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Journal of Bacteriology, December 2007, p. 9030-9036, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01161-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Orotate Phosphoribosyltransferase from Corynebacterium ammoniagenes Lacking a Conserved Lysine ^,

Xing Wang,^1,2 Cuiqing Ma,² Xiuwen Wang,² and Ping Xu^1,2*

Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China,¹ State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China²

Received 23 July 2007/ Accepted 26 September 2007

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The pyrE gene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting from Corynebacterium ammoniagenes ATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) in the C. ammoniagenes OPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinant Escherichia coli strain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn²⁺ and Co²⁺. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. The K_m values for the four substrates were determined to be 33 µM for orotate, 64 µM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 µM for orotidine-5-phosphate (OMP), and 36 µM for pyrophosphate. The K_m value for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.

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Orotate phosphoribosyltransferase (OPRTase) is one of 10 phosphoribosyltransferases (PRTases), which are required in de novo and salvage pathways of nucleotide synthesis, as well as histidine and tryptophan formation (10, 18, 35). The enzyme catalyzes the reaction between orotate and the ribose-5-phosphate donor 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5-phosphate (OMP) and pyrophosphate. In humans, the bifunctional OPRTase-OMP decarboxylase (UMP synthase) reaction serves to activate the prodrug 5-fluorouracil, and genetic defects in the enzyme result in human pathologies, such as orotic aciduria, 2,8-dihydroxyademine lithiasis, and Lesch-Nyhan syndrome (35). In prokaryotes, OPRTase represents an attractive target for the rational design of antimicrobial, antiparasitic, and anticancer theapeutics (15, 29). Furthermore, the gene encoding OPRTase (pyrE) can be used as a genetic handling marker in many microorganisms: strains deficient in pyrE become resistant to the bactericidal compound 5-fluoroorotic acid, while the deficient strains are uracil auxotrophs. Thus, both wild-type and pyrE-deficient strains can be positively selected (37). Because of these key roles in nucleotide metabolism and the ubiquitous distribution, the pyrE genes from many organisms have been cloned and sequenced (7, 9, 13, 14, 22-24, 33), and more than 13 homologous enzymes that carry out the OMP formation reaction have been identified in bacteria, fungi, insects, and mammals (26, 29). Although there is little similarity among these sequences, the OPRTases were considered to have a common fold of a conserved Rossman nucleotide binding fold with a variant "hood" structure and to belonged to the type I PRTases (26, 35).

The biochemical mechanism of catalysis by PRTases has been studied for decades and is perhaps best known for OPRTase (35). The kinetic mechanism (6, 17, 31, 35), active site (10, 12, 20), crystal structure (1, 21, 25-27), transition state structure (29), and motional dynamics of a catalytic loop (34, 35) have all been studied. However, there are still some questions that need to be answered, such as the essential roles of the conserved dicarboxylate motif and metal ion in catalysis (29), the relation between ligand binding and catalytic loop opening (34), and the different product release rates between forward and reverse reactions (34, 35). Therefore, knowledge about more properties of other OPRTases is required to provide answers for these questions.

Coryneform bacteria have been widely used in the large-scale production of amino acid and nucleotides. However, except for several entirely sequenced species, no research has been reported on the pyrE gene and neither is there information about the OPRTase. In addition, in the completely sequenced strains of the genus Corynebacterium, the amino acid sequences of OPRTase deduced from putative pyrE genes are all defective in two lysines (Lys 26 and Lys 73 in Salmonella enterica serovar Typhimurium OPRTase) which are conserved in the reported OPRTase and are essential to catalysis in S. enterica serovar Typhimurium OPRTase (20). The loss of the conserved residues in the active site may indicate a diversity of the properties of OPRTase in the genus Corynebacterium, and studies on these enzymes may be helpful in understanding the catalytic mechanism of OPRTase.

In this study, the pyrE gene of Corynebacterium ammoniagenes ATCC 6872, the industrial organism for pyrimidine nucleotide production (36), was cloned together with 63 nucleotide residues upstream of the coding frame. The OPRTase was purified from a recombinant Escherichia coli strain, in which the enzyme was overproduced and characterized.

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Enzymes and chemicals. Restriction enzymes and other DNA-modifying enzymes were obtained from TaKaRa Bio. Inc. (China). PRPP and OMP were purchased from Sigma-Aldrich. All other chemicals were of reagent grade and commercially available.

Bacterial strains, media, and culture conditions. The genotypes and descriptions of strains and plasmids are given in Table 1. Luria-Bertani (LB) medium was used as a liquid medium for both E. coli and C. ammoniagenes. C. ammoniagenes (type strain ATCC 6872) was grown aerobically at 30°C on a rotary shaker platform, while E. coli was grown at 37°C. Ampicillin was used at a concentration of 100 µg ml^–1, while chloramphenicol was used at 10 µg ml^–1 and isopropyl-thio-β-D-galactoside (IPTG) at 1 mM.

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TABLE 1. Strains, plasmids, and primers used in this work

Cloning of the pyrE gene. The pyrE gene from C. ammoniagenes ATCC 6872 was cloned by colony blotting methods, which were carried out with the ECL direct nucleic acid labeling and detection system (Amersham Biosciences Corp., Piscataway, NJ) in accordance with the manufacturer's recommendations. Nested PCR was performed to prepare the probe DNA with primers designed based on the completely sequenced genomes of several Corynebacterium spp., including C. glutamicum ATCC 13032 (accession no. BX927147), C. diphtheriae NCTC 13129 (BX248353), C. efficiens YS-314 (BA000035), and C. jeikeium K 411 (CR931997). Primers P1 and P2 (Table 1) were used for the first round of amplification. Total genomic DNA of C. ammoniagenes ATCC 6872 extracted with the Wizard genomic DNA purification kit (Promega, Madison, WI) was used as the template for the first round of PCR. One microliter of the PCR products from the reaction mixture was used as the template for the second round of PCR with primers P3 and P4. The final PCR product was purified with the Biospin gel extraction kit (BioFlux, Japan) and labeled as the probe DNA for colony screening. The DNA fragments produced from the first round of PCR were ligated together into a pMD18-T vector (TaKaRa) and transformed into E. coli DH5. Ampicillin-resistant colonies grown on agar plates were lifted by using a Hybond-N⁺ nylon hybridization transfer membrane (Amersham Biosciences Corp.) and were lysed with 0.5 M NaOH, followed by fixation and rinsing in 5x SSC (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Hybridization was then performed with horseradish peroxidase-labeled probe, and luminol generated by the enzyme was used for light detection. Southern hybridization experiments were performed using a digoxigenin DNA labeling and detection kit (Roche). Probes were prepared by random-primer labeling with digoxigenin according to the manufacturer's instructions. Hybridization was performed overnight at 68°C. Filters (positively charged nylon transfer membranes) were washed under conditions of high stringency.

Purification of OPRTase. The pyrE coding region was subcloned into the PstI/EcoRI site of pBV220 (Table 1), which is an expression vector induced at 42°C. The resulting plasmid was named pBVPYRE. The OPRTase was purified from a culture of E. coli DH5 containing the plasmid grown at 42°C. The harvested cells were resuspended in buffer T (50 mM Tris-HCl, 2 mM EDTA, 2 mM mercaptoethanol, pH 6.4) and disrupted by sonication. Lysates were centrifuged to remove debris. The turbid supernatant fraction was clarified by addition of 0.1% protamine sulfate (Sigma). The resultant precipitate was removed by centrifugation. The precipitation was followed by (NH₄)₂SO₄ fractionation (30 to 70% saturation) of the supernatant. The redissolved precipitate was then dialyzed overnight and applied to a DEAE-Sepharose Fast Flow column previously equilibrated with buffer T. The protein was eluted with a linear gradient of 0 to 0.3 M NaCl in buffer T. The active fractions were combined and concentrated by ultrafiltration, and the enzyme solution obtained was applied to a Superdex-75 column (Amersham Biosciences) which had been equilibrated with buffer T without EDTA. The column was washed with the same buffer. The pool with OPRTase activity was stored at 0 to 4°C.

Enzyme assay. Measurements of the initial velocities of the phosphoribosyl transfer reaction were carried out according to the method of Umezu et al. (30). The value for the conversion of orotate to OMP was determined to be 3,780 M^–1 cm^–1 at 298 nm under our experimental conditions. The OPRTase reaction was performed in a buffer containing citric acid, KH₂PO₄, boric acid, and barbital (CKBB buffer). The buffer was adjusted to the appropriate pH with 200 mM NaOH. One unit of enzyme activity is defined as the amount of OPRTase required to convert 1 µmol of orotate to OMP per minute. Protein was measured by the method of Lowry et al. (16).

Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using a 12.5% polyacrylamide resolving gel and a 4% polyacrylamide stacking gel.

Nucleotide sequence accession number. The nucleotide sequence of the pyrE gene has been deposited in the GenBank nucleotide sequence databases under accession no. EU123869.

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Cloning of pyrE. The entire genomes of four Corynebacterium strains, i.e., C. glutamicum ATCC 13032, C. diphtheriae NCTC 13129, C. efficiens YS-314, and C. jeikeium K 411, have been sequenced. The annotated pyrE genes have about 70% sequence homology among these strains. Based on the putative pyrE genes from the completely sequenced strains, primers P1 and P2 were designed to clone the pyrE gene, encoding OPRTase, from C. ammoniagenes ATCC 6872. However, the amplification generated several fragments near 600 bp, which were expected to be pyrE and hardly to be separated by excision from the gel. The presence of pyrE in these fragments was confirmed by nested PCR with primers P3 and P4, which were targeted to a relatively conserved region of pyrE in Corynebacterium strains. A single 274-bp fragment was obtained after the second PCR and was found to be identical to that of pyrE from C. glutamicum ATCC 13032, indicating the presence of pyrE of C. ammoniagenes in the amplification products generated in the first PCR with primers P1 and P2. From E. coli clones carrying fragments derived from the first PCR, a 623-bp fragment was identified by colony blotting. Sequence analysis revealed that the fragment (p26) consisted of a 555-bp coding region identical to the pyrE gene from C. glutamicum ATCC 13032 and a 63-bp region upstream from the initiation trinucleotide, ATG. Southern blot analysis performed under stringent conditions confirmed that this fragment indeed existed in C. ammoniagenes ATCC 6872 (see Fig. S1 in the supplemental material).

Sequence alignment. The cloned pyrE gene (555-bp coding region) from C. ammoniagenes showed almost 100% identity to that annotated in the C. glutamicum genome. However, for the gene of the latter organism, no functional data are known. Therefore, the amino acid sequence of the cloned OPRTase, as deduced from the nucleotide sequence, was compared with the reported OPRTase sequences from other organisms (Fig. 1).

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FIG. 1. Multiple alignment of the deduced amino acid sequences of OPRTase genes of selected species. Species and accession numbers (SwissProt) of sequences are as follows: E. coli (Ecol), P0A7E3; S. enterica serovar Typhimurium (Styp), P08870; Saccharomyces cerevisiae (Scer), P30402; C. ammoniagenes (Camm), this study; Thermus thermophilus (Tthe), P61499; and Rhizobium leguminosarum (Rleg), P42719. Relatively conserved residues are shaded. The active-site amino acid residues of S. enterica serovar Typhimurium OPRTase are marked as follows: :, orotate ring binding region; *, ribose ring binding region; +, 5'-phosphate binding region; =, PPi binding region; —, catalytic loop. Lys 73 is boxed.

Although the OPRTases from E. coli and Salmonella enterica serovar Typhimurium, which had been studied in detail, showed only 28% similarity with the cloned enzyme, a PRPP binding motif composed of two carboxyl residues and the flanking hydrophobic residues (29) is found in the protein (Val 121 to Gly 131) as expected, indicating that the protein belongs to the PRTases which contain this highly conserved motif. Comparative sequence analysis also revealed a solvent-exposed catalytic loop, the movement of which was proposed to be integral to catalysis by type I nucleotide synthase (35). The essential residues (Arg 99, Lys 100, and Lys 103) (10, 20, 21) are also found in the enzyme.

However, it is noteworthy that several amino acid residues in active site of the S. enterica serovar Typhimurium OPRTase (26) cannot be assigned in the C. ammoniagenes sequence by the alignment (Fig. 1). Some of these residues are displaced by other residues with similar side chains or similar polarity. For example, the Phe 34 in the orotate ring binding region of the OPRTase from S. enterica serovar Typhimurium (25) was replaced by another aromatic residue, Tyr 39, in the OPRTase of C. ammoniagenes. Lys 26 was replaced by another polar residue, Ser 31. Although these displacements may change the interaction between orotate and the protein or influence the k_cat, the roles of these residues in catalysis would not change very much. However, the loss of Lys 73, which should extend into the active site, interact with either the 5'-phosphate of OMP or the 2-hydroxyl and -phosphoryl oxygen of PRPP, and be essential for the enzyme activity, indicates that this OPRTase may exhibit properties different from those of the reported OPRTases. Therefore, the gene was expressed and the enzyme was purified to be characterized.

Purification of C. ammoniagenes OPRTase. The purification (Table 2) yielded an electrophoretically homogeneous enzyme (Fig. 2). The specific activity of the product was 423 ± 32 units mg^–1 protein, which was a nearly 20-fold increase compared to that in the crude extract. The purified enzyme was routinely stored at 4°C in buffer T, pH 6.4. Under these conditions, the enzyme was stable for months with no apparent loss of activity and was used throughout the studies described below.

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TABLE 2. Purification of OPRTase of C. ammoniagenes ATCC 6872a

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FIG. 2. Overexpression and purification of OPRTase. Lane M, marker proteins; lane 1, cell extract of induced culture sample of E. coli DH5(pBVPYRE); lane 2, after ammonium sulfate precipitation; lane 3, pooled fraction containing OPRTase after DEAE-Sepharose; lane 4, purified OPRTase after Superdex-75 (about 3.5 µg).

Properties of purified OPRTase. The OPRTase of C. ammoniagenes ATCC 6872 has a predicted molecular mass of 19.4 kDa and a slightly acidic pI of 5.40 according to the derived sequence. By electrophoresis in a 12.5% polyacrylamide gel containing sodium dodecyl sulfate, a molecular mass of 21.3 ± 0.6 kDa was found for the subunit of OPRTase (Fig. 2). The molecular mass of the native OPRTase was estimated by gel filtration on a Superdex 75 10/300 GL column (Amersham Biosciences) with blue dextran 2000, albumin (67 kDa), peroxidase (44 kDa), and RNase A (13.7 kDa) as markers. The enzyme was determined to be 45.4 ± 1.5 kDa, indicating a dimeric structure of the native enzyme like those in other organisms.

Divalent metal ions such as Mg²⁺ or Mn²⁺ are required for activation of all PRTase-catalyzed reactions (5, 32). For the C. ammoniagenes OPRTase, the maximal rate of 456 ± 23 units mg^–1 was achieved at 1 to 3 mM Mg²⁺ like that described by Bhatia and Grubmeyer (5), and then the OPRTase activity decreased slowly with increasing Mg²⁺ concentration (Fig. 3a).

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FIG. 3. Properties of C. ammoniagenes OPRTase. Enzyme reactions were carried out for forward or reverse reactions in CKBB buffer of the appropriate pH. For the forward reaction, the reaction mixture contained 250 µM orotate, 200 µM PRPP, 3 mM MgCl2, and 0.1 µg purified OPRTase. For the reverse reaction, the assay solution contained 2 mM pyrophosphate, 3 mM MgCl2, 1 mM OMP, and 0.1 µg purified OPRTase. The enzyme activity was determined by measuring the decrease in absorbance at 298 nm. (a) Effect of Mg2+ on enzyme activity. The concentration of MgCl2 was varied from 0 to 8 mM at pH 8.0. (b) Effect of pH on enzyme activity. The optimum pH was assessed for both forward and reverse reactions in CKBB buffer of various pHs (4.0 to 12.0). (c) Effect of temperature on OPRTase activity. Enzyme reactions were carried out at pH 8.0 for the forward reaction at different temperatures. (d) Effect of temperature on OPRTase stability. After the purified enzyme was preincubated at different temperatures (30°C, 40°C, 50°C, 60°C, and 70°C) for 30 min, enzyme reactions were carried out at pH 8.0 at room temperature. Error bars indicate standard deviations.

Ali and Sloan (2) have shown that the Mg²⁺ requirement in yeast hypoxanthine/guanine PRTase could be substituted for by Mn²⁺, Co²⁺, or Zn²⁺. However, the effects of these metal ions on OPRTase have not been investigated because the presence of these ions will influence the UV absorption of orotate and make detection difficult (30). Here we determined the extinction coefficient of orotate in the presence of 2 mM divalent metal ions at 298 nm. Under these conditions, the absorbance of OMP was not affected by the addition of metal ions. Enzyme activities were assayed using the corrected . The results (Table 3) showed that Mn²⁺ and Co²⁺ could activate the reaction, while other divalent metal ions (Zn²⁺, Ca²⁺, Ba²⁺, and Ni²⁺) could not substitute for Mg²⁺.

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TABLE 3. Effects of metal ions on enzyme activity

The effect of temperature on the activity of the OPRTase was investigated by use of the OMP-forming reaction (forward reaction) over the range of 25 to 50°C. Maximum activity was observed at 35°C (Fig. 3c), with specific activity of 509 ± 24 units mg^–1 protein. The heat stability of the enzyme was also investigated by incubating the enzyme at 30 to 80°C for 30 min. The results (Fig. 3d) showed that OPRTase activity decreased with the increase in incubation temperature, and more than 50% of the activity was lost upon incubation at 50°C for 30 min.

Although the reported optimum pHs of most OPRTases were determined only for the forward reaction (Table 4), Ashton et al. (3) have reported that the pH dependencies of the forward and reverse OPRTase-catalyzed reactions were dissimilar. Therefore, we determined the activity of the purified OPRTase for both the forward and reverse reactions. The maximal activities for the forward and reverse reactions were observed at pH 10.5 to 11.0 and pH 5.5 to 6.5, respectively (Fig. 3b).

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TABLE 4. Comparison of the enzymatic properties of C. ammoniagenes ATCC 6872 OPRTase with those of other OPRTases

Since the apparent K_m values for OPRTases from other organisms were usually investigated at pH 8.0, the initial velocity experiments for C. ammoniagenes OPRTase were also carried out at pH 8.0, by spectrophotometric assay on both the forward (phosphoribosyltransfer) and the reverse (pyrophosphorolysis) reactions. The apparent K_m values were determined from double-reciprocal plots (see Fig. S2 in the supplemental material) and are shown in Table 4. For the forward reaction, >150 µM of orotate resulted in a decrease in activity typical of substrate inhibition. Therefore, the forward reactions were carried out with orotate at concentrations under 120 µM. The K_m values calculated from double-reciprocal plots were 33 µM for orotate, 64 µM for PRPP, 45 µM for OMP, and 36 µM for PP_i. The maximum reaction velocities observed with OPRTase were about 1,150 units mg^–1 for the forward reaction and 550 units mg^–1 for the reverse reaction.

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For most OPRTases, the highest phosphoribosyl transfer activities were obtained at pHs of lower than 10.0, whereas in C. ammoniagenes, the optimal pH for the forward reaction is 10.5 to 11.5. As we know, a specific pH dependence of protein characteristics such as stability, enzymatic activity, and binding specificity is achieved by the deprotonation of amino acid side chains (19). The difference in the optimal pH indicates an alteration of the pK_a values of the active sites. Furthermore, the diversity of pH dependence between the forward reaction and the reverse reaction suggests that the OPRTase-catalyzed pyrophosphorolysis of OMP is not a simple reversal of its formation but that two different deprotonated residues may contribute to the pyrophosphorolysis and phosphoribosyl transfer reactions, respectively (3).

Previous work has revealed a region composed of active sites located at the dimer interface of OPRTase (12, 21, 26). A lysine from one subunit and an aspartic acid from the adjacent subunit are essential for the catalysis. In the C. ammoniagenes OPRTase, these two residues are well assigned by sequence alignment (Fig. 1). The common fold of OPRTases (26) makes us believe that the catalytic biochemistry of C. ammoniagenes OPRTase is similar to that of OPRTase from S. enterica serovar Typhimurium. Thus, the two residues (Lys 103 and Glu 125 or Asp 126 in C. ammoniagenes OPRTase) are likely the two different deprotonated residues that contribute to the phosphoribosyl transfer and pyrophosphorolysis reactions, respectively, as discussed above, and the roles of the two residues in catalysis of the forward reaction are different from those in catalysis of the reverse reactions. The discrepancy between the crystal structures of the OPRTase-orotate-PRPP complex and the OPRTase-OMP complex from S. enterica serovar Typhimurium proved this assumption: in the crystal structure of the S. enterica serovar Typhimurium OPRTase complex with orotate and PRPP, the side chain of residue Asp 124 interacts with the ribose of PRPP, while in the OPRTase-OMP complex, the two aspartic acid residues in the active site do not interact with groups of the bound substrate. Furthermore, Wang et al. (34, 35) have shown that in the forward reaction, a diffusion-controlled step is predominantly rate limiting, whereas the reverse reaction is less limited by diffusion, also indicating the different roles of active sites in the forward reaction and the reverse reaction.

Since the two catalytic residues were not changed in C. ammoniagenes OPRTase compared with those in S. enterica serovar Typhimurium OPRTase, the higher optimal pH for the forward reaction indicated that alterations occurred at the positions of other residues in the active region.

In the OPRTase from S. enterica serovar Typhimurium, Lys 73 is located in the active region and has been shown to be essential to catalysis (20). In the crystal complex of OPRTase with OMP, the residues contributed to interact with the 5'-phosphate of OMP through an H bond, whereas in the OPRTase-orotate-PRPP complex, the atoms interact with the side chain nitrogens of Lys 73 that are O2' of the ribose ring and the β-phosphate oxygen atoms of pyrophosphate. The different roles of Lys 73 in the two crystal structures were explained by Tao et al. (29) for the reverse reaction: the loss of the interaction between the 5'-phosphate of OMP and Lys 73 caused by the binding of pyrophosphate initiates the catalysis by representing the interaction which reorganizes ground-state enzyme-substrate interactions and initiates movement of the ribose 5'-phosphate ring away from the orotate ring to form an oxocarbonium ion in the transition state, thus preventing nonproductive hydrolysis via solvent capture of the oxocarbonium ion. The important role of this residue was also proved by specific amino acid substitutions, where mutation of Lys 73 produced a 50- to 100-fold decrease in k_cat (20). However, this important residue could not be found in the OPRTase from C. ammoniagenes or in those from Thermus thermophilus, Rhizobium leguminosarum (Fig. 1), and human. This "common" loss may suggest a different catalytic biochemistry, or some other residues near the (steric) position function as the Lys 73 in OPRTase from S. enterica serovar Typhimurium should.

Ozturk et al. (20) have reported that mutation of Lys 73 produced an 8- to 12-fold increase in the K_m value for PRPP. We show that the K_m value of C. ammoniagenes OPRTase for OMP is much larger than those of S. enterica serovar Typhimurium, E. coli, and yeast (Table 4). This may also be caused by the loss of this residue. In addition, as mentioned above, the higher optimal pH for the forward reaction is also likely caused by this distinct difference in the active site between C. ammoniagenes OPRTase and the other OPRTases. However, the lower optimal pH of 9.0 for the OPRTase from T. thermophilus, in which Lys 73 is also missing, indicates that another residue(s) near the active site influenced the deprotonation of active site (Lys 103). Although the exact catalytic biochemistry of OPRTase is still unclear, the unusual OPRTase from C. ammoniagenes provides a good native model for research on PRTases.

 
This work was supported by the Hundred-Talent Project of the Chinese Academy of Sciences and in part by a grant from the Ministry of Science and Technology of China (National Basic Research Program of China, 2007CB714303).

 
* Corresponding author. Mailing address: State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, People's Republic of China. Phone: 86(531)88564003. Fax: 86(531)88567250. E-mail: pingxu{at}sdu.edu.cn

Published ahead of print on 5 October 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

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Journal of Bacteriology, December 2007, p. 9030-9036, Vol. 189, No. 24
0021-9193/07/$08.00+0     doi:10.1128/JB.01161-07
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