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The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase.

ABSTRACT: BACKGROUND: The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. FINDINGS: We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1) is 68-fold larger than that for the mutant K69A (0.73 s-1). There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 microM; wild-type = 29 microM) and NADPH (K69A = 30 microM; wild-type = 11 microM). The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (+/- 4) microM and 134 (+/- 21), respectively. CONCLUSION: Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

SUBMITTER: Rodrigues VS

PROVIDER: S-EPMC2789096 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature

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The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase.

Rodrigues Valnês S VS Breda Ardala A Santos Diógenes S DS Basso Luiz A LA

BMC research notes 20091116

<h4>Background</h4>The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation ...[more]

PMID: 19917104

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Project description:KSI (ketosteroid isomerase) from Comamonas testosteroni is a homodimeric enzyme that catalyses the allylic isomerization of Delta5-3-ketosteroids to their conjugated Delta4-isomers at a reaction rate equivalent to the diffusion-controlled limit. Based on the structural analysis of KSI at a high resolution, the conserved cis-Pro39 residue was proposed to be involved in the proper positioning of Asp38, a critical catalytic residue, since the residue was found not only to be structurally associated with Asp38, but also to confer a structural rigidity on the local active-site geometry consisting of Asp38, Pro39, Val40, Gly41 and Ser42 at the flexible loop between b-strands B1 and B2. In order to investigate the structural role of the conserved cis-Pro39 residue near the active site of KSI, Pro39 was replaced with alanine or glycine. The free energy of activation for the P39A and P39G mutants increased by 10.5 and 16.7 kJ/mol (2.5 and 4.0 kcal/mol) respectively, while DG(U)H2O (the free-energy change for unfolding in the absence of urea at 25.00+/-0.02 degrees C) decreased by 31.0 and 35.6 kJ/mol (7.4 and 8.5 kcal/mol) respectively, compared with the wild-type enzyme. The crystal structure of the P39A mutant in complex with d-equilenin [d-1,3,5(10),6,8-estrapentaen-3-ol-17-one], a reaction intermediate analogue, determined at 2.3 A (0.23 nm) resolution revealed that the P39A mutation significantly disrupted the proper orientations of both d-equilenin and Asp38, as well as the local active-site geometry near Asp38, which resulted in substantial decreases in the activity and stability of KSI. Upon binding 1-anilinonaphthalene-8-sulphonic acid, the fluorescence intensities of the P39A and P39G mutants were increased drastically, with maximum wavelengths blue-shifted upon binding, indicating that the mutations might alter the hydrophobic active site of KSI. Taken together, our results demonstrate that the conserved cis-Pro39 residue plays a crucial role in the proper positioning of the critical catalytic base Asp38 and in the structural integrity of the active site in KSI.

Dataset Information

The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase.

Publications

The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase.

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