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

Abstract

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.

Figure 1

Amino acid sequence of anthranilate synthase of S. solfataricus, with secondary structural elements (defined by DSSP; ref. 22) indicated in blue (β-strands) and green (α-helices). The numbers of these elements appear above the sequences. ∗, invariant residues; ●, at least 85% conserved residues. (A) TrpG subunit. Red boxes mark the residues Cys-84, His-175, and Glu-177 of the catalytic triad. Residues in contact with the TrpE subunit are underlined. (B) TrpE subunit. Residues corresponding to catalytically important or feedback-sensitive residues in anthranilate synthase of S. typhimurium are enclosed in red and blue boxes, respectively. ≫≪, Sites of deletions compared with TrpE of S. typhimurium. Residues in contact with the TrpG subunit are underlined.

Figure 2

Topology diagram of the TrpE subunit of anthranilate synthase. The layout corresponds roughly to the view direction in Fig. 3A. β-Strands are depicted as black arrows and α-helices as cylinders. Domain I (green in Fig. 3A) is labeled EI; domain II (yellow in Fig. 3A) is subdivided into the subdomains EIIA and EIIB as described in the text.

Figure 3

Three-dimensional structure of anthranilate synthase of S. solfataricus. Residues 33–39 (loop β2β3) of the TrpE subunit are missing in the model. (A) Ribbon representation of the TrpG:TrpE protomer of anthranilate synthase. Domains I and II of the TrpE subunit are colored green and yellow, respectively. The secondary structural elements of TrpE are numbered. In the TrpG subunit (gray), the catalytic residues Cys-84, His-175, and Glu-177 are depicted in ball-and-stick. (B) Cα trace of the TrpE subunit. Every 20th residue (●) is numbered. Residues involved in catalysis and feedback inhibition by tryptophan are labeled by red and blue spheres, respectively. (C) The TrpG₂:TrpE₂ heterotetramer viewed along the crystallographic (and molecular) twofold axis. The top TrpG:TrpE protomer is depicted as ribbon diagram. The secondary structural elements of the TrpG subunit (gray) are numbered, and the catalytic residues Cys-84, His-175, and Glu-177 are depicted in ball-and-stick. The bottom protomer is given as a Cα trace, in which every 20th residue (●) of the TrpG subunit is numbered. Regions of the TrpE subunit involved in a crystal lattice contact through an additional crystallographic twofold axis are marked in red. Drawings produced with molscript (23).

Figure 4

(A) View of the TrpG:TrpE interface. The Cα trace of the TrpE subunit (green for domain I and yellow for domain II) is superimposed on the 2Fo-Fc electron-density map (orange) contoured at 1.0 σ. Secondary structural features of the TrpE subunit are labeled. The TrpG subunit is depicted as a transparent surface in light blue, in which a structural stick model of the contact region of the TrpG subunit is drawn in magenta and atom colors. Residues Cys-84, His-175, and Glu-177 (hidden behind helix α7 of the TrpE subunit) as well as their corresponding surface areas are green. (B) View of the open cleft of the TrpE subunit. To compare the orientation of B to that of A, it is helpful to use the relative positions of helices α4 and α7. Transparent surfaces of the TrpE (light gray) and the TrpG subunit (dark gray) are shown. Surface areas of the TrpE subunit corresponding to residues important for catalysis and feedback regulation are colored red and blue, respectively. The Cα trace of the TrpE subunit is shown (green for domain I and yellow for domain II) with helices α4, α7, and α9 labeled. Catalytically important residues of the TrpE subunit and residues Cys-84, His-175, and Glu-177 of the TrpG subunit are depicted in ball-and-stick with atom colors and are labeled. Drawings produced with dino (A. Philippsen, unpublished work; see http://www.bioz.unibas.ch/∼x-ray/dino).