Defining the Potassium Binding Region in an Apple Terpene Synthase

• Published in: The Journal of biological chemistry 2009-03, Vol.284 (13), p.8661-8669
• Main Authors: Green, Sol, Squire, Christopher J., Nieuwenhuizen, Niels J., Baker, Edward N., Laing, William
• Format: Article
• Language: English
• Subjects: Alkyl and Aryl Transferases - chemistry; Alkyl and Aryl Transferases - metabolism; Binding Sites - physiology; Enzyme Activation - physiology; Enzyme Catalysis and Regulation; Malus - enzymology; Models, Molecular; Plant Proteins - chemistry; Plant Proteins - metabolism; Potassium - chemistry; Potassium - metabolism; Protein Structure, Secondary; Sesquiterpenes - chemistry; Sesquiterpenes - metabolism; Structural Homology, Protein
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M807140200

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 α-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-αl 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-αl 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-αl loop region in terpene synthase catalysis.