Molecular characterization of the C-methyltransferase NovO of Streptomyces spheroides, a valuable enzyme for performing Friedel–Crafts alkylation

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.

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“…The activity assay was carried out according to Tengg et al . [ 4 ]. Soluble protein fractions of recombinant E .…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure and Catalytic Mechanism of CouO, a Versatile C-Methyltransferase from Streptomyces rishiriensis

et al. 2017

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“…Compared to the methyltransferases investigated previously in our labs, those described here show very restricted substrate specificity and do not accept nonrelated substrate scaffolds.…”

Section: Resultsmentioning

confidence: 99%

“…Several S ‐adenosyl‐ l ‐methionine (SAM) dependent methyltransferases able to perform the biocatalytic Friedel–Crafts reaction with non‐natural cofactors (SAM analogues) were investigated in this and previous studies. A comparison between these enzymes shows similarities and differences in this group of methyltransferases.…”

Section: Discussionmentioning

confidence: 99%

“…Although NovO, CouO, SfmM2, and Orf19 occur naturally in Streptomyces species, SibL was originally from Streptosporangium sibiricum , and SacF originates from Pseudomonas fluorescens , which are all bacterial sources. The enzyme NovO, which was characterized with regard to its kinetic data, shows an activity level comparable to known methyltransferases ( k cat =1 min −1 ). The substrate specificity of CouO and NovO can be described as moderate, whereas the other methyltransferases show high substrate specificity.…”

Section: Discussionmentioning

confidence: 99%

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Methyltransferases: Green Catalysts for Friedel–Crafts Alkylations

et al. 2016

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A set of S‐adenosyl‐l‐methionine (SAM) dependent methyltransferases has been characterized as versatile catalysts for the enzymatic Friedel–Crafts (alkylation) reaction. Although the substrate specificity of the enzymes range from high (in the case of SfmM2, SacF, and ORF19) to moderate (in the case of NovO and CouO), the cofactor spectrum is broad. Modified cofactors decorated with alkyl groups other than methyl were used for biocatalytic Friedel–Crafts alkylation, and conversions up to 99 % were achieved. In contrast to the classical chemical reaction the biotransformation is very selective and environmentally compatible.

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“…However, NovO clearly profited from slightly alkaline conditions. Benchmarked against previously used pH of 6.5, 22,34 the methylation rate roughly doubled to 0.23 µmol min…”

Section: Establishing Conditions For Joint Methylation and Glucosylationmentioning

confidence: 98%

An ortho C-methylation/O-glycosylation motif on a hydroxy-coumarin scaffold, selectively installed by biocatalysis

et al. 2017

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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-β-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.

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Molecular characterization of the C-methyltransferase NovO of Streptomyces spheroides, a valuable enzyme for performing Friedel–Crafts alkylation

Cited by 15 publications

References 40 publications

Crystal Structure and Catalytic Mechanism of CouO, a Versatile C-Methyltransferase from Streptomyces rishiriensis

Crystal Structure and Catalytic Mechanism of CouO, a Versatile C-Methyltransferase from Streptomyces rishiriensis

Methyltransferases: Green Catalysts for Friedel–Crafts Alkylations

An ortho C-methylation/O-glycosylation motif on a hydroxy-coumarin scaffold, selectively installed by biocatalysis

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