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Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase.
FEBS J. 2018 12; 285(23):4494-4511.FJ

Abstract

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.

Authors+Show Affiliations

Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Química de Rosario (IQUIR-CONICET), Universidad Nacional de Rosario, Argentina.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Química de Rosario (IQUIR-CONICET), Universidad Nacional de Rosario, Argentina.Manchester Centre of Fine and Specialty Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology (MIB), University of Manchester, UK.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET), Universidad Nacional de Rosario, Argentina.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30300504

Citation

Sabatini, Martin, et al. "Biochemical Characterization of the Minimal Domains of an Iterative Eukaryotic Polyketide Synthase." The FEBS Journal, vol. 285, no. 23, 2018, pp. 4494-4511.
Sabatini M, Comba S, Altabe S, et al. Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase. FEBS J. 2018;285(23):4494-4511.
Sabatini, M., Comba, S., Altabe, S., Recio-Balsells, A. I., Labadie, G. R., Takano, E., Gramajo, H., & Arabolaza, A. (2018). Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase. The FEBS Journal, 285(23), 4494-4511. https://doi.org/10.1111/febs.14675
Sabatini M, et al. Biochemical Characterization of the Minimal Domains of an Iterative Eukaryotic Polyketide Synthase. FEBS J. 2018;285(23):4494-4511. PubMed PMID: 30300504.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase. AU - Sabatini,Martin, AU - Comba,Santiago, AU - Altabe,Silvia, AU - Recio-Balsells,Alejandro I, AU - Labadie,Guillermo R, AU - Takano,Eriko, AU - Gramajo,Hugo, AU - Arabolaza,Ana, Y1 - 2018/10/25/ PY - 2018/05/15/received PY - 2018/08/03/revised PY - 2018/09/25/accepted PY - 2018/10/10/pubmed PY - 2019/6/8/medline PY - 2018/10/10/entrez KW - PKS biochemistry KW - domain deconstruction KW - iterative PKS KW - substrate specificity SP - 4494 EP - 4511 JF - The FEBS journal JO - FEBS J. VL - 285 IS - 23 N2 - Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs. SN - 1742-4658 UR - https://www.unboundmedicine.com/medline/citation/30300504/Biochemical_characterization_of_the_minimal_domains_of_an_iterative_eukaryotic_polyketide_synthase_ L2 - https://doi.org/10.1111/febs.14675 DB - PRIME DP - Unbound Medicine ER -