Penicillin and related beta-lactam antibiotics have been a mainstay in the treatment of infections for 50 years. Their effectiveness, however, like other known classes of antibiotics has come under increasing challenge from the rise of multiply drug-resistant pathogenic bacteria. Efforts have intensified to understand the mechanisms of resistance and to overcome them. Structural modification through genetic manipulation of their biosynthetic pathways is a promising approach to produce variants of known antibiotics by cost-effective fermentation and semi-synthetic methods. Continuation of a program to investigate beta-lactam antibiotic biosynthesis is proposed in this application. Three of the four known classes of these antibiotics will be studied: (1) clavulanic acid, a potent inhibitor/inactivator of beta-lactamase enzymes and a wide-spread source of resistance, (2) the nocardicins, a family of monocyclic beta-lactams, and the metabolically related monobactams, and (3) the carbapenems, represented clinically by thienamycin and its derivatives, but most simply by carbapen-2-em-3-carboxylic acid. Characterization of the biosynthetic gene clusters for at least one member of each of these principal groups has led to rapid advances in the current grant period using techniques ranging from organic synthesis and enzymology to molecular biology and macromolecular structural methods. It is proposed to pursue these discoveries through mechanistic and structural studies of N2-(carboxyethyl)-L-arginine synthase, site-specific mutatagenesis to examine the mechanism and engineer the function of beta-lactam synthetase, collaborative studies to characterize the iron center of clavaminate synthase, and substrate analogue and site-directed mutatagenesis experiments to examine its mechanism, and to investigate the """"""""enantiomerization that occurs in the penultimate step of clavulanic acid biosynthesis. Disruption and over-expression of nocardicin biosynthetic genes will be undertaken to delineate the pathway, understand the mechanism of monocyclic beta-lactam formation and determine the roles of two unusual non-ribosomal peptide synthetases. Investigation of the biosynthetically related monobactams will be initiated. Characterization of three key proteins that form the carbapenem nucleus will figure prominantly in studies of this group, and new experiments with thienamycin will be begun to understand the more complex members of this family and engineering of their synthesis.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Huntley, Clayton C
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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
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
Townsend, Craig A (2016) Convergent biosynthetic pathways to ?-lactam antibiotics. Curr Opin Chem Biol 35:97-108
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
Gaudelli, Nicole M; Townsend, Craig A (2014) Epimerization and substrate gating by a TE domain in ?-lactam antibiotic biosynthesis. Nat Chem Biol 10:251-8
Buller, Andrew R; Freeman, Michael F; Schildbach, Joel F et al. (2014) Exploring the role of conformational heterogeneity in cis-autoproteolytic activation of ThnT. Biochemistry 53:4273-81
Li, Rongfeng; Lloyd, Evan P; Moshos, Kristos A et al. (2014) Identification and characterization of the carbapenem MM 4550 and its gene cluster in Streptomyces argenteolus ATCC 11009. Chembiochem 15:320-31
Gaudelli, Nicole M; Townsend, Craig A (2013) Correction to stereocontrolled syntheses of Peptide thioesters containing modified seryl residues as probes of antibiotic biosynthesis. J Org Chem 78:11115
Gaudelli, Nicole M; Townsend, Craig A (2013) Stereocontrolled syntheses of peptide thioesters containing modified seryl residues as probes of antibiotic biosynthesis. J Org Chem 78:6412-26
Buller, Andrew R; Townsend, Craig A (2013) Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad. Proc Natl Acad Sci U S A 110:E653-61

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