An orthogonal active site identification system (OASIS) for proteomic profiling of natural product biosynthesis.
A significant gap exists between genetics-based investigations of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) biosynthetic pathways and our understanding of their regulation, interaction, and activity in living systems. To help bridge this gap, here we present an orthogonal active site identification system (OASIS) for the proteomic identification and analysis of PKS/NRPS biosynthetic enzymes. OASIS probes target conserved features of PKS/NRPS active sites to provide activity-based enrichment of modular synthases, followed by analysis through multidimensional protein identification technology (MudPIT) LC-MS/MS analysis. When applied to the model bacterium Bacillus subtilis, this functional proteomics method detects and quantifies all four modular synthases in the organism. Furthermore, tandem application of multiple OASIS probes enhances identification of specific PKS/NRPS modules from complex proteomic mixtures. By expanding the dynamic range of proteomic analysis for PKS/NRPS enzymes, OASIS offers a valuable tool for strain comparison, culture condition optimization, and enzyme discovery.
Meier
Jordan L
JL
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Drive, La Jolla, California 92037-0358, USA.
Niessen
Sherry
S
Hoover
Heather S
HS
Foley
Timothy L
TL
Cravatt
Benjamin F
BF
Burkart
Michael D
MD
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001688:Biological Products / Q000032:analysis* / Q000378:metabolism; D020134:Catalytic Domain; D008969:Molecular Sequence Data; D010453:Peptide Synthases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*
D001688:Biological Products; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase