Polyneuridine Aldehyde Esterase: Difference between revisions
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Up to now the phytochemical analysis of ''Rauvolfia'' in vitro systems resulted in the isolation of more than 30 monoterpenoid indole alkaloids. Many of them belong to the well known ajmalan-sarpagan type alkaloids. One of the most intriguing alkaloid was '''ajmaline''', a compound comprising a complex hexacyclic ring system bearing nine chiral carbon centers. | Up to now the phytochemical analysis of ''Rauvolfia'' in vitro systems resulted in the isolation of more than 30 monoterpenoid indole alkaloids. Many of them belong to the well known ajmalan-sarpagan type alkaloids. One of the most intriguing alkaloid was '''ajmaline''', a compound comprising a complex hexacyclic ring system bearing nine chiral carbon centers. | ||
'''Polyneuridine Aldehyde Esterase''''''(PNAE)''' is an important enzyme during Ajmaline Biosynthesis, transforming C-10 skeleton polyneuridine aldehyde into C-9 skeleton epi-vellosimine. | '''Polyneuridine Aldehyde Esterase''''''(PNAE)''' is an important enzyme during Ajmaline Biosynthesis, transforming C-10 skeleton polyneuridine aldehyde into C-9 skeleton epi-vellosimine.[[Image:pathway.jpg]] | ||
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''. | 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''. | ||
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'''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. | '''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 α/β 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. | Sequence analysis led to the preliminary classification of '''PNAE''' as a new candidate of the large α/β 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.[[Image:F1-1.jpg]] | ||
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. | 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. | ||