title: Epimerization and substrate gating by a TE domain in β-lactam antibiotic biosynthesis.
abstract: Nonribosomal peptide synthetases are versatile engines of bioactive natural product biosynthesis that function according to the multiple carrier thiotemplate mechanism. C-terminal thioesterase (TE) domains of these giant modular proteins typically catalyze product release by hydrolysis or macrocyclization. We now report an unprecedented, dual-function TE that is involved in the biosynthesis of nocardicin A, which is the paradigm monocyclic β-lactam antibiotic. Contrary to our expectation, a stereodefined series of potential peptide substrates for the nocardicin TE domain failed to undergo hydrolysis. The stringent discrimination against peptide intermediates was overcome by prior monocyclic β-lactam formation at an L-seryl site. Kinetic data are interpreted such that the TE domain acts as a gatekeeper to hold the assembling peptide on an upstream domain until β-lactam formation takes place and then rapidly catalyzes epimerization, which has not been observed previously as a TE catalytic function, and thioesterase cleavage to discharge a fully fledged pentapeptide β-lactam harboring nocardicin G, the universal precursor of the nocardicins.
authors: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, USA.
authors: Nicole M
authors: NM
authors: Gaudelli
authors: Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, USA.
authors: Craig A
authors: CA
authors: Townsend
mesh_terms: D000107:Acetylation; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003065:Coenzyme A / Q000378:metabolism; D003500:Cyclization; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D006868:Hydrolysis; D007700:Kinetics; D007769:Lactams / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D005810:Multigene Family; D010406:Penicillins / Q000096:biosynthesis; D010452:Peptide Biosynthesis; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D013237:Stereoisomerism; D013869:Thiolester Hydrolases / Q000378:metabolism*; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D007769:Lactams; D010406:Penicillins; D047090:beta-Lactams; C027318:nocardicin; D004247:DNA; D013869:Thiolester Hydrolases; D003065:Coenzyme A