Farnesyl diphosphate (FPP) synthase catalyzes the condensation of geranyl diphosphate (GPP) with isopentenyl diphosphate (IPP). Changes in the structure of GPP during the biosynthesis of FPP were studied by ^1H, ^<13>C and ^<31>P NMR measurements. (1) Substrate specificity: The ability of FPP synthase to identify the substrates for the biosynthetic process was tested by using GPP, linalyl diphosphate (LPP), dihydrolinalyl diphosphate (DHLPP) and citronellyl diphosphate (CPP) as substrates. LPP gives 4% of GPP by non-enzymatic isomerization, and then the so-formed GPP was transformed into FPP by condensation with IPP by FPP synthase. On the other hand, DHLPP and CPP remained unchanged. Thus, only the primary, allylic double bond-bearing GPP acts as substrate for FPP synthase. (2) Conformation changes of GPP by chelation of divalent metal ions: Mn^<2+> and Mg^<2+> were used as divalent metal ions. (i) Changes in the ^<13>C and ^<31>P NMR spectra indicated a one to one chelation of divalent metal ion to the diphosphate moiety, which in turn weakens the C-OP bond of GPP and facilitates the elimination of the diphosphate. (ii) ^1H and ^<31>P NMR relaxation measurements revealed that chelation of divalent metal ion induces a folding of the carbon chain of GPP. (3) NMR detection of geranyl cation: The condensation of [1-^<13>C]GPP with IPP under control of the FPP synthase activity by 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and dithiothreitol (DTT) was monitored by ^<13>C NMR. Besides the signals for C-1 of GPP and C-5 of FPP, a signal at δ 164.5 assigned to geranyl cation was observed (the intensity of the latter was 8.8% that of C-1 of GPP). This signal disappeared when the enzyme activity was inhibited by DTNB, but reappeared when activity was recuperated by addition of DTT. Therefore, geranyl cation is formed and acts as intermediate in the biosynthesis of FPP.
