Purification Of The Mammalian NgBR/hCIT cis-Prenyltransferase Complex: IdentificationOf A Conserved Carboxyterminal RxG Motif Crucial For Enzymatic Activity

cis -Prenyltransferases ( cis PTs) constitute a large family of enzymes conserved during evolution and present in all domains of life. In eukaryotes and archaea, cis PT is the first enzyme committed to the synthesis of dolichyl-phosphate (DolP). DolP is obligate lipid carrier in protein glycosylation reactions in mammalian cells. The homodimeric bacterial enzyme, undecaprenyl diphosphate synthase (UPPS) generates 11 isoprene units and has been structurally and mechanistically characterized in great detail. Recently our group discovered that unlike UPPS, mammalian cis PT is a heteromer consisting of NgBR (NUS1) and hCIT (DHDDS) subunits and this composition has been confirmed in plants and fungal cis PTs. Here, we establish the first purification system for heteromeric cis PT and show that both NgBR and hCIT subunits function in catalysis and substrate binding. Finally, we identified a critical RxG sequence in the C-terminal tail of NgBR that is conserved and essential for enzyme activity across phyla.