CYTOCHROME P-450(TYR) IS A MULTIFUNCTIONAL HEME-THIOLATE ENZYME CATALYZING THE CONVERSION OF L-TYROSINE TO P-HYDROXYPHENYLACETALDEHYDE OXIME IN THE BIOSYNTHESIS OF THE CYANOGENIC GLUCOSIDE DHURRIN IN SORGHUM-BICOLOR (L) MOENCH
O. Sibbesen et al., CYTOCHROME P-450(TYR) IS A MULTIFUNCTIONAL HEME-THIOLATE ENZYME CATALYZING THE CONVERSION OF L-TYROSINE TO P-HYDROXYPHENYLACETALDEHYDE OXIME IN THE BIOSYNTHESIS OF THE CYANOGENIC GLUCOSIDE DHURRIN IN SORGHUM-BICOLOR (L) MOENCH, The Journal of biological chemistry, 270(8), 1995, pp. 3506-3511
Cytochrome P-450(TYR), which catalyzes the N-hydroxylation of L-tyrosi
ne in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum
bicolor (L.) Moench has recently been isolated (Sibbesen, O., Koch, B.
, Halkier, B. A., and Moller, B. L. (1994) Proc. Natl. Acad. Sci. U. S
. A. 92, 9740-9744). Reconstitution of the enzyme activity in lipid mi
celles containing cytochrome P-450, and NADPH-cytochrome P-450 oxidore
ductase demonstrates that cytochrome P-450(TYR) catalyzes the conversi
on of L-tyrosine into p-hydroxyphenylacetaldehyde oxime. Earlier studi
es with microsomes have demonstrated that this conversion involves two
N-hydroxylation reactions of which the first produces N-hydroxytyrosi
ne. We propose that the product of the second N-hydroxylation reaction
is N,N-dihydroxytyrosine. N,N-dihydroxytyrosine is dehydrated to 2-ni
troso-3-(p-hydroxyphenyl) propionic acid which decarboxylates to p-hyd
roxyphenylacetaldehyde oxime. The dehydration and decarboxylation reac
tions may proceed non-enzymatically. The E/Z ratio of the p-hydroxyphe
nylacetaldehyde oxime produced by reconstituted cytochrome P-450(TYR)
is 69:31. Lipid micelles made from L-alpha-dilauroyl phosphatidylcholi
ne are more than twice as effective in reconstituting cytochrome P-450
(TYR) activity as compared to other lipids. The K-m and turnover numbe
r of the enzyme is 0.14 mM and 200 min(-1), respectively, when assayed
in the presence of 15 mM NaCl whereas the values are 0.21 mM and 230
min(-1) when assayed in the absence of added salt. The multifunctional
nature cytochrome P-450(TYR) is confirmed by demonstrating that bindi
ng of L-tyrosine or N-hydroxytyrosine mutually excludes binding of the
other substrate. These results explain why the conversion of tyrosine
to p-hydroxyphenylacetaldehyde oxime as earlier reported (Moller, B.
L., and Conn, E. E. (1980) J. Biol. Chem. 255, 3049-3056) shows the ph
enomenon of catalytic facilitation (''channeling''). Cytochrome P-450(
TYR) is the first isolated multifunctional heme-thiolate enzyme from p
lants. N-Hydroxylases of the cytochrome P-450 type with high substrate
specificity have not previously been reported.