Enzymic Synthesis of Juvenile Hormone in Locust Corpora Allata: Evidence for a Microsomal Cytochrome P‐450 Linked Methyl Farnesoate Epoxidase

Abstract

Homogenates of corpora allata from adult Locusta migratoria in phosphate‐buffered EDTA have been analysed by sucrose‐density‐gradient centrifugation. Succinate‐cytochrome c reductase activity (mitochondrial) bands between d204 1.13–1.15, whereas NADPH‐cytochrome c reductase and NADPH–dependent methyl farnesoate 10, 11‐epoxidase activities band identically between d204 1.06–1.12. We conclude that the methyl farnesoate epoxidase is exclusively microsomal. Farnesoic acid O‐methyltransferase is an exclusively soluble enzyme which stoichiometrically transfers the S‐methyl group from S‐adenosylmethionine to farnesoic acid. No carboxyl esterase activity was found. Isolated microsomes were used to obtain an apparent Km= 7.7 × 10−6 M for the epoxidase, although substrate solubility limits the rate to 0.5 V As expected, the product (juvenile hormone III) is chiral (10 R). The epoxidase is inhibited by excess NADP+ and oxidised cytochrome c, but neither inhibited nor synergised by NADH. NADH supports less than 10% of the NADPH rate of epoxidation. The epoxidase is inhibited by a carbon monoxide/oxygen atmosphere, half‐maximal inhibition occurring at a CO/O2 ratio of 4.0. This inhibition is reversed by white‐light irradiation.

Juvenile hormone acid methyltransferase: A key regulatory enzyme for insect metamorphosis

“…Although the occurrence of JHAMT in the CA has long been recognized by radiochemical assays (23,24), neither the protein nor its gene has been isolated from any insect. In this study, we have isolated by FDD a cDNA that encodes an SAM-dependent MTase from the CA of B. mori.…”

CYP15A1, the cytochrome P450 that catalyzes epoxidation of methyl farnesoate to juvenile hormone III in cockroach corpora allata

“…These differences are most likely due to the differences in P450 reductase interaction with the P450 in the two different reconstitution assays and͞or to the lipophilic nature of the substrate. The critical micelle concentration of methyl farnesoate is Ϸ10 M (18), favoring rapid partition into the membrane lipid bilayer, and this lipophilicity has hampered kinetic determinations before (9). Although we have not pursued an optimization of the reconstitution conditions in this study, the values obtained for the enzyme's velocity and affinity for its substrate are consistent with the physiological function of CYP15A1.…”

“…Its catalytic activity is supported by NADPH and house fly NADPH cytochrome P450 reductase. The microsomal P450 nature of the enzyme had been demonstrated in Blaberus giganteus and Locusta migratoria (8,9).…”

“…Less is known about the epoxidase, reported to be a microsomal cytochrome P450 enzyme in cockroach and locust CA (8,9). Although cytochrome P450 enzymes are ubiquitous and share common catalytic mechanisms, they are the products of a large family of genes (10) that can be highly divergent in sequence.…”

Comparative genomics of insect juvenile hormone biosynthesis☆

“…In contrast, the heterologously expressed crustacean cDNA has only traces of OMT activity, and moreover, does not contain a characteristic AdoMet binding site (Gunawardene et al, 2002). Yet esterification of the farnesoic acid carboxylic group is clearly dependent in a stoichiometric manner on AdoMet (Feyereisen et al, 1981). The exact function of the "crustacean OMT" thus remains to be discovered.…”