The loading module of rifamycin synthetase is an adenylation-thiolation didomain with substrate tolerance for substituted benzoates

The rifamycin synthetase is primed with a 3-amino-5-hydroxybenzoate starter unit by a loading module that contains domains homologous to the adenylati on and thiolation domains of nonribosomal peptide synthetases. Adenylation and thiolation activities of the loading module were reconstituted in vitro and shown to be independent of coenzyme A, countering literature proposals that the loading module is a coenzyme A ligase. Kinetic parameters for cov alent arylation of the loading module were measured directly for the unnatu ral substrates benzoate and 3-hydroxybenzoate. This analysis was extended t hrough competition experiments to determine the relative rates of incorpora tion of a series of substituted benzoates. Our results show that the loadin g module can accept a variety of substituted benzoates, although it exhibit s a preference for the 3-, 5-, and 3,5-disubstituted benzoates that most cl osely resemble its biological substrate. The considerable substrate toleran ce of the loading module of rifamycin synthetase suggests that the module h as potential as a tool for generating substituted derivatives of natural pr oducts.