TY - JOUR
T1 - Structural insight on assembly-line catalysis in terpene biosynthesis
AU - Faylo, Jacque L.
AU - van Eeuwen, Trevor
AU - Kim, Hee Jong
AU - Gorbea Colón, Jose J.
AU - Garcia, Benjamin A.
AU - Murakami, Kenji
AU - Christianson, David W.
N1 - Funding Information:
We thank the NIH for grants GM56838 (D.W.C.), GM123233 (K.M.), AI118891 (B.A.G.), and CA196539 (B.A.G.) in support of this research. In addition, we thank the Structural Biology and Molecular Biophysics NIH Training Grant T32-GM008275 (J.L.F. and T.v.E.) and the Chemistry-Biology Interface NIH Training Grant T32-GM071339 (H.J.K.) for support. J.J.G.C. thanks the NSF for a Graduate Research Fellowship Program award (DGE-1845298). We thank the Electron Microscopy Resource Lab of the Perelman School of Medicine, University of Pennsylvania, for instrument time and Dr. Sudheer Molugu for assistance. A portion of this research was supported by NIH grant U24GM129547 and performed at the Pacific Northwest Center for Cryo-EM at Oregon Health & Science University and accessed through EMSL (grid.436923.9), a DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research. We are especially grateful to Nancy Meyer at the PNCC for her assistance. This research was also supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National Laboratory for Cancer Research under contract HSSN261200800001E. We especially thank Adam Wier for data collection and support. XL-MS data analysis was performed in the Biochemistry & Biophysics department cluster supported by NIH grant S10OD023592. We are grateful to Professor Elizabeth Rhoades for helpful scientific discussions regarding intrinsically disordered peptides. Finally, we thank Michal Tycak for expert guidance with proSHADE. Molecular graphics and analyses were performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health grant R01-GM129325 and from the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12/1
Y1 - 2021/12/1
N2 - Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.
AB - Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.
UR - http://www.scopus.com/inward/record.url?scp=85107506824&partnerID=8YFLogxK
U2 - 10.1038/s41467-021-23589-9
DO - 10.1038/s41467-021-23589-9
M3 - Article
C2 - 34108468
AN - SCOPUS:85107506824
SN - 2041-1723
VL - 12
JO - Nature Communications
JF - Nature Communications
IS - 1
M1 - 3487
ER -