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Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.
The steroid hormone 20-hydroxyecdysone, which is produced from cholesterol via a series of oxidation steps, is the physiologically active molting hormone that controls insect development. The final step of its biosynthesis, 20-hydroxylation of ecdysone, has been reported to occur in the microsomal or mitochondrial fractions of fat bodies, Malpighian tubules and midguts of insects at certain developmental stages . The silkworm Bombyx mori has two types of egg in terms of developmental fates [2,3]. Embryonic development in the diapause egg stops at the gastrulation stage, whereas the nondiapause egg continues to develop, producing a larva on the 11th day after oviposition. We previously reported that the content of 20-hydroxyecdysone in the diapause egg remains low, whereas that in the nondiapause egg increases significantly during development [4–6], and suggested that characterization of the ecdysone 20-hydroxylation reaction would be important in understanding silkworm embryogenesis . Our subsequent studies have demonstrated that most of the 20-hydroxylation activity in the three-day-old nondiapause egg is associated with the microsomes , suggesting that the microsomal cytochrome P450-dependent hydroxylation system is involved in this metabolism.
NADPH-cytochrome P450 oxidoreductase (P450 reductase), a component of the microsomal P450 electron transport system, plays an essential role in the transfer of reducing equivalents from NADPH to various P450 molecules . The P450 reductase in insects was first purified from the housefly Musca domestica, and was shown to participate in P450-dependent drug metabolism . Antisera raised against the enzyme were used for isolating a cDNA from the abdominal tissue of phenobarbital-treated flies [12,13]. The role of P450 reductase involved in the ecdysteroid biosynthesis, however, has not been well understood. Here we report the isolation of a cDNA encoding P450 reductase from developing silkworm eggs, and discuss its physiological relevance to the expression of ecdysone 20-hydroxylation during embryonic development.
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Although more than 50 genes for insect cytochrome P450 have been reported to date, the physiological function of the gene products remains to be identified except for potential detoxification of xenobiotics. As to the genes involved in metabolism of ecdysteroids, important regulators of major developmental process in arthropods, only two P450 genes, CYP18 cloned from Drosophila and CYP6H1 cloned from Locusta migratoria, have been putatively related to ecdysteroid catabolism (26-hydroxylation) [19,20].
In silkworm eggs, ecdysone 20-hydroxylation activity was associated with the microsomes , suggesting the involvement of the microsomal P450 system in this activity. All the microsomal P450-mediated hydroxylation reactions are dependent upon the presence of the P450 reductase that supplies reducing equivalents from NADPH to the P450. Although several insect P450 reductase genes have been reported [10,21], very little is known about their physiological function. Molecular cloning of P450 reductase and production of its antibody would help us to understand better the role of P450s in insect physiology. In the present study, we described the molecular cloning and expression of P450 reductase cDNA and its involvement in ecdysone 20-hydroxylation during silkworm embryogenesis.
The deduced amino-acid sequence of silkworm P450 reductase shows several domains highly conserved among the enzymes of various species (Fig. 1). The domains indicated in the figure have been accounted for by the binding sites for flavin and NADPH molecular moieties. The extent of amino-acid identity within these domains was more than 80%, higher than that of overall structures. The recently reported three-dimensional structure of rat P450 reductase clearly showed the importance of several amino-acid residues conserved in these domains . The residues of particular interest are indicated in the figure. Y149 and Y187 are involved in FMN binding. The mutagenesis study of rat enzyme has shown that the former is necessary for efficient electron transfer whereas the latter is essential for FMN binding . The isoalloxazine ring of FMN is covered from both sides by the phenolic rings of tyrosines Y149 at the re-side and Y187 at the si-side. The re-side of the FAD ring is stacked with the W688 indole ring, and its si-side with Y467 . The N-terminal hydrophobic domain is responsible for anchoring the enzyme to the membrane and its cleavage at W45 by trypsin abolishes the capacity of the enzyme to reduce P450 proteins in reconstituted systems.
NADPH-dependent cytochrome c-reducing activity of GST-P450 reductase was 27 ± 1.4 µmol·min−1·mg−1 protein. This specific activity was consistent with the reported activity of purified rat liver enzyme (48.1 µmol·min−1·mg protein−1), when the difference in molecular masses between the two enzymes was considered. The GST-fused silkworm P450 reductase could be reconstituted with bovine 21-hydroxylase P450. The NADPH-dependent steroid 21-hydroxylation occurred with the specific activity 1.75 ± 0.3 pmol·min−1·pmol P450−1, which was a little lower than that reported in the reconstituted system of bovine P450 reductase and 21-hydroxylase P450 (9.0 pmol·min−1·pmol−1 P450) .
By examining the effect of actinomycin D or α-amanitin on 20-hydroxyecdysone biosynthesis in the silkworm egg, we have shown previously that gene transcription was required for the induction of ecdysone 20-hydroxylase activity . Our present study of ecdysone 20-hydroxylation in silkworm egg microsomes suggests that the activity depends upon the presence of both ecdysone 20-hydroxylase P450 and P450 reductase, and changes during the embryogenesis. Therefore, gene transcription events for both enzymes may be required for advancing embryonic development. However, a possibility still remains that the change in the activity we report here is due to post-translational regulation of the enzymatic activity.
The immunoblot study of nondiapause egg microsomes in various developmental stages demonstrated that the content of P450 reductase protein continuously increased during development (Fig. 6). It was also noted that little P450 reductase protein was detected in diapause eggs in which embryogenesis was stopped. These results suggest that P450 reductase plays an important role in the regulation or activation of ecdysone 20-hydroxylation during the early phase of embryonic development. The finding that the microsomal ecdysone 20-hydroxylation activity in the nondiapause eggs reached maximum at a certain stage and then decreased despite the continued presence of the P450 reductase protein could be explained by the temporary expression of microsomal ecdysone 20-hydroxylase P450.
Taken together, the amino-acid sequence and the molecular properties of P450 reductase of silkworm and its involvement in the ecdysone 20-hydroxylation during the insect's embryonic development was for the first time demonstrated in this paper.