Abstract
An apyrase (NTP/NDPase) implicated in the response of Mimosa pudica to stimuli, such as touch, has been cloned, sequenced and expressed in Escherichia coli. While purifying and characterizing this enzyme, it was observed that a chromophore is associated with it, having absorption in the ultraviolet-A/blue region of the spectrum. The absorbance maximum of the chromophore, purified from the enzyme complex by gel filtration and HPLC, is around 350 nm. The chromophore has been identified as N5,N10-methenyl tetrahydrofolate (MTHF) by comparing the excitation and emission spectra of synthetic MTHF and the isolated cofactor, and by reconstitution of the enzyme complex with synthetic MTHF. Upon excitation with light (350 nm), an increase of apyrase activity was observed in the purified or reconstituted holoenzyme but not in the apoenzyme. The wavelength dependence of the light stimulation matched well with the fluorescence excitation spectra of the cofactor, MTHF. Possible implications of the results for signal transduction in M. pudica have been discussed.
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