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Crystal structure of chorismate synthase from Aquifex aeolicus reveals a novel beta alpha beta sandwich topology

Introduction. Chorismate synthase (CS) (EC 4.6.1.4) catalyzes the last enzymatic step of the shikimate biosynthetic pathway involving the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate. Chorismate serves as a precursor for the biosynthesis of a large number of aromatic compounds, including the aromatic amino acids, and secondary metabolites in plants. This pathway is essential in microbes, fungi, parasites and plants, and is absent in mammals, which makes it an excellent target for the design of drug compounds and herbicides. The potential of such compounds has been exemplified by the herbicide glyphosate, which inhibits EPSP synthase, the enzyme preceding chorismate synthase in the pathway. The shikimate pathway has also recently been discovered in apicomplexan parasites such as Plasmodium falciparum, thus providing another avenue to improve the present treatment for devastating diseases such as malaria or toxoplasmosis. The CS-catalyzed reaction requires reduced FMN and substrate EPSP to produce chorismate, a phosphate molecule and reduced FMN. It is an unusual reaction in that the redox state of the functional cofactor (FMN) remains unchanged. Another unusual feature the of CS catalyzed reaction is the nonconcerted anti-1,4-elimination of the 3-phosphate and the C-(6-pro-R) hydrogen. It is expected that the three-dimensional structure of CS can significantly enhance our understanding of its enzymatic mechanism for this unusual chemistry. Experimental Procedures. Cloning, Protein Expression and Purification: The gene encoding CS from Aquifex aeolicus (AA) (gi16081332) was cloned from genomic AA DNA and its gene product was expressed, selenomethionine (SeMet)-labelled, and purified from a bacterial system as described elsewhere. Screening for crystallization conditions was also performed as described elsewhere. The final crystallization condition consists of 30% isopropanol, 0.2 M magnesium chloride and 0.1 M sodium HEPES at pH 7.5. The crystals chosen for X-ray data collection were flash-frozen in this solution containing 25% glycerol as cryoprotectant. X-Ray Diffraction and Structure Determination: Crystals of the AACS protein belong to the monoclinic space group C2 with the unit cell dimensions a 153.3 Å, b 95.8 Å, c 113.4 Å, 93.1 °. A single wavelength anomalous dispersion (SAD) experiment with selenium as the anomalous scatterer was carried out at 100 K at beamline ID14B, APS, using a MAR CCD detector. The 2.05 Å dataset was collected at 100 K at beamline BM14C, APS using a Q4 CCD detector. Both, the SAD and remote data were processed and scaled using the HKL2000 software package. The Selenium sites were identified using SOLVE and density modification and initial model building was carried out with RESOLVE. Automated model building and refinement were carried out with RESOLVE and Refmac 5.0 of the CCP4 package. With this approach, greater than 60% of the initial protein model was built. Further model building was done manually with the O software package and CNS was used for refinement of the model. All four molecules were built and refined using this approach without applying NCS constraints. Once both R values were below 30%, water molecules were identified with CNS and manually verified in O with the criteria: A peak of at least 2.5 in an Fo-Fc map and with expected interacting distances. The crystallographic and refinement data are shown in Table I.