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Journal of Bacteriology, December 2006, p. 8638-8648, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.00441-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Biochemical and Structural Characterization of the Secreted Chorismate Mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ Enzyme Not Regulated by the Aromatic Amino Acids

Sook-Kyung Kim,1 Sathyavelu K. Reddy,1,{dagger} Bryant C. Nelson,2 Gregory B. Vasquez,1 Andrew Davis,1 Andrew J. Howard,1,{ddagger} Sean Patterson,1 Gary L. Gilliland,1,§ Jane E. Ladner,1 and Prasad T. Reddy1*

Biochemical Science Division,1 Analytical Chemistry Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland 208992

Received 30 March 2006/ Accepted 24 July 2006

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 kcat 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 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.

* Corresponding author. Mailing address: National Institute of Standards and Technology, Mail stop 831.2, Bldg. 227, Room B244, Gaithersburg, MD 20899. Phone: (301) 975-4871. Fax: (301) 975-5449. E-mail: prasad.reddy{at}nist.gov.

{dagger} Present address: Department of Zoology, Sri Venkateswara University, Tirupati, Andhra Pradesh, India.

{ddagger} Present address: Illinois Institute of Technology, Chicago, IL 60616.

§ Present address: Centocor, Inc., 145 King of Prussia Rd., Radnor, PA 10987.

Journal of Bacteriology, December 2006, p. 8638-8648, Vol. 188, No. 24
0021-9193/06/$08.00+0     doi:10.1128/JB.00441-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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