Scientists have simplified a process that uses the promiscuity of post-translational enzymes to create and improve complex molecules.1 The team behind the work says it could be developed into a platform technology to generate libraries of compounds with the structural diversity of natural products that are also new to nature.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of natural products with great structural diversity. RiPP biosynthesis begins with the ribosome translating a structural gene to produce a precursor peptide. Enzymes then modify this in several steps to produce the final biologically active product. “The way the RiPP enzymes work is very interesting; it is different from how enzymes normally work. The enzymes bind to one bit, but catalyze elsewhere … the result of this is that many of these enzymes are very promiscuous, ”explains Jesko Koehnke from the University of Glasgow, UK.

Although it could potentially be used to build various libraries of compounds, RiPP engineering is currently limited by the need for a tailored precursor peptide with a leader segment to which the post-translational enzymes can bind. In many cases, the peptides must also contain a specific sequence of amino acid residues in order for enzymes to recognize them as a substrate – the enzyme then modifies another part of the peptide known as the core peptide. The resulting compound libraries are currently variations on a theme determined by the chemistry of the enzyme used. Koehnke wanted to investigate whether more diverse libraries would be possible if such a dependency on the leader peptide were eliminated.

“We tried different ways and it didn’t work; we tried chemically and with other enzymes. I ended up going on a series of conversations and there was one guy [Dirk Schwarzer at the University of Tübingen in Germany] who talked about sortase A and then suddenly it was easy, ”recalls Koehnke. Sortase A is a bacterial enzyme that links peptidoglycan chains that structural biologists have adopted to fuse proteins. Koehnke and Laura Franz from Saarland University have now shown that Sortase A can be used to exchange leader peptides between biosynthetic steps, making it much easier to instruct different enzymes to modify ribosomal products.

As a proof of concept for their leader peptide exchange technique, Koehnke and Franz combined enzymes from two unrelated biosynthetic pathways that come from different cyanobacteria. They began by modifying their precursor peptide with LynD, an enzyme that converts cysteine ​​residues into thiazoline heterocycles. The precursor peptide contained a leader segment to which LynD could bind, followed by the recognition motif of Sortase A and was linked to the core segment of the peptide by two glycines. They then introduced Sortase A, which cleaved two of the motif’s residues before catalyzing a new amide bond between that end of the peptide and the leader peptide for the next modifying enzyme, MdnC. MdnC-catalyzed ester bond formation between the side chains of certain residues. In a final step, Sortase A was reintroduced to release the desired hybrid product.

In 2017, scientists from the University of Illinois at Urbana-Champaign reported a method for the production of RiPP hybrid products using a chimeric leader peptide with recognition sequences for two different enzymes, so that two enzyme modification steps can occur in succession.2 This was a huge step forward in the creation of hybrid natural products, but users needed to know the recognition sequences associated with each enzyme. Koehnke and Franz’s method is less complicated; They say it has the potential to evolve into a plug-and-play approach, where compatible enzymatic steps can yield complex compounds that conventional synthetic chemistry could not make.

“RiPP natural products are a very exciting class of potential drugs that can fight anything from antibiotic resistance to cancer,” notes Michael Jewett, whose laboratory at Northwestern University in the United States also works on ribosomal synthesis. “Hence, this approach can enable the creation of new substance libraries that were previously inaccessible to identify unique biotherapeutic activities.”

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