Year: 2026
Type: dissertation
Abstract: AlkBH4 is an Fe(II)/2-oxoglutarate-dependent dioxygenase enzyme. It uses cofactors Fe(II), 2-oxoglutarate (2-OG), and oxygen to oxidize different putative substrates, such as DNA, RNA, and proteins. It plays important roles in DNA replication and embryonic development and has been linked to non-small-cell lung cancers, breast cancer, and hypoxic tumors. AlkBH4’s prime substrate is currently unknown. It was recently suggested to catalyze the hydroxylation of mcm5U to (R)-mchm5U on tRNAs and has been shown to demethylate N6-methyladenosine (6mA) to adenosine (A) on double-stranded DNA (dsDNA); however, none of its proposed substrates has been carefully validated with in vitro biochemistry and recombinant enzyme. We aimed to express and purify AlkBH4 and produce various tRNA and DNA substrates to investigate AlkBH4’s enzymatic activity and determine possible nucleic acid substrates for AlkBH4 through in vitro biochemical experiments. AlkBH4 was overexpressed in E. coli BL21 cells and purified. Binding assays with putative substrates 6mA-containing DNA and unmodified tRNA and activity assays with 6mA-containing DNA and mcm5U tRNA were performed. These results indicate that 6mA DNA is not a substrate for AlkBH4. However, tRNA(Arg)UCU and tRNA(Arg)UCG successfully bound AlkBH4 with binding affinities of 3.389±0.151μM and 2.442±0.062μM, respectively. AlkBH4 also appears to convert mcm5U to a currently unidentified product in activity assays, suggesting mcm5U-modified tRNA is indeed a substrate for AlkBH4.
Author: Driscoll, Cassidy M.
Language: English
Subfield: Inorganic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7219356155