Biochemical characterization of the Mycobacterium smegmatis rifampicin ADP-ribosylating enzyme ARR

Letseka, Helen Susannah
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ARR is an antibiotic resistance protein found in Mycobacterium smegmatis with several homologues in other species and genera. ARR inactivates rifampicin through the transfer of an ADP-ribose group from NAD+ to C23 of rifampicin. This work aimed to characterize ARR both biochemically and functionally. ARR was heterologously expressed and purified to a homogenous level. Using far-UV circular dichroism it was found that ARR has a mixed alpha helical and beta sheet secondary structure. It was also shown that ARR retains a high proportion of this structure after heating from 10 °C to 85 °C. Fluorescence spectroscopy showed the two tryptophans (W59 and W107) in ARR to be solvent exposed. These findings are congruous with the crystal structure of ARR. Study of cell wall morphology revealed that expression of ARR led to increased cell length in E. coli and branching in M. smegmatis. We suggest other possible targets for ARR in M. smegmatis based on the bld pathway in S. coelicolor which involves an ADP-ribosyltransfer reaction leading to changes in cell wall morphology. These targets are extracellular binding protein, sugar binding lipoprotein and D-xylose binding periplasmic protein. These targets suggest a role for ARR in nutrient sensing and stress response.