RESUMEN
The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: ⢠αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. ⢠The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. ⢠αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).
Asunto(s)
Acremonium , Hesperidina , Hesperidina/química , GlicerolRESUMEN
In the biosynthesis of menaquinone in bacteria, the thiamine diphosphate-dependent enzyme MenD catalyzes the decarboxylative carboligation of α-ketoglutarate and isochorismate to (1R,2S,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC). The regioisomer of SEPHCHC, namely (1R,5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-2-ene-1-carboxylate (iso-SEPHCHC), has been considered as a possible product, however, its existence has been doubtful due to a spontaneous elimination of pyruvate from SEPHCHC to 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC). In this work, the regioisomer iso-SEPHCHC was distinguished from SEPHCHC by liquid chromatography-tandem mass spectrometry. Iso-SEPHCHC was purified and identified by NMR spectroscopy. Just as SEPHCHC remained hidden as a MenD product for more than two decades, its regioisomer iso-SEPHCHC has remained until now.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Ciclohexenos , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Ácidos Cetoglutáricos , Piruvatos , Especificidad por Sustrato , Tiamina Pirofosfato/metabolismo , Vitamina K 2RESUMEN
Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg-Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3â g L-1 ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.
Asunto(s)
Ácidos Ciclohexanocarboxílicos/metabolismo , Proteínas de Escherichia coli/metabolismo , Piruvato Oxidasa/metabolismo , Dominio Catalítico , Ácido Corísmico/química , Ácido Corísmico/metabolismo , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Simulación del Acoplamiento Molecular , Mutación , Unión Proteica , Ingeniería de Proteínas , Piruvato Oxidasa/química , Piruvato Oxidasa/genética , Especificidad por SustratoRESUMEN
A wide range of thiamine diphosphate (ThDP)-dependent enzymes catalyze the benzoin-type carboligation of pyruvate with aldehydes. A few ThDP-dependent enzymes, such as YerE from Yersinia pseudotuberculosis (YpYerE), are known to accept ketones as acceptor substrates. Catalysis by YpYerE gives access to chiral tertiary alcohols, a group of products difficult to obtain in an enantioenriched form by other means. Hence, knowledge of the three-dimensional structure of the enzyme is crucial to identify structure-activity relationships. However, YpYerE has yet to be crystallized, despite several attempts. Herein, we show that a homologue of YpYerE, namely, PpYerE from Pseudomonas protegens (59 % amino acid identity), displays similar catalytic activity: benzaldehyde and its derivatives as well as ketones are converted into chiral 2-hydroxy ketones by using pyruvate as a donor. To enable comparison of aldehyde- and ketone-accepting enzymes and to guide site-directed mutagenesis studies, PpYerE was crystallized and its structure was determined to a resolution of 1.55â Å.
Asunto(s)
Proteínas Bacterianas/metabolismo , Benzaldehídos/metabolismo , Cetonas/metabolismo , Ligasas/metabolismo , Pseudomonas/enzimología , Ácido Pirúvico/metabolismo , Tiamina Pirofosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Humanos , Ligasas/química , Ligasas/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Pseudomonas/química , Pseudomonas/genética , Infecciones por Pseudomonas/microbiología , Estereoisomerismo , Especificidad por SustratoRESUMEN
Carboligations catalyzed by aldolases or thiamine diphosphate (ThDP)-dependent enzymes are well-known in biocatalysis to deliver enantioselective chain elongation reactions. A pyruvate-dependent aldolase (2-oxo-3-deoxy-6-phosphogluconate aldolase [EDA]) introduces a chiral center when reacting with the electrophile, glyoxylic acid, delivering the (S)-enantiomer of (4S)-4-hydroxy-2-oxoglutarate [(S)-HOG]. The ThDP-dependent enzyme MenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (SEPHCHC synthase)) enables access to highly functionalized substances by forming intermolecular C-C bonds with Michael acceptor compounds by a Stetter-like 1,4- or a benzoin-condensation 1,2-addition of activated succinyl semialdehyde (ThDP adduct formed by decarboxylation of 2-oxoglutarate). MenD-catalyzed reactions are characterized by high chemo- and regioselectivity. Here, we report (S)-HOG, in situ formed by EDA, to serve as new donor substrate for MenD in 1,4-addition reactions with 2,3-trans-CHD (2,3-trans-dihydroxy-cyclohexadiene carboxylate) and acrylic acid. Likewise, (S)-HOG serves as donor in 1,2-additions with aromatic (benzaldehyde) and aliphatic (hexanal) aldehydes. This enzyme cascade of two subsequent C-C bond formations (EDA aldolase and a ThDP-dependent carboligase, MenD) generates two new stereocenters.
Asunto(s)
Ácidos Ciclohexanocarboxílicos/metabolismo , Cetoácidos/metabolismo , Tiamina Pirofosfato/metabolismo , Biocatálisis , Ciclohexenos/metabolismo , Descarboxilación/fisiología , Especificidad por SustratoRESUMEN
S-Adenosylmethionine-dependent methyltransferases (MTs) play a decisive role in the biosynthesis of natural products and in epigenetic processes. MTs catalyze the methylation of heteroatoms and even of carbon atoms, which, in many cases, is a challenging reaction in conventional synthesis. However, C-MTs are often highly substrate-specific. Herein, we show that SgvM from Streptomyces griseoviridis features an extended substrate scope with respect to the nucleophile as well as the electrophile. Aside from its physiological substrate 4-methyl-2-oxovalerate, SgvM catalyzes the (di)methylation of pyruvate, 2-oxobutyrate, 2-oxovalerate, and phenylpyruvate at the ß-carbon atom. Chiral-phase HPLC analysis revealed that the methylation of 2-oxovalerate occurs with Râ selectivity while the ethylation of 2-oxobutyrate with S-adenosylethionine results in the Sâ enantiomer of 3-methyl-2-oxovalerate. Thus SgvM could be a valuable tool for asymmetric biocatalytic C-alkylation reactions.
RESUMEN
The hydroxylation of vitaminâ D3 (VD3, cholecalciferol) side chains to give 25-hydroxyvitaminâ D3 (25OHVD3) is a crucial reaction in the formation of the circulating and biologically active forms of VD3 . It is usually catalyzed by cytochrome P450 monooxygenases that depend on complex electron donor systems. Cell-free extracts and a purified Mo enzyme from a bacterium anaerobically grown with cholesterol were employed for the regioselective, ferricyanide-dependent hydroxylation of VD3 and proVD3 (7-dehydrocholesterol) into the corresponding tertiary alcohols with greater than 99 % yield. Hydroxylation of VD3 strictly depends on a cyclodextrin-assisted isomerization of VD3 into preVD3 , the actual enzymatic substrate. This facile and robust method developed for 25OHVD3 synthesis is a novel example for the concept of substrate-engineered catalysis and offers an attractive alternative to chemical or O2 /electron-donor-dependent enzymatic procedures.
Asunto(s)
Colecalciferol/síntesis química , Esteroides/química , Hidroxilación , Agua/químicaRESUMEN
For almost 100 years, phenoxy radical coupling has been known to proceed in nature. Because of the linkage of their molecular halves (regiochemistry) and the configuration of the biaryl axis (stereochemistry), biaryls are notoriously difficult to synthesize. Whereas the intramolecular enzymatic coupling has been elucidated in detail for several examples, the bimolecular intermolecular coupling could not be assigned to one single enzyme in the biosynthesis of axially chiral biaryls. As these transformations often take place regio- and stereoselectively, enzyme-catalyzed control is reasonable. We now report the identification and expression of fungal cytochrome P450 enzymes that catalyze regio- and stereoselective intermolecular phenol couplings. The cytochrome P450 enzyme KtnC from the kotanin biosynthetic pathway of Aspergillus niger was expressed in Saccharomyces cerevisiae. The recombinant cells catalyzed the coupling of the monomeric coumarin 7-demethylsiderin both regio- and stereoselectively to the 8,8'-dimer P-orlandin, a precursor of kotanin. The sequence information obtained from the kotanin biosynthetic gene cluster was used to identify in silico a similar gene cluster in the genome of Emericella desertorum, a producer of desertorin A, the 6,8'-regioisomer of orlandin. The cytochrome P450 enzyme DesC was also expressed in S. cerevisiae and was found to regio- and stereoselectively catalyze the coupling of 7-demethylsiderin to M-desertorin A. Our results show that fungi use highly specific cytochrome P450 enzymes for regio- and stereoselective phenol coupling. The enzymatic activities of KtnC and DesC are relevant for an understanding of the mechanism of this important biosynthetic step. These results suggest that bimolecular phenoxy radical couplings in nature can be catalyzed by phenol-coupling P450 heme enzymes, which might also apply to the plant kingdom.
Asunto(s)
Aspergillus niger/enzimología , Biocatálisis , Productos Biológicos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Emericella/enzimología , Fenol/metabolismo , Aspergillus niger/metabolismo , Productos Biológicos/química , Sistema Enzimático del Citocromo P-450/genética , Emericella/metabolismo , Conformación Molecular , Fenol/química , EstereoisomerismoRESUMEN
Asymmetric mixed carboligation reactions of α-ketoglutarate with different aldehydes were explored with the thiamine diphosphate dependent enzymes SucA from E. coli, Kgd from Mycobacterium tuberculosis, and MenD from E. coli. All three enzymes proved to be efficient biocatalysts to selectively deliver chiral δ-hydroxy-γ-keto acids with moderate to excellent stereoselectivity. The high regioselectivity is due to the preserved role of α-ketoglutarate as acyl donor for these enzyme-catalyzed reactions.
Asunto(s)
Ciclo del Ácido Cítrico/fisiología , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Catálisis , Ciclo del Ácido Cítrico/genética , Escherichia coli/enzimología , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ácidos Cetoglutáricos/química , Ácidos Cetoglutáricos/metabolismo , Mycobacterium tuberculosis/enzimología , Piruvato Oxidasa/metabolismo , Ácido gamma-Aminobutírico/análogos & derivados , Ácido gamma-Aminobutírico/metabolismoAsunto(s)
Cromonas/metabolismo , Oxigenasas/química , Proteínas Recombinantes/química , Streptomyces/enzimología , Ácido 4-Aminobenzoico/metabolismo , Biocatálisis , Clonación Molecular , Transporte de Electrón , Electrones , Escherichia coli , Enlace de Hidrógeno , Manganeso/metabolismo , Modelos Moleculares , Mutación , Nitrocompuestos/metabolismo , Oxidación-Reducción , Oxigenasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleótido Reductasas/química , Ribonucleótido Reductasas/metabolismo , Streptomyces/química , Streptomyces/genética , Homología Estructural de ProteínaRESUMEN
Site-directed mutagenesis based on the crystal structure of AurF, a nitro group forming monooxygenase from Streptomyces thioluteus, revealed that AurF variants are capable of selectively transforming guanidyl- and amidinyl-substituted anilines into the corresponding nitro compounds. Our results provide new insights into the biochemical basis of regioselective N-oxygenation.
Asunto(s)
Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Ácido 4-Aminobenzoico/química , Ácido 4-Aminobenzoico/metabolismo , Dominio Catalítico , Oxigenasas de Función Mixta/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Nitrógeno/metabolismo , Oxidación-Reducción , Estereoisomerismo , Streptomyces/enzimología , Especificidad por SustratoRESUMEN
Starting from (3S,4R,1'S)-3-amino-2-oxo-1-[1'-(4-methoxyphenylethyl)]pyrrolidine carboxylic acid (2), the first synthesis of a beta-foldamer containing pyrrolidin-2-one rings is described, whose 12-helix conformation is assigned by NMR analysis and confirmed by molecular dynamics (MD) simulations.
Asunto(s)
Aminoácidos/química , Ácidos Carboxílicos/química , Ciclohexanos/química , Pirrolidinas/química , Pirrolidinonas/química , Compuestos de Bencidrilo , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Sensibilidad y EspecificidadRESUMEN
Although there is now strong evidence confirming the efficacy of psychological therapies in schizophrenia, the therapeutic processes which they activate remain widely unknown. In order to effectively implement them in clinical practice, identification of these processes is essential. In a controlled study, the efficacy of a coping-oriented therapy approach for schizophrenia patients was tested. Furthermore, the study aimed at establishing preliminary hypotheses on the therapeutically relevant factors. Treatment effects were found in the prominence of psychopathology, the extent of cognizance of the disorder, and the level of social functioning. Moreover, a better psychopathological and social outcome as measured 12 and 18 months after completion of therapy was best predicted by the patients' mastery of active, problem-focused coping strategies immediately after completion of therapy. The findings underscore the clinical relevance of specific coping styles and corroborate the appropriateness of focusing on aspects of coping behavior in psychological interventions for schizophrenia patients.