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

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Tea Pavkov-Keller, Kerstin Steiner, Mario Faber, Martin Tengg, Helmut Schwab, Mandana Gruber-Khadjawi, Karl Gruber, Albert Jeltsch.


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.

Partial Text

Methylation is one of the most essential reactions in all living organisms and plays an important role in the expression, structure, and function of biological molecules such as proteins, DNA/RNA, and small molecules. The methyl groups are selectively introduced by methyltransferases (MTases), a large group of enzymes that can be divided into several subclasses based on their structural features. The most common class of MTases is class I, possessing a Rossmann-like fold and utilizing S-adenosyl-L-methionine (SAM) as a methyl donor [1]. For this group, a general SN2-like nucleophilic substitution mechanism for methyl transfer is proposed yielding S-adenosyl-L-homocysteine (SAH) and the methylated substrate. Natural-product MTases are the functionally most diverse class of MTases and methyl groups are added to S, N, O or C atoms. The proposed catalytic mechanisms include general acid-base catalysis, metal-based catalysis as well as proximity and desolvation effects not requiring catalytic amino acids [2].

The crystal structure of CouO was determined by molecular replacement using the truncated common core-structure obtained by superposition of available methyltransferase structures with similar sequences (maximum identity <30%), which proved to be a challenging task. An important step was truncating the common core-structure to only those parts that were really conserved and obtaining the correct initial solution with two common core-structures in the asymmetric unit. Less than 40% of the total residues were thus used for initial phasing. Still, we continued with the manual rebuilding and refinement by adding only a few extra residues (max. 4) per refinement cycle until the electron density for the whole molecule appeared. After extensive rebuilding and refinement, two CouO molecules in the asymmetric unit could be completely modeled into the residual electron density. In addition, one SAH cofactor molecule was present in each chain (S1 Fig). Detailed statistics of the structure determination and refinement are listed in Table 1. Since methylation is known to enhance the bioactivity of many natural products [34, 35] CouO and NovO offer substantial promise as biocatalysts for the chemoenzymatic synthesis of novel compounds with therapeutic potential. In particular due to their excellent chemo- and regioselectivity, which favors them over chemical methylation, MTases have a great potential for biotechnological and biomedical applications [36]. The integration of the MTases in a multistep enzyme cascade that addresses the cofactor regeneration limitation already showed to be successful with other enzymes of this family [37]. The gathered information about the three-dimensional structure and the enzymatic mechanism can serve as the basis for rationally engineered CouO/NovO variants with a broadened acceptance of SAM analogues that carry extended carbon chains, as well as the consecutive alkylation of preferable substrates.   Source:


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