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.2013 Mar 12;52(10):1765-75.
doi: 10.1021/bi400037w. Epub 2013 Feb 27.

Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from Mycobacterium tuberculosis

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Structural, kinetic and chemical mechanism of isocitrate dehydrogenase-1 from Mycobacterium tuberculosis

Christine E Quartararo et al. Biochemistry..

Abstract

Mycobacterium tuberculosis (Mtb) is the leading cause of death due to a bacterial infection. The success of the Mtb pathogen has largely been attributed to the nonreplicating, persistence phase of the life cycle, for which the glyoxylate shunt is required. In Escherichia coli, flux through the shunt is controlled by regulation of isocitrate dehydrogenase (ICDH). In Mtb, the mechanism of regulation is unknown, and currently, there is no mechanistic or structural information about ICDH. We optimized expression and purification to a yield sufficiently high to perform the first detailed kinetic and structural studies of Mtb ICDH-1. A large solvent kinetic isotope effect [(D2O)V = 3.0 ± 0.2, and (D2O)(V/Kisocitrate) = 1.5 ± 0.3] and a smaller primary kinetic isotope effect [(D)V = 1.3 ± 0.1, and (D)(V/K[2R-(2)H]isocitrate) = 1.5 ± 0.2] allowed us to perform the first multiple kinetic isotope effect studies on any ICDH and suggest a chemical mechanism. In this mechanism, protonation of the enolate to form product α-ketoglutarate is the rate-limiting step. We report the first structure of Mtb ICDH-1 to 2.18 Å by X-ray crystallography with NADPH and Mn(2+) bound. It is a homodimer in which each subunit has a Rossmann fold, and a common top domain of interlocking β sheets. Mtb ICDH-1 is most structurally similar to the R132H mutant human ICDH found in glioblastomas. Similar to human R132H ICDH, Mtb ICDH-1 also catalyzes the formation of α-hydroxyglutarate. Our data suggest that regulation of Mtb ICDH-1 is novel.

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Figures

FIGURE 1
FIGURE 1
“pH dependence of Mtb ICDH-1.” (A)kcat andkcat/KNADP+ determined at saturating isocitrate and varying NADP+. (A) (●) Logkcat fitting to eq 5 determined pKa = 5.6 ± 0.1. (■) Logkcat/KNADP+ fitting to eq 5 determined pKa = 6.3 ± 0.2. (B)kcat andkcat/Kisocitrate determined at saturating NADP+ and varying isocitrate. (●) Logkcat fitting to eq 5 determined pKa = 5.5 ± 0.2. (■) Logkcat/Kisocitrate fitting to the inverse of eq 5 determined pKa = 5.8 ± 0.1.
FIGURE 2
FIGURE 2
“Primary Kinetic Isotope Effects.” (A) Primary KIE at 500 μM NADP+ and varying (20-300 μM) (●) isocitrate or (■) [2R-2H]isocitrate.DV = 1.3 ± 0.1,D[V/K[2R-2H]isocitrate] = 1.5 ± 0.2. (B) pH dependence of (●)DV and (■)D[V/K[2R-2H]isocitrate] at 500 μM NADP+ and varying (5 – 150 μM) isocitrate or [2R-2H]isocitrate.
FIGURE 3
FIGURE 3
“Solvent and Multiple Kinetic Isotope Effects.” (A) Solvent KIE with 500 μM NADP+ varying (20-300 μM) isocitrate in (●) H2O or (■) 95 % D2O.D2OV = 3.0 ± 0.2,D2O[V/Kisocitrate] = 1.5 ± 0.3. (B) Proton inventory wherekcat was measured with 500 μM isocitrate and 500 μM NADP+ at 10% increments of D2O and fitted to a straight line. (C) Multiple KIE with 500 μM NADP+ varying (20-300 μM) (●) isocitrate or (■) [2R-2H]isocitrate in 95% D2O.DV = 1.2 ± 0.1,D[V/K[2R-2H]isocitrate] = 1.0 ± 0.2. (D) Multiple KIE with 500 μM NADP+ varying (20-300 μM) [2R-2H]isocitrate in (●) H O or (■) 95% D2O.D2OV = 1.7 ± 0.2,D2O[V/K[2R-2H]isocitrate] = 1.0 ± 0.2.
FIGURE 4
FIGURE 4
“Global Structure of Mtb ICDH-1.” Stereo view of Mtb ICDH-1. Each monomer is bound to one Mn2+, shown as a green sphere, and one NADPH, shown in stick form.
FIGURE 5
FIGURE 5
“Ligands in the Mtb ICDH-1 Structure.” (A) Electron density map for NADPH at 2σ. (B) A close up of one of the two the active sites showing Mn2+ as a green sphere and NADPH as sticks. Coordinating residues of the Mn2+ from the A chain are shown in pink, and the residue from the B chain is shown in blue. (C) Metal binding site.
SCHEME 1
SCHEME 1
Proposed Kinetic Mechanism of Mtb ICDH-1
SCHEME 2
SCHEME 2
Proposed Chemical Mechanism of Mtb ICDH-1
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