Enzymology of Alpha-Ketoglutarate-Dependent Hydroxylases

Our overarching goals are to characterize the detailed chemical mechanism of the Fe2+/alpha-ketoglutarate (aKG)-dependent hydroxylases and to further explore the versatility of this enzyme family. The specific objectives planned during the next five year period include: a. Maintain our efforts to characterize the best-studied member of this enzyme family, TauD, and its enzyme variants by using a wide range of biophysical spectroscopic and kinetic approaches. b. Carry out analogous spectroscopic and kinetic studies with one or more alternative family members to discern the commonality of catalytic intermediates. Of special interest is XanA, where the site undergoing hydroxylation is somewhat activated compared to other examples; it is possible that rather than the Fe(IV)-oxo intermediate, a less potent oxidizing species (e.g., Fe(III)-superoxo or Fe(IV)-peroxo) is adequate for catalyzing this reaction. c. Continue studies to characterize the mechanism of base J formation in trypanosomes. This might include studies following up our earlier J-binding protein efforts, but initially will focus on characterization of two gene products that are related in sequence to thymine 7-hydroxylase. d. Extend efforts to characterize the functions and properties of ABH1 and possibly ABH4-ABH8. Although we have shown that ABH1 has endonuclease activity at abasic sites, the function of this activity must still be unraveled. e. Hypothetical functions will be tested for the known family members CsiD of Escherichia coli, the TfdA-like protein from Bordetella pertusis, and other related proteins from animals, plants, and bacteria. We have created clones of the first two genes, overproduced those proteins, and tested some initial ideas already. The versatility of this vast enzyme family is only beginning to be explored and this work will add to it.