Biochemical and structural studies of AlkB: A 2-oxoglutarate iron(II) dependent dioxygenase

In order to understand structurally the mechanisms underlying substrate specificity and DNA binding, the crystal structure of a D135A mutant enzyme was determined to 2.2 A in complex with a 17-mer oligonucleotide with a 1-methylguanine lesion bound to the active site. Also, an unliganded wild type AlkB structure was also determined to 2.9 A. Comparisons of the nucleotide bound structure with the unliganded structure demonstrated that Y76 participates in a "gating action" that opens the substrate binding pocket for the methylated base to enter and upon binding it seals the substrate in the catalytic complex. Additionally, the DNA binding loop expands ∼2.5 A once DNA binds to allow the phosphodiester backbone to rest in the DNA binding groove. Structural comparison of the 1-methylguanine structure with 1-methyladenine structures demonstrate that AlkB binds both lesions identically, and it supports the biochemical data that the lower efficiency of AlkB for 1-methylguanine substrates is due to the selectivity that D135 imposes on DNA bases when they enter the binding pocket.;Analysis of the current AlkB crystal structures permitted the development of mutagenesis strategies to investigate amino acid residues that may be involved in substrate binding and catalysis. In vitro activity and DNA binding assays were performed on each mutant. Data obtained from these experiments demonstrated that the substrate binding pocket in AlkB created by W69 and D135 is critical for maintaining the catalytic complex through stabilization of the base for catalysis to occur. Additionally, it was discovered that D135 holds the key to the specificity of this enzyme for DNA methylation damage on adenine and cytosine due to an exocyclic nitrogen present on these bases as opposed to guanine and thymine. In vitro activity assays with this mutant displayed no detectable activity on 1-methyladenine substrates, but its activity on 1-methylguanine substrates increased ∼40-fold in comparison to the wild type enzyme. Also, equilibrium binding data using methylated and undamaged oligonucleotides provided evidence for AlkB searching each base for DNA damage in ssDNA substrates. Significant reduction in activity and DNA binding using a Y76A and T51A mutation suggested these residues also participate in substrate stabilization and DNA base searching through Y76 hydrogen bonding to the phosphate of the methylated base and T51 stabilizing the DNA binding loop which forms extensive contacts to the phosphodiester backbone.;Recent studies have linked two human AlkB homologues, hABH8 and FTO, to human bladder cancer, growth and development, and obesity. Preliminary experiments have been performed on an AlkB homologue from Drosophila melanogaster (DmAlkB) that has 60% sequence identity to hABH8. This enzyme is unique in that it contains an AlkB domain and a methyltransferase domain. It is hypothesized that this enzyme may be involved in protein translation and regulation through modification of specific tRNAs. Nucleic acid precipitation assays suggest that DmAlkB primarily interacts with RNA. Additionally, the human FTO protein has been cloned and purified in the laboratory. Structural studies and biochemical studies are currently being carried out on these two enzymes. The data presented in this dissertation have provided valuable insight into the mechanisms of substrate specificity and DNA binding of the AlkB enzyme that can be applied to other studies on AlkB homologues and may assist in the development of inhibitors of these proteins to increase the efficacy of current chemotherapeutics. (Abstract shortened by UMI.)...