Polyneuridine Aldehyde Esterase
Polyneuridine Aldehyde Esterase is an important enzyme during Ajmaline Biosynthesis, transforming C-10 skeleton polyneuridine aldehyde into C-9 skeleton epi-vellosimine.
For the biochemical characterization of PNAE, the enzyme was isolated from plant cell cultures of the Indian medicinal plant Rauvolfia serpentina and partially sequenced; the PNAE cDNA was then cloned and overexpressed in Escherichia coli.
PNAE showed extremely high specificity for its natural substrate polyneuridine aldehyde, out of 14 alkaloidal and aromatic esters only polyneuridine aldehyde and its ethylester were processed.
Sequence analysis led to the preliminary classification of PNAE as a new candidate of the large a/b hydrolase superfamily. This classification is based in particular on the catalytic triad Ser87, Asp216, and His244, which was previously verified by single mutations and homology modeling.
The structural data now available for PNAE support the overall mechanism, which undoubtedly represents the key reaction for the biosynthesis of C9-monoterpenoid Rauvolfia alkaloids. The data will also allow a rational, structure-based redesign of PNAE, similar what we have recently demonstrated for the Pictet-Spenglerase, strictosidine synthase, which is now applied for chemoenzymatic synthesis of novel alkaloid libraries.
Because the cap domain (see the Supporting Information) and the architecture of the binding pocket of PNAE determines its exceptional high substrate specificity, future systematic structure-based, second-sphere, and random mutations should give PNAE mutants with altered, especially low, substrate specificity. Such enzymes then could be useful multipurpose catalysts for generation of novel alkaloid structures for biological screening.
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