Cloning and functional expression of a cDNA encoding geranylgeranyl diphosphate synthase from Taxus canadensis and assessment of the role of this prenyltransferase in cells induced for taxol production.
Geranylgeranyl diphosphate synthase supplies the essential acyclic precursor for Taxol biosynthesis in methyl jasmonate-induced Taxus canadensis suspension cell cultures. A cDNA encoding this prenyltransferase was cloned from an induced T. canadensis cell library. The recombinant enzyme expressed in yeast was confirmed by radiochromatographic analysis to produce geranylgeranyl diphosphate from farnesyl diphosphate and [4-14C]isopentenyl diphosphate and was subjected to preliminary kinetic characterization. The deduced amino acid sequence of this gymnosperm geranylgeranyl diphosphate synthase (393 residues) resembles those of geranylgeranyl diphosphate synthases of angiosperm origin, except for the 90-100 N-terminal residues that correspond to the plastidial transit peptide. The full-length preprotein (42.6 kDa) and two truncated versions, corresponding to putative "mature proteins" from which the transit peptide was deleted, were transformed into a yeast mutant defective for the beta-subunit of type II geranylgeranyl transferase. Under conditions of regulated expression, both the full-length construct and the longest of the truncations (at Phe 99) were able to complement the mutant. However, when these two constructs were overexpressed in a wild-type yeast strain, they were apparently toxic, most probably due to depletion of endogenous farnesyl diphosphate as the cosubstrate for the geranylgeranyl diphosphate synthase reaction. In vitro activity of the corresponding recombinant enzymes paralleled the expression level of the constructs as determined by SDS-PAGE analysis of the appropriate proteins of predicted size, and was correlated with toxicity in the wild-type yeast strain and with ability to complement the mutant strain. Results from the analysis of geranylgeranyl diphosphate synthase activity levels and measurement of the corresponding steady-state mRNA levels during the time course of Taxol production in induced T. canadensis suspension cell cultures, and comparison to similar data for activity and message levels for taxadiene synthase, the committed step of the pathway, indicated that for each enzyme both the level of corresponding message and catalytic activity rapidly increased after methyl jasmonate induction.
Hefner
J
J
Institute of Biological Chemistry, and Department of Genetics and Cell Biology, Washington State University, Pullman, Washington, 99164-6340, USA.
Ketchum
R E
RE
Croteau
R
R
D000085:Acetates / Q000494:pharmacology; D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification*; D004122:Dimethylallyltranstransferase / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D051231:Farnesyltranstransferase; D015870:Gene Expression; D005816:Genetic Complementation Test; D007535:Isomerases / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D054883:Oxylipins; D017239:Paclitaxel / Q000096:biosynthesis*; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics*; D017368:Protein Prenylation; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014197:Trees / Q000201:enzymology* / Q000235:genetics
D000085:Acetates; D003517:Cyclopentanes; D018076:DNA, Complementary; D054883:Oxylipins; D010940:Plant Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C072239:methyl jasmonate; D019883:Alkyl and Aryl Transferases; D004122:Dimethylallyltranstransferase; D051231:Farnesyltranstransferase; D007535:Isomerases; C095926:taxa-4(5),11(12)-diene synthase; D017239:Paclitaxel