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.

Blavatnik Center for Drug Discovery Tel Aviv University Tel Aviv 6997801 Israel

Department of Biochemistry and Molecular Biology IMRIC Hadassah Medical School The Hebrew University Jerusalem 9112001 Israel

Department of Physiology and Pharmacology Sackler School of Medicine Tel Aviv University Tel Aviv 6997801 Israel

Institute of Microbiology of the Czech Academy of Sciences Division BioCeV Prumyslova 595 252 50 Vestec Czech Republic

Sagol School of Neuroscience Tel Aviv University Tel Aviv 6997801 Israel

Tel Aviv Sourasky Medical Center Tel Aviv 6423906 Israel

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