Defining the potassium binding region in an apple terpene synthase.

Abstract

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 (M2 ) metal ion-assisted electrophilic attack. In addition to M2 , 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 -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- l 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- l 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- l loop region in terpene synthase catalysis.