P167: Unlocking the secrets of the enigmatic nocardicin NRPS

Nocardicin A is a monocyclic β-lactam isolated from the actinomycete N. uniformis, which shows moderate activity against a broad spectrum of Gram-negative bacteria.  Thus, the monobactams are of renewed interest due to emerging resistant Gram-negative strains.  Analogous to the ACV tripeptide core of isopenicillin N, Nocardicin A also has a tripeptide core of nonribosomal origin.  However, the nocardicin A gene cluster encodes two nonribosomal peptide synthetases (NRPSs), NocA and NocB predicted to produce an unknown pentapeptide, unless module skipping or other non-linear reactions are occurring.  Following unsuccessful attempts to heterologously express NocA and NocB for in vitro characterization, in vivo mutagenesis experiments were performed that relied upon the development of a double replacement gene strategy in which point mutations were engineered into the NRPS genes without disruption of the nocABC operon.  A series of thiolation (T) domain mutants showed that all 5 modules are essential for nocardicin A biosynthesis.  Following heterologous expression of affinity-tagged multidomain constructs of each module in E.coli and isolation, the substrate specificity for each A domain was tested using the ATP/PPi exchange assay.  Three of the five A domains required the addition of stoichiometric amounts of MbtH-like protein NocI to the assay for observation of exchange activity.  Based on these analyses, the predicted product of the NocA+NocB NRPS is L-pHPG- L-Arg- D-pHPG-L-Ser- L-pHPG, a pentapeptide, in which the L-pHPG- L-Arg is cleaved during nocardicin A biosynthesis resulting in the tripeptide core.