Catalytic activity and expression of two flavin‐containing monooxygenases from Drosophila melanogaster

Abstract

Two flavin-containing monooxygenase genes occur in the Drosophila genome (named DmFMO-1 and DmFMO-2). Differences exist between these two FMOs in: (1) genomic DNA architecture and predicted post-translational modifications; (2) recombinant protein solubility, activity, and absorbance spectra; and (3) subcellular distribution and developmental transcription/translation profiles in wildtype flies. Characteristic FAD absorbance spectra and strong catalytic competence in methimazole sulfoxidation were observed for recombinant DmFMO-2. Alternatively, weak sulfoxidation was observed for DmFMO-1, which correlated with reduced solubility in the recombinant system. Western blot analyses using specific antisera raised to each FMO showed the two FMOs to be immunologically distinct. In addition, Western blot analyses revealed FMO protein expression in both the microsomal and cytosolic sub-cellular fractions. Interestingly, a larger form of DmFMO-1 occurs in the cytosol that is most strongly expressed in the adult head. These findings suggest divergent physiological roles for DmFMO-1 and DmFMO-2. More specifically, it appears that DmFMO-1 has a distinct developmental role, while DmFMO-2 may have a general housekeeping function.

Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

“…Positive colonies with sense inserts were identified by colony PCR using the vector T7 and Cell-1 reverse primers noted above. Recombinant proteins with C-terminal histidine tags were produced with selective culturing and IPTG induction at various temperatures (see Results), following manufacturer protocols (Novagen), as described previously (Scharf et al, 2004).…”

Two hexamerin genes from the termite Reticulitermes flavipes: Sequence, expression, and proposed functions in caste regulation

“…The membrane was blocked in PBST (PBS containing 0.1% Tween-20) + 15 mg/ml non-fat dry milk, cut vertically into 16 equal-sized strips, and then re-placed into blocking solution. Four treatments were compared: (i) anti-JH alone, (ii) anti-JH + competing JH III (Sigma), (iii) no primary antibody, and (iv) antibody raised in rabbits to an unrelated protein (Drosophila FMO-2) (Scharf et al, 2004). All incubations were in 1-ml volumes; in individual 15-ml screw cap plastic vials.…”

Flavin-Dependent Monooxygenases as a Detoxification Mechanism in Insects: New Insights from the Arctiids (Lepidoptera)

“…CYP enzymes play the dominant role in drug and xenobiotic metabolism in insects [11], possibly compensating the need for FMOs in these processes. Of note, the genome of Drosophila melanogaster contains only two genes for FMOs [1], [12] but 90 genes for CYPs, of which 86 seem to be functional [13]. The large number of CYPs in insect genomes has been suggested to be necessary to protect the insect from the diverse array of harmful compounds in its environment [14].…”