Screening the Mycobacterium tuberculosisMycobacterium tuberculosis H37Rv, the etiologic agent of tuberculosis, accounts for more human causalities than any other single infection (1). Pathogenesis of M. tuberculosis is not attributable to any single gene product. In other bacterial pathogens such as Bordetella pertussis and Bacillus anthrax, adenylyl cyclase, the enzyme responsible for the synthesis of adenosine 3Ј,5Ј-monophosphate (cAMP), plays a pivotal role in pathogenesis (2). The secreted form of adenylyl cyclase from B. pertussis (3) and B. anthrax (4) invades a variety of eukaryotic cells and is activated by the intracellular calmodulin of the host. This results in the unregulated conversion of ATP to a supraphysiological concentration of cAMP, which in turn has a profound effect on the metabolism and immune function of the host cell (5, 6). Thus, in infections caused by B. pertussis and B. anthrax, adenylyl cyclase plays a critical role in the onset of the disease. To examine whether adenylyl cyclase in M. tuberculosis has any role in infection, we report in this paper the cloning of cya gene from M. tuberculosis H37Rv, heterologous expression in Escherichia coli, and characterization of adenylyl cyclase (Mtb AC, 1

The Rv0948c gene from Mycobacterium tuberculosis H37Rv encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90‐MtCM, exhibits Michaelis–Menten kinetics with a kcat of 5.5 ± 0.2 s−1 and a Km of 1500 ± 100 μm at 37 °C and pH 7.5. The 2.0 Å X‐ray structure shows that 90‐MtCM is an all α‐helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C‐terminus helix 3 is shortened. The absence of two residues in the active site of 90‐MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low kcat. Hence, 90‐MtCM belongs to a subfamily of α‐helical AroQ CMs termed AroQδ. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N‐terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis–Menten kinetics with a kcat of 70 ± 5 s−1 and Km of 500 ± 50 μm at 37 °C and pH 7.5. The 2.1 Å X‐ray structure shows that *YpCM is an all α‐helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQγ class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.

The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg 134 . We provide evidence by site-directed mutagenesis that the residues Arg 49 , Lys 60 , Arg 72 , Thr 105 , Glu 109 , and Arg 134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

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