Stereochemical Studies on the Making and Unmaking of Isopentenyl Diphosphate in Different Biological Systems

To investigate the unknown stereochemical course of the reaction catalyzed by the type‐II isomerase, which interconverts isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), a sample of [1,2‐13C2]‐IPP stereospecifically labelled with 2H at C(2) was prepared by incubating a D2O solution of (E)‐4‐hydroxy‐3‐methyl[1,2‐13C2]but‐2‐enyl diphosphate with a recombinant IspH protein of Escherichia coli in the presence of NADH as a reducing agent and flavodoxin as well as flavodoxin reductase as auxiliary proteins. As monitored by 13C‐NMR spectroscopy, treatment of the deuterated IPP with either type‐I or type‐II IPP isomerase resulted in the formation of DMAPP molecules retaining all the 2H label of the starting material. From the known stereochemical course of the type‐I isomerase‐catalyzed reaction, one has to conclude that the label introduced from D2O in the course of the IspH reaction resides specifically in the HSiC(2) position of IPP and that the two isomerases mobilize specifically the same HReC(2) ligand of their common IPP substrate. The outcome of an additional experiment, in which unlabelled IPP was incubated in D2O with the type‐II enzyme, demonstrates that the two isomerases also share the same preference in selecting for their reaction the (E)‐methyl group of DMAPP.