Abstract
The reaction mechanism of ketone reduction by short chain dehydrogenase/reductase, (S)-1-phenylethanol dehydrogenase from Aromatoleum aromaticum, was studied with DFT methods using cluster model approach. The characteristics of the hydride transfer process were investigated based on reaction of acetophenone and its eight structural analogues. The results confirmed previously suggested concomitant transfer of hydride from NADH to carbonyl C atom of the substrate with proton transfer from Tyr to carbonyl O atom. However, additional coupled motion of the next proton in the proton-relay system, between O2′ ribose hydroxyl and Tyr154 was observed. The protonation of Lys158 seems not to affect the pKa of Tyr154, as the stable tyrosyl anion was observed only for a neutral Lys158 in the high pH model. The calculated reaction energies and reaction barriers were calibrated by calorimetric and kinetic methods. This allowed an excellent prediction of the reaction enthalpies (R2 = 0.93) and a good prediction of the reaction kinetics (R2 = 0.89). The observed relations were validated in prediction of log Keq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.
Highlights
Pure enantiomers of chiral alcohols are one of the most valuable synthons for the production of various biologically active compounds especially pharmaceuticals, agrochemicals or flavours [1,2,3,4,5,6]
Ketone reduction activity of phenylethanol dehydrogenase (PEDH) was initiated by addition of the selected substrate from stock solution in acetonitrile, and NADH oxidation was followed at 365 nm (Δε = 3.4 × 10−3 M−1 cm−1)
The amount of heat (Q) involved in converting of n moles of selected substrate to the product is expresses by the equation: Fig. 3 PEDH substrates used in the study
Summary
Pure enantiomers of chiral alcohols are one of the most valuable synthons for the production of various biologically active compounds especially pharmaceuticals, agrochemicals or flavours [1,2,3,4,5,6]. One of the successful examples of commercialized biocatalytic methods for synthesis of chiral alcohols is (S)-1-phenylethanol dehydrogenase (PEDH) from Aromatoleum aromaticum (strain EbN1) [7,8,9,10,11,12]. It catalyses the NAD+ dependent stereospecific oxidation of (S)-1-phenylethanol to acetophenone during anaerobic ethylbenzene mineralization as well as the reverse reaction: the NADH dependent enantioselective reduction of acetophenone to (S)-1-phenylethanol (Fig. 1). The latter reaction is interesting since such alcohols are important substances for pharmaceutical, fragrance or food industry [13]
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