Kinetic and chemical mechanisms of shikimate dehydrogenase from Mycobacterium tuberculosis

Fonseca, I.; Silva, Rafael G.; Fernandes, Cláudia Lemelle; Souza, Osmar Norberto de; Basso, Luiz Augusto; Santos, Daniel Silas Veras dos

doi:10.1016/j.abb.2006.11.015

Cited by 12 publications

(33 citation statements)

References 35 publications

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“…The values obtained for the apparent steady-state kinetic parameters were as follows: K m = 29 (±2 M) for DHS, K m = 11 (±1 M) for NADPH, k cat = 50 (±1) s −1 . These values are in agreement with previously reported parameters [25,26]. It should be noted that here we The results presented are for a purification protocol from 5 g of E. coli host cells.…”

Section: Resultssupporting

confidence: 93%

“…The pH-rate profile of the A. aeolicus SD also indicates that a residue, probably a lysine, functions as a general base during catalysis [32]. We have recently reported determination of kinetic and chemical mechanisms for MtbSD, including pH-rate profiles, and the data are consistent with the probable participation of a lysine residue in catalysis and/or substrate binding [26].…”

Section: Introductionmentioning

confidence: 55%

“…All kinetic experiments were performed in potassium phosphate 100 mM buffer since in phosphate [26] the catalytic rate value for this enzyme seems to be larger than when the assay is carried out in Tris-HCl buffer [25]. The values obtained for the apparent steady-state kinetic parameters were as follows: K m = 29 (±2 M) for DHS, K m = 11 (±1 M) for NADPH, k cat = 50 (±1) s −1 .…”

Section: Resultsmentioning

confidence: 99%

“…Recent report of specific inhibitors against the shikimate dehydrogenase from Helicobacter pylori has reinforced the importance of the target diversity and the introduction of novel effective antimicrobial agents [23]. Our group has previously reported cloning, expression, purification, kinetic properties, and structural studies of the aroEencoded shikimate dehydrogenase from M. tuberculosis (MtbSD) [24][25][26][27]. However, the molecular basis of DHS recognition and the catalytic mechanism for MtbSD are not yet known.…”

Section: Introductionmentioning

confidence: 99%

“…Multiple sequence alignment studies showed that the amino acids lysine in the position 69 and aspartate in the position 105 (Lys69 and Asp105, M. tuberculosis numbering) are, amongst other residues, fully conserved in the polypeptide chain of the following aligned enzymes: AroE from M. tuberculosis, Escherichia coli, H. influenzae, Methanococcus jannaschii, Thermus thermophilus, Neisseria meningitides, H. pylori, Archaeoglobus fulgidus, Aquiflex aeolicus, Pseudomonas aeruginosa, YdiB from E. coli, Corynebacterium glutamicum, SD-like from H. influenzae, SD parts of Arabidopsis thaliana and Lycopersicon esculentum DHQ-SDs [21,23,26,28,29]. In the following, we will refer to residues according to the M. tuberculosis AroE numbering.…”

Section: Introductionmentioning

confidence: 99%

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Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Rodrigues-Junior

Basso

Santos

2009

International Journal of Biological Macromolecules

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Mycobacterium tuberculosis shikimate dehydrogenase (MtbSD) catalyzes the forth reaction in the shikimate pathway. Here we describe production of K69A, K69H, K69I, K69Q, D105A, and D105N mutant proteins. Screening of several conditions was performed to optimize MtbSD production yield, and an improved purification protocol to obtain homogeneous MtbSD is presented. The rational design of new antitubercular drugs hinges on the availability of M. tuberculosis proteins. Our results show that optimization of expression, disruption, and purification protocols resulted in a higher yield of functional MtbSD enzyme.

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Section: Resultssupporting

confidence: 93%

Section: Introductionmentioning

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Rodrigues-Junior

Basso

Santos

2009

International Journal of Biological Macromolecules

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Structural studies of shikimate 5‐dehydrogenase from Mycobacterium tuberculosis

et al. 2008

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Tuberculosis (TB) remains the leading cause of mortality due to a single bacterial pathogen, Mycobacterium tuberculosis. The reemergence of TB as a potential public health threat, the high susceptibility of human immunodeficiency virus-infected persons to the disease, the proliferation of multi-drug-resistant strains (MDR-TB) and, more recently, of extensively drug resistant isolates (XDR-TB) have created a need for the development of new antimycobacterial agents. Amongst the several proteins and/or enzymes to be studied as potential targets to develop novel drugs against M. tuberculosis, the enzymes of the shikimate pathway are attractive targets because they are essential in algae, higher plants, bacteria, and fungi, but absent from mammals. The mycobacterial shikimate pathway leads to the biosynthesis of chorismate, which is a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Here we report the structural studies by homology modeling and circular dichroism spectroscopy of the shikimate dehydrogenase from M. tuberculosis (MtSDH), which catalyses the fourth step of the shikimate pathway. Our structural models show that the MtSDH has similar structure to other shikimate dehydrogenase structures previously reported either in presence or absence of NADP, despite the low amino acid sequence identity. The circular dichroism spectra corroborate the secondary structure content observed in the MtSDH models developed. The enzyme was stable up to 50 degrees C presenting a cooperative unfolding profile with the midpoint of the unfolding temperature value of approximately 63-64 degrees C, as observed in the unfolding experiment followed by circular dichroism. Our MtSDH structural models and circular dichroism data showed small conformational changes induced by NADP binding. We hope that the data presented here will assist the rational design of antitubercular agents.

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Characterization of shikimate dehydrogenase homologues of Corynebacterium glutamicum

Kubota

Tanaka

Hiraga

et al. 2013

Appl Microbiol Biotechnol

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The function of three Corynebacterium glutamicum shikimate dehydrogenase homologues, designated as qsuD (cgR_0495), cgR_1216, and aroE (cgR_1677), was investigated. A disruptant of aroE required shikimate for growth, whereas a qsuD-deficient strain did not grow in medium supplemented with either quinate or shikimate as sole carbon sources. There was no discernible difference in growth rate between wild-type and a cgR_1216-deficient strain. Enzymatic assays showed that AroE both reduced 3-dehydroshikimate, using NADPH as cofactor, and oxidized shikimate, the reverse reaction, using NADP(+) as cofactor. The reduction reaction was ten times faster than the oxidation. QsuD reduced 3-dehydroquinate using NADH and oxidized quinate using NAD(+) as cofactor. Different from the other two homologues, the product of cgR_1216 displayed considerably lower enzyme activity for both the reduction and the oxidation. The catalytic reaction of QsuD and AroE was highly susceptible to pH. Furthermore, reduction of 3-dehydroshikimate by AroE was inhibited by high concentrations of shikimate, but neither quinate nor aromatic amino acids had any effect on the reaction. Expression of qsuD mRNA was strongly enhanced in the presence of shikimate, whereas that of cgR_1216 and aroE decreased. We conclude that while AroE is the main catalyst for shikimate production in the shikimate pathway, QsuD is essential for quinate/shikimate utilization.

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Kinetic and chemical mechanisms of shikimate dehydrogenase from Mycobacterium tuberculosis

Cited by 12 publications

References 35 publications

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Homogeneous recombinant Mycobacterium tuberculosis shikimate dehydrogenase production: An essential step towards target-based drug design

Structural studies of shikimate 5‐dehydrogenase from Mycobacterium tuberculosis

Characterization of shikimate dehydrogenase homologues of Corynebacterium glutamicum

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