RESUMEN
Various bioactive natural products, like the aminocoumarin antibiotics novobiocin and coumermycin, exhibit an aromatic C-methyl group adjacent to a glycosylated phenolic hydroxyl group. Therefore, tailoring of basic phenolic scaffolds to contain the intricate C-methyl/O-glycosyl motif is of high interest for structural and functional diversification of natural products. We demonstrate site-selective 8-C-methylation and 7-O-ß-d-glucosylation of 4,5,7-trihydroxy-3-phenyl-coumarin (1) by S-adenosyl-l-methionine dependent C-methyltransferase (from Streptomyces niveus) and uridine 5'-diphosphate glucose dependent glycosyltransferase from apple (Malus × domestica). Both enzymes were characterized and shown to react readily with underivatized 1. However, glucosylation of the ortho-hydroxyl group prevented C-methylation, probably by precluding an essential substrate activation through deprotonation of 7-OH. Therefore, dual modification was only feasible when C-methylation occurred strictly before O-glucosylation. The target product was synthesized in near quantitative yield (98% conversion) from 500 µM 1 and its structure was confirmed by NMR. Combination of C-methyltransferase and O-glycosyltransferase reactions for synthetic tailoring of a natural product through biocatalysis was demonstrated for the first time.
Asunto(s)
Cumarinas/metabolismo , Glicosiltransferasas/metabolismo , Hidróxidos/metabolismo , Metiltransferasas/metabolismo , Biocatálisis , Cumarinas/química , Glicosilación , Hidróxidos/química , Malus/enzimología , Metilación , Estructura Molecular , Streptomyces/enzimologíaRESUMEN
Homology and similarity based approaches are most widely used for the identification of new enzymes for biocatalysis. However, they are not suitable to find truly novel scaffolds with a desired function and this averts options and diversity. Hydroxynitrile lyases (HNLs) are an example of non-homologous isofunctional enzymes for the synthesis of chiral cyanohydrins. Due to their convergent evolution, finding new representatives is challenging. Here we show the discovery of unique HNL enzymes from the fern Davallia tyermannii by coalescence of transcriptomics, proteomics and enzymatic screening. It is the first protein with a Bet v1-like protein fold exhibiting HNL activity, and has a new catalytic center, as shown by protein crystallography. Biochemical properties of D. tyermannii HNLs open perspectives for the development of a complementary class of biocatalysts for the stereoselective synthesis of cyanohydrins. This work shows that systematic integration of -omics data facilitates discovery of enzymes with unpredictable sequences and helps to extend our knowledge about enzyme diversity.
Asunto(s)
Aldehído-Liasas/metabolismo , Antígenos de Plantas/metabolismo , Helechos/enzimología , Nitrilos/metabolismo , Proteínas de Plantas/metabolismo , Aldehído-Liasas/química , Aldehído-Liasas/genética , Secuencia de Aminoácidos , Antígenos de Plantas/química , Antígenos de Plantas/genética , Secuencia de Bases , Biocatálisis , Cristalografía por Rayos X , Helechos/genética , Perfilación de la Expresión Génica/métodos , Modelos Moleculares , Nitrilos/química , Proteínas de Plantas/química , Proteínas de Plantas/genética , Conformación Proteica , Multimerización de Proteína , Proteómica/métodos , Homología de Secuencia de Aminoácido , EstereoisomerismoRESUMEN
This corrects the article DOI: 10.1038/srep46738.
RESUMEN
Friedel-Crafts alkylation of aromatic systems is a classic reaction in organic chemistry, for which regiospecific mono-alkylation, however, is generally difficult to achieve. In nature, methyltransferases catalyze the addition of methyl groups to a wide range of biomolecules thereby modulating the physico-chemical properties of these compounds. Specifically, S-adenosyl-L-methionine dependent C-methyltransferases possess a high potential to serve as biocatalysts in environmentally benign organic syntheses. Here, we report on the high resolution crystal structure of CouO, a C-methyltransferase from Streptomyces rishiriensis involved in the biosynthesis of the antibiotic coumermycin A1. Through molecular docking calculations, site-directed mutagenesis and the comparison with homologous enzymes we identified His120 and Arg121 as key functional residues for the enzymatic activity of this group of C-methyltransferases. The elucidation of the atomic structure and the insight into the catalytic mechanism provide the basis for the (semi)-rational engineering of the enzyme in order to increase the substrate scope as well as to facilitate the acceptance of SAM-analogues as alternative cofactors.
Asunto(s)
Metiltransferasas/química , Metiltransferasas/metabolismo , Streptomyces/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Metiltransferasas/genética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Conformación Proteica , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismoRESUMEN
The residue-specific labeling of proteins with non-canonical amino acids (ncAA) is well established in shake flask cultures. A key aspect for the transfer of the methodology to larger scales for biotechnological applications is the cost of the supplemented ncAAs. Therefore, we established a scalable bioprocess using an engineered host strain for the biosynthesis of the methionine analog norleucine at titers appropriate for the efficient and economic labeling of proteins. To enhance the biosynthesis of norleucine, which is a side-product of the branched chain amino acid pathway, we deleted all three acetolactate synthase isoforms of the methionine auxotrophic Escherichia coli expression strain B834(DE3). Additionally, we overexpressed leuABCD to boost the biosynthesis of norleucine. We systematically analyzed the production of norleucine under the conditions for its residue-specific incorporation in bioreactor cultures that had a 30-fold higher cell density than shake flask cultures. Under optimized conditions, 5g/L norleucine was biosynthesized. This titer is two times higher than the standard supplementation with norleucine of a culture with comparable cell density. We expect that our metabolically engineered strain for the improved biosynthesis of norleucine in combination with the proposed bioprocess will facilitate the efficient residue-specific labeling of proteins at a reasonable price in scales beyond the shake flask.
Asunto(s)
Escherichia coli/metabolismo , Ingeniería Metabólica/métodos , Norleucina , Proteínas Recombinantes , Acetolactato Sintasa/metabolismo , Escherichia coli/genética , Norleucina/análisis , Norleucina/química , Norleucina/metabolismo , Proteínas Recombinantes/análisis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Hydroxynitrile lyases (HNLs) are powerful carbon-carbon bond forming enzymes. The reverse of their natural reaction - the stereoselective addition of hydrogen cyanide (HCN) to carbonyls - yields chiral cyanohydrins, versatile building blocks for the pharmaceutical and chemical industry. Recently, bacterial HNLs have been discovered, which represent a completely new type: HNLs with a cupin fold. Due to various benefits of cupins (e.g. high yield recombinant expression in Escherichia coli), the class of cupin HNLs provides a new source for interesting, powerful hydroxynitrile lyases in the ongoing search for HNLs with improved activity, enantioselectivity, stability and substrate scope. In this study, database mining revealed a novel cupin HNL from Acidobacterium capsulatum ATCC 51196 (AcHNL), which was able to catalyse the (R)-selective synthesis of mandelonitrile with significantly better conversion (97%) and enantioselectivity (96.7%) than other cupin HNLs.
RESUMEN
Recombinant Q9F8T9 protein from Streptomyces rishiriensis (CouO), an S-adenosyl-L-methionine-dependent C-methyltransferase, has been successfully cloned, expressed and purified. CouO was crystallized from a single condition in the Morpheus crystallization screen. A vitrified crystal diffracted to 2.05 Å resolution and belonged to space group P2(1), with unit-cell parameters a = 33.02, b = 82.87, c = 76.77 Å, ß = 96.93°.