A recombinant enzyme (also known as a monoterpene synthase or cyclase) from the grand fir (Abies grandis) requires Mn2+ and K+ for activity. Mg2+ is essentially ineffective as the divalent metal ion cofactor.
bifunctional enzyme: geranyldiphosphate synthase (EC 22.214.171.124)/myrcene synthase (EC 126.96.36.199). When the recombinant enzyme is incubated with dimethylallyl diphosphate and isopentenyl diphosphate as cosubstrates, the level of geranyl diphosphate formed is approximately tenfold higher than the level of myrcene formed. The data suggest that geranyl diphosphate is a free intermediate in myrcene production
mutated enzymes are generated using the QuickChange II site-directed mutagenesis kit. The PCR-based mutagenesis protocol is performed using pET200-D-TOPO harbouring the respective AcTPS2 cDNAs as template. The single-site mutant enzymes overexpressed in Escherichia coli.