Two ORFs Rv1885c and Rv0948c from the genome of Mycobacterium tuberculsosis H37Rv were identified as putative chorismate mutases. We reported earlier that Rv1885c is indeed a chorismate mutase (CM) with a 33 amino acid cleavable signal sequence in a heterologous E. coli expression system and thus is an extracytoplasmic protein (American Society of Microbiology Biodefense Meeting 2004, Abstract No:173, www.asmbiodefense.org
). The Rv1885c CM mature protein, termed hereafter MtCM1, was purified from the periplasmic fluid of E. coli. By mass spectral analysis and amino terminal sequence analysis of MtCM1 mature protein, the signal peptidase sequence site was confirmed to be between Ala33 and Asp34. MtCM1 has only chorismate mutase activity with no associated prephenate dehydrogenase or prephenate dehydratase activity and hence is a monofunctional enzyme and belongs to the aroQ subclass of CMs. MtCM1 is a highly active enzyme, functions as a dimer, and the minimal protein concentration for a fully functional dimer was calculated to be 25 nmolar. MtCM1 is not allosterically regulated by the aromatic amino acids. Based on the homology alignment with other CMs and by site-directed mutagenesis, we have identified Arg49, Lys60, and Arg72 as catalytic amino acid residues, since a point mutation of these residues to alanine resulted in complete loss of activity. The Rv0948c gene product is a cytoplasmic chorismate mutase (MtCM2), also functions as a dimer. MtCM2 has two logs lower catalytic efficiency compared to MtCM1. Both MtCM1 and MtCM2 exhibit simple Michaelis-Menten kinetics with a Km of 0.5 mM and a Vmax of 110 moles of phenylpyruvate/min/mg protein for MtCM1 and a Km of 5.0 mM with a Vmax of 0.9 moles of phenylpyruvate /min/mg protein for MtCM2. Consistent with the translocation of heterologously expressed MtCM1 from the cytoplasmic to periplasmic compartment in E. coli and the absence of a discrete peripalsmic compartment in M. tubercuclosis, MtCM1 was found in the culture filtrate of M. tubercuclosis. The rational for the human pathogen M. tuberculosis to have two monofunctional CMs, one highly active extracellular enzyme and the other very low active intracellular enzyme, is not obvious.