Fusion of farnesyldiphosphate synthase and epi-aristolochene synthase, a sesquiterpene cyclase involved in capsidiol biosynthesis in Nicotiana tabacum

被引:58
作者
Brodelius, M [1 ]
Lundgren, A
Mercke, P
Brodelius, PE
机构
[1] Univ Kalmar, Dept Chem & Biomed Sci, S-39182 Kalmar, Sweden
[2] Lund Univ, Dept Plant Biochem, S-22100 Lund, Sweden
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 2002年 / 269卷 / 14期
关键词
bifunctional enzyme; epi-aristolochene synthase; farnesyl diphosphate synthase; gene fusion; recombinant expression;
D O I
10.1046/j.1432-1033.2002.03044.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
A clone encoding farnesyl diphosphate synthase (FPPS) was obtained by PCR from a cDNA library made from young leaves of Artemisia annua . A cDNA clone encoding the tobacco epi -aristolochene synthase (e AS) was kindly supplied by J. Chappell (University of Kentucky, Lexington, KY, USA). Two fusions were constructed, i.e. FPPS/e AS and e AS/FPPS. The stop codon of the N-terminal enzyme was removed and replaced by a short peptide (Gly-Ser-Gly) to introduce a linker between the two ORFs. These two fusions and the two single cDNA clones were separately introduced into a bacterial expression vector (pET32). Escherichia coli was transformed with the expression vectors and enzymatically active soluble proteins were obtained after induction with isopropyl thio-beta-d-thiogalactoside. The recombinant enzymes were purified using immobilized metal affinity chromatography on Co2+ columns. The fusion enzymes produced epi- aristolochene from isopentenyl diphosphate through a coupled reaction. The K (m) values of FPPS and e AS for isopentenyl diphosphate and farnesyl diphosphate, respectively, were essentially the same for the single and fused enzymes. The bifunctional enzymes showed a more efficient conversion of isopentenyl diphosphate to epi -aristolochene than the corresponding amount of single enzymes.
引用
收藏
页码:3570 / 3577
页数:8
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