Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes.

Bioorg Med Chem

Faculty of Pharmacy, Kindai University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502, Japan. Electronic address:

Published: August 2024

AI Article Synopsis

  • The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) activates substrate amino acids using ATP, and specialized photoaffinity probes have been developed to selectively detect these A-domains.
  • Researchers designed seven different photoaffinity probes to study binding affinities and labeling efficiencies of the A-domain from gramicidin S synthetase A (GrsA) in live cells.
  • Findings revealed that the efficiency of these probes is more influenced by their binding affinities rather than their structural features like linker length or flexibility, with probe 2 showing the highest efficiency and selectivity in targeting GrsA.

Article Abstract

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

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http://dx.doi.org/10.1016/j.bmc.2024.117815DOI Listing

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