-1-phenylethanol to its pure (R)-enantiomer was also successfully achieved, thus providing a biocatalytic alternative to the chemical Mitsunobu inversion reaction.
Controlling Substrate Specificity and Stereospecificity of Alcohol Dehydrogenases
ACS Catalysis, 2015
The ability to control the substrate specificity and stereochemistry of enzymatic reactions is of increasing interest in biocatalysis. As this review highlights, this control can be achieved through various means, including mutagenesis of active site residues, alteration of physical variables like temperature and pressure, as well as through changing the reaction medium. While the focus of this article is on alcohol dehydrogenase reactions, each of these techniques can be readily applied towards other enzyme classes as well.
Bioresources and Bioprocessing, 2019
Background: A thermostable alcohol dehydrogenase from Thermoanaerobacter brockii (TbSADH) has been repurposed to perform asymmetric reduction of a series of prochiral ketones with the formation of enantio-pure secondary alcohols, which are crucial chiral synthons needed in the preparation of various pharmaceuticals. However, it is incapable of asymmetric reduction when applied to bulky ketones. Recently, mutations at two key residues A85 and I86 were shown to be crucial for reshaping the substrate binding pocket. Increased flexibility of the active site loop appears to be beneficial in the directed evolution of TbSADH towards difficult-to-reduce ketones. Methods: Using the reported mutant A85G/I86A as template, double-code saturation mutagenesis (DCSM) was applied at selected residues lining the substrate binding pocket with a 2-membered reduced amino acid alphabet. Results and conclusions: The mutant A85G/I86A was first tested for activity in the reaction of the model substrate (4-chlorophenyl)-(pyridin-2-yl)methanone, which showed a total turnover number (TTN) of 3071. In order to further improve the turnovers, a small and smart mutant library covering a set of mutations at Q101, W110, L294, and C295 was created. Eventually, a triple-mutant A85G/I86A/Q101A was identified to be a superior catalyst that gave S-selective product with 99% ee and 6555 TTN. Docking computations explain the source of enhanced activity. Some of the best variants are also excellent catalysts in the reduction of other difficult-to-reduce ketones.
Applied Microbiology and Biotechnology, 2014
Enzyme-catalyzed enantioselective reductions of ketones and keto esters have become popular for the production of homochiral building blocks which are valuable synthons for the preparation of biologically active compounds at industrial scale. Among many kinds of biocatalysts, dehydrogenases/reductases from various microorganisms have been used to prepare optically pure enantiomers from carbonyl compounds. (S)-1-phenylethanol dehydrogenase (PEDH) was found in the denitrifying bacterium Aromatoleum aromaticum (strain EbN1) and belongs to the short-chain dehydrogenase/reductase family. It catalyzes the stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization, but also the reverse reaction, i.e., NADH-dependent enantioselective reduction of acetophenone to (S)-1phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 β-keto esters to enantiopure secondary alcohols. The high enantioselectivity of the reaction is explained by docking experiments and analysis of the interaction and binding energies of the theoretical enzyme-substrate complexes leading to the respective (S)-or (R)-alcohols. The conversions were carried out in a batch reactor using Escherichia coli cells with heterologously produced PEDH as whole-cell catalysts and isopropanol as reaction solvent and cosubstrate for NADH recovery. Ketones were converted to the respective secondary alcohols with excellent enantiomeric excesses and high productivities. Moreover, the progress of product formation was studied for nine para-substituted acetophenone derivatives and described by neural network models, which allow to predict reactor behavior and provides insight on enzyme reactivity. Finally, equilibrium constants for conversion of these substrates were derived from the progress curves of the reactions. The obtained values matched very well with theoretical predictions.
Advanced Synthesis & Catalysis, 2009
Regio-and stereoselective reductions of several diketones to afford enantiopure hydroxy ketones or diols were accomplished using isolated alcohol dehydrogenases (ADHs). Results could be rationalised taking into account different (promiscuous) substrate-binding modes in the active site of the enzyme. Furthermore, interesting natural cyclic diketones were also reduced with high regio-and stereoselectivity. Some of the 1,2-and 1,3-diketones used in this study were reduced by employing a low excess of the hydrogen donor (2-propanol) due to the quasi-irreversibility of these ADH-catalysed processes. Thus, using lower quantities of co-substrate, scale-up could be easily achieved.
Applied Microbiology and Biotechnology, 1990
A new alcohol dehydrogenase catalysing the enantioselective reduction of acetophenone to R ( + )phenylethanol was found in a strain of Lactobacillus kefir. A 70-fold enrichment of the enzyme with an overall yield of 76% was obtained in two steps. The addition of Mg 2+ ions was found to be necessary to prevent rapid deactivation. The enzyme depends essentially on N A D P H and was inactive when supplied with N A D H as the coenzyme. Important enzymological data of the dehydrogenase are: Km (acetophenone) 0.6 raM, K~ (NADPH) 0.14 mM, and a pH optimum for acetophenone reduction at 7.0. Addition of EDTA leads to complete deactivation of the enzyme activity. Added iodoacetamide or p-hydroxymercuribenzoate cause only slight inhibition, revealing that the active centre of the enzyme contains no essential SH-group. Besides acetophenone several other aromatic and long-chain aliphatic secondary ketones are substrates for this enzyme. Batch production of phenylethanol was examined using three different methods for the regeneration of N A D P H : glucose/glucose dehydrogenase, glucose-6-phosphate/glucose-6-phosphate dehydrogenase, and isopropanol.
Advanced Synthesis & Catalysis, 2003
An NAD‐dependent and widely applicable (S)‐alcohol dehydrogenase is isolated and described as a novel enzyme. It is expressed in an E. coli strain with a high production potential. The application of this enzyme in asymmetric biocatalytic reduction is presented as well. This enzyme shows a broad substrate range comprising aliphatic and aromatic ketones as well as β‐keto esters. The synthetic application of this new (S)‐alcohol dehydrogenase led to optically active alcohols in high conversion rates accompanied by enantioselectivities of up to >99% ee. Compared to the wild‐type enzyme significant advantages are observed besides the improved availability, such as a high enantioselectivity independent of the reaction time. Furthermore, the alcohol dehydrogenase was cloned and successfully overexpressed in E. coli resulting in a biocatalyst with a potential to be available on technical scale in the future. The development of a large‐scale available and widely applicable (S)‐alcohol de...
Expansion of the Catalytic Repertoire of Alcohol Dehydrogenases in Plant Metabolism
bioRxiv (Cold Spring Harbor Laboratory), 2022
Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism. Alcohol dehydrogenases (ADHs EC 1.1.1.1) are NAD-(P)H-dependent medium-chain oxidoreductases found in all kingdoms of life. These enzymes typically catalyse the reversible reduction of aldehydes or ketones to the corresponding alcohol (Figure 1A). [1-4] The structural motifs of ADHs are highly conserved in all known eukaryotic examples; most notably, a zinc ion involved in catalysis, a second zinc ion involved in maintaining protein structure, and the Rossmann peptide-fold involved in cofactor binding (Figures S1). ADHs have been shown to catalyse many complex biochemical transformations in plant natural product biosynthesis, suggesting that their catalytic repertoire has been expanded. For example, we recently reported the discovery of dihydroprecondylocarpine acetate synthase (DPAS), an ADH involved in vinblastine biosynthesis in the plant Catharanthus roseus [5] and in ibogaine biosynthesis in the phylogenetically related species Tabernanthe iboga. [6] Since the product of DPAS is unstable and either immediately decomposes or rearranges in the presence of a downstream cyclase enzyme, the reaction remained unsubstantiated (Figure 1B). However, the cyclised products suggest that DPAS catalyses the 1,4-reduction of an α,β-unsaturated iminium which is an hitherto unprecedented reaction for an ADH. Here, we use isotopic labelling to definitively establish that DPAS catalyses this unusual 1,4-reduction. We report four crystal structures of apo-and substratebound DPAS from two phylogenetically related species. These structures reveal, surprisingly, the loss of the catalytic zinc ion from the DPAS active site, indicating that zinc is not strictly required for reduction by ADHs. We also report the structure of the ADH geissoschizine synthase (GS) that catalyses an atypical 1,2-iminium reduction. Comparison of the active site of DPAS and GS with other highly similar ADHs that catalyse either 1,2-aldehyde reduction or 1,2reduction of an iminium moiety suggests that changes in the proton relay system are also implicated in modulating ADH reactivity. The mechanism and structure of DPAS highlights the catalytic versatility of ADHs. Overall, these findings demonstrate how the active site of ADHs have been