title: Identification of Middle Chain Fatty Acyl-CoA Ligase Responsible for the Biosynthesis of 2-Alkylmalonyl-CoAs for Polyketide Extender Unit.
abstract: Understanding the biosynthetic mechanism of the atypical polyketide extender unit is important for the development of bioactive natural products. Reveromycin (RM) derivatives produced by Streptomyces sp. SN-593 possess several aliphatic extender units. Here, we studied the molecular basis of 2-alkylmalonyl-CoA formation by analyzing the revR and revS genes, which form a transcriptional unit with the revT gene, a crotonyl-CoA carboxylase/reductase homolog. We mainly focused on the uncharacterized adenylate-forming enzyme (RevS). revS gene disruption resulted in the reduction of all RM derivatives, whereas reintroduction of the gene restored the yield of RMs. Although RevS was classified in the fatty acyl-AMP ligase clade based on phylogenetic analysis, biochemical characterization revealed that the enzyme catalyzed the middle chain fatty acyl-CoA ligase (FACL) but not the fatty acyl-AMP ligase activity, suggesting the molecular evolution for acyl-CoA biosynthesis. Moreover, we examined the in vitro conversion of fatty acid into 2-alkylmalonyl-CoA using purified RevS and RevT. The coupling reaction showed efficient conversion of hexenoic acid into butylmalonyl-CoA. RevS efficiently catalyzed C8-C10 middle chain FACL activity; therefore, we speculated that the acyl-CoA precursor was truncated via β-oxidation and converted into (E)-2-enoyl-CoA, a RevT substrate. To determine whether the β-oxidation process is involved between the RevS and RevT reaction, we performed the feeding experiment using [1,2,3,4-(13)C]octanoic acid. (13)C NMR analysis clearly demonstrated incorporation of the [3,4-(13)C]octanoic acid moiety into the structure of RM-A. Our results provide insight into the role of uncharacterized RevS homologs that may catalyze middle chain FACL to produce a unique polyketide extender unit.
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and; the Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Miyazawa
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
authors.forename: Shunji
authors.initials: S
authors.lastname: Takahashi
authors.affiliation: the Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kawata
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
authors.forename: Suresh
authors.initials: S
authors.lastname: Panthee
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
authors.forename: Teruo
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shimizu
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Nogawa
authors.affiliation: RIKEN Center for Sustainable Resource Science, Chemical Biology Research Group, Saitama 351-0198 and; the Graduate School of Science and Engineering, Saitama University, Saitama 338-8570, Japan. Electronic address: shunjitaka@riken.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D008316:Malonyl Coenzyme A / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D061065:Polyketides / Q000737:chemistry / Q000378:metabolism; D011714:Pyrans / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D061065:Polyketides; D011714:Pyrans; D011994:Recombinant Proteins; D013141:Spiro Compounds; C068098:reveromycin A; D008316:Malonyl Coenzyme A; D003066:Coenzyme A Ligases
keywords: Streptomyces; acyl-CoA ligase; alkylmalonyl-CoA; biosynthesis; enzyme catalysis; natural product; polyketide; reveromycin; secondary metabolism