Characterization of the secreted chorismate mutase from the pathogen Mycobacterium tuberculosis.
The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM-deficient E. coli strain, but only if produced without the predicted N-terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis-Menten kinetics, having a k(cat) of 50 s(-1) and a K(m) of 180 microM (at 30 degrees C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size-exclusion chromatography. Secondary-structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all-alpha-helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram-positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.
Sasso
Severin
S
Laboratorium für Organische Chemie, Swiss Federal Institute of Technology, CH-8093 Zürich (ETH), Switzerland.
Ramakrishnan
Chandra
C
Gamper
Marianne
M
Hilvert
Donald
D
Kast
Peter
P
D000595:Amino Acid Sequence; D002826:Chorismate Mutase / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*
D002826:Chorismate Mutase