Conversion of a plant oxidosqualene-cycloartenol synthase to an oxidosqualene-lanosterol cyclase by random mutagenesis

Tung-Kung Wu*, John H. Griffin

*Corresponding author for this work

研究成果: Article同行評審

36 引文 斯高帕斯(Scopus)

摘要

A random mutagenesis/in vivo selection approach was applied to generate and identify mutations that alter the product specificity of oxidosqualene-cycloartenol synthase (CAS) from Arabidopsis thaliana. This work complements previous studies of triterpene cyclase enzymes and was undertaken to provide knowledge of the frequency and locations at which point mutations can alter cyclase product specificity. Random mutations were introduced by treatment with hydroxylamine or passage through a mutator strain of bacteria. Libraries of mutated plasmids carrying the cas1 gene were transformed into a cyclase-deficient strain of Saccharomyces cerevisiae (CBY57) bearing a complementing plasmid (pZS 11) carrying an Erg7 gene that encodes wild-type yeast oxidosqualene-lanosterol cyclase and a URA3 marker that could be counterselected by growth in media containing 5-fluoroorotic acid (5-FOA). This allowed use of a plasmid shuffle to select for cas1 mutants that could substitute for ERG7 activity. Five of ∼73000 transformants were observed to grow in media containing 5-FOA but lacking ergosterol. pTKP5-derived plasmids isolated from these transformants were sequenced, revealing five distinct and unique point mutations: Tyr410Cys, Ala469Val, His477Tyr, Ile481Thr, and Tyr532His. Analysis of the nonsaponifiable lipids from CBY57 cells expressing these mutants suggests that the Tyr410Cys and His477Tyr mutants produce lanosterol as the dominant product, whereas the Ala469Val, Ile481Thr, and Tyr532His mutants produce a mixture of lanosterol and achilleol A, a product of monocyclization. Sequence and structural homology modeling of CAS indicate that the observed product specificity-altering mutations occur both within (Tyr410Cys, Ile481Thr, and Tyr532His) and outside of (Ala469Val and His477Tyr) the cyclase active site.

原文English
頁(從 - 到)8238-8244
頁數7
期刊Biochemistry
41
發行號26
DOIs
出版狀態Published - 2 七月 2002

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