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

Abstract

The molecular analysis of insect hormone biosynthesis has long been hampered by the minute size of the endocrine glands producing them. Expressed sequence tags from the corpora allata of the cockroach Diploptera punctata yielded a new cytochrome P450, CYP15A1. Its full-length cDNA encoded a 493-aa protein that has only 34% amino acid identity with CYP4C7, a terpenoid omega-hydroxylase previously cloned from this tissue. Heterologous expression of the cDNA in Escherichia coli produced >300 nmol of CYP15A1 per liter of culture. After purification, its catalytic activity was reconstituted by using phospholipids and house fly P450 reductase. CYP15A1 metabolizes methyl (2E,6E)-3,7,11-trimethyl-2,6-dodecatrienoate (methyl farnesoate) to methyl (2E,6E)-(10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate [juvenile hormone III, JH III] with a turnover of 3-5 nmol/min/nmol P450. The enzyme produces JH III with a ratio of approximately 98:2 in favor of the natural (10R)-epoxide enantiomer. This result is in contrast to other insect P450s, such as CYP6A1, that epoxidize methyl farnesoate with lower regio- and stereoselectivity. RT-PCR experiments show that the CYP15A1 gene is expressed selectively in the corpora allata of D. punctata, at the time of maximal JH production by the glands. We thus report the cloning and functional expression of a gene involved in an insect-specific step of juvenile hormone biosynthesis. Heterologously expressed CYP15A1 from D. punctata or its ortholog from economically important species may be useful in the design and screening of selective insect control agents.

Fig. 1.

SDS/PAGE analysis of recombinant CYP15A1 produced in E. coli. (A) Membrane fraction. (B) CYP15A1 after purification on a nickel-nitrilotriacetic acid column. The migration distance of molecular weight (MW) markers (Bio-Rad) is indicated (MW × 10^–3).

Fig. 2.

Conversion of methyl (2E,6E) farnesoate to JH III by CYP15A1. Molecular ion and fragmentation patterns observed by mass spectrometry are indicated.

Fig. 3.

Chiral HPLC elution profile of the CYP15A1 metabolite (dotted line) and of the racemic synthetic methyl (2E,6E)-10,11 epoxy-farnesoate (solid line). The 10R enantiomer is the faster eluting compound.

Fig. 4.

Comparative expression in the CA (day 5, CA5; day 7, CA7), fat body (FB), and brain (BR) of CYP15A1 (1,490 bp), CYP4C7 (781 bp), and CYP9E1 (645 bp) by RT-PCR. The lower migrating band (301 bp) is an amplicon of actin serving as control.

Fig. 5.

CYP15A1 and related sequences. Sequences were aligned by clustal x, and a tree was generated by phylip protpars. Species abbreviations are as follows: Dpu, D. punctata; Aga, A. gambiae; Bmo, B. mori; Dme, D. melanogaster; Has, Homo sapiens; Mdo, Musca domestica; and Bdi, Blaberus discoidalis.