617-12-9Relevant articles and documents
A HR-MS based method for the determination of chorismate synthase activity
Khera, Harvinder K.,Singh, Susheel K.,Mir, Rafia,Bharadwaj, Vikram,Singh, Subhash
, p. 229 - 234 (2017/02/15)
Chorismate synthase (Cs) catalyzes the last step of Shikimate pathway involving a unique biochemical reaction of anti-1,4 elimination of 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate-3-phosphate (EPSP) leading to the formation of chorismate, which is the common precursor for aromatic amino acid, ubiquinone, and folate biosynthesis in plants and several bacterial, fungal, and parasitic pathogens. Absence of Shikimate pathway in the vertebrate host, make Cs an appealing target for drug discovery against these pathogens. Here, we report a new method for detection of chorismate through a specific liquid chromatography, coupled with negative electrospray ionization high-resolution tandem mass spectrometry (ESI-HRMS) for determination of Cs enzyme activity. For this, we used a coupled enzyme reaction consisting of purified recombinant MtbEPSPs (EPSP synthase from Mycobacterium tuberculosis) for biosynthesis of EPSP, which is the substrate for Chorismate synthase along with MtbCs (Chorismate synthase both from Mycobacterium tuberculosis) for the formation of chorismate, followed by its detection through LC/HRMS. Since, the reaction components of Cs enzyme activity assay which otherwise may interfere with the other known spectrophotometric methods of checking Cs enzyme activity have no effect on this LC/HRMS based method, this method offer advantages over other existing methods for detection of Cs activity. Further, this LC/HRMS based method could be applicable for detection of enzyme activity of both monofunctional and bifunctional Cs from different species irrespective of their specific requirements of anaerobic or aerobic reaction conditions.
Isotope effects on the enzymatic and nonenzymatic reactions of chorismate
Wright, S. Kirk,Declue, Michael S.,Mandal, Ajay,Lee, Lac,Wiest, Olaf,Cleland, W. Wallace,Hilvert, Donald
, p. 12957 - 12964 (2007/10/03)
The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4- 2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 °C. Moreover, a 1.846 ± 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 ± 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 ± 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 ± 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionary unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.
Spectrophotometric detection of a modified flavin mononucleotide (FMN) intermediate formed during the catalytic cycle of chorismate synthase
Ramjee, Manoj N.,Coggins, John R.,Hawkes, Timothy R.,Lowe, David J.,Thorneley, Roger N. F.
, p. 8566 - 8567 (2007/10/02)
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