title: Cloning and characterization of Ginkgo biloba levopimaradiene synthase which catalyzes the first committed step in ginkgolide biosynthesis.
abstract: 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.
authors.affiliation: Department of Chemistry, Rice University, Houston, Texas 77005, USA.
authors.forename: H G
authors.initials: HG
authors.lastname: Schepmann
authors.forename: J
authors.initials: J
authors.lastname: Pang
authors.forename: S P
authors.initials: SP
authors.lastname: Matsuda
mesh_terms: 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
chemical_list: D018076:DNA, Complementary; D011106:Polyisoprenyl Phosphates; D012333:RNA, Messenger; D001224:Aspartic Acid; D019883:Alkyl and Aryl Transferases; C434663:levopimaradiene synthase; C002963:geranylgeranyl pyrophosphate