Figure 7.

Enzymatic Activity Assays Using the FaNES1 and FaNES2 Recombinant Enzymes Produced in E. coli Cells.

(A) Seven different constructs (C1 to C7) used for the production of recombinant proteins and tested for enzyme activity. In C3, the stop codon present between the two Met residues (Met1 and Met2) in FaNES1 was removed by site-directed mutagenesis, allowing translation from Met1. All proteins were produced as fusions with a His-tag at their N termini (marked in gray). Results of enzyme activity assays with GPP and FPP are shown on the right-hand side of the construct schemes. lin, linalool; ner, nerolidol; npf, no product formed.

(B) Radio-GC analyses of radiolabeled products formed from 20 μM [³H]-geranyl diphosphate (top two panels) or [³H]-farnesyl diphosphate (bottom two panels) by heterologously produced, His-tag purified FaNES1 protein. Panel 1, flame ionization detector (F.I.D.) signal of coinjected, unlabeled linalool. Panel 3, unlabeled standards of (Z)-nerolidol and (E)-nerolidol. Panels 2 and 4, radio traces showing radiolabeled products. For further details, see Methods.