Reaction #467348

ord-21ac1516ddd141008844e67fa55522ce

Reaction equation

NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
O=C(O)CNC(CO)(CO)CO
Tricine
ON=CCc1ccc(O)cc1
p-hydroxyphenylacetaldehyde oxime
→
O=Cc1ccc(O)cc1
p-hydroxybenzaldehyde

Reactants

—
dilaurylphosphatidylcholine
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
NC(=O)C1=CN([C@@H]2O[C@H](COP(=O)(O)OP(=O)(O)OC[C@H]3O[C@@H](n4cnc5c(N)ncnc54)[C@H](OP(=O)(O)O)[C@@H]3O)[C@@H](O)[C@H]2O)C=CC1
NADPH
O=C(O)CNC(CO)(CO)CO
Tricine
ON=CCc1ccc(O)cc1
p-hydroxyphenylacetaldehyde oxime

Reagents

None

Conditions

Detailed conditions
See reaction.notes.procedure_details.

Workup

  1. 1
    Other5 μl membranes isolated

Procedure

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.

Source

DOI: 10.6084/m9.figshare.5104873.v1Patent: US06300544B1uspto-grants-2001_10