Structure of shikimate kinase fromMycobacterium tuberculosisreveals the binding of shikimic acid

Abstract

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

Structural Basis for Shikimate-Binding Specificity of Helicobacter pylori Shikimate Kinase

“…The mean B factor of protein atoms in the complex is higher than that of the apo structure (50.3 versus 27.5 Å 2 , respectively). Notably, high B factors are seen for residues 99 to 110 and 113 to 122, which display significant conformational changes upon substrate binding, similar to those observed in the complex form of EcSK or MtSK (7,12,18,36). The overall root mean square deviation in C␣ atom positions between the superimposed HpSK and HpSK-shikimate-PO 4 structures is 0.68 Å.…”

“…In the Walker B motif of HpSK (VISTGGG) (residues 75 to 81), the conserved Gly80 has an orientation that may interact with the ␥-phosphate of a bound ATP. Shikimate kinase Walker B motifs lack an Asp residue involved in coordinating the active site Mg 2ϩ , and, instead, shikimate kinases have the DT/SD motif at residues 31 to 33 (24,36).…”

“…The determined apo EcSK and EcSK-MgADP complex structures reveal an opento-closed induced-fit movement of the enzyme upon substrate binding (19), as also observed in NMP kinases such as adenylate kinase (9,42). Other determined shikimate kinase structures include Escherichia coli shikimate kinase I (39), Campylobacter jejuni shikimate kinase (CjSK) (not published; PDB code, 1VIA), Mycobacterium tuberculosis shikimate kinase (MtSK), the MtSK-MgADP complex (12), and the ternary MtSK-MgADP-shikimate complex (7,36).…”

The use of biodiversity as source of new chemical entities against defined molecular targets for treatment of malaria, tuberculosis, and T-cell mediated diseases: a review

“…Another requirement is that protein stocks should be fairly concentrated (> 0.5 mg ml -1 ). These requirements are met by a number of reports on recombinant proteins we have published , Azevedo et al 2003a,b, Oliveira et al 2003, Dias et al 2004, Nolasco et al 2004, Pereira et al 2004a,b, Rizzi et al 2005.…”

“…The proteins to be studied have to be crystallized using vapour diffusion and crystallization conditions as previously described (Azevedo et al 2003a,b,c, Santos et al 2003, Dias et al 2004, Nolasco et al 2004, Pereira et al 2004a. Usually, the solution containing ligands is dissolved and the native crystals are soaked in these solutions 48 h prior to data collection.…”