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

Abstract

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.

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

“…Most importantly, however, C-methylation had to occur strictly before ortho O-glucosylation. In agreement with recent mechanistic proposals, [40][41][42] 7-O-β-D-glucosylation precluded subsequent C-methylation at the adjacent position 8, probably by preventing acceptor activation through deprotonation of 7-OH. Therefore ortho C-methylation and O-glucosylation have to be performed sequentially, independent of the acceptor and the pair of enzymes used.…”

A Tandem Enzymatic sp2‐C‐Methylation Process: Coupling in Situ S‐Adenosyl‐l‐Methionine Formation with Methyl Transfer

“…One C‐MT exemplar is NovO, which methylates the C‐8 position of 1 a in the biosynthesis of the antibiotic novobiocin to form 2 a (Scheme A) . We and others have shown that regiospecific methylation of the 8‐position of 1 b is catalysed by a novel His‐Arg motif, which facilitates the deprotonation of the phenolic proton in the 7‐position, followed by methylation at position 8 by SAM to form 2 b . NovO is also effective in catalysing Friedel–Crafts alkylations by using non‐natural SAM analogues to alkylate 1 b – d regiospecifically .…”

S‐Adenosyl Methionine Cofactor Modifications Enhance the Biocatalytic Repertoire of Small Molecule C‐Alkylation

“…[11][12][13] However,o btaining regiospecificity,p articularly when this is required at alate-stage in asynthetic workflow,is an enduring challenge. [14][15][16][17] Ther epertoire of C-methylation extends to small molecules,which opens up opportunities to tailor these enzymes as ag eneral platform for biocatalytic C À Cb ond formation (Figure 1a). [14][15][16][17] Ther epertoire of C-methylation extends to small molecules,which opens up opportunities to tailor these enzymes as ag eneral platform for biocatalytic C À Cb ond formation (Figure 1a).…”

“…[27,28] Am ore step-and atom-efficient strategy is to couple cofactor formation with C-alkyl transfer. [15,27,29,35,36] Although in-depth knowledge of the substrate promiscuity of C-MTases has been garnered from structural and mutagenesis studies, [17,19,20,37] little is known about how the structural features of the SAM cofactor itself influences the yield and scope of C-alkylation. [34] ClDAi sashelf-stable,a tom-economical adenosine source for such aprocess catalyzed by SalL compared to ATP, which is asubstrate for SAM production by methionine adenosyltransferase (MAT).…”

“…[3] In contrast, MTases catalyze regiospecific C-methylation of biomolecules using the SAM cofactor as the corresponding methyl donor. [14][15][16][17] Ther epertoire of C-methylation extends to small molecules,which opens up opportunities to tailor these enzymes as ag eneral platform for biocatalytic C À Cb ond formation ( Figure 1a). Ar epresentative example is NovO,w hich catalyzes the C-alkylation of coumarins (e.g., 1), and forms akey step in the biosynthesis of novobiocin.…”