Abstract

? Electrospray fourier transfer mass spectrometry (ESI-FTMS) provides a powerful tool for analysis of covalent modifications of proteins. This proposal applies ESI-FTMS for the detection of transient covalent modifications of non-ribosomal-petide synthetases (NRPS) and polyketide synthases. Initially, a general protocol will be developed for the rapid identification of the acyl domains. Subsequently, this method will be extended to the identification of the in vivo or in vitro """"""""preferred"""""""" substrates for acyl domains. This method will then be applied to the identification of the substrate(s) for the calE8 gene product involved in the biosynthesis of the antitumor agent calicheamycin, a PKS for which we do not know the substrate. Finally, ESI-FTMS will be used for the kinetic characterization of the NRPS found in the biosynthesis of the aminocoumarin antibiotics coumeramycin and clorbiocin while the substrates are still connected to their acyl domains. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM073323-01
Application #
6883837
Study Section
Special Emphasis Panel (ZRG1-F04A (20))
Program Officer
Flicker, Paula F
Project Start
2005-03-16
Project End
2008-03-15
Budget Start
2005-03-16
Budget End
2006-03-15
Support Year
1
Fiscal Year
2005
Total Cost
$42,068
Indirect Cost

  Institution

Name
University of Illinois Urbana-Champaign
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820

  Publications

Zhang, Jian; Van Lanen, Steven G; Ju, Jianhua et al. (2008) A phosphopantetheinylating polyketide synthase producing a linear polyene to initiate enediyne antitumor antibiotic biosynthesis. Proc Natl Acad Sci U S A 105:1460-5
Calderone, Christopher T; Iwig, David F; Dorrestein, Pieter C et al. (2007) Incorporation of nonmethyl branches by isoprenoid-like logic: multiple beta-alkylation events in the biosynthesis of myxovirescin A1. Chem Biol 14:835-46
Van Lanen, Steven G; Lin, Shuangjun; Dorrestein, Pieter C et al. (2006) Substrate specificity of the adenylation enzyme SgcC1 involved in the biosynthesis of the enediyne antitumor antibiotic C-1027. J Biol Chem 281:29633-40
Garneau-Tsodikova, Sylvie; Dorrestein, Pieter C; Kelleher, Neil L et al. (2006) Protein assembly line components in prodigiosin biosynthesis: characterization of PigA,G,H,I,J. J Am Chem Soc 128:12600-1
Calderone, Christopher T; Kowtoniuk, Walter E; Kelleher, Neil L et al. (2006) Convergence of isoprene and polyketide biosynthetic machinery: isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis. Proc Natl Acad Sci U S A 103:8977-82
Dorrestein, Pieter C; Kelleher, Neil L (2006) Dissecting non-ribosomal and polyketide biosynthetic machineries using electrospray ionization Fourier-Transform mass spectrometry. Nat Prod Rep 23:893-918
Hicks, Leslie M; Mazur, Matthew T; Miller, Leah M et al. (2006) Investigating nonribosomal peptide and polyketide biosynthesis by direct detection of intermediates on >70 kDa polypeptides by using Fourier-transform mass spectrometry. Chembiochem 7:904-7
Van Lanen, Steven G; Dorrestein, Pieter C; Christenson, Steve D et al. (2005) Biosynthesis of the beta-amino acid moiety of the enediyne antitumor antibiotic C-1027 featuring beta-amino acyl-S-carrier protein intermediates. J Am Chem Soc 127:11594-5
Dorrestein, Pieter C; Yeh, Ellen; Garneau-Tsodikova, Sylvie et al. (2005) Dichlorination of a pyrrolyl-S-carrier protein by FADH2-dependent halogenase PltA during pyoluteorin biosynthesis. Proc Natl Acad Sci U S A 102:13843-8
Aron, Zachary D; Dorrestein, Pieter C; Blackhall, Jonathan R et al. (2005) Characterization of a new tailoring domain in polyketide biogenesis: the amine transferase domain of MycA in the mycosubtilin gene cluster. J Am Chem Soc 127:14986-7