RESUMEN
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.
Asunto(s)
Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Ginkgo biloba/enzimología , Plantas Medicinales , Secuencia de Aminoácidos , Ácido Aspártico/química , Catálisis , Clonación Molecular , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Eliminación de Gen , Biblioteca de Genes , Modelos Químicos , Datos de Secuencia Molecular , Fosfatos de Poliisoprenilo/metabolismo , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Cycloartenol synthase converts oxidosqualene to the pentacyclic sterol precursor cycloartenol. An Arabidopsis thaliana cycloartenol synthase Ile481Val mutant was previously shown to produce lanosterol and parkeol in addition to its native product cycloartenol. Experiments are described here to construct Phe, Leu, Ala, and Gly mutants at position 481 and to determine their cyclization product profiles. The Phe mutant was inactive, and the Leu mutant produced cycloartenol and parkeol. The Ala and Gly mutants formed lanosterol, cycloartenol, parkeol, achilleol A, and camelliol C. Monocycles comprise most of the Gly mutant product, showing that an alternate cyclization route can be made the major pathway by a single nonpolar mutation.