RESUMEN
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 K eq obtained for real whole-cell reactor systems that modelled industrial synthesis of S-alcohols.
Asunto(s)
Alcoholes/química , Oxidorreductasas/química , Catálisis , Análisis por Conglomerados , Concentración de Iones de Hidrógeno , Cetonas/química , Cinética , Lisina/química , Simulación del Acoplamiento Molecular , NAD/química , Oxidación-Reducción , Teoría Cuántica , Rhodocyclaceae/enzimología , Estereoisomerismo , Tirosina/químicaRESUMEN
Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S is a molybdenum oxidoreductase belonging to the so-called ethylbenzene dehydrogenase (EBDH)-like subclass of DMSO reductases capable of the regioselective hydroxylation of cholesterol or cholecalciferol to 25-hydroxy products. Both products are important biologically active molecules: 25-hydroxycholesterol is responsible for a complex regulatory function in the immunological system, while 25-hydroxycholecalciferol (calcifediol) is the activated form of vitamin D3 used in the treatment of rickets and other calcium disorders. Studies revealed that the optimal enzymatic synthesis proceeds in fed-batch reactors under anaerobic conditions, with 6-9 % (w/v) 2-hydroxypropyl-ß-cyclodextrin as a solubilizer and 1.25-5 % (v/v) 2-methoxyethanol as an organic co-solvent, both adjusted to the substrate type, and 8-15 mM K3[Fe(CN)6] as an electron acceptor. Such thorough optimization of the reaction conditions resulted in high product concentrations: 0.8 g/L for 25-hydroxycholesterol, 1.4 g/L for calcifediol and 2.2 g/L for 25-hydroxy-3-ketosterols. Although the purification protocol yields approximately 2.3 mg of pure S25DH from 30 g of wet cell mass (specific activity of 14 nmol min-1 mg-1), the non-purified crude extract or enzyme preparation can be readily used for the regioselective hydroxylation of both cholesterol and cholecalciferol. On the other hand, pure S25DH can be efficiently immobilized either on powder or a monolithic silica support functionalized with an organic linker providing NH2 groups for enzyme covalent binding. Although such immobilization reduced the enzyme initial activity more than twofold it extended S25DH catalytic lifetime under working conditions at least 3.5 times.
Asunto(s)
Colecalciferol/metabolismo , Colesterol/metabolismo , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Esteroles/metabolismo , Betaproteobacteria/enzimología , Biocatálisis , Reactores Biológicos , Calcifediol/metabolismo , Hidroxicolesteroles/metabolismo , Hidroxilación , Ingeniería Metabólica , Oxidorreductasas/químicaRESUMEN
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)-1-phenylethanol. In this work, we present the application of PEDH for asymmetric reduction of 42 prochiral ketones and 11 ß-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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ésteres/metabolismo , Cetonas/metabolismo , Oxidorreductasas/metabolismo , Rhodocyclaceae/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Catálisis , Desnitrificación , Cetonas/química , Cinética , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas/genética , Rhodocyclaceae/química , Rhodocyclaceae/genética , Estereoisomerismo , Especificidad por SustratoRESUMEN
The molybdenum/iron-sulfur/heme protein ethylbenzene dehydrogenase (EbDH) was successfully applied to catalyze enantiospecific hydroxylation of alkylaromatic and alkylheterocyclic compounds. The optimization of the synthetic procedure involves use of the enzyme in a crude purification state that saves significant preparation effort and is more stable than purified EbDH without exhibiting unwanted side reactions. Moreover, immobilization of the enzyme on a crystalline cellulose support and changes in reaction conditions were introduced in order to increase the amounts of product formed (anaerobic atmosphere, electrochemical electron acceptor recycling or utilization of ferricyanide as alternative electron acceptor in high concentrations). We report here on an extension of effective enzyme activity from 4h to more than 10 days and final product yields of up to 0.4-0.5g/l, which represent a decent starting point for further optimization. Therefore, we expect that the hydrocarbon-hydroxylation capabilities of EbDH may be developed into a new process of industrial production of chiral alcohols.