title: Alteration of a single amino acid residue reverses fosfomycin resistance of recombinant MurA from Mycobacterium tuberculosis.
abstract: 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.
authors.affiliation: Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
authors.forename: Koen A L
authors.initials: KAL
authors.lastname: De Smet
authors.affiliation: Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage SG1 2NY, UK2.
authors.forename: Karen E
authors.initials: KE
authors.lastname: Kempsell
authors.affiliation: Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
authors.forename: Alex
authors.initials: A
authors.lastname: Gallagher
authors.affiliation: Glaxo Wellcome Research and Development, Medicines Research Centre, Stevenage SG1 2NY, UK2.
authors.forename: Ken
authors.initials: K
authors.lastname: Duncan
authors.affiliation: Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, St Mary's Campus, Norfolk Place, London W2 1PG, UK1.
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Young
mesh_terms: 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
chemical_list: D000900:Anti-Bacterial Agents; D005578:Fosfomycin; D019883:Alkyl and Aryl Transferases; C022566:UDP-N-acetylglucosamine 1-carboxyvinyltransferase