In Vivo Characterization of Nonribosomal Peptide Synthetases NocA and NocB in the Biosynthesis of Nocardicin A

Davidsen, Jeanne M.; Townsend, Craig A.

doi:10.1016/j.chembiol.2011.10.020

Cited by 26 publications

(31 citation statements)

References 57 publications

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“…As noted earlier, the structure of the nocardicins is characterized by the nonribosomal tripeptide core D- p HPG–L-Ser–D- p HPG biosynthesized by NRPSs NocA and NocB comprising five modules, all essential for nocardicin A biosynthesis. 46 The heterologous expression and ATP/PPi exchange data for the modules of NocA and NocB validates the previous assumption that the tripeptide core derives from modules 3, 4 and 5. 24 The substrate specificities for MbtH-dependent A1 and A2, L- p HPG and L-Arg, respectively, have been of particular interest due to their absence in nocardicin A, and in the case of A2, the inability to predict its substrate using bioinformatics or establish it experimentally.…”

Section: Discussionsupporting

confidence: 83%

“…46 N. uniformis is maintained on ISP2 solid medium (Difco Laboratories, Detroit, MI) at 28 °C. Seed cultures were prepared in TSB medium (Difco Laboratories) and grown to saturation at 28 °C with shaking.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…HPLC analysis to detect nocardicin A was performed as described previously. 46 A second HPLC analysis for the detection of novel metabolites was performed using the same HPLC system and Luna C18(2) column (Phenomenex, Torrance, CA). For this analysis, the binary mobile phase solutions were: A: 0.1% trifluoroacetic acid (TFA) in water and B: 90:10 acetonitrile:water with 0.1% TFA.…”

Section: Experimental Methodsmentioning

confidence: 99%

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Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI

2013

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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 nonribosomal origin. Paradoxically, the nocardicin A gene cluster encodes two nonribosomal 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 non-linear 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. Multi-domain 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. Based on these analyses, the predicted product of the NocA+NocB NRPSs is L-pHPG–L-Arg–D-pHPG–L-Ser–L-pHPG, a pentapeptide. Despite being flanked by nonproteinogenic 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 nonribosomal natural products which is supported by the identification of trypsin-encoding genes in N. uniformis.

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Section: Discussionsupporting

confidence: 83%

Section: Experimental Methodsmentioning

confidence: 99%

Section: Experimental Methodsmentioning

confidence: 99%

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Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI

2013

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“…171,175–178 The noc biosynthetic gene cluster encodes an NRPS that builds a β-lactam-containing tripeptide scaffold. 179,180 The biosynthesis of 37 has been studied extensively, and a unique role for the NRPS in promoting β-lactam formation has been elucidated. 181–184 …”

Section: N–o Bond Forming Enzymesmentioning

confidence: 99%

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

et al. 2017

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Natural products that contain functional groups with heteroatom-heteroatom linkages (X–X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X–X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X–X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X–X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

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“…This possibility was strengthened by inserts of short, repeated sequences not seen in other NRPSs comprising ~40 residues in M1 and ~90 in M2 [40]. Later after developing a double gene replacement strategy, each with its own selection, it was possible to introduce “scarless” mutations in vivo and thereby establish that all five modules are required for antibiotic biosynthesis despite the long insertions and other anomalies evident in the sequences of NocA and NocB [45]. In time A3 and A5 were demonstrated to give PP i exchange with L-pHPG (but not D-pHPG), as predicted.…”

Section: Nrps-dependent Pathways Nocardicin and Monobactamsmentioning

confidence: 99%

Convergent biosynthetic pathways to β-lactam antibiotics

Townsend

2016

Current Opinion in Chemical Biology

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Five naturally-occurring β-lactams have inspired a class of drugs that constitute >60% of the antimicrobials used in human medicine. Their biosynthetic pathways reveal highly individualized synthetic strategies that yet converge on a common azetidinone ring assembled in structural contexts that confer selective binding and inhibition of D,D-transpeptidases that play essential roles in bacterial cell wall (peptidoglycan) biosynthesis. These enzymes belong to a single “clan” of evolutionarily distinct serine hydrolases whose active site geometry and mechanism of action is specifically matched by these antibiotics for inactivation that is kinetically competitive with their native function. Unusual enzyme-mediated reactions and catalytic multitasking in these pathways are discussed with particular attention to the diverse ways the β-lactam itself is generated, and more broadly how the intrinsic reactivity of this core structural element is modulated in natural systems through the introduction of ring strain and electronic effects.

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In Vivo Characterization of Nonribosomal Peptide Synthetases NocA and NocB in the Biosynthesis of Nocardicin A

Cited by 26 publications

References 57 publications

Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI

Non-ribosomal Propeptide Precursor in Nocardicin A Biosynthesis Predicted from Adenylation Domain Specificity Dependent on the MbtH Family Protein NocI

Heteroatom–Heteroatom Bond Formation in Natural Product Biosynthesis

Convergent biosynthetic pathways to β-lactam antibiotics

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