Elaborations of unactivated carbon centers are among the most demanding reactions that enzymes catalyze. These reactions generally involve radical intermediates, often produced by strategic abstraction of substrate hydrogen atoms (H). A prevalent strategy to cleave C?H bonds possessing homolytic bond-dissociation energies (BDEs) in excess of 95 kcal/mol involves intermediates derived from the reaction of O2 with transition metal cofactors. A distinct strategy, predominant in the anaerobic world and still important in aerobes, employs a 5'-deoxyadenosyl 5'-radical as the H abstractor. This radical is generated via the homolysis of adenosylcobalamin (AdoCbl) or the reductive cleavage of S-adenosylmethionine (SAM). Those enzymes employing SAM to catalyze radical-dependent reactions belong to the so-called radical SAM (RS) superfamily, which contains almost 114,000 individual sequences that encompass at least 65 distinct reactions. Moreover, the number of enzymes and reactions catalyzed by members of the superfamily are increasing at an amazing pace as sequences of new genomes become available. The work described herein builds on and advances work from our laboratory on the characterization of some of the most novel reactions within the superfamily, including those involved in tRNA and ribosome modification, lipoic acid biosynthesis, the biosynthesis of several antibiotics, and antibiotic resistance. Specific objectives will be to i) elucidate how methylation of unactivated carbon and phosphorus atoms takes place, and provide rationale for the strategy employed for each type of methyl acceptor; ii) formulate methods to determine substrates for the many unannotated radical SAM methylases; iii) elucidate how iron-sulfur clusters are used as sources of sulfur atoms during sulfur insertion reactions and to determine how they are resynthesized after each turnover; iv) elucidate the pathway for the biosynthesis of the thiopeptide antibiotic, nosiheptide; and v) begin to characterize several radical SAM enzymes from humans that play important roles in health and disease.

Public Health Relevance

Iron-sulfur-dependent proteins catalyze numerous essential cellular reactions using free radicals mechanisms. Many of these reactions are involved in myriad essential processes, such as antibiotic biosynthesis and resistance, the biosynthesis of essential cofactors, DNA biosynthesis and repair, and viral defense.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZGM1-TRN-Y (MR))
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Anderson, Vernon
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Pennsylvania State University
Schools of Arts and Sciences
University Park
United States
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Lanz, Nicholas D; Blaszczyk, Anthony J; McCarthy, Erin L et al. (2018) Enhanced Solubilization of Class B Radical S-Adenosylmethionine Methylases by Improved Cobalamin Uptake in Escherichia coli. Biochemistry 57:1475-1490
Blaszczyk, Anthony J; Booker, Squire J (2018) A (Re)Discovery of the Fom3 Substrate. Biochemistry 57:891-892
Badding, Edward D; Grove, Tyler L; Gadsby, Lauren K et al. (2017) Rerouting the Pathway for the Biosynthesis of the Side Ring System of Nosiheptide: The Roles of NosI, NosJ, and NosK. J Am Chem Soc 139:5896-5905
LaMattina, Joseph W; Wang, Bo; Badding, Edward D et al. (2017) NosN, a Radical S-Adenosylmethionine Methylase, Catalyzes Both C1 Transfer and Formation of the Ester Linkage of the Side-Ring System during the Biosynthesis of Nosiheptide. J Am Chem Soc 139:17438-17445
McCarthy, Erin L; Booker, Squire J (2017) Destruction and reformation of an iron-sulfur cluster during catalysis by lipoyl synthase. Science 358:373-377
Blaszczyk, Anthony J; Wang, Bo; Silakov, Alexey et al. (2017) Efficient methylation of C2 in l-tryptophan by the cobalamin-dependent radical S-adenosylmethionine methylase TsrM requires an unmodified N1 amine. J Biol Chem 292:15456-15467
Blaszczyk, Anthony J; Wang, Roy X; Booker, Squire J (2017) TsrM as a Model for Purifying and Characterizing Cobalamin-Dependent Radical S-Adenosylmethionine Methylases. Methods Enzymol 595:303-329