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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 ?-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.

SUBMITTER: Davidsen JM

PROVIDER: S-EPMC3571714 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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

Davidsen Jeanne M JM Bartley David M DM Townsend Craig A CA

Journal of the American Chemical Society 20130118 5

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 (NR ...[more]

PMID: 23330869

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Project description:BackgroundCreating designer molecules using a combination of select domains from polyketide synthases and/or nonribosomal peptide synthetases (NRPS) continues to be a synthetic goal. However, an incomplete understanding of how protein-protein interactions and dynamics affect each of the domain functions stands as a major obstacle in the field. Of particular interest is understanding the basis for a class of methyltransferase domains (MT) that are found embedded within the adenylation domain (A) of fungal NRPS systems instead of in an end-to-end architecture.ResultsThe MT domain from bassianolide synthetase (BSLS) was removed and the truncated enzyme BSLS-ΔMT was recombinantly expressed. The biosynthesis of bassianolide was abolished and N-desmethylbassianolide was produced in low yields. Co-expression of BSLS-ΔMT with standalone MT did not recover bassianolide biosynthesis. In order to address the functional implications of the protein insertion, we characterized the N-methyltransferase activity of the MT domain as both the isolated domain (MTBSLS) and as part of the full NRPS megaenzyme. Surprisingly, the MTBSLS construct demonstrated a relaxed substrate specificity and preferentially methylated an amino acid (L-Phe-SNAC) that is rarely incorporated into the final product. By testing the preference of a series of MT constructs (BSLS, MTBSLS, cMT, XLcMT, and aMT) to L-Phe-SNAC and L-Leu-SNAC, we further showed that restricting and/or fixing the termini of the MTBSLS by crosslinking or embedding the MT within an A domain narrowed the substrate specificity of the methyltransferase toward L-Leu-SNAC, the preferred substrate for the BSLS megaenzyme.ConclusionsThe embedding of MT into the A2 domain of BSLS is not required for the product assembly, but is critical for the overall yields of the final products. The substrate specificity of MT is significantly affected by the protein context within which it is present. While A domains are known to be responsible for selecting and activating the biosynthetic precursors for NRPS systems, our results suggest that embedding the MT acts as a secondary gatekeeper for the assembly line. This work thus provides new insights into the embedded MT domain in NRPSs, which will facilitate further engineering of this type of biosynthetic machinery to create structural diversity in natural products.

Dataset Information

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

Publications

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

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