Reaktionsgleichung
C1N=C(N)C2N=CN([C@@H]3[O:14][C@H](COP(OP(OC[C@H]4O[C@@H](N5C=C(C(N)=O)CC=C5)[C@H](O)[C@@H]4O)(O)=O)(O)=O)[C@@H](O)[C@H]3OP(O)(O)=O)C=2N=1.[OH:49][C:50]1[CH:55]=[CH:54][C:53]([CH2:56]C=NO)=[CH:52][CH:51]=1.N(C(CO)(CO)CO)CC(O)=O>>[OH:49][C:50]1[CH:55]=[CH:54][C:53]([CH:56]=[O:14])=[CH:52][CH:51]=1Komponenten
Versuchsvorschrift
5 μl membranes isolated as described in Halkier et al, Archives of Biochemistry and Biophysics 322: 369-377, 1995, from E. coli expressing “12” was reconstituted with 0.225 units NADPH-cytochrome P450-reductase, 50 μg NADPH, 42 nCi p-hydroxyphenylacetaldehyde oxime, and 100 μg dilaurylphosphatidylcholine in a total volume of 100 μl of 30 mM Tricine pH 7.9. After incubation at 30° C. for 30 minutes the reaction mixture was applied to a TLC plate and analyzed as described above. Reconstitution of membranes from E. coli expressing “12” resulted in the accumulation of p-hydroxybenzaldehyde which is the stable dissociation product of p-hydroxymandelonitrile, the last intermediate in the biosynthesis of the cyanogenic glucoside dhurrin. This shows that demonstrates that “12” is the cytochrome P450 that catalyzes the conversion of p-hydroxyphenylacetaldehyde oxime to p-hydroxymandelonitrile. The cDNA is designated P450OX. A clone comprising the described cDNA of P450OX has been deposited on Jan. 10, 1997 with the DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, under the accession number DSM 11367.
Aufarbeitung
- custom5 μl membranes isolated
Quellen und Nachweise
- ORD-Eintrag
- ord-40aa030ae29a41a4bf51d13dde9fe52a
- Quelldatensatz
- uspto-grants-2003_11
- Patentnummer
- US06649814B2
Verknüpfte Notizen
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