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Darobactin Substrate Engineering and Computation Show Radical Stability Governs Ether versus C-C Bond Formation.
Austin M Woodard1,2, Francesca Peccati3, Claudio D Navo3
1Department of Chemistry, University of Illinois at Urbana─Champaign, Urbana, Illinois 61801, United States.
Journal of the American Chemical Society
|May 10, 2024
Summary
Researchers explored the substrate tolerance of the enzyme DarE, which modifies the antibiotic darobactin. They created 51 variants, revealing DarE
Area of Science:
- Biochemistry
- Chemical Biology
- Structural Biology
Background:
- Darobactin A is a Gram-negative selective antibiotic with a unique fused bicyclic structure.
- It targets the outer membrane protein BamA, crucial for bacterial survival.
- Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP) produced by the radical S-adenosyl methionine (rSAM)-dependent enzyme DarE.
Purpose of the Study:
- To analyze the substrate tolerance of the rSAM enzyme DarE.
- To elucidate the catalytic principles governing ether and C-C cross-link formation in darobactin biosynthesis.
- To understand the structural requirements for high-affinity BamA engagement by darobactin variants.
Main Methods:
- Enzymatic modification of darobactin precursors using DarE to generate variants.
- Characterization of novel darobactin variants, including W3Y and K5F.
- Quantum mechanical modeling and molecular docking to investigate reaction mechanisms and binding interactions.
- Mutational analysis and protein structural predictions to identify substrate-binding determinants.
Main Results:
- DarE demonstrated broad substrate tolerance, installing ether and C-C cross-links independently and at various positions.
- Fifty-one enzymatically modified darobactin variants were produced, including novel structures like darobactin W3Y and K5F.
- Computational analysis revealed that radical stability influences ether formation, and specific indole connectivities correlate with ether (Trp-C7) and C-C (Trp-C6) cross-links.
- Experimental and computational data supported a proposed DarE mechanism and identified key substrate residues for enzyme engagement.
Conclusions:
- DarE exhibits remarkable flexibility in catalyzing ether and C-C cross-links, enabling extensive scaffold engineering.
- Understanding DarE's catalytic mechanism provides insights into radical SAM enzyme catalysis.
- The study identified structural features critical for BamA interaction, informing future antibiotic development.
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