The biosynthesis of the non-classical clans of the ?-lactam antibiotics will be investigated. Together with the fourth, or classical penicillins and cephalosporins, these drugs collectively constitute >65% of the world antibiotic market and account for >$25B/yr in economic value. They remain vital mainstays of human health and longevity, but with their widespread use has come the inevitable rise of antibiotic-resistant infections. Structural modifications have slowed these effects, but there is increased reliance on the newer, non-classical families, for example, the ?-lactamase inhibitor clavulanic acid and the potent, broad-spectrum carbapenems like Imipenem(r) and Meropenem,(r) inspired by the natural product thienamycin. The ?-lactams are instructive examples of convergent evolution where the pathways to the four known classes exemplify remarkably different biosynthetic strategies, evolution of enzyme function to new tasks and impressive synthetic efficiency. Detailed examination of these versatile catalysts will be undertaken using tools ranging from organic synthesis to enzymology, directed evolution and protein X-ray crystallography to understand their mechanisms and structures in detail, and to explore their potential uses and targeted evolution for the chemo-enzymatic synthesis of variants that may be of practical use. A series of experimental breakthroughs has revealed unprecedented non-ribosomal peptide synthetase activities among which is the ability to synthesize the internal 4-membered ring of monocyclic ?-lactam antibiotics. This understanding will guide experiments with the biosynthetically related monobactams, distinct for their terminal N-sulfonated ?-lactam rings that confer the critically important property of resistance to the Class B ?-lactamases (metalloproteases). Yet a fourth mechanism of ?-lactam formation is likely in play and one with exciting possibilities for chemo-enzymatic synthesis. The mechanisms of the unusual oxidative enzyme carbapenem synthase and three centrally-acting methylcobalamin-dependent radical-SAM enzymes in thienamycin biosynthesis will be examined. Collaborative work toward the first crystal structure of one of the latter is underway. With an efficient fluorescent screen for ?-lactam synthesis in hand, directed evolution of CarC and CarA is aimed to a new chemo-enzymatic synthesis of complex carbapenems. A first-rate, interdisciplinary training environment is sought for students encompassing scientific disciplines from organic synthesis and a fundamental interest in mechanism, to protein chemistry and enzymology to structural biology and protein engineering.

Public Health Relevance

The 'non-classical' ?-lactam antibiotics, notably the potent, broad-spectrum carbapenems and the ?-lactamase inhibitor clavulanic acid, have become increasingly important in healthcare to overcome resistant bacterial infections. There is renewed interest in the monocyclic ?-lactams, notably the N-sulfonated monobactams owing to their resistance to metallo-?-lactamases. This grant will focus on the biosynthetic study of these classes and examine the potential of biosynthetic enzymes for chemo-enzymatic synthesis of analog structures potentially having increased potency, spectrum and ability to overcome resistance mechanisms

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Xu, Zuoyu
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Johns Hopkins University
Schools of Arts and Sciences
United States
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Long, Darcie H; Townsend, Craig A (2018) Mechanism of Integrated ?-Lactam Formation by a Nonribosomal Peptide Synthetase during Antibiotic Synthesis. Biochemistry 57:3353-3358
Oliver, Ryan A; Li, Rongfeng; Townsend, Craig A (2018) Monobactam formation in sulfazecin by a nonribosomal peptide synthetase thioesterase. Nat Chem Biol 14:5-7
Li, Rongfeng; Oliver, Ryan A; Townsend, Craig A (2017) Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster. Cell Chem Biol 24:24-34
Horsman, Mark E; Marous, Daniel R; Li, Rongfeng et al. (2017) Whole-Genome Shotgun Sequencing of Two ?-Proteobacterial Species in Search of the Bulgecin Biosynthetic Cluster. ACS Chem Biol 12:2552-2557
Townsend, Craig A (2016) Convergent biosynthetic pathways to ?-lactam antibiotics. Curr Opin Chem Biol 35:97-108
Marous, Daniel R; Lloyd, Evan P; Buller, Andrew R et al. (2015) Consecutive radical S-adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis. Proc Natl Acad Sci U S A 112:10354-8
Gaudelli, Nicole M; Long, Darcie H; Townsend, Craig A (2015) ?-Lactam formation by a non-ribosomal peptide synthetase during antibiotic biosynthesis. Nature 520:383-7