Anthranilate phosphoribosyltransferase from the hyperthermophilic archaeon Thermococcus kodakarensis shows maximum activity with zinc and forms a unique dimeric structure

Anthranilate phosphoribosyltransferase (TrpD) is involved in tryptophan biosynthesis, catalyzing the transfer of a phosphoribosyl group to anthranilate, leading to the generation of phosphoribosyl anthranilate. TrpD belongs to the phosphoribosyltransferase (PRT) superfamily and is the only member of the structural class IV. X‐ray structures of TrpD from seven species have been solved to date. Here, functional and structural characterization of a recombinant TrpD from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkTrpD) was carried out. Contrary to previously characterized Mg2+‐dependent TrpD enzymes, TkTrpD was found to have a unique divalent cation dependency characterized by maximum activity in the presence of Zn2+ (1580 μmol·min−1·mg−1, the highest reported for any TrpD) followed by Ca2+ (948 μmol·min−1·mg−1) and Mg2+ (711 μmol·min−1·mg−1). TkTrpD displayed an unusually low thermostability compared to other previously characterized proteins from T. kodakarensis KOD1. The crystal structure of TkTrpD was determined in free form and in the presence of Zn2+ to 1.9 and 2.4 Å resolutions, respectively. TkTrpD structure displayed the typical PRT fold similar to other class IV PRTs, with a small N‐terminal α‐helical domain and a larger C‐terminal α/β domain. Electron densities for Zn2+ were identified at the expected zinc‐binding motif, DE(217–218), of the enzyme in each subunit of the dimer. Two additional Zn2+ were found at a new dimer interface formed in the presence of Zn2+. A fifth Zn2+ was found bound to Glu118 at crystal lattice contacts and a sixth one was ligated with Glu235. Based on the TkTrpD–Zn2+ structure, it is suggested that the formation of a new dimer may be responsible for the higher enzyme activity of TkTrpD in the presence of Zn2+ ions.

Anthranilate phosphoribosyltransferase (TrpD) is involved in tryptophan biosynthesis, catalyzing the transfer of a phosphoribosyl group to anthranilate, leading to the generation of phosphoribosyl anthranilate. TrpD belongs to the phosphoribosyltransferase (PRT) superfamily and is the only member of the structural class IV. X-ray structures of TrpD from seven species have been solved to date. Here, functional and structural characterization of a recombinant TrpD from hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (TkTrpD) was carried out. Contrary to previously characterized Mg 2+ -dependent TrpD enzymes, TkTrpD was found to have a unique divalent cation dependency characterized by maximum activity in the presence of Zn 2+ (1580 lmolÁmin À1 Ámg À1 , the highest reported for any TrpD) followed by Ca 2+ (948 lmolÁmin À1 Ámg À1 ) and Mg 2+ (711 lmolÁmin À1 Ámg À1 ). TkTrpD displayed an unusually low thermostability compared to other previously characterized proteins from T. kodakarensis KOD1. The crystal structure of TkTrpD was determined in free form and in the presence of Zn 2+ to 1.9 and 2. 4 A resolutions, respectively. TkTrpD structure displayed the typical PRT fold similar to other class IV PRTs, with a small N-terminal a-helical domain and a larger C-terminal a/b domain. Electron densities for Zn 2+ were identified at the expected zinc-binding motif, DE(217-218), of the enzyme in each subunit of the dimer. Two additional Zn 2+ were found at a new dimer interface formed in the presence of Zn 2+ . A fifth Zn 2+ was found bound to Glu118 at crystal lattice contacts and a sixth one was ligated with Glu235. Based on the TkTrpD-Zn 2+ structure, it is suggested that the formation of a new dimer may be responsible for the higher enzyme activity of TkTrpD in the presence of Zn 2+ ions.
Anthranilate phosphoribosyltransferase (TrpD, EC 2. 4.2.18) catalyzes the second step in tryptophan biosynthesis, which involves the transfer of a phosphoribosyl group to anthranilate to generate phosphoribosyl anthranilate (PRA), the basic skeleton of tryptophan (Fig. S1). TrpD belongs to the functional superfamily of phosphoribosyltransferases (PRTs) [1], which play important role in the metabolism of nucleotides and amino acids [2].
Phosphoribosyltransferases have been divided into four different classes on the basis of their tertiary structures [3,4]. Class I has a common a/b fold and comprises uracil, orotate, and purine PRTs. Class II has an N-terminal a/b sandwich domain and a Cterminal a/b TIM barrel domain. This class includes the quinolinate and nicotinic acid PRTs. Class III has a unique domain structure and includes ATP-PRTase. Class IV PRTs are limited to TrpD [5] and exhibit a homodimeric structure and a novel PRT fold, consisting of a small N-terminal a-helical domain connected to a large C-terminal a/b domain by a hinge region [6]. The X-ray structures of TrpD enzymes from Sulfolobus solfataricus (SsTrpD; PDB entry 2GVQ) [7], Pectobacterium carotovorum (PcTrpD; PDB entry 1KHD) [8], Mycobacterium tuberculosis (MtbTrpD; PDB entry 4X5B) [ [8,10]. These divalent cations have been implicated in phosphoribosyl pyrophosphate (PRPP) complexation, which induces prominent ordering of a conserved Gly-rich loop GTGGD in TrpD [7].
Here, we report the biochemical and structural characterization of TrpD (TkTrpD) from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1, an obligate heterotroph that grows optimally at 85°C and pH 6.5 [11]. The gene encoding TkTrpD was expressed in Escherichia coli and the recombinant gene product was purified, characterized, crystallized and its crystal structure was determined in free form as well as in the presence of Zn 2+ to 1.9 and 2.4 A resolutions, respectively. The results would provide a better understanding of the TrpD family of enzymes and help in biotechnological applications to synthesize compounds for use in biochemical assays [12,13]. Moreover, TrpD has also emerged as a potential candidate for biomedical applications. The importance of TrpD has been emphasized by a genome-wide transposon mutagenesis study in M. tuberculosis [14], which showed that the enzymes responsible for the biosynthesis of PRPP as well as biosynthetic enzymes that use PRPP, such as TrpD, are essential for mycobacterial growth [14,15].

Results and Discussion
Production and purification of TkTrpD The TkTrpD gene (KEGG entry: TK0253) consists of an open reading frame (ORF) of 978 nucleotides, encoding for a polypeptide of 325 amino acid residues with a theoretical molecular mass of 34346.16 Da and pI of 4.9. TkTrpD was produced in E. coli and purified to homogeneity using heat treatment and ion-exchange chromatography. Purified recombinant TkTrpD exhibited a molecular weight of about 36 kDa (Fig. 1), matching the molecular weight calculated from the amino acid sequence. By gel filtration chromatography, the molecular mass of TkTrpD was estimated to be 70 kDa, indicating that TkTrpD is a homodimer in solution (Fig. S2).

Effect of pH and temperature
The optimal pH for TkTrpD activity was found to be 8.5-9.0 (Fig. S3). The effect of temperature on TkTrpD activity was examined at optimal pH. TkTrpD exhibited highest activity at 55°C (Fig. 2) although the optimal growth temperature of T. kodakarensis is 85°C. This result is in contrast to most of the enzymes from hyperthermophiles but similar to ribose-5-phosphate pyrophosphokinases from T. kodakarensis [16] and Pyrobaculum calidifontis [12], and phosphoribosyl diphosphate synthase from S. solfataricus [17]. It should be noted that a protective mechanism of protein stabilization in hyperthermophiles has been suggested involving the secretion of small-molecule osmolytes in stressful conditions [18]. A similar mechanism may apply to TkTrpD to increase its stability and prevent unfolding at elevated temperatures.

Cation dependency
Anthranilate phosphoribosyltransferase enzymes from E. coli, S. typhimurium, Saccharomyces cerevisiae, S. solfataricus, and M. tuberculosis have been reported to be dependent on Mg 2+ for enzymatic activity [15,19,20]. Pectobacterium carotovorum and S. typhimurium TrpDs have been reported to be activated by Mn 2+ [8,10]. Addition of EDTA completely inhibited the enzymatic activity of TkTrpD, indicating the dependency of the enzyme on metal cations. The effect of various cations on TkTrpD was therefore examined (Fig. 3). Surprisingly, addition of Zn 2+ and Ca 2+ led to higher specific activities than Mg 2+ , whereas Cu 2+ , Ni 2+ , Co 2+ , and Mn 2+ showed lower activities. The decrease in enzyme activity in the presence of Co 2+ and Mn 2+ may be attributed to the slight precipitation of TkTrpD in the presence of these metal ions.

Effect of cation concentration
TkTrpD activity increased with the addition of Zn 2+ until the Zn 2+ concentration reached 100 lM. Higher concentrations of Zn 2+ significantly inhibited the reaction (Fig. 4A). In the case of Mg 2+ , the activity was maximal at 200 lM; however, concentrations above 200 lM also decreased enzyme activity (Fig. 4B).

Kinetic parameters
The effect of substrate concentration on TkTrpD activity was investigated in the presence of Zn 2+ . Anthranilate and PRPP were the two substrates used in the assays. The first substrate, PRPP, was kept constant at 1 mM during the measurement of the kinetic parameters toward anthranilate. Similarly, the second substrate, anthranilate, was kept constant at 4 lM when the kinetic parameters toward PRPP were measured. Anthranilate concentrations above 4 lM resulted in reduced enzymatic activity, suggesting substrate inhibition by anthranilate as observed also in M. tuberculosis TrpD [21]. Apparent K m values for anthranilate and PRPP were 2.2 lM and 250 lM, respectively (Fig. 5). TkTrpD was highly active with specific activity of 1580 lmolÁmin À1 Ámg À1 . To the best of our knowledge, this is the highest enzyme activity for any TrpD reported so far. A comparison of kinetic  parameters and specific activities of characterized TrpDs from various sources is shown in Table 1.

Quality of the TkTrpD structure
TkTrpD crystallizes with four molecules (A, B, C, and D) in the asymmetric unit that form two homodimers (A-C and B-D) (Fig. 6). The Matthews coefficient V M [22] for four molecules in the asymmetric unit is 2.4 A 3Á Da À1 , corresponding to a solvent content of $ 48.5%. The refined structure shows a root mean square deviation (rmsd) of 0.012 A and 1.15°from the ideal values of bond lengths and angles, respectively. The observed crystal form of TkTrpD soaked with ZnCl 2 has a dimer (A, B) in the asymmetric unit. The Matthews coefficient V M for two molecules in the asymmetric unit is 2.3 A 3 ÁDa À1 , corresponding to a solvent content of~46.2%. As the crystals used for the Zn 2+ soaking had been grown in different conditions, unsoaked crystals were tested and found to have similar space group and cell dimensions as those of the free TkTrpD, suggesting that soaking with Zn 2+ induced a rearrangement of the crystal packing. The refined structure shows an rmsd of 0.010 A and 1.41°f rom the ideal values of bond lengths and bond angles, respectively. Detailed statistics of data collection and refinement for both structures are presented in Table 2.  seven b-strands (six parallel and one antiparallel) surrounded by eight a-helices. A hinge region (a4-b1, b3-a8, and a9-b4) connects the two domains. The N-terminal domain is involved in dimer formation in TrpD enzymes (SsTrpD, MtbTrpD, TtTrpD, AsTrpD, XcTrpD, NsTrpD, and PcTrpD). Similarly, TkTrpD subunits also associate with each other at their N-terminal ends through their small a-helical domains (C-terminal end of a1, a3, and a8). In SsTrpD, residues Ile36 and Met47 have been shown to be involved in dimerization. Mutations of these residues resulted in loss of dimeric form with decreased thermal stability [2]. Both of these residues are not conserved in TrpD family. The corresponding residues in TkTrpD are Val31 and Thr42. Analysis of proteinprotein interactions with PDBsum [23] shows that Ala35, Thr42 (located at the N and C termini of a3, respectively) and Leu162 (C-terminal end of a8) are found to form highest number of inter-subunit interactions, showing that mostly hydrophobic residues are involved in inter-subunit interactions in TkTrpD dimer formation in agreement with dimer formation in other TrpD enzymes.

Active site
Each monomer has an active site in a cleft found in the hinge region. In TkTrpD, substrate (anthranilate + PRPP) binding positions were found conserved as in other TrpDs. A conserved anthranilate binding motif (KHGN(101-104)) was found in b2-a6 loop. Lys101, in particular, is involved in anthranilate binding and HGN(102-104) in PRPP binding. This motif has been determined to be involved in catalysis in previously determined TrpD structures (e.g., SsTrpD, MtbTrpD). Arg159 in TkTrpD found on helix a8 is also conserved and in previous structures [7,15] has been shown to be involved in anthranilate binding by forming hydrogen bond to it and is essential for catalytic function. The corresponding residues in MtbTrpD and SsTrpD are Arg193 and Arg164, respectively. A highly conserved Gly-rich sequence GTGGD(74-78) found in TkTrpD in b1-a5 loop is considered as a signature motif of TrpD family and is involved in PRPP binding. Identical sequences have been found in MtbTrpD (GTGGD(107-111)) and in SsTrpD (GTGGD(79-83)). The first Gly of this region, in particular, is known to interact with the PPi group of PRPP via its peptide amino group and also with the amino group of anthranilate.

Divalent ion binding sites
Metal ions bind to two sites in the TrpD family. The first metal ion binds to pyrophosphate and ribose oxygen atoms of PRPP, and this site is common in PRT superfamily. The second site is specific for TrpD family and involves a conserved DE motif whose residues are key to metal binding and are invariant in all TrpD enzymes structurally characterized until now ( Table 3).
Soaking of TkTrpD with a ZnCl 2 solution resulted in the identification of a total of six Zn 2+ ions in the dimer, while in other TrpDs only four Zn 2+ ions per dimer are present. In each subunit of TkTrpD, one Zn 2+ ion was found in the primary metal binding site, involving the conserved DE(217-218) motif and Asp78. As PRPP is not present in the structure, no additional Zn 2+ ion was found in the vicinity of the primary metal binding site. The structure of TkTrpD soaked with zinc has a dimer in the asymmetric unit. Gel filtration has also shown that in the presence of Zn 2+ , TkTrpD exists as a dimer in solution (Fig. S2). Structural comparison of the Zn 2+ -free and Zn 2+bound structures at subunit level shows low rmsd between Ca atoms (0.63 A), suggesting no significant changes. Notable changes, however, were identified in the position of helices a8 and a9 that move toward the active site in the Zn 2+ -bound structure. Most importantly, following superposition with the Zn 2+ -free TkTrpD (Fig. 9A), the structure solution of the Zn 2+bound TkTrpD revealed a different arrangement of the two subunits compared to the typical dimer found in other TrpD enzymes. Interestingly, two Zn 2+ ions (V and VI) were found at the interface between Glu48 and Glu198 of subunit A and ED(295-296) of subunit B in the dimer. The two zinc ions are close to each other with a distance of 2.9 A (Fig. 9B) and have similar B-factors (43.7 and 46.0 A 2 , respectively). These two additional Zn 2+ binding sites in TkTrpD may therefore explain the effect of Zn 2+ on TkTrpD by promoting a different dimer formation. At present, we cannot conclude whether this property is shared by this enzyme from other sources as well, or whether it is a unique property of TkTrpD. However, the structure-based alignment (Fig. 8) shows that the ED (295-296) motif is not conserved, and therefore, other TrpDs may be unable to adopt the same dimer arrangement. Sequence variations are also evident for Glu48 and Glu198.
In conclusion, the biochemical and structural characterization of TkTrpD reported here may lead to new strategies to alter TrpD enzymatic activity. The new subunit-subunit interface may play a role in the increased activity of TkTrpD in the presence of Zn 2+ . For example, Glu198 belongs to helix a10 and slight alterations upon Zn 2+ binding and dimer rearrangement could be traversed to the active site through the a8 and a9 helices. Alternatively, formation of the new dimer may affect the position of helix a8, which in the typical TrpD dimer is part of the conventional interface. In the new dimer, helix a8 becomes free from any interactions with a neighboring subunit, and therefore, it may be able to adopt more favorable positions for substrate binding. Further studies, however, are needed to elucidate the precise role of the Zn 2+ -binding sites and their potential direct and indirect effects on the active site of the enzyme.

Gene cloning
Gene encoding TkTrpD was amplified from genomic DNA of T. kodakarensis, using sequence-specific forward FTk-TrpD (CATATGAGCCTTCTTGCGAAGATCGTCGAT GG), which include a NdeI recognition site (shown in boldface) and reverse RTkTrpD (TCAGCTTTTTGAGA GGCATGCTATCTCCTC) primers. PCR-amplified gene product was ligated to cloning vector pTZ57R/T. The resultant recombinant plasmid PTZ-TkTrpD was digested with NdeI and HindIII to liberate TkTrpD, which was cloned into the expression vector pET21a(+) using the same restriction sites. pET-TkTrpD name was assigned to the resultant recombinant expression plasmid. Presence of TkTrpD in the expression plasmid was subsequently confirmed by restriction analysis and DNA sequencing.  Ribbon diagram of X-ray crystal structure of TkTrpD. Only one subunit of the homodimer is shown, with the amino acid chain colored from blue at the N terminus to red at the C terminus. Each subunit consists of a small a-helical domain containing four helices (a1, a2, a3, and a4) and a larger C-terminal a/b domain with a central b-sheet containing seven b-strands (six parallel and one antiparallel) surrounded by eight a-helices. Figure was created using UCSF Chimera.  [9]. Figure was created using ESPRIPT 3.0 [51]. Conserved residues are indicated by white letters on a red background (strictly conserved) or red letters on a white background (global similarity score, 0.7) and framed in blue boxes. Markers indicate residues postulated to be involved in PRPP binding (magenta arrows), anthranilate binding (green stars), metal binding (blue ovals), and dimerization (red boxes). Residues involved in Zn 2+ binding at the TrpD-Zn 2+ dimer interface are shown with brown ovals. were harvested and resuspended in 50 mM Tris/HCl pH 8.5 buffer containing 1 mM DTT, 1 mM PMSF, and 20% v/v glycerol. For purification, soluble portion obtained after sonication was heat-treated at 65°C for 25 min and centrifuged (15 000 g for 15 min). € AKTA Purifier chromatography system (GE Healthcare, Uppsala, Sweden) was used for further purification. Heat-treated supernatant was applied to anionexchange QFF (6 mL) column (GE Healthcare) and the recombinant TkTrpD was eluted with a linear gradient of 0-1 M NaCl. Fractions containing TkTrpD were desalted by dialysis against 50 mM Tris/HCl (pH 8.5) buffer containing 1 mM DTT, 1 mM PMSF, and 20% v/v glycerol. Dialyzed TkTrpD samples were applied to Resource Q (1 mL) column (GE Healthcare), and the protein was eluted with a linear gradient of 0-1 M NaCl. Analysis of the purified TkTrpD was performed by SDS/PAGE. Molecular weight and oligomeric nature of TkTrpD were determined by gel filtration chromatography column Superdex 75 10/300 GL attached to € AKTA purifier (GE Healthcare). The standard curve was obtained with bovine pancreas chymotrypsinogen A (25 kDa), chicken egg white ovalbumin (48 kDa), and bovine serum albumin (63 kDa). Their gel-phase distribution coefficient (K av ) values were calculated and plotted against the log of their molecular weight (Fig. S3). Protein concentration was determined spectrophotometrically at every step of purification by Bradford reagent [24].

Enzyme assays
TkTrpD activity was determined fluorometrically by measuring the decrease in the concentration of anthranilic acid. Anthranilic acid reacts with PRPP, leading to the production of PRA (Fig. S1). The initial rate of decrease in anthranilate was measured, as anthranilate is utilized by TkTrpD to form PRA, resulting in a decrease in emission/ fluorescence at 390 nm. The activation and emission wavelengths for anthranilate were 315 and 390 nm, respectively. A standard curve was then used to convert fluorescent intensity to anthranilate concentration. The reaction mixture contained 4 lM anthranilate, 1 mM PRPP, 100 lM ZnCl 2 , 100 mM Tris/HCl buffer (pH 8.5), and 5 lg of TkTrpD. The reaction mixture without PRPP was incubated at 55°C for 5 min. The reaction started by adding PRPP at 55°C and continued for 2.5 min. Two control experiments were carried out: one without enzyme and one without PRPP. The half-life of PRPP is 56 min at 60°C [20], suggesting that at 55°C and for the time used for the reaction, no significant hydrolysis of PRPP is expected.

Effect of temperature, pH, and metal ions
For the measurement of optimal temperature, enzyme assays were performed at various temperatures ranging from 35 to 85°C keeping the pH constant. For the estimation of optimal pH, assays were performed at various pH values keeping the temperature unchanged at 55°C. The following buffers were used: Na-phosphate (pH: 6.0-7.0), Tris/HCl (pH: 7.0-9.0), and Na-bicarbonate (pH: 9.0-10.0). The effect of divalent metal ions on the enzyme activity was investigated in the presence of either 50 or 100 lM of ZnCl 2 , MgCl 2 , CaCl 2 , MnCl 2 , NiCl 2 , CoCl 2 , and CuCl 2 . In case of EDTA, the final EDTA concentration was 100 and 2.5 mM. The effect of Zn 2+ and Mg 2+ concentration on the enzyme activity was measured in the range of 0-1 mM.

Crystallization
Purified TkTrpD was concentrated to 12 mgÁmL À1 in 10 mM Tris/HCl (pH 8.0) buffer containing 0.1 M NaCl and 0.002% (w/v) NaN 3 . PACT screen (Molecular Dimensions, Suffolk, UK) was performed in 96-well plate using the sitting drop vapor diffusion method. Promising crystals found in solution 79 (0.2 M sodium acetate (pH 7.5), 20% (w/v) PEG 3350) were optimized by the hanging-drop vapor diffusion method at 16°C in Linbro 24-well cell culture plates. The reservoir solution consisted of 0.6 mL of condition 79 mixed with 0.2 mL of MilliQ water and the drops comprised 2 lL of 12 mgÁmL À1 TkTrpD mixed with 2 lL of reservoir solution. Crystals appeared after 1 day and were harvested after~4 weeks for X-ray data collection. Crystals were transferred to a reservoir solution supplemented with 20% v/v glycerol and flash-cooled in liquid N 2 .

Ions
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Data collection and structure determination
Diffraction data for the free TkTrpD were collected at ESRF (Grenoble) on the fully automatic high-throughput MASSIF-1 beamline [25] from a crystal that diffracted to 1.9 A. XDS [26] was used to index and integrate the data and AIMLESS [27] for merging and scaling. The crystal was found to belong to the P2 1 2 1 2 1 space group. SsTrpD (PDB entry 2GVQ) [7] was found to be the best matched search model by MOLREP [28] as implemented in MRBUMP [29] from CCP4 [30] and was used to obtain initial phases. After the solution was found, BUCCANEER [31] was employed for initial model building and automatic refinement with REFMAC5 [32]. Further refinement was carried out using PHENIX and water molecules were added with tools in PHENIX [33]. Manual rebuilding and structure visualization was performed by COOT [34]. The progress of refinement was monitored using the R free [35] with 5% of the reflections set aside.

ZnCl 2 soaking
Data from a crystal soaked with ZnCl 2 were collected at EMBL Hamburg (c/o DESY, Hamburg, Germany) on the P13 beamline at PETRA III and processed as before. Chain A of free TkTrpD crystal structure was used as search model in PHASER for structure determination by molecular replacement. The best solution was found in the orthorhombic P22 1 2 1 space group. Refinement was initially carried out using PHENIX and water molecules were added with tools in PHENIX. At the final stages of refinement, PDB_REDO [36] was employed and REFMAC [32] was used. Manual rebuilding and structure visualization was performed by COOT [34]. The progress of refinement was monitored with the R free .

Structure analysis
Interfaces were analyzed by PDBePISA [37]. Structural superpositions were performed with PDBeFold [38] as implemented in COOT [34]. The superimposed structures were visually inspected using COOT.

Supporting information
Additional Supporting Information may be found online in the supporting information tab for this article: Fig. S1. Reaction catalyzed by TrpD. Fig. S2. Gel filtration elution profile of TkTrpD with and without Zn 2+ . Fig. S3. Determination of optimal pH for TkTrpD enzymatic activity.