The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the α/β hydrolase super family
The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the α/β hydrolase super family
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DOI：
10.1046/j.1432-1327.2000.01136.x
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发表时间：
2000-03-01
期刊：
影响因子：
--
通讯作者：
Stöckigt, J
中科院分区：
文献类型：
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作者：
Dogru, E;Warzecha, H;Stöckigt, J
抗寄生虫生物碱ajaxine的生物合成由10多种特异性酶催化。在该多步骤过程中，多聚神经苷醛酯酶（PNAE）通过将多聚神经苷醛转化为表维洛司明（epi-vellosiminine）来催化中心反应，表维洛司明是合成阿齐马拉烷骨架的直接前体。PNAE是从蛇萝芙木的细胞悬浮培养物中纯化的。纯化蛋白的N-末端序列和内切蛋白酶LysC片段用于引物设计和特异性PCR产物的扩增，导致从R. serpentina图书馆将PNAE cDNA与C-末端His-标签融合，在大肠杆菌中表达并使用Ni-亲和层析纯化至均一。纯酶显示出非凡的底物特异性，与其他酯酶完全不同。序列比对表明PNAE是α/β水解酶超家族的新成员。
The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family.