Geranyl diphosphate synthase from Abies grandis: cDNA isolation, functional expression, and characterization.
Geranyl diphosphate synthase catalyzes the condensation of dimethylallyl diphosphate and isopentenyl diphosphate to generate geranyl diphosphate, the essential precursor of monoterpene biosynthesis. Using geranylgeranyl diphosphate synthase from Taxus canadensis as a hybridization probe, four full length cDNA clones, sharing high sequence identity to each other (>69%) and to the Taxus geranylgeranyl diphosphate synthase (>66%), were isolated from a grand fir (Abies grandis) cDNA library. When expressed in Escherichia coli, three of the recombinant enzymes produced geranyl diphosphate and one produced geranylgeranyl diphosphate as the dominant product when supplied with isopentenyl diphosphate and dimethylallyl diphosphate as cosubstrates. One enzyme (AgGPPS2) was confirmed as a specific geranyl diphosphate synthase, in that it accepted only dimethylallyl diphosphate as the allylic cosubstrate and it produced exclusively geranyl diphosphate as product, with a k(cat) of 1.8s(-1). Gel filtration experiments performed on the recombinant geranyl diphosphate synthases, in which the plastidial targeting sequences had been deleted, revealed that these enzymes are homodimers similar to other short-chain prenyltransferases but different from the heterotetrameric geranyl diphosphate synthase of mint.
Burke
Charles
C
Institute of Biological Chemistry and Program in Plant Physiology, Washington State University, Pullman, Washington 99164-6340, USA.
Croteau
Rodney
R
D028202:Abies / Q000201:enzymology*; D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000378:metabolism; D004122:Dimethylallyltranstransferase / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D017343:Genes, Plant; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D018076:DNA, Complementary; D004122:Dimethylallyltranstransferase