title: Probing the selectivity and protein·protein interactions of a nonreducing fungal polyketide synthase using mechanism-based crosslinkers.
abstract: Protein·protein interactions, which often involve interactions among an acyl carrier protein (ACP) and ACP partner enzymes, are important for coordinating polyketide biosynthesis. However, the nature of such interactions is not well understood, especially in the fungal nonreducing polyketide synthases (NR-PKSs) that biosynthesize toxic and pharmaceutically important polyketides. Here, we employ mechanism-based crosslinkers to successfully probe ACP and ketosynthase (KS) domain interactions in NR-PKSs. We found that crosslinking efficiency is closely correlated with the strength of ACP·KS interactions and that KS demonstrates strong starter unit selectivity. We further identified positively charged surface residues by KS mutagenesis, which mediates key interactions with the negatively charged ACP surface. Such complementary/matching contact pairs can serve as "adapter surfaces" for future efforts to generate new polyketides using NR-PKSs.
authors.affiliation: Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697, USA; Department of Chemistry, University of California, Irvine, Irvine, CA 92697, USA; Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, CA 92697, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Bruegger
authors.forename: Robert W
authors.initials: RW
authors.lastname: Haushalter
authors.forename: Bob
authors.initials: B
authors.lastname: Haushalter
authors.forename: Anna L
authors.initials: AL
authors.lastname: Vagstad
authors.forename: Anna
authors.initials: A
authors.lastname: Vagstad
authors.forename: Gaurav
authors.initials: G
authors.lastname: Shakya
authors.forename: Nathan
authors.initials: N
authors.lastname: Mih
authors.forename: Craig A
authors.initials: CA
authors.lastname: Townsend
authors.forename: Michael D
authors.initials: MD
authors.lastname: Burkart
authors.forename: Shiou-Chuan
authors.initials: SC
authors.lastname: Tsai
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010204:Pantetheine / Q000737:chemistry; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment
chemical_list: D000213:Acyl Carrier Protein; D003432:Cross-Linking Reagents; D061065:Polyketides; D011994:Recombinant Proteins; D010204:Pantetheine; D048630:Polyketide Synthases