Structural and Biochemical Characterization of a Novel Regulatory Site of Bacillus Subtilis Class IB Ribonucleotide Reductase

Abstract

Ribonucleotide reductase (RNR) is an enzyme critical to DNA replication and repair in all organisms. Recently, it was shown that the class Ib RNR from Bacillus subtilis is subject to a form of allosteric activity regulation that had previously only been observed in RNRs containing a structural motif known as the ATP-cone domain. Interestingly, this regulation appeared to involve an adenine-containing nucleotide that co-purifies with one of the enzyme subunits. In this thesis, I apply biochemical techniques including high-resolution mass spectrometry, isothermal titration calorimetry, and high-pressure liquid chromatography along with structural techniques such as small-angle X-ray scattering and X-ray crystallography as part of an integrated workflow in order to determine the relevance of tightly-bound nucleotides to the activity regulation of B. subtilis RNR. I show that this nucleotide is a mixture of deoxyadenosine 5'-monophosphate (dAMP) with a small amount of the diphosphate and show that dAMP binding is competitive with the binding of both deoxyadenosine 5'-triphosphate (dATP), a known inhibitor, and adenosine 5'-triphosphate (ATP), a known activator. I demonstrate that both dAMP and dATP binding are weakened by a mutation to a residue located within a proposed binding site, thus providing support for an earlier dAMP-bound crystal structure that was initially not thought to be biologically relevant. Additionally, co-crystallization of the enzyme with ATP reveals disruption of a dimeric interface that is present in the dAMP-bound crystal structure. From these results, I conclude that dAMP may play a significant biological role in the activity regulation of B. subtilis class Ib RNR and that the dAMP binding site is also a site of dATP and perhaps ATP binding, identifying it as the likely site at which overall enzyme activity is regulated.