Defining the Potassium Binding Region in an Apple Terpene Synthase

Sol Green,Christopher J Squire,Niels J Nieuwenhuizen,Edward N Baker,William Laing

doi:10.1074/jbc.m807140200

Sol Green, Christopher J Squire + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m807140200

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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 (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.

Highlights

Introduction of a Conserved Terpene SynthaseLysine Restores Activity Independent of Kϩ in MdAFS1—Previous phylogenetic comparisons of MdAFS1 [21, 54] indicated that it clustered with the terpene synthases (TPS)-b synthases (Fig. 5A), a subgroup dominated by angiosperm mono-TPS enzymes that are not reported to have any Mϩ dependence
The model was generated by threading the MdAFS1 amino acid sequence onto the three-dimensional structure of its closest structural homologue, a bornyldiphosphate synthase (BPPS) structure complexed with a geranyl diphosphate (GDP) analogue [36]
Introduction of a Conserved Terpene Synthase Lysine Restores Activity Independent of Kϩ in MdAFS1—Previous phylogenetic comparisons of MdAFS1 [21, 54] indicated that it clustered with the TPS-b synthases (Fig. 5A), a subgroup dominated by angiosperm mono-TPS enzymes that are not reported to have any Mϩ dependence

Summary

EXPERIMENTAL PROCEDURES

Sequence Analysis—Multiple amino acid sequence alignments of TPS were performed with ClustalX [38], using default parameters, and were manually adjusted in GeneDoc (nrbsc.org/gfx/genedoc/). Production of Recombinant Proteins—The WT and mutated PsTPS2 were re-amplified from the original pET101/D-TOPO vectors and recloned into the pET200/D-TOPO vector using identical forward and reverse primers to McKay et al [44] This was carried out to enable the purification and analysis of N-terminal His-tagged PsTPS2 proteins rather than using non-purified cleared bacterial lysates as previously described [44]. Recombinant MdAFS1, PsTPS2, and AcTPS2 proteins were extracted and purified according to previous methods [21]. FDP Activity Assays—Assays (50 ␮l) based on previous methods [46] were conducted in quadruplicate, and typically contained 300 nM recombinant protein in assay buffer (50 mM Bistris propane (pH 7.5), 50 mM KCl (omitted as appropriate), 10 mM MgCl2, and 5 mM dithiothreitol). Authentic compound retention time standards used were E-␤-farnesene (Givaudan, Switzerland), and Z,E- and E,E␣-farnesene obtained from “Granny Smith” apples [47, 48], and quantification was against hexadecane

RESULTS

DISCUSSION

The almost complete loss of activity following its removal in