Discovery and Biosynthesis of Nitrilobacillins by Post-translational Introduction of C-Terminal Nitrile Groups

Technical Summary: Discovery and Biosynthesis of Nitrilobacillins

1. Problem Statement
Nitrile-containing natural products are ubiquitous across all kingdoms of life and are highly valued in medicinal chemistry for their utility as peptide scaffolds. However, a significant gap existed in the understanding of ribosomally synthesized and post-translationally modified peptides (RiPPs): the nitrile functional group had never been observed in this class of natural products. The scientific community lacked knowledge regarding the genetic machinery and enzymatic mechanisms responsible for introducing C-terminal nitrile groups into RiPPs, limiting the potential for discovering novel bioactive compounds with this specific warhead.

2. Methodology
The researchers employed a combination of genomic mining, biochemical characterization, and structural analysis to investigate a specific biosynthetic gene cluster (BGC).

• Genomic Analysis: They identified a novel BGC encoding a precursor peptide and several tailoring enzymes.
• Enzymatic Characterization: The study focused on three key enzymatic activities:
  • An asparagine synthetase-like (AS-like) protein hypothesized to convert the C-terminal carboxylate to a nitrile.
  • A multinuclear nonheme iron-dependent oxidative enzyme (MNIO) for hydroxylation.
  • An $\alpha$-ketoglutarate-dependent HExxH motif-containing enzyme (KG-HExxH) for stereoselective modifications.
• Functional Assays: The team characterized the substrates and products of these enzymes to map the biosynthetic pathway and confirmed the chemical structure of the final metabolite.
• Biological Activity Testing: The final product was evaluated for its inhibitory activity against cysteine proteases.

3. Key Contributions

• First Identification of Nitrile RiPPs: This work reports the first discovery of a RiPP biosynthetic pathway that generates a C-terminal nitrile group, breaking the precedent that nitriles were absent in this class of natural products.
• Elucidation of a Novel Biosynthetic Mechanism: The study defines a unique enzymatic strategy where an AS-like protein performs the dehydration of a C-terminal carboxylate to form a nitrile, a mechanism distinct from previously known nitrile-forming pathways in non-RiPP contexts.
• Discovery of Specific Tailoring Enzymes: The authors characterized two distinct oxidative enzymes within the cluster:
  • The MNIO enzyme performs stereoselective $\beta$-hydroxylation of aspartate residues.
  • The KG-HExxH enzyme performs stereoselective $\beta$-hydroxylation of proline residues.
• Structural Mimicry of Synthetic Drugs: The research highlights that Nature utilizes the same "warhead" (the nitrile group) found in synthetic therapeutics, specifically drawing a parallel to the active ingredient in Paxlovid (nirmatrelvir), a protease inhibitor used to treat COVID-19.

4. Results

• Product Identification: The biosynthetic pathway yields a novel peptide family named nitrilobacillins.
• Chemical Structure: The final product features a C-terminal nitrile group, $\beta$-hydroxylated aspartate, and $\beta$-hydroxylated proline residues, all installed by the specific enzymes identified in the BGC.
• Biological Activity: Nitrilobacillins function as potent cysteine protease inhibitors. The presence of the nitrile warhead allows the molecule to covalently modify the active site cysteine of target proteases, similar to the mechanism of action of synthetic nitrile-based drugs.

5. Significance

• Expansion of RiPP Diversity: These findings significantly broaden the known structural and functional diversity of RiPPs, proving that this class of natural products can access chemical space (nitriles) previously thought inaccessible via ribosomal synthesis.
• Biocatalytic Potential: The discovery of the AS-like nitrile-forming enzyme and the specific hydroxylases provides new biocatalytic tools for the semi-synthetic production of nitrile-containing peptides, which are valuable in drug discovery.
• Drug Discovery Implications: By demonstrating that Nature produces cysteine protease inhibitors with nitrile warheads analogous to successful synthetic drugs like Paxlovid, this work suggests that RiPPs are a rich, under-explored source for developing new therapeutics against viral and parasitic diseases. It bridges the gap between natural product chemistry and modern medicinal chemistry strategies.