Re-engineering a transferase scaffold for indole C3 methylation in diketopiperazines

Area of Science:

• Biochemistry and Organic Chemistry
• Enzyme Engineering and Biocatalysis

Background:

• The pyrroloindole (hexahydropyrrolo[2,3-b]indole, HPI) motif is crucial in biologically active natural products.
• Chemical synthesis of HPIs, especially C3-methylation, is challenging; nature utilizes S-adenosyl methionine (SAM)-dependent methyltransferases.

Purpose of the Study:

• To investigate and engineer a putative methyltransferase, SeMT, from Saccharopolyspora erythraea for HPI synthesis.
• To understand the structure-function relationship of indole C3-methyltransferases.

Main Methods:

• X-ray crystallography to determine the three-dimensional structure of SeMT.
• Site-directed mutagenesis to enhance the catalytic activity of SeMT on diketopiperazine (DKP) substrates.
• Comprehensive characterization of the engineered enzyme variant.

Main Results:

• SeMT shares structural similarity with known methyltransferases but has a divergent catalytic center.
• Mutagenesis efforts yielded a SeMT variant with significant methyltransferase activity.
• A key mutation in SeMT's catalytic triad influenced its function and its homolog SgMT's temperature-activity profile.

Conclusions:

• Engineered SeMT demonstrates potential as a biocatalyst for synthesizing indole C3-methylated compounds.
• Findings offer critical insights into the active site architecture of indole C3-methyltransferases.
• This study paves the way for developing improved biocatalysts for synthetic applications.