Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases

Moshe Giladi; Michal Lisnyansky Bar-El; Pavla Vaňková; Alisa Ferofontov; Emelia Melvin; Suha Alkaderi; Daniel Kavan; Boris Redko; Elvira Haimov; Reuven Wiener; Petr Man; Yoni Haitin

doi:10.1126/sciadv.abn1171

Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases

Moshe Giladi^*, Michal Lisnyansky Bar-El, Pavla Vaňková, Alisa Ferofontov, Emelia Melvin, Suha Alkaderi, Daniel Kavan, Boris Redko, Elvira Haimov, Reuven Wiener, Petr Man, Yoni Haitin^*

^*Corresponding author for this work

Department of Biochemistry and Molecular Biology

Research output: Contribution to journal › Article › peer-review

3 Scopus citations

Abstract

Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

Original language	American English
Article number	eabn1171
Journal	Science advances
Volume	8
Issue number	20
DOIs	https://doi.org/10.1126/sciadv.abn1171
State	Published - May 2022

Bibliographical note

Funding Information:
We acknowledge the ESRF for provision of synchrotron radiation facilities, and we would like to thank M. Soler-López and C. Mueller-Dieckmann for assistance in using beamlines ID-23-1 and ID-30B, respectively. We also acknowledge the Diamond Light Source for provision of synchrotron radiation facilities, and we would like to thank F. Bertram for assistance in using beamline I03. The KG405 yeast strain and DHDDS.GW1 and NgBR.GW2 expression vectors were provided by W. C. Sessa. This work was supported by the Israel Science Foundation grant 1721/16 (to Y.H.); Israel Science Foundation grant 1653/21 (to Y.H.); Israel Cancer Research Fund grant 01214 (to Y.H.); Israel Cancer Research Fund grant 19202 (to M.G.); Israel Cancer Association grant 20200037 (to M.G. and Y.H.); German-Israeli Foundation for Scientific Research and Development grant I-2425-418.13/2016 (to Y.H.); I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation grant 1775/12 (to Y.H.); Kahn foundation’s Orion project, Tel Aviv Medical Center, Israel (to M.G.); Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University (to Y.H. and M.G.); EU Horizon 2020 grant EU_FT–ICR_MS project number 731077 (to P.M.); CIISB grant LM2018127 (to P.M.); and MEYS CZ grant CZ.1.05/1.1.00/02.0109 (to P.M. and P.V.).

Publisher Copyright:
Copyright © 2022 The Authors,

Access to Document

10.1126/sciadv.abn1171

Cite this

@article{755acc2f441a480990f0c56ac5b134c8,

title = "Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases",

abstract = "Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.",

author = "Moshe Giladi and Bar-El, {Michal Lisnyansky} and Pavla Va{\v n}kov{\'a} and Alisa Ferofontov and Emelia Melvin and Suha Alkaderi and Daniel Kavan and Boris Redko and Elvira Haimov and Reuven Wiener and Petr Man and Yoni Haitin",

note = "Funding Information: We acknowledge the ESRF for provision of synchrotron radiation facilities, and we would like to thank M. Soler-L{\'o}pez and C. Mueller-Dieckmann for assistance in using beamlines ID-23-1 and ID-30B, respectively. We also acknowledge the Diamond Light Source for provision of synchrotron radiation facilities, and we would like to thank F. Bertram for assistance in using beamline I03. The KG405 yeast strain and DHDDS.GW1 and NgBR.GW2 expression vectors were provided by W. C. Sessa. This work was supported by the Israel Science Foundation grant 1721/16 (to Y.H.); Israel Science Foundation grant 1653/21 (to Y.H.); Israel Cancer Research Fund grant 01214 (to Y.H.); Israel Cancer Research Fund grant 19202 (to M.G.); Israel Cancer Association grant 20200037 (to M.G. and Y.H.); German-Israeli Foundation for Scientific Research and Development grant I-2425-418.13/2016 (to Y.H.); I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation grant 1775/12 (to Y.H.); Kahn foundation{\textquoteright}s Orion project, Tel Aviv Medical Center, Israel (to M.G.); Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University (to Y.H. and M.G.); EU Horizon 2020 grant EU_FT–ICR_MS project number 731077 (to P.M.); CIISB grant LM2018127 (to P.M.); and MEYS CZ grant CZ.1.05/1.1.00/02.0109 (to P.M. and P.V.). Publisher Copyright: Copyright {\textcopyright} 2022 The Authors,",

year = "2022",

month = may,

doi = "10.1126/sciadv.abn1171",

language = "American English",

volume = "8",

journal = "Science advances",

issn = "2375-2548",

publisher = "American Association for the Advancement of Science",

number = "20",

}

TY - JOUR

T1 - Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases

AU - Giladi, Moshe

AU - Bar-El, Michal Lisnyansky

AU - Vaňková, Pavla

AU - Ferofontov, Alisa

AU - Melvin, Emelia

AU - Alkaderi, Suha

AU - Kavan, Daniel

AU - Redko, Boris

AU - Haimov, Elvira

AU - Wiener, Reuven

AU - Man, Petr

AU - Haitin, Yoni

N1 - Funding Information: We acknowledge the ESRF for provision of synchrotron radiation facilities, and we would like to thank M. Soler-López and C. Mueller-Dieckmann for assistance in using beamlines ID-23-1 and ID-30B, respectively. We also acknowledge the Diamond Light Source for provision of synchrotron radiation facilities, and we would like to thank F. Bertram for assistance in using beamline I03. The KG405 yeast strain and DHDDS.GW1 and NgBR.GW2 expression vectors were provided by W. C. Sessa. This work was supported by the Israel Science Foundation grant 1721/16 (to Y.H.); Israel Science Foundation grant 1653/21 (to Y.H.); Israel Cancer Research Fund grant 01214 (to Y.H.); Israel Cancer Research Fund grant 19202 (to M.G.); Israel Cancer Association grant 20200037 (to M.G. and Y.H.); German-Israeli Foundation for Scientific Research and Development grant I-2425-418.13/2016 (to Y.H.); I-CORE Program of the Planning and Budgeting Committee and The Israel Science Foundation grant 1775/12 (to Y.H.); Kahn foundation’s Orion project, Tel Aviv Medical Center, Israel (to M.G.); Claire and Amedee Maratier Institute for the Study of Blindness and Visual Disorders, Sackler Faculty of Medicine, Tel-Aviv University (to Y.H. and M.G.); EU Horizon 2020 grant EU_FT–ICR_MS project number 731077 (to P.M.); CIISB grant LM2018127 (to P.M.); and MEYS CZ grant CZ.1.05/1.1.00/02.0109 (to P.M. and P.V.). Publisher Copyright: Copyright © 2022 The Authors,

PY - 2022/5

Y1 - 2022/5

N2 - Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

AB - Isoprenoids are synthesized by the prenyltransferase superfamily, which is subdivided according to the product stereoisomerism and length. In short- and medium-chain isoprenoids, product length correlates with active site volume. However, enzymes synthesizing long-chain products and rubber synthases fail to conform to this paradigm, because of an unexpectedly small active site. Here, we focused on the human cis-prenyltransferase complex (hcis-PT), residing at the endoplasmic reticulum membrane and playing a crucial role in protein glycosylation. Crystallographic investigation of hcis-PT along the reaction cycle revealed an outlet for the elongating product. Hydrogen-deuterium exchange mass spectrometry analysis showed that the hydrophobic active site core is flanked by dynamic regions consistent with separate inlet and outlet orifices. Last, using a fluorescence substrate analog, we show that product elongation and membrane association are closely correlated. Together, our results support direct membrane insertion of the elongating isoprenoid during catalysis, uncoupling active site volume from product length.

UR - http://www.scopus.com/inward/record.url?scp=85130283475&partnerID=8YFLogxK

U2 - 10.1126/sciadv.abn1171

DO - 10.1126/sciadv.abn1171

M3 - Article

C2 - 35584224

AN - SCOPUS:85130283475

SN - 2375-2548

VL - 8

JO - Science advances

JF - Science advances

IS - 20

M1 - eabn1171

ER -

Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases

Abstract

Bibliographical note

Access to Document

Other files and links

Fingerprint

Cite this