Science Advances 2022
Overview of hcis-PT structure and reaction scheme
(A) Cartoon representation of a single DHDDS-NgBR heterodimer in complex with FPP [Protein Data Bank (PDB) 6Z1N]. DHDDS and NgBR are colored blue and yellow, respectively. Surface representations of the FPP and IPP [placed by superposition with PDB 6W2L] molecules are colored pink and green, respectively. The residue W3, at the DHDDS N terminus, is shown as spheres. (B) Condensation reaction scheme. At the first cycle, the allylic diphosphate primer, FPP (C15, pink), undergoes a condensation with IPP (C5, green) to produce geranylgeranyl-diphosphate (GGPP) (C20). The cycle repeats with further condensations (14–17) of the allylic diphosphate at S1, ultimately leading to a final product length of C85–100.
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.
Giladi, M., Bar-El, ML., Lisnyansky, M., Vankova, P., Ferofontov, A., Melvin, E., Alkaderi, S., Kavan, D., Redko, B., Haimov, E., Wiener, R., Man, P., and Haitin, Y.: Structural basis for long-chain isoprenoid synthesis by cis-prenyltransferases, Sci. Adv. 2022, 7, eabn1171, https://doi.10.1126/sciadv.abn1171