A Detoxification Enzyme for Apis mellifera Newly Characterized by Recombinant Expression: 10-Formyl Tetrahydrofolate Dehydrogenase

Abstract

Honeybees are important managed pollinators that perform important ecological and economic functions. In recent decades, the obligate ectoparasite Varroa destructor severely affected survival of honeybees as it either feeds on hemolymph and fat bodies or acts as a vector for viruses. A common treatment against the varroa mite is formic acid, which has been used for many years by beekeepers. This treatment is known to be effective, but the therapeutic index is very narrow. Many beekeepers report negative effects of formic acid on bees, which include damage to brood, worker bee mortality, and queen loss. Little is yet known about the molecular mechanisms of formic acid detoxification in honeybees. Our previous study shows the upregulation of predicted 10-formyl tetrahydrofolate dehydrogenase (10-FTHFDH) transcripts in honeybees exposed to formic acid. Here, the predicted honeybee-specific 10-FTHFDH is recombinantly expressed, and its hydrolase and dehydrogenase activities are investigated. As a result, the enzyme shows similar dehydrogenase activity in comparison to known 10-FTHFDHs. This study provides further knowledge to better understand the detoxification mechanisms of formic acid in Apis mellifera.

The Active Site of the Enzyme 10-Formyl-THFDH in the Honey Bee Apis mellifera—A Key Player in Formic Acid Detoxification

“…The dehydrogenase activity was measured by monitoring the increase in absorbance at 340 nm. The recombinantly expressed wildtype protein showed an enzyme activity with a Vmax of 0.1846 nM•min −1 to 0.4253 nM•min −1 at different pH and Km values of 2.455 µ M up to 9.596 µ M (according to previously published data [22]). The mutations lost their activity almost entirely, with values ranging between 0.007 nM•min −1 and 0.12 nM•min −1 for their Vmax values and between The superimposed structure of the AlphaFold2 model and the bound-form homology model differed in the loop position (Figure 3B).…”

“…On one hand, Tsybovsky and Krupenko [46] have already shown that the residues C707 and E677 are important for the degradation of 10-formyl tetrahydrofolate in Rattus norvegicus. However, on the other hand, the conservation of this enzyme between the two species A. mellifera and R. norvegicus is so low [22] that confirmation of the active site is necessary to make further functional statements. The expected molecular mode of action is as follows: first, glutamate E673 and cysteine C707 are hydrogen-bonded.…”

“…Even though this treatment has been used by beekeepers for decades, its mode of action and the molecular basis of detoxification in honey bees are mainly unknown. Two of our recent studies showed transcriptional upregulation of the mRNA of the enzyme 10-formyl tetrahydrofolate dehydrogenase (10-formyl-THFDH) in honey bees treated with formic acid [21] and the detoxification capability of the recombinantly expressed enzyme [22]. The most recent study also revealed that the enzyme itself is poorly conserved between Apis mellifera and the well-described mammalian orthologs.…”