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Potential active-site residues in polyneuridine aldehyde esterase, a central enzyme of indole alkaloid biosynthesis, by modelling and site-directed mutagenesis

Mattern-Dogru, E.; Ma, X.; Hartmann, J.; Decker, H.; Stöckigt, J.

European Journal of Biochemistry 269(12): 2889-2896

2002

ISSN/ISBN: 0014-2956
PMID: 12071952
DOI: 10.1046/j.1432-1033.2002.02956.x
Accession: 011176435
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In the biosynthesis of the antiarrhythmic alkaloid ajmaline, polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epivellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. The PNAE cDNA was previously heterologously expressed in E. coli. Sequence alignments indicated that PNAE has a 43% identity to a hydroxynitrile lyase from Hevea brasiliensis, which is a member of the alpha/beta hydrolase superfamily. The catalytic triad, which is typical for this family, is conserved. By site-directed mutagenesis, the members of the catalytic triad were identified. For further detection of the active residues, a model of PNAE was constructed based on the X-ray crystallographic structure of hydroxynitrile lyase. The potential active site residues were selected on this model, and were mutated in order to better understand the relationship of PNAE with the alpha/beta hydrolases, and as well its mechanism of action. The results showed that PNAE is a novel member of the alpha/beta hydrolase enzyme superfamily.

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Related References

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