Defining the Potassium Binding Region in an Apple Terpene Synthase

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Terpene synthases are a family of enzymes largely responsible for synthesizing the vast array of terpenoid compounds known to exist in nature. Formation of terpenoids from their respective 10-, 15-, or 20-carbon atom prenyl diphosphate precursors is initiated by divalent (M(2+)) metal ion-assisted electrophilic attack. In addition to M(2+), monovalent cations (M(+)) have also been shown to be essential for the activity of certain terpene synthases most likely by facilitating substrate binding or catalysis. An apple alpha-farnesene synthase (MdAFS1), which has a dependence upon potassium (K(+)), was used to identify active site regions that may be important for M(+) binding. Protein homology modeling revealed a surface-exposed loop (H-alphal loop) in MdAFS1 that fulfilled the necessary requirements for a K(+) binding region. Site-directed mutagenesis analysis of specific residues within this loop then revealed their crucial importance to this K(+) response and strongly implicated specific residues in direct K(+) binding. The role of the H-alphal loop in terpene synthase K(+) coordination was confirmed in a Conifer pinene synthase also using site-directed mutagenesis. These findings provide the first direct evidence for a specific M(+) binding region in two functionally and phylogenetically divergent terpene synthases. They also provide a basis for understanding K(+) activation in other terpene synthases and establish a new role for the H-alphal loop region in terpene synthase catalysis.