Substrate specificity of the cytochrome P450 enzymes CYP79A1 and CYP71E1 involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.
The two multifunctional cytochrome P450 enzymes, CYP79A1 and CYP71E1, involved in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench have been characterized with respect to substrate specificity and cofactor requirements using reconstituted, recombinant enzymes and sorghum microsomes. CYP79A1 has a very high substrate specificity, tyrosine being the only substrate found. CYP71E1 has less stringent substrate requirements and metabolizes aromatic oximes efficiently, whereas aliphatic oximes are slowly metabolized. Neither CYP79A1 nor CYP71E1 catalyze the metabolism of a range of different herbicides. The reported resistance of sorghum to bentazon is therefore not linked to the presence of CYP79A1 or CYP71E1. NADPH is a much better cofactor than NADH although NADH does support the entire catalytic cycle of both P450 enzymes. Km and Vmax values for NADPH when supporting CYP71E1 activity are 0.013 mM and 111 nmol/mg protein/s. For NADH, the corresponding values are 0. 3 mM and 42 nmol/mg protein/s. CYP79A1 is a fairly stable enzyme. In contrast, CYP71E1 is labile and prone to rapid denaturation at room temperature. CYP71E1 is isolated in the low spin form. CYP71E1 catalyzes an unusual dehydration reaction of an oxime to the corresponding nitrile which subsequently is C-hydroxylated. The oxime forms a peculiar reverse Type I spectrum, whereas the nitrile forms a Type I spectrum. Several compounds which do not serve as substrates formed Type I substrate binding spectra with the two P450 enzymes.
Department of Plant Biology, The Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Copenhagen, DK-1871 Frederiksberg C, Denmark.
R A
RA
Kahn
T
T
Fahrendorf
B A
BA
Halkier
B L
BL
Møller
D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004795:Enzyme Stability / Q000502:physiology; D006540:Herbicides / Q000494:pharmacology; D006900:Hydroxylation / Q000187:drug effects; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000378:metabolism; D009570:Nitriles / Q000378:metabolism*; D010944:Plants / Q000201:enzymology; D006109:Poaceae / Q000201:enzymology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D006540:Herbicides; D009097:Multienzyme Complexes; D009570:Nitriles; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C089817:cytochrome P450TYR; C110889:cytochrome P-450 CYP71E1 (sorghum); C011220:dhurrin