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

• DOI: 10.1002/prot.10592
• Publication Date: 2003-12-29T22:00:39Z
• Authors: Cristina M. Viola; Vivian Saridakis; Dinesh Christendat
• Keywords: Shikimate pathway; Aquifex aeolicus; Transferase; Shikimic acid

Chorismate synthase (CS) (EC 4.6.1.4) catalyzes the last enzymatic step of shikimate biosynthetic pathway involving conversion 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate.1 serves as a precursor for biosynthesis large number aromatic compounds, including amino acids, and secondary metabolites in plants. This is essential microbes, fungi, parasites plants, absent mammals, which makes it an excellent target design drug compounds herbicides. The potential such has been exemplified by herbicide glyphosate, inhibits EPSP synthase, enzyme preceding chorismate pathway.2 also recently discovered apicomplexan Plasmodium falciparum, thus providing another avenue improve present treatment devastating diseases malaria or toxoplasmosis.3 CS-catalyzed reaction requires reduced FMN substrate produce chorismate, phosphate molecule FMN. It unusual that redox state functional cofactor (FMN) remains unchanged. Another feature CS catalyzed nonconcerted anti-1,4-elimination C-(6-pro-R) hydrogen.4 expected three-dimensional structure can significantly enhance our understanding its mechanism this chemistry. gene encoding from Aquifex aeolicus (AA) (gi16081332) was cloned genomic AA DNA product expressed, selenomethionine (SeMet)-labelled, purified bacterial system described elsewhere.5, 6 Screening crystallization conditions performed elsewhere.5 final condition consists 30% isopropanol, 0.2 M magnesium chloride 0.1 sodium HEPES at pH 7.5. crystals chosen X-ray data collection were flash-frozen solution containing 25% glycerol cryoprotectant. Crystals AACS protein belong monoclinic space group C2 with unit cell dimensions = 153.3 Å, b 95.8 c 113.4 β 93.1 °. A single wavelength anomalous dispersion (SAD) experiment selenium scatterer carried out 100 K beamline ID14B, APS, using MAR CCD detector. 2.05 Å dataset collected BM14C, APS Q4 Both, SAD remote processed scaled HKL2000 software package.7 Selenium sites identified SOLVE8 density modification initial model building RESOLVE.9 Automated refinement RESOLVE Refmac 5.0 CCP4 package.9, 10 With approach, greater than 60% built. Further done manually O package11 CNS12 used model. All four molecules built refined approach without applying NCS constraints. Once both R values below 30%, water CNS verified criteria: peak least 2.5 σ Fo-Fc map interacting distances. crystallographic are shown Table I. crystal solved method signal Se energy. atomic models good quality indicated factors small deviations ideal bond length angle parameters. contains 331 × 4 acids (residues 2–49, 56–86, 118–325 351–398) 559 molecules. Refinement resulted 0.209 Rfree 0.250. According Procheck, 99.4 % residues most favored additionally allowed regions Ramachandran plot.13 Two (Arg84A Leu116D) disallowed regions. dominant structural topology beta-alpha-beta sandwich, each monomer central helical core, helix order α1, α4, α9 α7, sandwiched between two four-stranded antiparallel beta sheets, strand β1, β2, β7 β4 sheet “one” β8, β10, β17 β12 “two” (Fig. 1). core helices staggered cris-cross manner about 45° away other. sheets further characterized golf club topology, handle short three-stranded continuation individual strands separated loops seen In addition composed other features. hairpin formed β13 β14 observed extend monomer. helices, α5 α6, flank Packing within stabilized mainly sandwich produces tightly packed molecule. long C-terminal helix, α9, hairpin, β14, H-bonding neighboring protein. a: Ribbon diagram α colored green gold, respectively. β-α-β located on left right. b: Topology CS. (β 1–β17) alpha (α1–α10) dark gray arrows light cylinders, reveals internal 2-fold symmetry equivalent major 4-stranded anti-parallel minor 3-stranded surrounding core. disrupted extension (between β8 β10) (β13, β14). interacts α10. c: tetramer separately. constructed dimer dimers AD BC 90°. putative binding site interface BC. d: molecular surface representation showing electrostatic (same orientation part c). Acidic red, basic blue neutral grey. pockets positive (blue) front represent site. determined be gel filtration chromatography, consistent obtained AACS. AACS, depicts intermolecular interactions D form first B C second eight-stranded combining subunit, β8A, β10A, β17A, β12A, β12D, β17D, β10D β8D. case, 6116 A2 total 25592 buried upon dimerization (A C). hydrophobic; ionic more prominent solvent-exposed via their N-terminal faces 90° rotation, results subunit perpendicular axis meshed pattern sets 9 C. same D. involve combination hydrophobic, polar ionizable residues. networks, R7 E24 (from A) D246 R247(from B) D379 K375 B). Such hydrophobic known have stabilizing effect may provide added stability oligomeric Comparison proteins database DALI seven Z scores 3.2 2.0, Bira (PDB ID 1BIA) being closest match. Structure superposition these revealed β-sheet only conserved element. analysis different any flavin proteins. Visual inspection βαβ-sandwich motif confirmed indeed unique. active searching cavity fingerprint sequence signatures CS, 1) R-P-[GS]-H-[AG]-D-x(5)-K 2) R-x-S-[AG]-R-[EV]-[ST]-x(3)-V-x(2)-G-x(6)-L.14 pocket approximate dimension 28 A3 signature identified. (S14, S135, H15, R44, R137, D59, D85, K55, E57, N261, Y48, Q45, N83) pocket. disordered 50–56, 87–117 326–350) location shared subunits; Using motif, we propose Additional information, complexes analogues analogs required characterize role reaction.