Fluorescent profiling of modular biosynthetic enzymes by complementary metabolic and activity based probes.
The study of the enzymes responsible for natural product biosynthesis has proven a valuable source of new enzymatic activities and been applied to a number of biotechnology applications. Protein profiling could prove highly complementary to genetics based approaches by allowing us to understand the activity, transcriptional control, and post-translational modification of these enzymes in their native and dynamic proteomic environments. Here we present a method for the fluorescent profiling of PKS, NRPS, and FAS multidomain modular synthases in their whole proteomes using complementary metabolic and activity based probes. After first examining the reactivity of these activity based probes with a variety of purified recombinant PKS, NRPS, and FAS enzymes in vitro, we apply this duel labeling strategy to the analysis of modular synthases in a human breast cancer cell line and two strains of the natural product producer Bacillus subtilis. Collectively, these studies demonstrate that complementary protein profiling approaches can prove highly useful in the identification and assignment of inhibitor specificity and domain structure of these modular biosynthetic enzymes.
Meier
Jordan L
JL
Department of Chemistry and Biochemistry, University of California-San Diego, 9500 Gilman Drive, La Jolla, California 92093-0358, USA.
Mercer
Andrew C
AC
Burkart
Michael D
MD
D000345:Affinity Labels / Q000737:chemistry* / Q000378:metabolism; D001412:Bacillus subtilis / Q000201:enzymology; D001709:Biotechnology / Q000379:methods*; D001943:Breast Neoplasms / Q000201:enzymology / Q000473:pathology; D064429:Fatty Acid Synthases / Q000096:biosynthesis* / Q000235:genetics; D005260:Female; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010453:Peptide Synthases / Q000096:biosynthesis* / Q000235:genetics; D048630:Polyketide Synthases / Q000096:biosynthesis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D005456:Fluorescent Dyes; D048630:Polyketide Synthases; D064429:Fatty Acid Synthases; D010453:Peptide Synthases