title: Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster.
abstract: The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the β-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and β-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic β-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.
authors.affiliation: Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Rongfeng
authors.initials: R
authors.lastname: Li
authors.affiliation: Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Oliver
authors.affiliation: Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA. Electronic address: ctownsend@jhu.edu.
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
mesh_terms: D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D008968:Molecular Conformation; D008997:Monobactams / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology; D005810:Multigene Family / Q000235:genetics*; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism; D011549:Pseudomonas / Q000235:genetics / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism
chemical_list: D008997:Monobactams; D047090:beta-Lactams; D010453:Peptide Synthases; C028547:sulfazecin
keywords: antibiotic; biosynthesis; metallo-β-lactamase; natural product; non-ribosomal peptide synthetase; sulfazecin; sulfotransferase; β-lactam