Biochemical characterization of the Mycobacterium smegmatis rifampicin ADP-ribosylating enzyme ARR
Date
2011-03-30
Authors
Letseka, Helen Susannah
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Abstract
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.