title: Mass spectrometric interrogation of thioester-bound intermediates in the initial stages of epothilone biosynthesis.
abstract: Direct detection of thioester intermediate mixtures bound to EpoC, a 195 kDa polyketide synthase, has been achieved using limited proteolysis and Fourier-transform mass spectrometry (FTMS). Incubation with various N-acetylcysteamine thioester (S-NAC) substrate mimics produced mass shifts on the EpoC ACP domain consistent with their condensation with an enzyme-bound carbanion produced by the decarboxylation of methylmalonyl-S-EpoC. Reconstitution of EpoA-ACP, EpoB, and EpoC gave a +165.0 Da mass shift consistent with the formation of the methylthiazolyl-methacrylyl product by incorporation of acetyl-CoA, cysteine, and methylmalonyl-CoA. Thioester-templated reaction intermediates and products are typically characterized by quantifying radioactive substrates, either enzyme bound or chemically hydrolyzed. In contrast, the MS-based methodology described here provides semiquantifiable ratios of free enzyme, intermediate, and product occupancy and reveals that certain substrates result in a >50% formation of nonproductive intermediates.
authors.affiliation: Department of Chemistry, 600 South Mathews Avenue, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Leslie M
authors.initials: LM
authors.lastname: Hicks
authors.forename: Sarah E
authors.initials: SE
authors.lastname: O'Connor
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Mazur
authors.forename: Christopher T
authors.initials: CT
authors.lastname: Walsh
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D034261:Epothilones / Q000096:biosynthesis* / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004952:Esters / Q000737:chemistry*; D005583:Fourier Analysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: D034261:Epothilones; D004952:Esters; D009097:Multienzyme Complexes; D013438:Sulfhydryl Compounds; D012694:Serine