Cloning and heterologous expression of a second (+)-delta-cadinene synthase from Gossypium arboreum.
Screening of a Gossypium arboreum L. cv. Nanking cDNA library resulted in the identification and cloning of a second (+)-delta-cadinene synthase. A probe for the screens was prepared by PCR using primers based on conserved sequences in farnesyl diphosphate cyclases and genomic DNA as a template. This second cDNA clone encodes a protein that is 80% identical to the recently described (+)-delta-cadinene synthases CAD1-C1 and C14 from G. arboreum and maintains a significant degree of homology to the other known mono-, sesqui-, and diterpene synthases. As in the case of CAD1-C1 (+)-delta-cadinene synthase from cultured cotton cells, the synthesis of the second CAD1-A mRNA was induced by treatment of cotton cell suspension cultures with a partially purified elicitor preparation from the phytopathogenic fungus Verticillium dahliae. Expression of CAD1-A mRNA was quantitated with reverse transcription PCR and showed that CAD1-A mRNA was maximally increased 8-fold, 6 h after addition of elicitor. Heterologous expression of this second cDNA produced a 64 kD protein that catalyzed the cyclization of farnesyl diphosphate to (+)-delta-cadinene, the identical product produced by CAD1-C1. The steady-state kinetic parameters of CAD1-A were similar to CAD1-C, showing a Km of 7 mM farnesyl diphosphate and kcat of 0.039 s-1 at 30 degrees C. However, the optimal pH and Mg2+ concentration for CAD1-A activity were significantly higher than those observed for CAD1-C.
Chen
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Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333, USA.
Wang
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Chen
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Davisson
V J
VJ
Heinstein
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P
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D003904:Mitosporic Fungi / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010946:Plants, Medicinal / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D007535:Isomerases; C097608:delta-cadinene synthase