title: Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases.
abstract: Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from Taxus brevifolia (TbTS, αβγ), fusicoccadiene synthase from Phomopsis amygdali (PaFS, (αα)6), and ophiobolin F synthase from Aspergillus clavatus (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [ Chen , M. , Chou , W. K. W. , Toyomasu , T. , Cane , D. E. , and Christianson , D. W. ( 2016 ) ACS Chem. Biol. 11 , 889 - 899 ]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C20diterpene products and three C25sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products.
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Travis A
authors.initials: TA
authors.lastname: Pemberton
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Mengbin
authors.initials: M
authors.lastname: Chen
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Golda G
authors.initials: GG
authors.lastname: Harris
authors.affiliation: Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912, United States.
authors.forename: Wayne K W
authors.initials: WK
authors.lastname: Chou
authors.affiliation: Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912, United States.
authors.forename: Lian
authors.initials: L
authors.lastname: Duan
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Mustafa
authors.initials: M
authors.lastname: Köksal
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: Alex S
authors.initials: AS
authors.lastname: Genshaft
authors.affiliation: Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912, United States.
authors.forename: David E
authors.identifier: 0000-0002-1492-9529
authors.initials: DE
authors.lastname: Cane
authors.affiliation: Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania , 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, United States.
authors.forename: David W
authors.identifier: 0000-0002-0194-5212
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D003500:Cyclization; D004224:Diterpenes / Q000378:metabolism; D015870:Gene Expression; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D050506:Mutant Chimeric Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072417:Protein Domains; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D004718:Saccharomycetales / Q000201:enzymology / Q000235:genetics*; D054830:Sesterterpenes / Q000096:biosynthesis; D020946:Taxus / Q000201:enzymology / Q000235:genetics*
chemical_list: D004224:Diterpenes; D050506:Mutant Chimeric Proteins; D054830:Sesterterpenes; C542617:fusicoccadiene; D019883:Alkyl and Aryl Transferases; C581422:ophiobolin F synthase, Aspergillus clavatus; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase