title: Probing the promiscuity of ent-kaurene oxidases via combinatorial biosynthesis.
abstract: The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis.
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Sibongile
authors.initials: S
authors.lastname: Mafu
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Meirong
authors.initials: M
authors.lastname: Jia
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Jiachen
authors.initials: J
authors.lastname: Zi
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Dana
authors.initials: D
authors.lastname: Morrone
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Yisheng
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Meimei
authors.initials: M
authors.lastname: Xu
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Hillwig
authors.affiliation: Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, IA 50011 rjpeters@iastate.edu.
authors.forename: Reuben J
authors.identifier: http://orcid.org/0000-0003-4691-8477
authors.initials: RJ
authors.lastname: Peters
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005875:Gibberellins / Q000096:biosynthesis*; D015335:Molecular Probes; D010802:Phylogeny; D010944:Plants / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D005875:Gibberellins; D015335:Molecular Probes; D003577:Cytochrome P-450 Enzyme System; C066398:ent-kaurene oxidase
keywords: cytochrome P450 monooxygenases; enzyme specificity; gibberellins; labdane-related diterpenoids; natural products