Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry

Abstract

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 flexible 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 processes--the ribosylation and deribosylation of metabolic pathway intermediates. These relationships establish new links for enzymes involved in nucleotide and amino acid metabolism.

Fig. 1. Reactions catalysed by (A) AnPRT and (B) TNP.

Fig. 2. Structure-based sequence alignment, using the sequences of the structures of AnPRT from S.solfataricus (ssAnPRT, PDB code: 1K8E), pyrimidine nucleoside phosphorylase from B.stearothermophilus (bsPyNP, PDB code: 1BRW) and thymidine phosphorylase from E.coli (ecTNP, PDB code: 1OTP). The numbers above the alignment correspond to the sequence of ssAnPRT. The positions of the α-helices and β-strands of the structure of ssAnPRT are shown by cylinders and arrows, respectively, and are labelled as in Figure 3A. The shared secondary structural elements of the small α-helical domain are in yellow, and those of the large mixed α/β domain are in red (α-helices) and cyan (β-strands). Strand β6, which is not topologically equivalent in the structures of ssAnPRT and bsPyNP/ecTNP but is structurally superimposable, is shown in magenta. Strand β7, which is unique in the ssAnPRT structure, is in grey. For comparison, the locations of the secondary structural elements of all corresponding structures are indicated by an underline. Residues that are conserved among each of the available sequence sets of AnPRT and NP-II families (data not shown) are highlighted in green if identical in at least 80% of the sequences. Conserved residues in structurally equivalent positions in the AnPRT and NP-II sequences are shown in bold, and are indexed below the alignment. The following ssAnPRT residue positions are labelled: *, specific inter-domain contacts between the small α-helical domain and the large mixed α/β domain; #, specific dimer contacts; A, predicted anthranilate-binding site; P, pyrophosphate- binding site; M, metal-binding site.

Fig. 2. Structure-based sequence alignment, using the sequences of the structures of AnPRT from S.solfataricus (ssAnPRT, PDB code: 1K8E), pyrimidine nucleoside phosphorylase from B.stearothermophilus (bsPyNP, PDB code: 1BRW) and thymidine phosphorylase from E.coli (ecTNP, PDB code: 1OTP). The numbers above the alignment correspond to the sequence of ssAnPRT. The positions of the α-helices and β-strands of the structure of ssAnPRT are shown by cylinders and arrows, respectively, and are labelled as in Figure 3A. The shared secondary structural elements of the small α-helical domain are in yellow, and those of the large mixed α/β domain are in red (α-helices) and cyan (β-strands). Strand β6, which is not topologically equivalent in the structures of ssAnPRT and bsPyNP/ecTNP but is structurally superimposable, is shown in magenta. Strand β7, which is unique in the ssAnPRT structure, is in grey. For comparison, the locations of the secondary structural elements of all corresponding structures are indicated by an underline. Residues that are conserved among each of the available sequence sets of AnPRT and NP-II families (data not shown) are highlighted in green if identical in at least 80% of the sequences. Conserved residues in structurally equivalent positions in the AnPRT and NP-II sequences are shown in bold, and are indexed below the alignment. The following ssAnPRT residue positions are labelled: *, specific inter-domain contacts between the small α-helical domain and the large mixed α/β domain; #, specific dimer contacts; A, predicted anthranilate-binding site; P, pyrophosphate- binding site; M, metal-binding site.

Fig. 3. The structure of the protomeric unit of ssAnPRT. (A) Ribbon presentation coloured in a blue-to-red gradient. The secondary structural elements and the termini are labelled as in Figure 2. (B) Ribbon, using the colour code of Figure 2. The side chains of the residues that are involved in specific inter-domain interactions are shown in blue. Those residues involved in specific interactions that are conserved among the structures of ssAnPRT, PyNP and TNP are shown in green and numbered as in Figure 2. (C) Superposition of two monomeric units of ssAnPRT, where one is in the closed hinge conformation (magenta) and the other is in the open hinge conformation (green).

Fig. 4. Structural and topological similarity in the structures of ssAnPRT (left) and bsPyNP (right). The colour code is as in Figure 2. (A) Monomeric unit; (B) asymmetric dimer; (C) topology diagram (for ease of comparison, only major secondary structure elements are included). The α-helices and β-strands are shown as circles and triangles, respectively.

Fig. 5. Location of the active site in the AnPRT structure. (A) Ribbon diagram. The colour code is as in Figures 2 and 3B. Side chains of residues that may be involved in PRPP/metal or anthranilate binding are shown in blue and magenta, respectively. The pyrophosphate group of PRPP is shown in green, the bound metal-binding ion as a yellow sphere and the modelled solvent molecule as a red sphere. (B) Surface presentation. Ligands follow the same colour code as in (A). The residues involved in binding of the ligands are mapped in the respective colours on the interior protein surface.

Fig. 6. (2F_obs – F_calc)α_calc electron density map corresponding to the pyrophosphate/metal-binding site of the Mg²⁺/pyrophosphate complex in the ssAnPRT protomer C at 2.7 Å resolution. The magnesium ion (yellow) is coordinated by D223, E224, S91 (not shown) and the two phosphate groups of pyrophosphate. Phosphate groups are bound further by the side chains of T92 and K106. In the molecule copies in the closed hinge conformation (protomers C and D), additional electron density bridging residues D223 and E224 have been modelled as a solvent molecule (red). The electron density associated with the residues involved in binding of the metal/pyrophosphate group is contoured at 1.0σ in dark violet. The density associated with the metal/pyrophosphate group and the solvent molecule is contoured at 1.5σ in dark green.

Fig. 7. Substrate-binding sites in ssAnPRT (left) and bsPyNP (right). (A) Pyrophosphate/metal- and phosphate-binding sites, respectively (Pugmire and Ealick, 1998). (B) Predicted base-binding site in ssAnPRT and as observed in the bsPyNP–pseudouridine complex (Pugmire and Ealick, 1998). The binding site for anthranilate is predicted to be formed by R164 and by three further residues (H107, H152 and N174) that are conserved within the AnPRT sequences (Figure 2). The invariant arginine in the PyNP–pseudouridine complex aligns with R164 of the ssAnPRT sequence (Figure 2). Numbering is as in Figure 2.