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.
|Advisor:||Townsend, Craig A.|
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 69/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology, Biochemistry|
|Keywords:||Beta-lactams, Carbapenems, Coenzyme A, Pantetheine, Thienamycin|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be