RESUMEN
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.
Asunto(s)
Arabidopsis/enzimología , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Sustitución de Aminoácidos , Arabidopsis/genética , Ciclización , Cromatografía de Gases y Espectrometría de Masas , Transferasas Intramoleculares/genética , Lanosterol/análogos & derivados , Lanosterol/biosíntesis , Lanosterol/metabolismo , Espectroscopía de Resonancia Magnética , Mutación , Fitosteroles/biosíntesis , Fitosteroles/metabolismo , Protones , Relación Estructura-Actividad , TriterpenosRESUMEN
A 2274 bp Arabidopsis thaliana cDNA was isolated that encodes a protein 57% identical to cycloartenol synthase from the same organism. The expressed recombinant protein encodes lupeol synthase, which converts oxidosqualene to the triterpene lupeol as the major product. Lupeol synthase is a multifunctional enzyme that forms other triterpene alcohols, including beta-amyrin, as minor products. Sequence analysis suggests that lupeol synthase diverged from cycloartenol synthase after plants diverged from fungi and animals. This evolutionary order is the reason that fungi and animals do not make lupeol.