title: Structure and mechanism of the trans-acting acyltransferase from the disorazole synthase.
abstract: The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the "AT-less" disorazole synthase (DSZS) and that of its acetate complex at 1.35 Å resolution are reported. Separately, comprehensive alanine-scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. With a goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking the active site Ser → Cys mutant of the DSZS AT to its ACP substrate and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a transacylation efficiency 200-fold lower than that of its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.
authors.affiliation: Department of Chemical Engineering, Department of Chemistry, and §Department of Biochemistry, Stanford University , Stanford, California 94305, United States.
authors.forename: Fong T
authors.initials: FT
authors.lastname: Wong
authors.forename: Xi
authors.initials: X
authors.lastname: Jin
authors.forename: Irimpan I
authors.initials: II
authors.lastname: Mathews
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 / Q000737:chemistry / Q000235:genetics; D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D003460:Crystallization; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics; D018942:Macrolides / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010080:Oxazoles / Q000737:chemistry*; D017434:Protein Structure, Tertiary / Q000235:genetics; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D000213:Acyl Carrier Protein; D029968:Escherichia coli Proteins; D018942:Macrolides; D010080:Oxazoles; D000217:Acyltransferases; D064429:Fatty Acid Synthases; D000409:Alanine