Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis [H.sub.37][R.sub.v]: an [sup.*]AroQ enzyme not regulated by the aromatic amino acids

The gene Rv1885c from the genome of Mycobacterium tuberculosis [H.sub.37][R.sub.v] encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the [sup.*]AroQ class of chorismate mutases. Consistent with the heterologously expressed [sup.*]MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis, we show here that [sup.*]MtCM secretes into the culture filtrate of M. tuberculosis. [sup.*]MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. [sup.*]MtCM exhibits simple Michaelis-Menten kinetics with a [K.sub.m] of 0.5 [+ or -] 0.05 mM and a [k.sub.cat] of 60 [s.sup.-1] per active site (at 37[degrees]C and pH 7.5). The crystal structure of [sup.*]MtCM has been determined at 1.7 [Angstrom] resolution (Protein Data Bank identifier 2F6L). 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.sub.134]. We provide evidence by site-directed mutagenesis that the residues [Arg.sub.49], [Lys.sub.60], [Arg.sub.72], [Thr.sub.105], [Glu.sub.109], and [Arg.sub.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 [sup.*]MtCM shows that [sup.*]MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of [sup.*]MtCM does not have an allosteric regulatory site.