Characterization of four members of the ferrous ion and alpha-ketoglutarate dependent hydroxylase family from Trypanosoma brucei: Two thymine hydroxylase-like proteins, J-binding protein 1, and an ALKB homolog

Trypanosomes are eukaryotic parasites and the causative agents of several mammalian diseases. They are phylogenetically divergent making study of their biology pivotal to understanding their relationships to both 'lower' and 'higher' organisms. Trypanosomes contain many open reading frames predicted to encode proteins of the FeII/alpha-ketoglutarate (aKG) hydroxylase family including two thymine 7-hydroxylase-like proteins (TLP5 & TLP7), J-binding protein 1 (JBP1), and an AIkB homolog (TbABH).;TLP5 & TLP7 transcript levels are 4 and 2.5 fold higher in the bloodstream form over the procyclic form parasites, indicating a role for the respective proteins in the former life stage. Protein production was verified in both life stages by western blot with a polyclonal anti-TLP5 antibody and suggested that these proteins may undergo some posttranslational modification in vivo. Recombinant TLP5 & TLP7 were confirmed to be members of the FeII/alphaKG hydroxylase family based upon formation of a characteristic metal-to-ligand charge-transfer (MLCT) chromophore when FII and alphaKG were added to anaerobic samples. A homology model of TLP5 was created and a library of small molecules docked with this model, indicating a propensity of the protein to bind heterocyclic molecules resembling nucleotides. Unfortunately, no DNA binding or enzymatic activity was observed for these proteins, leaving their cellular function unknown.;JBP 1 is known to be involved in the hypermodification of thymine in nuclear DNA resulting in the formation of (3-D-glucosylhydroxymethyldeoxyuracil, or base J. The first step of base J synthesis involves hydroxylation of the CS methyl group of thymidine, a reaction type known to be catalyzed by the FII/alphaKG hydroxylases in other organisms. Recombinant JBP1 was confirmed to be a member of this enzyme family based on the characteristic MLCT chromophore formation. Unfortunately, no enzymatic turnover was observed by the in vitro methods employed leaving the direct detection of activity elusive.;TbABH is a homolog of AlkB from Escherichia coli, an enzyme that oxidatively repairs alkylated DNA, consistent with such a mechanism of DNA repair existing in trypanosomatids. Sequence analysis suggested that TbABH is a nuclear, DNA-binding protein, and electrophoretic mobility shift assays using recombinant TbABH demonstrated preferential binding to alkylated double-stranded DNA. Membership in the FII/alphaKG hydroxylase family was verified by MLCT chromophore formation, however, attempts to detect in vitro activity were unsuccessful. TbABH was shown to be capable of complementing an alkB mutant of E. coli subjected to alkylation stress. The complemented cell line was approximately 2-fold more resistant to alkylation-induced growth inhibition than the mutant, confirming the assignment of TbABH as a functional AlkB-like DNA repair protein in T. brucei.