Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.
The gene Rv1885c from the genome of Mycobacterium tuberculosis H37Rv 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 Km of 0.5 +/- 0.05 mM and a k(cat) of 60 s(-1) per active site (at 37 degrees C and pH 7.5). The crystal structure of *MtCM has been determined at 1.7 A 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 Arg134. We provide evidence by site-directed mutagenesis that the residues Arg49, Lys60, Arg72, Thr105, Glu109, and Arg134 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.
Kim
Sook-Kyung
SK
Biochemical Science Division, National Institute of Standards and Technology, Mail stop 831.2, Bldg. 227, Room B244, Gaithersburg, MD 20899, USA.
Reddy
Sathyavelu K
SK
Nelson
Bryant C
BC
Vasquez
Gregory B
GB
Davis
Andrew
A
Howard
Andrew J
AJ
Patterson
Sean
S
Gilliland
Gary L
GL
Ladner
Jane E
JE
Reddy
Prasad T
PT
D024322:Amino Acids, Aromatic / Q000494:pharmacology*; D020134:Catalytic Domain; D002826:Chorismate Mutase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics
D024322:Amino Acids, Aromatic; D002826:Chorismate Mutase