FIG. 5.

(A) [¹⁴C]IPP incorporation into the isoprenoid fraction of the supernatant of WT is stimulated by FR6P and GA3P, indicating DMAPP production. (B) In the supernatant of the Δsll1556 mutant the same substrates failed to stimulate [¹⁴C]IPP incorporation. (C) Reconstitution of activity by addition of Sll1556 protein at an 0.15-μg/ml final concentration in the reaction mixture. Symbols: ▴, FR6P (500 μM); ▪, DMAPP (2 μM); ×, GA3P (1 mM); •, GA3P (1 mM) plus PYR (500 μM); ○, MEP (500 μM); ⧫, incubation cofactor mixture (500 μM ATP, 250 μM CTP, 100 μM thiamine PP, 1 mM NADPH, 500 μM NADP, 1 mM FAD, 5 mM glutathione, 5 mM MgCl₂, 2.5 mM MnCl₂, 10 μM coenzyme B₁₂, 100 mM HEPES-KOH, pH 7.7) incubated at 37°C. Incorporation with radiolabeled [¹⁴C]IPP (8.5 μM) followed preincubation with IPP (3 μM) (Materials and Methods).