title: A crotonyl-CoA reductase-carboxylase independent pathway for assembly of unusual alkylmalonyl-CoA polyketide synthase extender units.
abstract: Type I modular polyketide synthases assemble diverse bioactive natural products. Such multienzymes typically use malonyl and methylmalonyl-CoA building blocks for polyketide chain assembly. However, in several cases more exotic alkylmalonyl-CoA extender units are also known to be incorporated. In all examples studied to date, such unusual extender units are biosynthesized via reductive carboxylation of α, β-unsaturated thioesters catalysed by crotonyl-CoA reductase/carboxylase (CCRC) homologues. Here we show using a chemically-synthesized deuterium-labelled mechanistic probe, and heterologous gene expression experiments that the unusual alkylmalonyl-CoA extender units incorporated into the stambomycin family of polyketide antibiotics are assembled by direct carboxylation of medium chain acyl-CoA thioesters. X-ray crystal structures of the unusual β-subunit of the acyl-CoA carboxylase (YCC) responsible for this reaction, alone and in complex with hexanoyl-CoA, reveal the molecular basis for substrate recognition, inspiring the development of methodology for polyketide bio-orthogonal tagging via incorporation of 6-azidohexanoic acid and 8-nonynoic acid into novel stambomycin analogues.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Lauren
authors.initials: L
authors.lastname: Ray
authors.affiliation: Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697, USA.
authors.forename: Timothy R
authors.initials: TR
authors.lastname: Valentic
authors.affiliation: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.|Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazawa
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: David M
authors.initials: DM
authors.lastname: Withall
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Lijiang
authors.initials: L
authors.lastname: Song
authors.affiliation: Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697, USA.
authors.forename: Jacob C
authors.initials: JC
authors.lastname: Milligan
authors.affiliation: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.|Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
authors.affiliation: Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science, Saitama 351-0198, Japan.
authors.forename: Shunji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: Departments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, California 92697, USA.
authors.forename: Shiou-Chuan
authors.initials: SC
authors.lastname: Tsai
authors.affiliation: Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
authors.forename: Gregory L
authors.initials: GL
authors.lastname: Challis
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044944:Acyl-CoA Dehydrogenases / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019735:Carbon-Carbon Ligases / Q000378:metabolism; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D015394:Molecular Structure; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D061065:Polyketides; C015357:methylmalonyl-coenzyme A; D048630:Polyketide Synthases; D044944:Acyl-CoA Dehydrogenases; C100154:acyl-CoA dehydrogenase (NADP+); D019735:Carbon-Carbon Ligases; C018757:acyl-CoA carboxylase