Non-ribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI

Abstract

Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis that shows moderate antibiotic activity against a broad spectrum of gram-negative bacteria. The monobactams are of renewed interest due to emerging gram-negative strains resistant to clinically available penicillins and cephalosporins. Like isopenicillin N, nocardicin A has a tripeptide core of non-ribosomal origin. Paradoxically, the nocardicin A gene cluster encodes two non-ribosomal peptide synthetases (NRPSs), NocA and NocB, predicted to encode five modules pointing to a pentapeptide precursor in nocardicin A biosynthesis, unless module skipping or other nonlinear reactions are occurring. Previous radiochemical incorporation experiments and bioinformatic analyses predict the incorporation of p-hydroxy-L-phenylglycine (L-pHPG) into positions 1, 3, and 5 and L-serine into position 4. No prediction could be made for position 2. Multidomain constructs of each module were heterologous expressed in Escherichia coli for determination of the adenylation domain (A-domain) substrate specificity using the ATP/PPi exchange assay. Three of the five A-domains, from modules 1, 2, and 4, required the addition of stoichiometric amounts of MbtH family protein NocI to detect exchange activity. On the basis of these analyses, the predicted product of the NocA and NocB NRPSs is L-pHPG-L-Arg-D-pHPG-L-Ser-L-pHPG, a pentapeptide. Despite being flanked by non-proteinogenic amino acids, proteolysis of this pentapeptide by trypsin yields two fragments from cleavage at the C terminus of the L-Arg residue. Thus, a proteolytic step is likely involved in the biosynthesis of nocardicin A, a rare but precedented editing event in the formation of non-ribosomal natural products that is supported by the identification of trypsin-encoding genes in N. uniformis.

Figure 1

Representative results of the ATP/PPi exchange assay for L-pHPG activating modules A₁T₁-His₆, A₃T₃ E₃C₄-His₆ and C₅A₅T₅TE-His₆ and L-serine activating module, A₄T₄-His₆ at 0.5uM. For modules A₁T₁-His₆ and A₄T₄-His₆ which require addition of a MbtH protein for activity, His₆-NocI was added at 2 uM final concentration. All assays were performed at room temperature.

Figure 2

Representative results of the ATP/PPi exchange assay for 5 uM of the L-Arg activating MBP-A₂T₂-His₆ performed at 30 °C. A. Data plot for activity with and without addition of 10 uM His₆-NocI. B. Difference plot – the activity of MBP-A₂T₂-His₆ with His₆-NocI with the background activity seen in the absence His₆-NocI subtracted.

Figure 3

ATP/PPi exchange rate for modules A₁T₁-His₆ (A) and A₄T₄-His₆ (B) as a function of increasing NocI (untagged) concentration. The fitted line was generated by curve fitting to the Box Lucas model equation: y = a(1 − b^x) using OriginLab software version 8.6.

Figure 4

HPLC chromatogram and SDS-Page gel (inset) of A₄T₄-His6–NocI (untagged) coexpression, purified by NTA affinity chromatography.

Scheme 1