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

J Bacteriol. 2006 Dec;188(24):8638-48. doi: 10.1128/JB.00441-06.

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.

MeSH terms

  • Amino Acids, Aromatic / pharmacology*
  • Catalytic Domain
  • Chorismate Mutase* / chemistry
  • Chorismate Mutase* / genetics
  • Chorismate Mutase* / metabolism
  • Gene Expression Regulation, Bacterial*
  • Humans
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mycobacterium tuberculosis / drug effects*
  • Mycobacterium tuberculosis / enzymology*
  • Mycobacterium tuberculosis / genetics

Substances

  • Amino Acids, Aromatic
  • Chorismate Mutase