Research to understand the enzymatic machinery underlying the biosynthesis of red prodigiosin, a potent secondary metabolite with significant biological activity, led to the identification of HapC, an enzyme from the marine bacterium Hahella chejuensis. In this study, we demonstrated the catalytic capability of HapC in synthesizing a diverse array of short-chain prodiginines through the condensation of various 2-methyl-3-amylpyrrole (MAP) analogs with 4-methoxy-2,2'-bipyrrole-5-carboxaldehyde (MBC). Six prodiginines, all non-native to HapC, were enzymatically synthesized in vitro, including two analogs, 3,4-dimethyl-6-methoxyprodiginine and 2-ethyl-6-methoxyprodiginine, that have not been successfully produced by any prodigiosin-forming enzyme to date, demonstrating the subtle differences in the products formed by HapC. Constructing a structural model of HapC and performing ligand docking simulations with representative ligands corresponding to different reaction steps, including an ATP analog, a phosphorylated MBC, and a prodiginine product, revealed catalytic residues in the two active sites of HapC. To extend from local catalytic interactions to the global mechanistic level, a comparative structural analysis with the homologous rifampin phosphotransferase from Listeria monocytogenes was applied to infer domain dynamics in HapC. A well-defined three-phase catalytic cycle was delineated, highlighting the role of domain rearrangements and phosphotransfer mediated by the conserved histidine residue H859. These findings enhance our understanding of HapC, with the potential to diversify prodiginine libraries and guide rational drug design.
Chan et al. (Mon,) studied this question.