Investigation of the Linker‐Length Preferences of Pantetheine Probes in the Cross‐Linking Reactions Between Adenylation Enzymes and Carrier Proteins

Adenylation enzymes transfer acyl substrates selectively onto carrier proteins (CPs) in natural product biosynthesis. Despite the importance of adenylation enzyme-CP interactions, structural information on these transient complexes remains limited. Previously, we developed a pantetheine cross-linking probe (named C2Br), which contains an ethylenediamine linker with a reactive bromoacetamide group, and determined the structure of the cross-linked complex of the adenylation enzyme HitB with the CP HitD. Here, we investigated the linker-length effects of pantetheine probes in the cross-linking reactions of two adenylation enzymes, HitB and EntE, with CPs using probes with different diamine linkers, such as C2Br and C4Br, the latter containing a longer butanediamine linker moiety. Both adenylation enzymes formed cross-linked complexes with CPs irrespective of the probe used, but the reaction efficiencies depended on the linker length. Crystal structural analysis showed that the HitB-HitD interface interactions in the HitB-C4Br-HitD complex are essentially identical to those in the HitB-C2Br-HitD complex. In contrast, the diamine moieties of probes adopt different interaction modes, accounting for the observed variations in cross-linking efficiencies. A repertoire of pantetheine probes with varying linker lengths will facilitate structural studies on adenylation enzyme-CP interactions by enabling optimization for each adenylation enzyme.