Abstract
Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.
Highlights
Occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins
In the course of this study, we came across several unique properties of Mycobacterium tuberculosis (Mtb) chorismate mutase that are important in the context of differentiating the enzyme from the bacterial and fungal counterparts
The first and foremost observation with respect to the regulation of Mtb chorismate mutase by aromatic amino acids revealed an unusual property of the enzyme
Summary
Mycobacterium tuberculosis; ORF, open reading frame; ss, signal sequence; aa, amino acid(s); XP, 5-bromo4-chloro-3-indolyl phosphate; CPB, citrate phosphate buffer; MES, 4-morpholineethanesulfonic acid; CM, chorismate mutase; rRv1885c, sented by the host and to acquire essential nutrients from this adverse environment (1–3). For the development of new therapeutic intervention strategies, there is a need for identification of novel targets that are unique to Mtb but blocking of which would either prove lethal to the bacterium or render it extremely susceptible to the host immune response In this context, understanding the mechanism of action of the aromatic amino acid pathway enzymes of Mtb assumes the utmost importance because most of the corresponding genes have been proven essential for the bacterium and have no human or mammalian counterpart (7, 8). The low similarity at the amino acid sequence level among known chorismate mutases makes the enzyme one of the finest examples of convergent evolution of enzyme reaction mechanisms (24). Our study provides sufficient evidence to conclusively place the protein coded by ORF Rv1885c of Mtb in the AroQ class of periplasmic chorismate mutases
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