EARLY STEPS IN ECDYSTEROID BIOSYNTHESIS - EVIDENCE FOR THE INVOLVEMENT OF CYTOCHROME-P-450 ENZYMES

The first step in the biosynthesis of ecdysteroids by Manduca sexta pr othoracic glands, the conversion of cholesterol to 7-dehydrocholestero l, is mediated by an enzyme with characteristics of a microsomal cytoc hrome P-450, i.e. sensitivity to CO and fenarimol, and a requirement f or NADPH. The enzyme responsible for hydroxylation at C-25 of the puta tive 3-dehydroecdysone precursor, 14-hydroxy-5beta-cholest-7-en-3,6-di one, is also microsomal, while those mediating hydroxylations at C-22 and C-2 of 3,14,25-trihydroxy-5beta-cholest-7-en-6-one are mitochondri al. Indirect evidence revealed that the steps between 7-dehydrocholest erol and the trideoxyecdysteroids occur in the mitochondria, suggestin g that extensive shuttling of intermediates between the endoplasmic re ticulum and mitochondria takes place in the prothoracic gland cell dur ing ecdysteroid biosynthesis. During the fifth larval instar, choleste rol 7,8-dehydrogenase activity is evident from days 2 to 9, while the conversion to [H-3]ecdysteroids is not significant prior to the ecdyst eroid commitment peak on day 4. Terminal hydroxylase activity shows li ttle change throughout the instar. These data support the hypothesis t hat regulation of the biosynthetic pathway by PTTH occurs at the step immediately following the formation of 7-dehydrocholesterol. The stero id biosynthesis inhibitor, fenarimol, has been shown to inhibit each o f these P-450 enzymes, as well as fat body ecdysone 20-monooxygenase, with an 1, of 10(-4) M in disrupted glands, suggesting that it is a ge neral P-450 inhibitor. The secretion of ecdysteroids by the glands in vitro is very sensitive to fenarimol, i.e. I50 of 10(-6) M. RH5849, 1, 2-dibenzoyl-1-tert-butylhydrazine, fails to inhibit any of these proth oracic gland reactions, yet strongly inhibits fat body ecdysone 20-mon ooxygenase activity. This suggests that RH5849 is a specific ecdystero id substrate/product mimic in this reaction.