Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis.
Levopimaradiene synthase, which catalyzes the initial cyclization step in ginkgolide biosynthesis, was cloned and functionally characterized. A Ginkgo biloba cDNA library was prepared from seedling roots and a probe was amplified using primers corresponding to conserved gymnosperm terpene synthase sequences. Colony hybridization and rapid amplification of cDNA ends yielded a full-length clone encoding a predicted protein (873 amino acids, 100,289 Da) similar to known gymnosperm diterpene synthases. The sequence includes a putative N-terminal plastid transit peptide and three aspartate-rich regions. The full-length protein expressed in Escherichia coli cyclized geranylgeranyl diphosphate to levopimaradiene, which was identical to a synthetic standard by GC/MS analysis. Removing 60 or 79 N-terminal residues increased levopimaradiene production, but a 128-residue N-terminal deletion lacked detectable activity. This is the first cloned ginkgolide biosynthetic gene and the first in vitro observation of an isolated ginkgolide biosynthetic enzyme.
Schepmann
H G
HG
Department of Chemistry, Rice University, Houston, Texas 77005, USA.
Pang
J
J
Matsuda
S P
SP
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D002384:Catalysis; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D017353:Gene Deletion; D015723:Gene Library; D020441:Ginkgo biloba / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011106:Polyisoprenyl Phosphates; D012333:RNA, Messenger; D001224:Aspartic Acid; D019883:Alkyl and Aryl Transferases; C434663:levopimaradiene synthase; C002963:geranylgeranyl pyrophosphate