RESUMO
The major components of the isoprenoid-rich essential oil of Artemisia annua L. accumulate in the subcuticular sac of glandular secretory trichomes. As part of an effort to understand isoprenoid biosynthesis in A. annua, an expressed sequence tag (EST) collection was investigated for evidence of genes encoding trichome-specific enzymes. This analysis established that a gene denoted Adh2, encodes an alcohol dehydrogenase and shows a high expression level in glandular trichomes relative to other tissues. The gene product, ADH2, has up to 61% amino acid identity to members of the short chain alcohol dehydrogenase/reductase (SDR) superfamily, including Forsythia x intermedia secoisolariciresinol dehydrogenase (49.8% identity). Through in vitro biochemical analysis, ADH2 was found to show a strong preference for monoterpenoid secondary alcohols including carveol, borneol and artemisia alcohol. These results indicate a role for ADH2 in monoterpenoid ketone biosynthesis in A. annua glandular trichomes.
Assuntos
Álcool Desidrogenase/metabolismo , Artemisia annua/enzimologia , Monoterpenos/química , Álcool Desidrogenase/genética , Artemisia annua/química , Artemisia annua/genética , Monoterpenos Cicloexânicos , Estrutura Molecular , Monoterpenos/isolamento & purificação , Óleos Voláteis/química , Sementes/químicaRESUMO
Artemisinin, a sesquiterpene lactone endoperoxide derived from the glandular secretory trichomes (GSTs) of Artemisia annua, provides the basis for the most effective treatments of malaria. The biology and biochemistry of GSTs of the Asteraceae and their biosynthesis of isoprenoids is reviewed. Recent efforts to understand the biosynthesis of artemisinin in A. annua GSTs are discussed in detail. This includes the development in the authors' laboratory of an expressed sequence tag (EST) approach to identifying the relevant biosynthetic genes using isolated GST as a source of mRNA. This has lead to the isolation of a cDNA encoding CYP71AV1, a multifunctional cytochrome P450 which catalyzes multiple oxidations of the sesquiterpene intermediate amorpha-4,11-diene to artemisinic acid. Further biochemical and molecular genetic work is required to elucidate the precise route from artemisinic alcohol to artemisinin and to engineer more efficient low cost production of artemisinin-based antimalarial drugs.
Assuntos
Artemisininas/metabolismo , Vias Biossintéticas , Animais , Artemisininas/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Genômica , Estrutura Molecular , Oxigenases/genética , Oxigenases/metabolismo , Sesquiterpenos Policíclicos , Sesquiterpenos/metabolismoRESUMO
Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.
Assuntos
Antimaláricos/síntese química , Artemisia annua/enzimologia , Artemisininas/síntese química , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Sesquiterpenos/síntese química , Artemisia annua/citologia , Clonagem Molecular , DNA Complementar , DNA de Plantas , Células Epiteliais/enzimologia , Oxirredução , Plantas Medicinais/citologia , Plantas Medicinais/enzimologiaRESUMO
The mechanism by which the fatty acid acetylenase of Crepis alpina catalyzes crepenynic acid ((9Z)-octadeca-9-en-12-ynoic acid) production from linoleic acid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of regiospecifically deuterated isotopomers with a strain of Saccharomyces cerevisiae expressing a functional acetylenase. LC/MS analysis of crepenynic acid obtained in these experiments showed that the oxidation of linoleate occurs in two discrete steps, since the cleavage of the C12-H bond is very sensitive to isotopic substitution (k(H)/k(D) = 14.6 +/- 3.0) while a minimal isotope effect (k(H)/k(D) = 1.25 +/- 0.08) was observed for the C13-H bond breaking step. These data suggest that crepenynic acid is produced via initial H-atom abstraction at C12 of a linoleoyl substrate. The relationship between the mechanism of enzymatic acetylenation and epoxidation is discussed.
