Unusual features of a recombinant apple alpha-farnesene synthase.
A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.
Green
Sol
S
HortResearch, Mt Albert Research Centre, Horticultural and Food Research Institute of New Zealand, Private Bag 92169, Auckland, New Zealand. sol.green@hortresearch.co.nz
Friel
Ellen N
EN
Matich
Adam
A
Beuning
Lesley L
LL
Cooney
Janine M
JM
Rowan
Daryl D
DD
MacRae
Elspeth
E
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D001665:Binding Sites; D004122:Dimethylallyltranstransferase / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006153:Guanosine Diphosphate / Q000378:metabolism; D007290:Inosine Diphosphate / Q000378:metabolism; D019684:Magnoliopsida / Q000201:enzymology; D027845:Malus / Q000201:enzymology*; D008670:Metals; D039821:Monoterpenes / Q000032:analysis; D016296:Mutagenesis; D050505:Mutant Proteins / Q000378:metabolism; D010802:Phylogeny; D011106:Polyisoprenyl Phosphates / Q000032:analysis / Q000737:chemistry; D011755:Pyrophosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012717:Sesquiterpenes / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
D008670:Metals; D039821:Monoterpenes; D050505:Mutant Proteins; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D006153:Guanosine Diphosphate; C062672:alpha-farnesene; C004808:farnesyl pyrophosphate; D007290:Inosine Diphosphate; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase; C108877:terpene synthase; D004122:Dimethylallyltranstransferase; D011755:Pyrophosphatases