Abstract
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.
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