MECHANISTIC AND STRUCTURAL STUDIES OF THE THIOESTERASE DOMAIN IN THE TERMINATION MODULE OF THE NOCARDICIN NRPS
Gaudelli, Nicole Marie
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The nocardicins are monocyclic β-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp., tsuyamanensis ATCC 21806. In 2004 the gene cluster responsible for the biosynthesis of the flagship antibiotic, nocardicin A, was identified. This gene cluster accommodates a pair of non-ribosomal peptide synthetase (NRPS) whose five modules are indispensible for antibiotic production. In accordance with the prevailing co-linearity model of NRPS function, a linear L,L,D,L,L pentapeptide was predicted to be synthesized. Contrary to expectation and precedent, however, a stereodefined series of synthesized potential peptide substrates for the nocardicin thioesterase (NocTE) domain failed to undergo hydrolysis. The stringent discrimination against peptide intermediates was dramatically overcome by prior monocyclic β-lactam formation at an L-seryl site to render now facile substrates for C-terminal epimerization and hydrolytic release. It was concluded through biochemical and kinetic experimentation 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 and hydrolysis to discharge a fully-fledged pentapeptide β-lactam harboring nocardicin G, the simplest member of the nocardicin family. An x-ray crystal structure of the TE domain revealed a catalytic center containing the expected Asp, His, Ser triad. Mutational analysis of these catalytic residues along with a proximal His established that the His of the catalytic triad was likely responsible for the epimerization activity rendered by the domain. The in vitro reconstitution of the termination module, or module 5, established that the β-lactam core present in the nocardicins is formed within module 5 of the NRPS. This discovery has implications regarding the formation of the azetidinone moiety present in the entire class of monobactam antibiotics.