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-Å 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.Anthranilate synthase (AnthS) from bacteria and yeast is a multifunctional enzyme composed of small TrpG and large TrpE subunits or domains (1). TrpG belongs to the family of ''triad'' glutamine amidotransferases (2, 3), which hydrolyze glutamine and transfer nascent ammonia through an intramolecular channel to a synthase active site.The TrpE subunit is a bifunctional enzyme (4). It catalyzes the synthesis of anthranilate in two steps (Scheme 1): the reversible reaction of chorismate with ammonia to 2-amino 2-deoxyisochorismate (ADIC synthase reaction) followed by the irreversible elimination of pyruvate from ADIC (ADIC lyase reaction). Both reactions require Mg 2ϩ ions, and ADIC is not released into the solvent. The TrpG 2 :TrpE 2 complex mediates communication between three distinct ligandbinding sites on the two subunits (1): (i) chorismate binding to the TrpE subunit activates the release of ammonia from glutamine bound to the TrpG subunit; (ii) nascent ammonia is transferred intramolecularly from the TrpG to the TrpE subunit, in preference to ammonia from the bulk solvent (1), and (iii) tryptophan binding to a distinct site on the TrpE subunit (5) inhibits all partial reactions of the TrpG 2 :TrpE 2 complex. The strictly ordered addition of chorismate before glutamine (6) and the cooperative binding of both chorismate and tryptophan (7) to the complex suggest that conformational changes mediate the communication between the various ligand-binding sites.Here, we report the crystal structure of the unliganded AnthS complex from the hyperthermophile Sulfolobus solfataricus. The TrpE subun...

The crystal structure of the dimeric anthranilate phosphoribosyltransferase (AnPRT) reveals a new category of phosphoribosyltransferases, designated as class III. The active site of this enzyme is located within the¯exible hinge region of its two-domain structure. The pyrophosphate moiety of phosphoribosylpyrophosphate is co-ordinated by a metal ion and is bound by two conserved loop regions within this hinge region. With the structure of AnPRT available, structural analysis of all enzymatic activities of the tryptophan biosynthesis pathway is complete, thereby connecting the evolution of its enzyme members to the general development of metabolic processes. Its structure reveals it to have the same fold, topology, active site location and type of association as class II nucleoside phosphorylases. At the level of sequences, this relationship is mirrored by 13 structurally invariant residues common to both enzyme families. Taken together, these data imply common ancestry of enzymes catalysing reverse biological processesÐthe ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism. Keywords: enzyme evolution/nucleoside phosphorylase/ nucleotide salvage/phosphoribosyltransferase/tryptophan biosynthesis IntroductionThe transfer of a ribosyl group between an aromatic base and phosphate groups is one of the most fundamental biochemical reactions in the metabolism of nucleotides and amino acids (Craig and Eakin, 2000;Pugmire and Ealick, 2002). This transfer is generally reversible, leading to either the addition or the removal of a ribosyl unit from metabolic compounds. The addition of ribosyl groups is catalysed by phosphoribosyltransferases (PRTs), which displace the 1¢-pyrophosphate moiety from the substrate 5-phosphoribosyl-1¢-pyrophosphate (PRPP), forming a 1¢-glycosidic±nitrogen bond between a nitrogenated base and a phosphoribosyl group. The reverse process is catalysed by nucleoside phosphorylases (NPs) and is associated speci®cally with the removal of ribose from nucleosides by the phosphorolytic cleavage of the N-1¢-glycosidic bond (Figure 1). PRT catalysis generally requires the presence of a divalent metal ion, whereas no metal ion is needed for NP catalysis. Both types of reactions are believed to follow a sequential S N 1 mechanism, although alternative mechanisms are not ruled out (Craig and Eakin, 2000;Pugmire and Ealick, 2002).PRTs are known to be involved in nucleotide salvage and synthesis pathways as well as in the biosynthesis of the amino acids histidine and tryptophan, and the co-factor NAD. Most of the PRTs with known three-dimensional structure share the same two-domain architecture, known as the PRT-I fold (Craig and Eakin, 2000;Sinha and Smith, 2001), the only exception being quinolate PRT, which has been classi®ed as the PRT-II fold (Eads et al., 1997). The currently available NP structures are also categorized into two unrelated folds. The ®rst class is involved in the cleavage of...