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• W1997194941 abstract "Article1 July 2002free access Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry Olga Mayans Olga Mayans EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Present address: Department of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Andreas Ivens Andreas Ivens Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Present address: Universität zu Köln, Institut für Biochemie, Otto-Fischer-Strasse 12–14, D-50674 Köln, Germany Search for more papers by this author L.Johan Nissen L.Johan Nissen EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Search for more papers by this author Kasper Kirschner Kasper Kirschner Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Matthias Wilmanns Corresponding Author Matthias Wilmanns EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Search for more papers by this author Olga Mayans Olga Mayans EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Present address: Department of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Andreas Ivens Andreas Ivens Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Present address: Universität zu Köln, Institut für Biochemie, Otto-Fischer-Strasse 12–14, D-50674 Köln, Germany Search for more papers by this author L.Johan Nissen L.Johan Nissen EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Search for more papers by this author Kasper Kirschner Kasper Kirschner Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author Matthias Wilmanns Corresponding Author Matthias Wilmanns EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Search for more papers by this author Author Information Olga Mayans1,2, Andreas Ivens3,4, L.Johan Nissen1, Kasper Kirschner3 and Matthias Wilmanns 1 1EMBL Hamburg Outstation c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany 2Present address: Department of Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 3Department of Biophysical Chemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland 4Present address: Universität zu Köln, Institut für Biochemie, Otto-Fischer-Strasse 12–14, D-50674 Köln, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3245-3254https://doi.org/10.1093/emboj/cdf298 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The 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 specifically 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 SN1 mechanism, although alternative mechanisms are not ruled out (Craig and Eakin, 2000; Pugmire and Ealick, 2002). Figure 1.Reactions catalysed by (A) AnPRT and (B) TNP. Download figure Download PowerPoint 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 classified as the PRT-II fold (Eads et al., 1997). The currently available NP structures are also categorized into two unrelated folds. The first class is involved in the cleavage of all purine and some pyrimidine nucleosides, whereas the second class (referred as NP-II throughout this text) catalyses the lysis of pyrimidine nucleosides only (Pugmire and Ealick, 2002). Despite the close mechanistic relationship between PRT and NP catalysis, there has been no direct evidence for the common evolutionary ancestry of these enzymes to date. Anthranilate phosphoribosyltransferase (AnPRT) is involved in the biosynthesis pathway of the aromatic amino acid tryptophan. It catalyses the formation of a carbon–nitrogen bond between PRPP and anthranilate, thus establishing the precursor skeleton of tryptophan. Subsequently, the anthranilate group is converted into an indole side chain by two isomerization reactions and a cyclization reaction. Depending on the microrganism, AnPRT is either a monofunctional enzyme or is fused to other enzymes of the tryptophan biosynthesis pathway. Recently, it has been demonstrated that AnPRT mutants of Mycobacterium tuberculosis present attenuated virulence, allowing protection in the mouse model that is equal to vaccination with the virulent strain BCG (Smith et al., 2001). Therefore, AnPRT has gained considerable interest as a target with which to develop superior strains of M.tuberculosis that could be used for vaccination. Previous studies have noted that, in contrast to other PRTs, AnPRT shares similarity within three sequence segments with pyrimidine nucleoside phosphorylases (PyNPs) that comprise the NP-II fold (Mushegian and Koonin, 1994; Pugmire and Ealick, 2002). Lacking an experimental three-dimensional structure for AnPRT, it has remained uncertain whether members of the NP-II family and AnPRT share the same overall structure. Here we present the crystal structure of AnPRT from the hyperthermophile Sulfolobus solfataricus (ssAnPRT), revealing that indeed AnPRT has an NP-II-like fold. With this structure, we are able to show that there could be an evolutionary link between members of the NP-II family and AnPRT. The structure of ssAnPRT, however, is unrelated to those of other PRTs, thus establishing a third PRT fold, designated here as PRT-III. The new ssAnPRT structure completes the structural inventory of all seven enzymes of the tryptophan biosynthesis pathway. Results Overall architecture of ssAnPRT The crystal structure of the dimeric AnPRT from S.solfataricus (Ivens et al., 2001) has been determined at 2.0 Å resolution using multiple anomalous dispersion (MAD) phases from a selenomethionine variant. This crystal form contains in its asymmetric unit four ssAnPRT protomers as two pairs of dimers. Each ssAnPRT protomer is composed of two domains (Figures 2, 3 and 4). The small α-helical domain comprises six helices (α1–α4 and α8–α9). The large α/β domain is formed by a central β-sheet (strands β1–β7) and a C-terminal cluster of eight α-helices (α5–α7 and α10–α15), of which helix α10 is largely positioned towards the small α-helical domain (Figure 3A and B). All strands in the β-sheet are parallel except for β6 (Figures 3A and 4). The two domains within this bilobal fold are not separated topologically. The inter-domain connections are formed by three loops α4–β1, β3–α8 and α9–β4 (Figure 4C). Figure 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. Download figure Download PowerPoint Figure 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). Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint The structure shows that ssAnPRT dimerizes via the small α-helical domain in a head-to-head fashion (Figure 4B). The dimer interface follows an approximate 2-fold symmetry and is formed predominantly by surface patches of hydrophobic residues from helices α1 and α3 and the C-terminus of α8. Symmetrical contacts occur: (i) between helices α3 of both protomers; and (ii) between helix α1 from one protomer and helix α8 from the other. The only specific interaction observed at the dimer interface is one hydrogen bond between the main chain oxygen of L37 from the first molecule and the side chain hydroxyl group of S39 from the second. Due to this scarcity of directed interactions, dimer formation imposes few restraints on sequence conservation for the residues involved. The 2-fold symmetry observed at the dimer interface is maintained by the two small α-helical domains, but does not extend throughout the entire dimer due to the presence of an intramolecular hinge region. Both ssAnPRT dimers comprise one protomer in the open and another in the closed conformation. Taking the structure of the larger α/β domain as reference, the small α-helical domain is rotated by ∼8° in one conformation with respect to the other, as calculated with the program DynDom (Hayward et al., 1997). The hinge opening is of the same magnitude in both dimers in this crystal form. This rotational difference translates only into small coordinate deviations within the hinge region but into amplified differences in the order of 4.5 Å at the peripheral regions of the two ssAnPRT domains (Figure 3C). Reflecting the observed flexibility in the domain–domain arrangement, there are only a small number of residue-specific interactions at the hinge interface between the two ssAnPRT domains (Figure 3B). One hydrogen bond is formed between a conserved residue pair, D76 (strand β1) and N181 (helix α9). Both residues are located at different segments connecting the small α-helical domain and the large mixed α/β domain via the hinge region. However, the other, non-conserved inter-domain interactions are from K52 (helix α4) to E207 (helix α10) and from R60 (helix α4) to the main chain carbonyl group of E207 (helix α10). These contacts are found in all four copies of ssAnPRT. Their different conformations indicate that there is inherent flexibility within the hinge region and that this property could be involved in the catalytic function of ssAnPRT or its regulation, possibly allowing even larger domain motions. AnPRT has an NP-II like fold There is no structural relationship between ssAnPRT and any other PRT of known structure. Thus, the structure of ssAnPRT constitutes a new PRT fold, designated here PRT-III. In contrast, the ssAnPRT structure reveals that its fold is related to that of the NP-II family (Figure 4), thus confirming a previous prediction from sequence data (Mushegian and Koonin, 1994). These data were based on the identification of amino acid similarities within three sequence segments in the NP-II family and AnPRT, including an N-terminal 60 residue segment and the TGGxG phosphate-binding motif, whereas no significant sequence similarity was detectable for the large α/β domain. Comparison of the new ssAnPRT structure with the two currently available NP-II structures, thymidine phosphorylase from Escherichia coli (ecTNP) (Walter et al., 1990; Pugmire et al., 1998) and pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus (bsPyNP) (Pugmire and Ealick, 1998), demonstrates that the fold similarity indeed extends to this domain. Although large, overall structural deviations originate from the different orientations of the small α-helical and large mixed α/β domains, the separated domains unambiguously show structural similarity. The small α-helical domains of ssAnPRT and bsPyNP superimpose with a root-mean-square deviation (r.m.s.d.) of 1.7 Å, using all matching main chain atoms of the coordinate sets in the program ALIGN_PDB (Cohen, 1997). The structures of the large mixed α/β domains deviate by ∼2.0 Å. Despite the structural resemblance, a structure-based sequence alignment confirms the lack of overall sequence similarity within the large α/β domain (Figure 2). The common fold translates into an identical topology of the shared secondary structural elements and their inter-domain connections, except for two inserts, which are specific to each one of the two families (Figures 2 and 4C). In ssAnPRT, the large mixed α/β domain contains a 40 residue insertion between strand β5 and helix α11. It folds into two additional β-strands, β6 and β7, which extend the shared β-sheet β1–β5. Conversely, the members of the NP-II family contain a C-terminal 100 residue extension beyond helix α15, which folds into an additional subdomain of unknown function (Figure 4C). Remarkably, the very C-terminus in the NP-II fold forms a β-strand, β6, which extends the shared β-sheet β1–β5 by one strand, instead of two (β6–β7) as observed in the structure of the ssAnPRT. Strand β6 is the only one in antiparallel orientation with respect to the shared β-sheet and it is structurally equivalent in NP-II and AnPRT. A structure-based sequence alignment reveals that 13 residues are structurally identical between these PRT and NP-II members (Figure 2). These residues are confined to segments covering helices α3, α4 and α8 from the small α-helical domain and to two loop regions, β1–α5 and β2–α6, from the large α/β domain. Interestingly, none of them are involved in dimer formation. One of the structurally conserved residues is E54 (helix α4), which is involved in salt bridge formation in all structures available. Another residue (D76), located at the hinge interface, is also conserved across ssAnPRT and the NP-II members of known structure. In the bsPyNP structure, this residue forms a salt bridge to K183 (numbering as for ssAnPRT, cf. Figure 2), a side chain, which is structurally equivalent to N181 in ssAnPRT. Furthermore, the structures of members of the NP-II family and ssAnPRT share the same type of dimeric arrangement (Figure 4B), by head-to-head interactions between equivalent secondary structure elements of the small α-helical domain. In the dimeric structure of bsPyNP, asymmetric binding of a substrate analogue to only one of the two protomers is observed. The small α-helical subunit of the liganded protomer is rotated by ∼21° when compared with the open conformation of the free protomer (Pugmire and Ealick, 1998). In contrast, the uncomplexed ecTNP structure presents a symmetric dimer (Pugmire et al., 1998), although differences in the hinge conformation of a few degrees were observed among different crystal forms of this structure, confirming limited hinge flexibility. The active site of ssAnPRT The active site of ssAnPRT needs to accommodate both PRPP and anthranilate, in a structurally appropriate arrangement to catalyse the replacement of the 1′-pyrophosphate group of PRPP by anthranilate (Figure 1). In the reaction catalysed by NP-II, the nucleoside base is reversibly replaced by a phosphate group in the 1′-position. The leaving group, RP, is closely related to the PRPP substrate of AnPRT. The similarity between the aromatic bases, namely pyrimidines and anthranilate, is more tenuous. Only the PRT reaction requires divalent metal ions (Bauerle et al., 1987; Yanofsky et al., 1999). By mapping residues conserved among members of the AnPRT family onto the crystal structure of ssAnPRT, it is possible to locate the active site to the hinge region between the small α-helical domain and the large mixed α/β domain. A deep groove opens to the protein surface, similar to the location of the active site in the available NP-II structures (Figures 5, 6 and 7). Figure 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. Download figure Download PowerPoint Figure 6.(2Fobs − Fcalc)αcalc 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. Download figure Download PowerPoint Figure 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. Download figure Download PowerPoint PRPP/metal-binding site To determine the precise binding site of PRPP, we have soaked crystals from ssAnPRT with an excess of PRPP, magnesium chloride and an anthranilate analogue. The diffraction limit of these complexed crystals was 2.7 Å resolution. A difference Fourier map calculated using X-ray data from complexed and unliganded crystals reveals the presence of a pyrophosphate group associated with a metal ion within the binding groove of the four molecule copies of this crystal form (Figures 6 and 7). The ribose-5-phosphate moiety of PRPP has, however, remained invisible, as well as the base analogue, suggesting that PRPP hydrolysis has occurred. A magnesium ion is bound between the pyrophosphate moiety and the D223–E224 motif, which is invariant among all AnPRT sequences (Figure 2), and S91 from helix α5. In the unliganded structure at 2.0 Å resolution, no solvent molecules are located between the carboxylate groups of D223 and E224, providing further indirect evidence for specific metal binding. In addition, the two ssAnPRT copies with a closed hinge conformation present another electron density peak between the carboxylate groups of D223 and E224. In the absence of further experimental evidence, this density has been interpreted to originate from a solvent molecule, although a second magnesium-binding site is possible. This solvent molecule is not involved in specific contacts with the pyrophosphate group. In one of the ssAnPRT copies, in the open conformation, there is only weak electron density at this site (data not shown). Co-ordination of the pyrophosphate group via metal binding is in agreement with observations from other PRTs for which complexed structures are available (Craig and Eakin, 2000; Sinha and Smith, 2001). This suggests that in AnPRT catalysis as well, the metal ion plays a role in activation of the pyrophosphate. The DE binding motif is located in the loop connecting strands β5 and β6, which is part of the AnPRT-specific insert (Figures 2 and 4). Thus, the presence of this insert establishes a specific function in AnPRT, PRPP-associated metal binding, which is neither present nor required in NP catalysis. In ssAnPRT, the pyrophosphate group is bound by the side chains of T92 (loop β1–α5) and K106 (strand β2) (Figure 7A). S118 (loop β2–α6), albeit in close proximity to pyrophosphate, is not involved in direct binding. In the structure of bsPyNP, a lysine residue structurally equivalent to K106 also provides a salt bridge to the phosphate substrate. In addition, a threonine, equivalent to S118 of ssAnPRT, provides a hydrogen bond to the phosphate (Figures 2 and 7). This comparison indicates that the pyrophosphate- and phosphate-binding sites in ssAnPRT and in the NP-II family, respectively, are shared, in particular, with respect to two loops involving their flanking secondary structural elements β1–α5 and β2–α6 from the large α/β domain (Figures 2, 4 and 7). The first loop contains a glycine-rich sequence motif DxxxTGG that was identified previously as the phosphate-binding motif in other unrelated enzymes (Kinoshita et al., 1999). Predicted anthranilate-binding site To date, no experimental data on the binding of anthranilate by AnPRT are available. However, in the structures of ecTNP and bsPyNP, the known binding sites of the respective thymidine or uridine substrate analogue base moieties (Walter et al., 1990; Pugmire and Ealick, 1998) allow conclusions on anthranilate binding by AnPRT to be drawn by analogy (Figure 7). In members of the NP-II family, one of the key residues in base recognition is an arginine on helix α8 that interacts with the lateral O-4 group of the pyrimidine (Figure 7B). The equivalent residue position in the AnPRT sequences, R164 (Figure 2), is invariant, suggesting a similar role in binding of anthranilate by possibly forming a salt bridge with its o-carboxylate group. This prediction permits the approximate localization of the anthranilate-binding pocket in ssAnPRT. Other conserved residues involved in pyrimidine binding in NP-II are, however, different in the AnPRT sequences. On the other hand, there are a number of residues in this pocket, such as H107, H152 and N174, which are invariant among the AnPRT sequences (Figure 2), implying a role in recognition of anthranilate (Figure 7). In the current crystal form of ssAnPRT, the binding sites for PRPP and anthranilate are separated by 14 Å (approximate distance between the guanidinium group of R164 and the proximal phosphate group of pyrophosphate) and are, most likely, too distant for catalysis. We, therefore, speculate that ssAnPRT may undergo further domain movements via the flexible hinge region during productive substrate binding, exceeding the conformational flexibility observed in the current structures. Discussion Common evolutionary ancestry of AnPRT and the NP-II family Comparison of the structures of ssAnPRT and the NP-II family demonstrates that they are related, confirming previous predictions (Mushegian and Koonin, 1994). Their relationship is manifested by the following: (i) the overall structural similarity of the two domains; (ii) their identical topologies, except strands β6 and β7; (iii) the identical location of 13 invariant residues across NP-II and AnPRT sequences, providing a number of inter- and intra-domain interactions common to both families; (iv) the observation of structurally similar and topologically related binding sites for the phosphate substrate of NP-II and the pyrophosphate moiety within the PRPP substrate of AnPRTs; and (v) the prediction of the same binding site for the base substrate, in which the invariant R164 (ssAnPRT sequence numbering) is expected to play a key role in anthranilate binding, indicating an equivalent function to the structurally identical arginine in the NP-II structures. Taken together, the multilevel similarities in topology, structure and sequence unambiguously indicate that AnPRT and members of the NP-II family are related, and thus imply a common ancestry. With these data, AnPRT constitutes the first evolutionary link between reverse, but distinct, metabolic ribosylation and deribosylation processes. In contrast, there are no detectable overall relationships between ssAnPRT and other PRTs. Members of the PRT-I family are involved in nucleotide biosynthesis or salvage pathways and consist of a major mixed α/β domain and a flanking hood domain, which is poorly conserved in sequence and structure (Craig and Eakin, 2000; Sinha and Smith, 2001). These PRTs contain a conserved 13 residue metal/PRPP-binding motif. Remarkably, this motif contains a loop with the sequence signature DxxxTGG (Phillips et al., 1999), which is identical to that of the first PRPP-binding loop in ssAnPRT as well as to a related glycine-rich motif in the NP-II family. Variations of this motif are also found in other phosphate group-binding enzymes (Kinoshita et al., 1999). In members of the PRT-I family and AnPRT sequences, even the less conserved residue positions ‘xxx’ within this loop are shared. In both sequence families, the first two residue positions are generally hydrophobic, whereas the third position is largely conserved as an alanine in PRT-I and as a glycine in the AnPRT sequences (Figure 2). Another PRT of known structure is quinolinic acid PRT, which is involved in de novo NAD biosynthesis and quinolinic acid metabolism. This enzyme comprises a two-domain structure, which was designated as PRT-II, and includes an N-terminal mixed α/β domain and an incomplete β/α-barrel (Eads et al., 1997). Its active site is located at the C-ter" @default.
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• W1997194941 title "Structural analysis of two enzymes catalysing reverse metabolic reactions implies common ancestry" @default.
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