DIFFERENTIAL REGULATION OF FE(II)- AND 2-OXOGLUTARATE DEPENDENT DIOXYGENASES BY COFACTOR ASCORBATE

Abstract

The Fe(II)- and 2-oxoglutarate-dependent dioxygenase (FODD) enzyme superfamily is responsible for the oxidation of RNA, DNA, and peptide substrates in countless biological pathways. The necessity for cofactor ascorbate in some FODD catalysis has been known for over 50 years, but its usage across the family and even its putative role as a reducing agent have remained nebulous and understudied. Herein, we use model RNA demethylases FTO and AlkBH5 to show that ascorbate dependence can vary greatly, even in very similar enzymes and reactions. Indeed, ascorbate requirements can even change when a single enzyme is acting on different substrate species. Our findings allow us to propose how ascorbate dependence can relate to catalytic efficiency between enzyme and substrate, but also to an enzyme’s ability to properly couple the main oxidation reaction to the secondary decarboxylation of 2-OG. Driven by our data showing ascorbate’s specific reducing capabilities, we determined the first structure of an FODD bound with ascorbate in its active site, revealing coordination lead by the metal center, but also nearby Asn205. Together, our data suggests that ascorbate could selectively activate hydroxylation by RNA demethylases and, more globally, tune oxidation events in different cell-types and disease states from all members of the FODD superfamily of enzymes.