ISOLATION OF THE HEME-THIOLATE ENZYME CYTOCHROME P-450(TYR), WHICH CATALYZES THE COMMITTED STEP IN THE BIOSYNTHESIS OF THE CYANOGENIC GLUCOSIDE DHURRIN IN SORGHUM-BICOLOR (L) MOENCH

The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step-i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450(TYR) enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450(TYR) enzyme is estimated to constitute approximate to 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450(TYR) enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450(TYR) enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450(TYR) enzyme exhibits high, substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its abilty to separate cytochrome P-450(TYR) from the cytochrome P-450 enzyme catalyzing the C-hydroxylation of p-hydroxyphenylacetonitrile and from cinnamic acid 4-hydroxylase.