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.
Bibliographical noteFunding 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.).
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