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

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.