Biochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.

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TitleBiochemical and structural characterization of the secreted chorismate mutase (Rv1885c) from Mycobacterium tuberculosis H37Rv: an *AroQ enzyme not regulated by the aromatic amino acids.
Publication TypeJournal Article
Year of Publication2006
AuthorsKim, S-K, Reddy, SK, Nelson, BC, Vasquez, GB, Davis, A, Howard, AJ, Patterson, S, Gilliland, GL, Ladner, JE, Reddy, PT
JournalJ Bacteriol
Volume188
Issue24
Pagination8638-48
Date Published2006 Dec
ISSN0021-9193
KeywordsAmino Acids, Aromatic, Catalytic Domain, Chorismate Mutase, Gene Expression Regulation, Bacterial, Humans, Kinetics, Models, Molecular, Mutagenesis, Site-Directed, Mycobacterium tuberculosis
Abstract

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.

DOI10.1128/JB.00441-06
Alternate JournalJ. Bacteriol.
PubMed ID17146044
PubMed Central IDPMC1698256