title: In vivo characterization of nonribosomal peptide synthetases NocA and NocB in the biosynthesis of nocardicin A.
abstract: Two nonribosomal peptide synthetases (NRPS), NocA and NocB, together comprising five modules, are essential for the biosynthesis of the D,L,D configured tripeptide backbone of the monocyclic β-lactam nocardicin A. We report a double replacement gene strategy in which point mutations were engineered in the two encoding NRPS genes without disruption of the nocABC operon by placing selective markers in adjacent genes. A series of mutants was constructed to inactivate the thiolation (T) domain of each module and to evaluate an HHxxxDR catalytic motif in NocA and an atypical extended histidine motif in NocB. The loss of nocardicin A production in each of the T domain mutants indicates that all five modules are essential for its biosynthesis. Conversely, production of nocardicin A was not affected by mutation of the NocB histidine motif or the R828G mutation in NocA.
authors.affiliation: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
authors.forename: Jeanne M
authors.initials: JM
authors.lastname: Davidsen
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D007769:Lactams / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D005810:Multigene Family; D009615:Nocardia / Q000235:genetics / Q000378:metabolism; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation
chemical_list: D001426:Bacterial Proteins; D007769:Lactams; C027318:nocardicin; D010453:Peptide Synthases