Structural basis for selectivity in a highly reducing type II polyketide synthase

In type II polyketide synthases (PKSs), the ketosynthase–chain length factor (KS–CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here,...

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Veröffentlicht in:	Nature chemical biology Jg. 16; H. 7; S. 776 - 782
Hauptverfasser:	Du, Danyao, Katsuyama, Yohei, Horiuchi, Masanobu, Fushinobu, Shinya, Chen, Aochiu, Davis, Tony D., Burkart, Michael D., Ohnishi, Yasuo
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	New York Nature Publishing Group US 01.07.2020 Nature Publishing Group
Schlagworte:	631/535/1266 631/92/60 631/92/607 Acyl carrier protein Acyl Carrier Protein - chemistry Acyl Carrier Protein - genetics Acyl Carrier Protein - metabolism Antifungal agents Biocatalysis Biochemical Engineering Biochemistry Bioorganic Chemistry Biosynthesis Catalytic Domain Cell Biology Chemistry Chemistry and Materials Science Chemistry/Food Science Cloning, Molecular Condensates Condensation Crosslinking Crystal structure Crystallography, X-Ray E coli Elongation Escherichia coli - enzymology Escherichia coli - genetics Escherichia coli Proteins - chemistry Escherichia coli Proteins - genetics Escherichia coli Proteins - metabolism Fatty Acid Synthases - chemistry Fatty Acid Synthases - genetics Fatty Acid Synthases - metabolism Fatty acids Fatty-acid synthase Gene Expression Genetic Vectors - chemistry Genetic Vectors - metabolism Models, Molecular Molecular interactions Molecular structure Mutagenesis Mutagenesis, Site-Directed Polyketide synthase Polyketide Synthases - chemistry Polyketide Synthases - genetics Polyketide Synthases - metabolism Polyketides - chemistry Polyketides - metabolism Protein Binding Protein Conformation, alpha-Helical Protein Conformation, beta-Strand Protein Interaction Domains and Motifs Protein Multimerization Proteins Recombinant Proteins - chemistry Recombinant Proteins - genetics Recombinant Proteins - metabolism Selectivity Site-directed mutagenesis Streptomyces - enzymology Streptomyces - genetics Substrate Specificity Substrates
ISSN:	1552-4450, 1552-4469, 1552-4469
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Zusammenfassung:	In type II polyketide synthases (PKSs), the ketosynthase–chain length factor (KS–CLF) complex catalyzes polyketide chain elongation with the acyl carrier protein (ACP). Highly reducing type II PKSs, represented by IgaPKS, produce polyene structures instead of the well-known aromatic skeletons. Here, we report the crystal structures of the Iga11–Iga12 (KS–CLF) heterodimer and the covalently cross-linked Iga10=Iga11–Iga12 (ACP=KS–CLF) tripartite complex. The latter structure revealed the molecular basis of the interaction between Iga10 and Iga11–Iga12, which differs from that between the ACP and KS of Escherichia coli fatty acid synthase. Furthermore, the reaction pocket structure and site-directed mutagenesis revealed that the negative charge of Asp 113 of Iga11 prevents further condensation using a β-ketoacyl product as a substrate, which distinguishes IgaPKS from typical type II PKSs. This work will facilitate the future rational design of PKSs. Structures of the ketosynthase–chain length factor complex from ishigamide biosynthesis, cross-linked to the acyl carrier protein, reveal the molecular interactions between these domains and how the reaction pocket limits rounds of product extension.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 Author contributions Y.K., M.D.B. and D.D. designed the research. T.D.D. synthesized the chemical probes. The crystallization of the proteins was done by D.D. The X-ray diffraction experiment was carried out by Y.K. and D.D. The experimental phasing was done by Y.K. Refinement and validation of the structure was done by D.D., Y.K. and S.F. Site-directed mutagenesis and analysis of mutants were done by D.D. and M.H. The cross-linked complex was prepared by D.D. and A.C. D.D. and Y.K. wrote the draft manuscript. A.C., T.D.D., M.D.B., S.F. and Y.O. commented on the draft. Y.K. and Y.O. finalized the manuscript and all authors approved it. Y.K., M.D.B. and Y.O. directed the research.
ISSN:	1552-4450 1552-4469 1552-4469
DOI:	10.1038/s41589-020-0530-0