RESUMEN
Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.
Asunto(s)
Piruvato Descarboxilasa/metabolismo , Acetona/análogos & derivados , Acetona/química , Acetona/metabolismo , Sitios de Unión , Alcoholes Grasos/química , Alcoholes Grasos/metabolismo , Simulación de Dinámica Molecular , Mutagénesis , Estructura Terciaria de Proteína , Piruvato Descarboxilasa/química , Piruvato Descarboxilasa/genética , Estereoisomerismo , Zymomonas/enzimologíaRESUMEN
The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIPâ OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIPâ OR was expanded to chiral tertiary α-hydroxy ketones through the rare aldehyde-ketone cross-carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIPâ OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP-dependent enzymes. The combination of methylacetoin as acyl anion synthon and novel ThDP-dependent enzymes considerably expands the available range of C-C bond formations in asymmetric synthesis.
Asunto(s)
Aldehídos/química , Cetonas/química , Oxidorreductasas/metabolismo , Alcoholes/química , Alcoholes/metabolismo , Bacillus/enzimología , Biocatálisis , Escherichia coli/metabolismo , Oxidorreductasas/química , Oxidorreductasas/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , EstereoisomerismoRESUMEN
ThDP-dependent cyclohexane-1,2-dione hydrolase (CDH) catalyzes the CC bond cleavage of cyclohexane-1,2-dione to 6-oxohexanoate, and the asymmetric benzoin condensation between benzaldehyde and pyruvate. One of the two reactivities of CDH was selectively knocked down by mutation experiments. CDH-H28A is much less able to catalyze the CC bond formation, while the ability for CC bond cleavage is still intact. The double variant CDH-H28A/N484A shows the opposite behavior and catalyzes the addition of pyruvate to cyclohexane-1,2-dione, resulting in the formation of a tertiary alcohol. Several acyloins of tertiary alcohols are formed with 54-94 % enantiomeric excess. In addition to pyruvate, methyl pyruvate and butane-2,3-dione are alternative donor substrates for CC bond formation. Thus, the very rare aldehyde-ketone cross-benzoin reaction has been solved by design of an enzyme variant.
Asunto(s)
Hidrolasas/metabolismo , Tiamina Pirofosfato/química , Sustitución de Aminoácidos , Azoarcus/enzimología , Benzoína/química , Biocatálisis , Carbono/química , Dominio Catalítico , Ciclohexanonas/química , Ciclohexanonas/metabolismo , Hidrolasas/química , Hidrolasas/genética , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
The thiamine diphosphate (ThDP)-dependent enzyme cyclohexane-1,2-dione hydrolase (CDH) was expressed in Escherichia coli and purified by affinity chromatography (Ni-NTA). Recombinant CDH showed the same C-C bond-cleavage and C-C bond-formation activities as the native enzyme. Furthermore, we have shown that CDH catalyzes the asymmetric cross-benzoin reaction of aromatic aldehydes and (decarboxylated) pyruvate (up to quantitative conversion, 92-99 % ee). CDH accepts also hydroxybenzaldehydes and nitrobenzaldehydes; these previously have not (or only in rare cases) been known as substrates of other ThDP-dependent enzymes. On a semipreparative scale, sterically demanding 4-(tert-butyl)benzaldehyde and 2-naphthaldehyde were transformed into the corresponding 2-hydroxy ketone products in high yields. Additionally, certain benzaldehydes with electron withdrawing substituents were identified as potential inhibitors of the ligase activity of CDH.
Asunto(s)
Enzimas Multifuncionales/metabolismo , Tiamina/metabolismo , Azoarcus/enzimología , Benzaldehídos/química , Benzaldehídos/metabolismo , Benzoína/química , Benzoína/metabolismo , Biocatálisis , Enzimas Multifuncionales/genética , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Tiamina/químicaRESUMEN
The thiamine diphosphate (ThDP) dependent MenD catalyzes the reaction of α-ketoglutarate with pyruvate to selectively form 4-hydroxy-5-oxohexanoic acid 2, which seems to be inconsistent with the assumed acyl donor role of the physiological substrate α-KG. In contrast the reaction of α-ketoglutarate with acetaldehyde gives exclusively the expected 5-hydroxy-4-oxo regioisomer 1. These reactions were studied by NMR and CD spectroscopy, which revealed that with pyruvate the observed regioselectivity is due to the rearrangement-decarboxylation of the initially formed α-hydroxy-ß-keto acid rather than a donor-acceptor substrate role variation. Further experiments with other ThDP-dependent enzymes, YerE, SucA, and CDH, verified that this degenerate decarboxylation can be linked to the reduced enantioselectivity of acyloins often observed in ThDP-dependent enzymatic transformations.