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Structure of Escherichia coli uridine phosphorylase


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Acta Crystallographica Section D

Biological Crystallography
ISSN 0907-4449

Structure of Escherichia coli uridine phosphorylase ? at 2.0 A

F. Temple Burling,? Ryan Kniewel, John A. Buglino, Tanya Chadha, Andrew Beckwith and Christopher D. Lima*
Biochemistry Department and Structural Biology Program, Weill Medical College of Cornell University, New York, NY 10021, USA

? Current address: Department of Physics, Carthage College, Kenosha, WI 53140, USA.

? The 2.0 A crystal structure has been determined for Escherichia coli uridine phosphorylase (UP), an essential enzyme in nucleotide biosynthesis that catalyzes the phosphorolytic cleavage of the C?N glycosidic bond of uridine to ribose-1-phosphate and uracil. The structure determination of two independent monomers in the asymmetric unit revealed the residue composition and atomic details of the apo con?gurations of each active site. The native hexameric UP enzyme was revealed by applying threefold crystallographic ? symmetry to the contents of the asymmetric unit. The 2.0 A model reveals a closer structural relationship to other nucleotide phosphorylase enzymes than was previously appreciated.

Received 30 July 2002 Accepted 15 October 2002

PDB Reference: uridine phosphorylase, 1lx7, r1lx7sf.

Correspondence e-mail: lima@pinky.med.cornell.edu

1. Introduction
Uridine phosphorylase catalyzes the phosphorolytic cleavage of the C?N glycosidic bond of uridine to ribose-1-phosphate and uracil (Leer et al., 1977; Vita et al., 1986). Uridine phosphorylase is a member of the pyrimidine nucleoside phosphorylase (PyNP) class of enzymes that catalyze the general reaction pyrimidine nucleoside ? phosphate 2 3 ribose-1-phosphate ? pyrimidine baseX Functionally related to the PyNP proteins are the purine nucleoside phosphorylase (PNP) enzymes that catalyze the analogous reaction purine nucleoside ? phosphate 2 3 ribose-1-phosphate ? purine baseX Together, these two enzyme types comprise the nucleoside phosphorylase (NP) class of proteins. Nucleoside phosphorylases are involved in essential biochemical salvage pathways in the cell that provide free purine and pyrimidine bases for subsequent nucleotide biosynthesis, enabling a less costly alternative to de novo nucleotide biosynthesis. Nucleoside phosphorylases are also able to inactivate certain purine and pyrimidine nucleoside analogs that posses anti-tumor activity (Morgunova et al., 1995; Pugmire & Ealick, 2002). Thus, the discovery of selective inhibitors for both PyNP and PNP enzymes could lead to enhanced therapeutic activity for these nucleoside analogs. More detailed views provided by high-resolution structures of the active sites for both PyNP and PNP enzymes could aid in the development of such compounds. To this end, we report the determination of a ? 2.0 A apo structure of Escherichia coli uridine phosphorylase (UP; EC 2.4.2.3). The New York Structural Genomics Research Consortium (NYSGRC; http://www.nysgrc.org) has
Burling et al.


# 2003 International Union of Crystallography Printed in Denmark ± all rights reserved

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targeted highly conserved enzyme families for structure determination as part of the national effort in structural genomics. At the time of target selection, UP represented a highly conserved protein family not represented in the PDB, so it was selected as a target for crystallographic structure determination.
Table 1
Crystallographic data and re?nement statistics.
Crystal characteristics and data-collection statistics ? Unit-cell parameters (A,  ) a = b = 151.4, c = 48.2,  =  =  = 90 Space group R3 Molecules per asymmetric unit 2 X-ray source NSLS X9A beamline !1 (SeMet peak) ? Wavelength (A) 0.9790 ? Resolution (A) 20.0±2.0 No. of observations 747216 No. of re?ections? 49742 Completeness? (%) 89.2 (78.6) Mean I/'(I)? 27.8 (15.6) Rmerge on I?§ 3.8 (8.0) Cutoff criteria I < 0'(I) ? SOLVE ?gure of merit} 0.29 (20.0±2.0 A resolution) for 24381 re?ections ? RESOLVE ?gure of merit} 0.53 (20.0±2.0 A resolution) for 24381 re?ections Model and re?nement statistics Data set used in structure re?nement Structure factors derived from SOLVE ? Resolution range (A) 20.0±2.0 No. of re?ections 26441 (25126 in working set; 1315 in test set) Completeness (%) 95.1 (90.3 in working set; 4.7 in test set) Cutoff criterion |F| > 0.0 No. of amino-acid residues/atoms 474/3512 No. of water atoms 361 Rcryst???? 0.182 (0.204) Rfree?? 0.215 (0.234) Root-mean-square deviations ? Bond lengths (A) 0.006 ? Bond angles (A) 1.20 2 ? B factor main chain/side chain (A ) 1.26/2.34 Ramachandran plot statistics§§ Residues in most favored regions 366 (90.1%) Residues in additional allowed regions 38 (9.4%) Residues in generously allowed regions 2 (0.5%) Residues in disallowed regions 0 (0.0%)
? ? MAD data completeness treats Bijvoet mates independently. ? Values in paren? theses ? ? ? ? are for the highest resolution shell (2.07±2.0 A). § Rmerge = } Figure of?merit calculated using i jI?hkl?i ? hI?hkl?ija i hI?hkl?i i. hkl hkl ? SOLVE/RESOLVE. ?? Rcryst = hkl jFo ?hkl? ? Fc ?hkl?ja hkl jFo ?hkl?j, where Fo and Fc are the observed and calculated structure factors, respectively. ?? Values in ? ). §§ Computed with parentheses are for the highest resolution shell (2.13±2.0 A PROCHECK (CCP4 suite; Collaborative Computational Project, Number 4, 1994).

2. Materials and methods
2.1. Isolation, expression, crystallization and structure determination

UP was selected as a unique target for our structural genomics effort by virtue of its conservation (>25% sequence identity) between several bacterial, human and mouse genomes. The coding region for the E. coli UP enzyme was ampli?ed from E. coli genomic DNA by PCR, ligated into a modi?ed version of pET28b, expressed in E. coli BL21 DE3 Codon Plus RIL (Stratagene) and puri?ed using Ni±NTA± agarose resin (Qiagen). UP was further puri?ed by gel ?ltration (Superdex200, Pharmacia), eluting with an apparent molecular weight consistent with a hexamer. Fractions were analyzed by SDS±PAGE, pooled and concentrated to 10.0 mg ml?1 (10 mM Tris±HCl pH 8, 50 mM NaCl, 1 mM DTT). Selenomethionine-substituted (SeMet) UP was generated by expressing UP in B834(DE3) cells (Hendrickson et al., 1990). 96-well crystallization trials were conducted that produced diffraction-quality crystals. SeMet UP protein crystals were re?ned and grown by hanging-drop vapor diffusion against a well solution containing 10% PEG 4K, 0.1 M MES pH 6.5 and 5% glycerol to ?nal dimensions of 0.3 ? 0.3 ? 0.3 mm. The data presented here were obtained from UP crystallized in ? space group R3 (unit-cell parameters a = b = 151.4, c = 48.2 A,  =  = 90,  = 120 ). Diffraction data collection was accomplished using cryopreserved crystals (30% glycerol in mother liquor). Diffraction experiments took place at beamline X9A at the National Synchrotron Light Source and the data were processed with DENZO and SCALEPACK (Otwinowski & Minor, 1997) and input to SOLVE, RESOLVE (Terwilliger & Berendzen, 1999) and the CCP4 suite (Collaborative ? Computational Project, Number 4, 1994) to calculate a 2.0 A SAD phase set. RESOLVE automatically determined the twofold NCS operators relating the A and B monomers and this solvent-modi?ed NCS-averaged electron density was manually traced using O (Jones et al., 1991; see Table 1). The protomer model was initially ?tted into the two respective positions within the asymmetric unit using BRUTPTF (see http://www.nysgrc.org/molrep for details) and the subsequent model containing two monomers was re?ned without NCS ? restraints in CNS (Brunger et al., 1998). The ?nal model contained 474 amino-acid residues comprised of residues 4±253 from monomer A and residues 3±162, 183±221 and 233±253 from monomer B. The structure revealed two independent determinations of the active site since the model contained two monomers in the asymmetric

unit. The native oligomeric state for E. coli UP is a hexamer and this oligomeric state can be reconstructed by transforming the A and B monomers within the asymmetric unit by the R3 crystallographic threefold axis (for details, see PDB code 1lx7). A similar oligomeric state for E. coli UP has been described in previous reports (Zhao, 1991; Morgunova et al., 1995). Additional information on the expression and crystallization of E. coli UP can be found at http://www.nysgrc.org under target-identi?cation code T24.

3. Results and discussion
3.1. Structure of E. coli UP

E. coli UP was expressed, puri?ed, crystallized and char? acterized by X-ray crystallography (see x2). A previous 2.5 A structure of E. coli UP was determined in a monoclinic space group and published (Morgunova et al., 1995), although the
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in similar conformations to those in the E. coli PNP structure. data were not deposited in the PDB until this year (PDB code ? These observations are in contrast to the structure of the 1k3f). Additionally, a 3.0 A structure of E. coli UP determined ? active-site residues observed in the 2.5 A E. coli UP structure. in a trigonal space group was described in a PhD thesis, but the Greater structural homology observed here between E. coli coordinates were never deposited in the PDB (Zhao, 1991). ? UP and E. coli PNP lends further support to the hypothesis of The 2.5 A structure of E. coli UP was utilized in a comparison Pugmire and Ealick that these enzyme classes evolved from a with several purine nucleoside phosphorylase structures in a common ancestor. recent review (Pugmire & Ealick, 2002) including E. coli PNP (Mao et al., 1998; PDB code 1ecp), bovine PNP (bPNP) and human PNP (hPNP) (Pugmire & Ealick, 2002). E. coli UP and 3.2. E. coli UP active site E. coli PNP share only 26.7% sequence identity and E. coli UP The additional structural homologies observed between the and E. coli PNP both align with less than 20% sequence ? 2.0 A structure of E. coli UP and E. coli PNP are as follows. In identity to either bPNP or hPNP (Pugmire & Ealick, 2002). ? the previous 2.5 A E. coli UP structure, the authors note that Nonetheless, these four proteins share a common fold and are Arg48 is disordered. The corresponding Arg43 in E. coli PNP part of a family of proteins that Pugmire and Ealick term the is ordered and reaches into the phosphate-binding site of an nucleoside phosphorylase-I (NP-I) family. The striking simi? adjacent subunit. In the 2.0 A structure reported here, Arg48 larity in fold between E. coli UP (a PyNP) and E. coli PNP led appears well ordered and in a conformation similar to that of Pugmire and Ealick to propose that these two proteins Arg43 in the E. coli PNP structure. In addition, His8 from evolved from a common ancestor. It is interesting to note that monomer B was observed in a conformation essentially a second family of nucleoside phosphorylases, termed the ? identical to that of His4 in E. coli PNP. The 2.0 A structure NP-II family by Pugmire and Ealick, are structurally distinct also suggests that Glu196 has a similar conformation to from the NP-I enzymes. The NP-II family consists of enzymes ? Glu179, as observed in E. coli PNP. The previous 2.5 A E. coli that are speci?c for thymine in higher organisms, but will UP structure revealed Glu196 to be in a different conformacatalyze nucleoside phosphorylation reactions of both ? tion from that observed in the 2.0 A structure. thymine and uridine in lower organisms (Pugmire & Ealick, The most striking differences observed between the two 2002). ? E. coli UP structures are the divergent conformations between Neither the 2.0 A E. coli UP structure reported here nor the ? the loops comprised of amino-acid residues 163±177. The loop previously reported 2.5 A E. coli UP structure contains ? conformation observed in the 2.0 A structure positions two substrate at the active site. The E. coli PNP structure is also residues, Gln166 and Arg168, into the putative active-site unbound. However, there are several examples of substratecleft, amino acids that point out of and away from the binding bound and substrate-analog-bound structures of bPNP site in the previous structure. Tyr163 is also located within this (Morgunova et al., 1995; Pugmire & Ealick, 2002). The ? loop. In the 2.0 A structure, Tyr163 superimposes well with exceptional conservation of protein folds between the ? E. coli PNP Tyr160, whereas Tyr163 in the 2.5 A structure bacterial NPs and the mammalian PNPs permits identi?cation of the probable active-site residues of E. coli UP and E. coli PNP based on threedimensional alignment with substratebound bPNP structures. ? We aligned our 2.0 A UP structure to ? the 2.5 A UP structure and the structure of E. coli PNP using DALI (Holm & Sander, 1993). The root-mean-square deviations (r.m.s.d.) between monomers ? of the 2.5 A UP structure and monomer A from our structure ranged from 1.1 to ? 1.5 A2 over 236 amino acids (excluding disordered loops). The alignment of monomer A from our E. coli UP structure and monomer A from the E. coli PNP structure revealed an overall r.m.s.d. ? of 2.0 A2 over 223 amino acids. Based on these alignments, we ?nd several striking ? differences between the 2.0 A structure of E. coli UP reported here and the Figure 1 ? Stereo diagram of a representative UP active site. Three NP-I family members are represented: previous 2.5 A E. coli UP structure in the ? ? the 2.0 A structure of E. coli UP reported here (in grey thick lines), the previously reported 2.5 A vicinity of the putative active site (Fig. 1). ? structure of E. coli UP (in thin black lines) and the 2.0 A structure of E. coli PNP (in thick black Speci?cally, four critical active-site resi? lines). Speci?c amino-acid residues mentioned in the text are numbered for the 2.0 A structure of ? dues in the 2.0 A structure are observed E. coli UP. This ?gure was prepared using SETOR (Evans, 1993).
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? superimposes onto Gln166 of the 2.0 A structure and would be predicted to make very close contacts to a modeled pyrimidine ring within the putative ligand-binding site.

References
? Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T. & Warren, G. L. (1998). Acta Cryst. D54, 905±921. Collaborative Computational Project, Number 4 (1994). Acta Cryst. D50, 760±763. Evans, S. V. (1993). J. Mol. Graph. 11, 134±138. Hendrickson, W. A., Horton, J. R. & LeMaster, D. M. (1990). EMBO J. 9, 1665±1672. Holm, L. & Sander, C. (1993). J. Mol. Biol. 233, 123±138. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Acta Cryst. A47, 110±118. Leer, J. C., Hammer-Jespersen, K. & Schwartz, M. (1977). Eur. J. Biochem. 75, 217±224. Mao, C., Cook, W. J., Zhou, M., Federov, A. A., Almo, S. C. & Ealick, S. E. (1998). Biochemistry, 37, 7135±7146. Morgunova, E. Y., Mkhailov, A. M., Popov, A. N., Blagova, E. V., Smirnova, E. A., Vainshtein, B. K., Mao, C., Armstrong, S. R., Ealick, S. E., Komissarov, A. A., Linkova, E. V., Burlakova, A. A., Mironov, A. S. & Debabov, V. G. (1995). FEBS Lett. 367, 183±187. Otwinowski, Z. & Minor, W. (1997). Methods Enzymol. 276, 307±326. Pugmire, M. J. & Ealick, S.A. (2002). Biochem. J. 361, 1±25. Terwilliger, T. C. & Berendzen, J. (1999). Acta Cryst. D55, 849±861. Vita, A., Amici, A., Cacciamani, T., Lanciotti, M. & Magni, G. (1986). Int. J. Biochem. 18, 431±436. Zhao, B. (1991). PhD dissertation, University of Alabama at Birmingham, USA.

4. Conclusions
Homology-modeling studies of E. coli and mammalian purine phosphorylase along with E. coli uridine phosphorylase structures reveal a common fold and permit identi?cation of active-site residues in the apo uridine phosphorylase (Pugmire ? & Ealick, 2002). The 2.0 A resolution structure of the E. coli UP suggests greater structural similarity to E. coli purine phosphorylase than was previously surmised based on an ? earlier 2.5 A structure of E. coli uridine phosphorylase (Morgunova et al., 1995). Combined with previous observations, the high-resolution structure of E. coli UP lends further support to the hypothesis that members of the related but functionally distinct NP-I enzyme class evolved from a common ancestor. We thank the staff of beamline X9A at the National Synchrotron Light Source for their support. This work was supported in part by a Young Investigator award made to CDL from the Arnold and Mabel Beckman Foundation and NIH structural genomics pilot center grant 1P50 GM62529.

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Acta Cryst. (2003). D59, 73±76


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