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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI014937-35
Application #
8445239
Study Section
Special Emphasis Panel (NSS)
Program Officer
Huntley, Clayton C
Project Start
1978-09-01
Project End
2014-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
35
Fiscal Year
2013
Total Cost
$511,420
Indirect Cost
$187,792
Name
Johns Hopkins University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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 (2014) Epimerization and substrate gating by a TE domain in ?-lactam antibiotic biosynthesis. Nat Chem Biol 10:251-8
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
Phelan, Ryan M; Townsend, Craig A (2013) Mechanistic insights into the bifunctional non-heme iron oxygenase carbapenem synthase by active site saturation mutagenesis. J Am Chem Soc 135:7496-502
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
Davidsen, Jeanne M; Bartley, David M; Townsend, Craig A (2013) Non-ribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI. J Am Chem Soc 135:1749-59
Bodner, Micah J; Li, Rongfeng; Phelan, Ryan M et al. (2011) Definition of the common and divergent steps in carbapenem ?-lactam antibiotic biosynthesis. Chembiochem 12:2159-65
Bodner, Micah J; Phelan, Ryan M; Freeman, Michael F et al. (2010) Non-heme iron oxygenases generate natural structural diversity in carbapenem antibiotics. J Am Chem Soc 132:12-3
Phelan, Ryan M; Ostermeier, Marc; Townsend, Craig A (2009) Design and synthesis of a beta-lactamase activated 5-fluorouracil prodrug. Bioorg Med Chem Lett 19:1261-3
Raber, Mary L; Arnett, Samantha O; Townsend, Craig A (2009) A conserved tyrosyl-glutamyl catalytic dyad in evolutionarily linked enzymes: carbapenam synthetase and beta-lactam synthetase. Biochemistry 48:4959-71

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