Cation induced differential effect on structural and functional properties of Mycobacterium tuberculosis alpha-isopropylmalate synthase

Abstract

Background: Alpha-isopropylmalate synthase (MtalphaIPMS), an enzyme that catalyzes the first committed step of the leucine biosynthetic pathway of Mycobacterium tuberculosis is a potential drug target for the anti-tuberculosis drugs. Cations induce differential effect of activation and inhibition of MtalphaIPMS. To date no concrete mechanism for such an opposite effect of similarly charged cations on the functional activity of enzyme has been presented.

Results: Effect of cations on the structure and function of the MtalphaIPMS has been studied in detail. The studies for the first time demonstrate that different cations interact specifically at different sites in the enzyme and modulate the enzyme structure differentially. The inhibitors Zn2+ and Cd2+ ions interact directly with the catalytic domain of the enzyme and induce unfolding/denaturation of the domain. The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure. Studies with isolated catalytic TIM barrel domain showed that it can carry out the catalytic function on its own but probably requires the non-catalytic C-terminal domain for optimum functioning. An important observation was that divalent cations induce significant interaction between the regulatory and the catalytic domain of MtalphaIPMS thus inducing structural cooperativity in the enzyme. This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.

Conclusion: The studies for the first time demonstrate that different cations bind at different sites in the enzyme leading to their differential effects on the structure and functional activity of the enzyme.

Figure 1

Over-expression and purification of MtαIPMS. SDS-PAGE analysis of cell lysate over-expressing MtαIPMS and purified protein. Lanes 1–4 represent molecular weight markers, supernatant of un-induced cell lysate, supernatant of induced cell lysate and purified protein, respectively.

Figure 2

Cations influence enzymatic activity of both MtαIPMS and the TIM barrel domain. Effect of KCl, MgCl₂, ZnCl₂ or CdCl₂ on enzymatic activity of MtαIPMS (Panel A) and the catalytic TIM barrel Domain (Panel B). In both the panels the bars 1 to 4 represent samples of protein in presence of 20 mM KCl, 20 mM KCl + 5 mM MgCl₂, 20 mM KCl + 5 mM MgCl₂ +2 mM ZnCl₂ and 20 mM KCl + 5 mM MgCl₂ + 2 mM CdCl₂, respectively. The data is represented as percent activity with the activity of the protein in presence of 20 mM KCl taken as 100%. The data is represented as mean ± SD of three values.

Figure 3

Divalent cations have differential effects on the secondary structure of MtαIPMS and TIM barrel domain. A. Effect of increasing concentration of KCl (circle) and MgCl₂ (square) on the CD ellipticity at 222 nm of MtαIPMS (open symbols) and TIM barrel domain (closed symbol). B. Effect of increasing concentration of ZnCl₂ (up triangle) and CdCl₂ (down triangle) on the CD ellipticity at 222 nm of MtαIPMS (open symbols) and TIM barrel domain (closed symbols).

Figure 4

Electrostatic potential of molecular surface of MtαIPMS dimer. The colors blue and red represents negative and positive potential. Panels A and B represent MtαIPMS dimer molecule in two different orientations. The molecular surface was displayed using GRASP.

Figure 5

Limited proteolysis of MtαIPMS with α-chymotrypsin at pH 7.5 and 25°C. SDS-PAGE profile of the protein fragment(s) obtained on limited proteolysis of recombinant MtαIPMS with α-chymotrypsin. Lanes 1–4 represent molecular weight markers, undigested MtαIPMS, protein digested with α-chymotrypsin and purified Band I, respectively.

Figure 6

MOLSCRIPT model of MtαIPMS (Panel A) and the TIM barrel domain (Panel B). The TIM barrel domain (magenta), sub-domain I (yellow), sub-domain II (red) and regulatory domain (cyan). The model has been generated using program UCSF Chimera.

Figure 7

Structural and functional properties of MtαIPMS and the TIM barrel domain. A. Far-UV CD spectra of MtαIPMS (profile 1) and the TIM barrel domain (profile 2). B. Tryptophan emission spectra of MtαIPMS (profile 1) and the TIM barrel domain (profile 2). C. Thermal unfolding of MtαIPMS and the TIM barrel domain. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1) and the TIM barrel domain (profile 2), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively. D. Enzymatic activity of MtαIPMS and the TIM barrel domain. Relative enzymatic activity of MtαIPMS (Bar 1 and 2) and the TIM barrel domain (Bar 3 and 4) at 25 and 65°C, respectively. The values have been represented as percent activity with the value observed for MtαIPMS at 25°C taken as 100%. The data is represented as mean ± SD of three values. Inset shows the loss of enzymatic activity of the MtαIPMS (closed symbols) and the TIM barrel domain (open symbols) on incubation with increasing concentration of L-lecucine. The data has been represented as percentage with the activity of MtαIPMS or the TIM barrel domain in absence of Leucine taken as 100 percent. E. SDS-PAGE profile of glutaraldehyde cross-linked TIM barrel domain. Lanes 1–3 represent molecular weight markers, uncrosslinked and glutaraldehyde cross-linked TIM barrel domain, respectively.

Figure 8

Thermal unfolding of salt-treated MtαIPMS. Effect of increasing temperature on the CD ellipticity at 222 nm of MtαIPMS (profile 1), MtαIMPS in presence of 5 mM KCl (profile 2), 5 mM MgCl₂ (profile 3), 0.25 mM ZnCl₂ (profile 4) and 0.25 mM CdCl₂ (profile 5), respectively. The values have been represented as percentage with the value obtained at 25°C for each sample taken as 100 percent, respectively.