The crystal structure of anthranilate synthase from Sulfolobus solfataricus: functional implications.

Anthranilate synthase catalyzes the synthesis of anthranilate from chorismate and glutamine and is feedback-inhibited by tryptophan. The enzyme of the hyperthermophile Sulfolobus solfataricus has been crystallized in the absence of physiological ligands, and its three-dimensional structure has been determined at 2.5-A resolution with x-ray crystallography. It is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits, in which two TrpG:TrpE protomers associate mainly via the TrpG subunits. The small TrpG subunit (195 residues) has the known "triad" glutamine amidotransferase fold. The large TrpE subunit (421 residues) has a novel fold. It displays a cleft between two domains, the tips of which contact the TrpG subunit across its active site. Clusters of catalytically essential residues are located inside the cleft, spatially separated from clustered residues involved in feedback inhibition. The structure suggests a model in which chorismate binding triggers a relative movement of the two domain tips of the TrpE subunit, activating the TrpG subunit and creating a channel for passage of ammonia toward the active site of the TrpE subunit. Tryptophan presumably blocks this rearrangement, thus stabilizing the inactive states of both subunits. The structure of the TrpE subunit is a likely prototype for the related enzymes 4-amino 4-deoxychorismate synthase and isochorismate synthase.

The hyperthermostable indoleglycerol phosphate synthase from Thermotoga maritima is destabilized by mutational disruption of two solvent-exposed salt bridges.

The recombinantly expressed protein indoleglycerol phosphate synthase from the hyperthermophilic bacterium Thermotoga maritima (tIGPS) was purified and characterized with respect to oligomerization state, catalytic properties and thermostability. This enzyme from the biosynthetic pathway of tryptophan is a monomer in solution. In contrast to IGPS from the hyperthermophilic archaeon Sulfolobus solfataricus, tIGPS shows high catalytic activity at room temperature and only weak product inhibition. In order to test the hypothesis that salt bridges in a critical context contribute to the high thermostability of tIGPS, two solvent-exposed salt bridges were selected, based on its three-dimensional structure, for individual disruption by site-directed mutagenesis. The first salt bridge fixes the N terminus to the core of the protein, and the second serves as a clamp between helices alpha1 and alpha8, which are widely separated in sequence but adjacent in the (betaalpha)8-barrel. Kinetics of irreversible heat inactivation reveal that the salt bridge crosslinking helices alpha1 and alpha8 stabilizes tIGPS more strongly than that tethering the N terminus.

The crystal structure of indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus in three different crystal forms: effects of ionic strength.

Indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus is a monomeric enzyme with the common (beta/alpha)8-fold. Recently, its three-dimensional structure was solved in an orthorhombic crystal form, grown by using 1.3 M ammonium sulfate as precipitating agent. Here we describe the X-ray structure analysis of two new crystal forms of this enzyme that were obtained at medium and low ionic strength, respectively. Hexagonal crystals with space group P3(1)21 and cell dimensions a = 62.4 A, b = 62.4 A, c = 122.9 A, gamma = 120 degrees grew in 0.1 M Mes buffer at pH 6.0 with 30% polyethylene glycol monomethylether as precipitant and 0.2 M ammonium sulfate as co-precipitant. A second crystal form with space group P2(1)2(1)2(1) and cell constants a = 62.6 A, b = 74.0 A, c = 74.2 A was obtained using polyethylene glycol and ethylene glycol as precipitants in 0.1 M Mes buffer at pH 6.5. Both structures were solved by molecular replacement and refined at 2.5 A and 2.0 A resolution, respectively. Although the global folds are almost identical, alternative conformations are observed in flexible loop regions, mostly stabilized by crystal contacts. In none of the three crystal forms is the so-called phosphate binding site empty, suggesting that this position has high affinity for anions with tetrahedrally arranged oxygen atoms. Differences in ionic strength of the crystallization buffer have only minor effects on number and specificity of intramolecular salt bridges. The crystal packing, on the other hand, seems to be influenced by the ionic strength of the solvent, since the number of intermolecular salt bridges in the low ionic strength crystal forms is significantly higher.