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

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

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.

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Related in: MedlinePlus

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.
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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.

Mentions: The Band I showed no affinity for the nickel-nitrilotriacetic acid-agarose matrix and anti-His antibody on western (Data not shown) suggesting that it corresponds to portion of MtαIPMS in which N-terminus has been removed as the histidine tag is attached to the N-terminus of the full-length protein. For determining the site of cleavage of protease in the protein, N-terminal sequencing of the obtained fragment was carried out. It had a sequence RPFAE. Based on N-terminal sequence and the observed molecular mass, Band I corresponds to the protein fragment Arg47 to Phe457 of the MtαIPMS. This fragment consists of the TIM barrel portion, sub domain I and a small part of sub domain II of the full-length enzyme (Figure 6). It has been designated as the TIM barrel domain in this study.


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

Singh K, Bhakuni V - BMC Struct. Biol. (2007)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919377&req=5

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.
Mentions: The Band I showed no affinity for the nickel-nitrilotriacetic acid-agarose matrix and anti-His antibody on western (Data not shown) suggesting that it corresponds to portion of MtαIPMS in which N-terminus has been removed as the histidine tag is attached to the N-terminus of the full-length protein. For determining the site of cleavage of protease in the protein, N-terminal sequencing of the obtained fragment was carried out. It had a sequence RPFAE. Based on N-terminal sequence and the observed molecular mass, Band I corresponds to the protein fragment Arg47 to Phe457 of the MtαIPMS. This fragment consists of the TIM barrel portion, sub domain I and a small part of sub domain II of the full-length enzyme (Figure 6). It has been designated as the TIM barrel domain in this study.

Bottom Line: The activator K+ also interacts with the catalytic TIM barrel domain however, it does not induce any significant effect on the enzyme structure.This divalent cation induced structural cooperativity might result in modulation of activity of the catalytic domain by regulatory domain.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow, India. ksinghcdri@gmail.com <ksinghcdri@gmail.com>

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.

Show MeSH
Related in: MedlinePlus