Chrysanthemyl diphosphate synthase: isolation of the gene and characterization of the recombinant non-head-to-tail monoterpene synthase from Chrysanthemum cinerariaefolium.

نویسندگان

  • S B Rivera
  • B D Swedlund
  • G J King
  • R N Bell
  • C E Hussey
  • D M Shattuck-Eidens
  • W M Wrobel
  • G D Peiser
  • C D Poulter
چکیده

Chrysanthemyl diphosphate synthase (CPPase) catalyzes the condensation of two molecules of dimethylallyl diphosphate to produce chrysanthemyl diphosphate (CPP), a monoterpene with a non-head-to-tail or irregular c1'-2-3 linkage between isoprenoid units. Irregular monoterpenes are common in Chrysanthemum cinerariaefolium and related members of the Asteraceae family. In C. cinerariaefolium, CPP is an intermediate in the biosynthesis of the pyrethrin ester insecticides. CPPase was purified from immature chrysanthemum flowers, and the N terminus of the protein was sequenced. A C. cinerariaefolium lambda cDNA library was screened by using degenerate oligonucleotide probes based on the amino acid sequence to identify a CPPase clone that encoded a 45-kDa preprotein. The first 50 aa of the ORF constitute a putative plastidial targeting sequence. Recombinant CPPase bearing an N-terminal polyhistidine affinity tag in place of the targeting sequence was purified to homogeneity from an overproducing Escherichia coli strain by Ni(2+) chromatography. Incubation of recombinant CPPase with dimethylallyl diphosphate produced CPP. The diphosphate ester was hydrolyzed by alkaline phosphatase, and the resulting monoterpene alcohol was analyzed by GC/MS to confirm its structure. The amino acid sequence of CPPase aligns closely with that of the chain elongation prenyltransferase farnesyl diphosphate synthase rather than squalene synthase or phytoene synthase, which catalyze c1'-2-3 cyclopropanation reactions similar to the CPPase reaction.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Isolation of Monoterpene Synthase Gene (NsTPS2) and Evaluation of Terpenoid Compounds in Black Cumin Medicinal Plant (Nigella sativa L.)

Black cumin (Nigella sativa) is a medicinal plant of the Ranunculacea family which raised attention due to its pharmaceutical properties. Medical significance of N. sativa mainly attributed to its oxygenated monoterpenes which are biosynthesized via the methyl erythritol phosphate (MEP) pathway located in plastids. In this study, the essential oil components of leaves, flowers, and developmenta...

متن کامل

Monoterpene synthase from Dracocephalum kotschyi and SPME-GC-MS analysis of its aroma profile

Dracocephalum kotschyi (Lamiaceae), as one of the remarkable aromatic plants, widely grows and also is cultivated in various temperate regions of Iran. There are diverse reports about the composition of the oil of this plant representing limonene derivatives as its major compounds. There is no report on cloning of mono- or sesquiterpene synthases from this plant. In the present study, ...

متن کامل

The effect of drought stress on the expression of some genes involved in monoterpene and sesquiterpanes biosynthesis and essential oil compounds in basil

Background: Basil (Ocimum basillicum L.) is one of the most important medicinal plants belong to Lamiaceae family. Basil essential oil contains important terpenoid compounds, including monoterpanes and sesquiterpanes widely used in drug industries. Objective: In the current investigation, a completely randomized design-based experiment was conducted in greenhouse with three replications and tre...

متن کامل

(3R)-Linalool synthase from Artemisia annua L.: cDNA isolation, characterization, and wound induction.

Artemisia annua is an annual herb used in traditional Chinese medicine. A cDNA library was constructed from leaves of A. annua seedlings and target sequences were amplified by PCR using degenerate primers derived from a consensus sequence of angiosperm terpene synthases. Two clones, QH1 and QH5, with high sequence similarity to plant monoterpene synthases were ultimately obtained and expressed ...

متن کامل

Monoterpene Synthases from Grand Fir (Abies grandis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF MYRCENE SYNTHASE, (2)- (4S)-LIMONENE SYNTHASE, AND (2)-(1S,5S)-PINENE SYNTHASE*

Grand fir (Abies grandis) has been developed as a model system for studying defensive oleoresin formation in conifers in response to insect attack or other injury. The turpentine fraction of the oleoresin is a complex mixture of monoterpene (C10) olefins in which (2)limonene and (2)-aand (2)-b-pinene are prominent components; (2)-limonene and (2)-pinene synthase activities are also induced upon...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Proceedings of the National Academy of Sciences of the United States of America

دوره 98 8  شماره 

صفحات  -

تاریخ انتشار 2001