Dissertation/Thesis Abstract

Characterization of enzymes involved in thienamycin biosynthesis
by Freeman, Michael F., Ph.D., The Johns Hopkins University, 2009, 229; 3340042
Abstract (Summary)

The enzymatic activities of three proteins (ThnR, ThnH, and ThnT) encoded by the thienamycin gene cluster of Streptomyces cattleya have been shown to incrementally cleave coenzyme A (CoA) to afford the active side chain component of the β-lactam antibiotic thienamycin. These results supersede proposals based on earlier radiochemical incorporation experiments. For 20 years it has been thought that cysteine was directly incorporated into the antibiotic. Specific, stepwise truncation of CoA to 4-phosphopantetheine, pantetheine, and finally cysteamine was observed with ThnR, ThnH, and ThnT, respectively, in a series of coupled enzymatic assays. Pantetheinylated carbapenams were synthesized to address possible thienamycin biosynthetic intermediates and were shown to be effective substrates for the pantetheine-cleaving enzyme ThnT. A fourth gene, thnF, was shown to encode a protein capable of N-acetylating a model compound containing cysteamine in the presence of acetyl-CoA, consistent with the production of the S. cattleya co-metabolite, N-acetylthienamycin. Taken together, these four enzymes are proposed to siphon CoA from primary metabolism to create the side chains for the predominant S. cattleya carbapenems, thienamycin and N-acetylthienamycin, in a process likely to be general for the broader class of these antibiotics. In addition, the remaining putative biosynthetic enzymes encoded within the thienamycin gene cluster were expressed, purified (with the exception of ThnK, ThnL, and ThnP), and tested for a variety of activities proposed to occur in the biosynthesis of thienamycin.

Indexing (document details)
Advisor: Townsend, Craig A.
Commitee:
School: The Johns Hopkins University
School Location: United States -- Maryland
Source: DAI-B 69/12, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Molecular biology, Microbiology, Biochemistry
Keywords: Beta-lactams, Carbapenems, Coenzyme A, Pantetheine, Thienamycin
Publication Number: 3340042
ISBN: 9780549940227
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