title: Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry.
abstract: Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California 94305, USA.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Schnarr
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Chen
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000213:Acyl Carrier Protein / Q000378:metabolism; D000215:Acylation; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D007700:Kinetics; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000378:metabolism*; D048630:Polyketide Synthases / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D010446:Peptide Fragments; D048630:Polyketide Synthases; D014357:Trypsin