Lawrence Berkeley National Laboratory

The understudied nonribosomal-peptide-synthetase-independent siderophore (NIS) synthetase family has been increasingly associated with virulence in bacterial species due to its key role in the synthesis of hydroxamate and carboxylate "stealth" siderophores. We have identified a model family member, DesD, from Streptomyces coelicolor, to structurally characterize using a combination of a wild-type and a Arg306Gln variant in apo, cofactor product AMP-bound, and cofactor reactant ATP-bound complexes. The kinetics in the family has been limited by solubility and reporter assays, so we have developed a label-free kinetics assay utilizing a single-injection isothermal-titration-calorimetry-based method. We report second-order rate constants that are 50 times higher than the previous estimations for DesD. Our Arg306Gln DesD variant was also tested under identical buffer and substrate conditions, and its undetectable activity was confirmed. These are the first reported structures for DesD, and they describe the critical cofactor coordination. This is also the first label-free assay to unambiguously determine the kinetics for an NIS synthetase.