Listeria monocytogenes (Lmo) is a pathogenic bacterium responsible for food-borne illness. The bacterium uses the key enzyme, ribonucleotide reductase (RNR), to make deoxynucleotides, the building blocks of DNA, and to reproduce inside the host. The Lmo RNR comprises two homodimeric subunits, α2 and β2. α2 has a catalytic site that reduces nucleotides (NDPs) to deoxynucleotides (dNDPs) and β2 houses a metal cofactor that is essential for RNR activity. Lmo RNR has been previously suggested to utilize a diferric metal cofactor, but sequence similarity network (SSN) analysis and genetic context suggest that iron may not be the biologically relevant cofactor. In this project, proteins homologous to the Lmo β2 from five organisms were expressed and purified to understand general features of the Lmo SSN cluster. These proteins were purified by anion exchange and size exclusion chromatography, and inductively coupled plasma mass spectroscopy (ICP-MS) was conducted to determine the metal composition of the proteins as purified from the cell. Out of the five homologs, two did not purify with our methods and new approaches should be taken for these homologs. For the three purified homologs, unexpected differences in metal loading were observed.

Purification of homologs of Listeria monocytogenes ribonucleotide reductase

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