Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H 37 R v : an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids

Abstract

ABSTRACT The gene Rv1885c from the genome of Mycobacterium tuberculosis H 37 R v encodes a monofunctional and secreted chorismate mutase (*MtCM) with a 33-amino-acid cleavable signal sequence; hence, it belongs to the *AroQ class of chorismate mutases. Consistent with the heterologously expressed *MtCM having periplasmic destination in Escherichia coli and the absence of a discrete periplasmic compartment in M. tuberculosis , we show here that *MtCM secretes into the culture filtrate of M. tuberculosis . *MtCM functions as a homodimer and exhibits a dimeric state of the protein at a concentration as low as 5 nM. *MtCM exhibits simple Michaelis-Menten kinetics with a K m of 0.5 ± 0.05 mM and a k cat of 60 s −1 per active site (at 37°C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 Å 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 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.