Defining the potassium binding region in an apple terpene synthase.
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.
Green
Sol
S
The New Zealand Institute for Plant and Food Research, Mt. Albert, Private Bag 92169, Auckland, New Zeland. sol.green@hortresearch.co.nz
Squire
Christopher J
CJ
Nieuwenhuizen
Niels J
NJ
Baker
Edward N
EN
Laing
William
W
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites / Q000502:physiology; D004789:Enzyme Activation / Q000502:physiology; D027845:Malus / Q000201:enzymology*; D008958:Models, Molecular; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011188:Potassium / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D012717:Sesquiterpenes / Q000737:chemistry / Q000378:metabolism; D040681:Structural Homology, Protein
D010940:Plant Proteins; D012717:Sesquiterpenes; C062672:alpha-farnesene; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D011188:Potassium