Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis.
Mycobacterium tuberculosis has innate resistance to a range of broad-spectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the M. tuberculosis MurA enzyme might confer resistance. In Escherichia coli, fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the M. tuberculosis MurA. In the present study, it is demonstrated that the wild-type M. tuberculosis MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an M. tuberculosis enzyme that is unstable during constitutive expression.
De Smet
Koen A L
KAL
Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
Kempsell
Karen E
KE
Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage SG1 2NY, UK2.
Gallagher
Alex
A
Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
Duncan
Ken
K
Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage SG1 2NY, UK2.
Young
Douglas B
DB
Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
D019883:Alkyl and Aryl Transferases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004352:Drug Resistance, Microbial / Q000235:genetics; D005578:Fosfomycin / Q000494:pharmacology*; D005798:Genes, Bacterial; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D005578:Fosfomycin; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase