From the
Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041,
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9041
Received for publication, January 8, 2003
, and in revised form, March 27, 2003.
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ABSTRACT |
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INTRODUCTION |
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X-ray structural analysis of the native enzymes from mouse (6), T. brucei (7, 8), and human (9) demonstrates that the ODC monomer consists of two domains, an N-terminal /
-barrel domain and a C-terminal
-sheet domain. The T. brucei structure has also been solved in complex with the product putrescine and with DFMO, identifying important active site residues (7, 8). ODC is an obligate homodimer, and the two identical active sites are formed at the dimer interface, encompassing the
/
barrel domain from one subunit and
sheet domain from the other. Catalytic residues are contributed to the active site from both monomers. Both putrescine and DFMO form a covalent Schiff base complex with the PLP cofactor, and the side-chain amino groups of both compounds bind in a well conserved pocket that bridges between subunits. In addition DFMO has undergone decarboxylation and has formed a covalent bond to Cys-360 as expected based on its mechanism of inhibition (10).
The ODC active site is invariant between the host and parasite enzymes; thus, selective toxicity of DFMO for the parasite is not achieved through differential inhibitor binding to the enzyme active site (7). Instead, it is thought to arise from metabolic differences between the host and parasite, which include the rapid turnover of host ODC when compared with that of the parasite (2, 11), and the requirement for the polyamine spermidine for the formation of the novel cofactor trypanothione to maintain reduced thiol pools in the parasite (12).
The discovery that DFMO was curative against T. brucei gambiense infections provided the first alternative to the highly toxic melarsoprol for the treatment of late stage disease (13). However, utilization of DFMO is hampered by the large dose requirement and by poor activity against the T. brucei rhodesiense strain of the parasite (2). A number of small molecule inhibitors of ODC have been reported, but like DFMO they target the highly conserved active site (1416). The discovery of novel ODC inhibitors with chemical properties that differ from substrate may overcome the poor pharmacology of DFMO. We had observed previously (17) that mutations in the dimer interface of ODC distant from the active site decreased catalytic activity. Thus we sought to explore whether ODC could be inhibited allosterically by binding inhibitors to sites outside of the active site.
We have identified an inhibitor of ODC, G418 sulfate (Geneticin), which is non-competitive with substrate. Although the inhibitor has a high KI (58 mM) it was sufficiently inhibitory to allow us to observe D-Orn bound in the active site of ODC. Previous attempts to co-crystallize D-Orn and ODC were unsuccessful, because the substrate analog was unexpectedly decarboxylated to putrescine (8). Through crystallographic analysis of T. brucei ODC, G418, and D-Orn, we have determined the binding orientation of a carboxylated ligand. The structure reported here, in combination with the DFMO-bound structure and with mutagenic analysis, supports a model where the substrate carboxylate is buried on the re face of PLP on the same face as Lys-69. The inhibitor G418 binds outside the active site of ODC in the interface between the two domains of the ODC monomer. A loop region (residues 392401) at the symmetry-related center of the dimer interface, which contributes essential residues to the active site pocket, is disordered in this structure. The disorder in this region of the active site may explain the basis for inhibition of ODC by G418. This conformation, apparently available at low energy, is inactive and could be useful in inhibitor design.
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EXPERIMENTAL PROCEDURES |
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Methods
ODC Expression and PurificationODC was expressed from a T7 promoter in the BL21 (DE3) strain of Escherichia coli as described for T. brucei (7, 8, 18) and human (9) ODC. The protein was produced as an N-terminal His6-tagged fusion that included a tobacco etch virus (TEV) protease cleavage site to allow removal of the tag. Protein was purified by column chromatography over nickel-nitrilotriacetic acid-agarose and Sephadex 200. For crystallography, the His tag was removed from purified ODC by digestion with tobacco etch virus (TEV) protease immobilized on glutathione-agarose beads as described (19). The purified protein for crystallization experiments was concentrated to 2025 mg/ml.
Site-directed MutagenesisD243A, L339A, and E384A mutants of T. brucei ODC were created using PCR-based mutagenesis of the pODC29 expression plasmid using the QuikChangeTM site-directed mutagenesis kit from Stratagene (La Jolla, CA). The primers listed below contain the desired mutation (bold), and D243A and L339A also contain silent mutations (bold italics), which remove a restriction site (BseDI and StuI, respectively) for diagnostic purposes: L339A forward, 5'-GACCACGCAGTCGTCAGACCTGCGCCCCAGAGGGAGCC-3'; D243A forward, 5'-GTGGGTTTCCAGGTACGAGGGCTGCACCACTTAAATTTG3'; E384A forward, 5'-GGCTGCTCTTTGCGGATATGGGTGCC-3'.
Inhibitor IdentificationG418 and other aminoglycosides were identified as potential ODC inhibitors by computational methods using the Available Chemicals Directory (1998 version).
Enzyme AssaysSteady-state spectrophotometric assay of the decarboxylation of L-Orn (0.1 to 16 mM) by ODC (2080 nM) was followed at 37 °C as described (18) using the Sigma Diagnostics carbon dioxide detection kit (Sigma) in the presence of 50 µM PLP. In this assay CO2 production is linked to the oxidation of NADH (max·NADH = 340 nm) by coupling the ODC reaction to phosphoenolpyruvate carboxylase and malate dehydrogenase.
Direct assay of L-Orn decarboxylation by ODC was performed by a radioactive assay of 1-14C-L-Orn. For this assay, a 2x substrate solution, 1-14C-L-Orn (32 µM) mixed with cold L-Orn (0.8 mM) in Buffer A (0.2 M Hepes, pH 7.5, 0.05 mM PLP), was added to a culture tube by injection with a Hamilton syringe through a rubber stopper cap into a 2x ODC enzyme solution (0.25 µM ODC in Buffer A). After mixing, the reaction was allowed to proceed at 37 °C for varied times (0 to 8 min) and then quenched with 20% (final) trichloroacetic acid. Sample chambers were incubated for another hour at 37 °C to evolve liberated 1-14CO2 onto paper strips soaked with a saturated Ba(OH)2 solution that were hung in the chamber. The paper strips were submerged in Cytoscint scintillation mixture and counted for radioactive decay.
The KI and IC50 values for the inhibition of ODC by G418 sulfate were determined by spectroscopic assay over a range of inhibitor concentrations (016 mM). The inhibitors (at 1x) were preincubated with ODC (at 50x) in assay buffer for 15 min at 37 °C before addition to the assay mixture. The assay mixture was also allowed to pre-incubate for 5 min with G418 before the addition of pre-incubated enzyme. Salt controls (NaCl and KSO4) were run in the same concentration range as inhibitor. IC50 values were determined by fitting to Equation 1.
![]() | (Eq. 1) |
The KI values were determined by varying both L-Orn and inhibitor concentration, and KI values for the non-competitive inhibitors were determined by fitting to Equation 2.
![]() | (Eq. 2) |
Kinetic data were fitted to the appropriate equation with Sigma Plot 2001 version 7.0 (SPSS Inc., Chicago, IL) to determine the kinetic constants.
CrystallizationT. brucei ODC was co-crystallized with D-Orn and G418 sulfate under the following conditions: T. brucei ODC (23 mg/ml in 10 mM Hepes, pH 7.2, 50 mM NaCl, 10 mM dithiothreitol, 0.5 mM EDTA, 0.01% Brij-20) was preincubated with 25 mM D-Orn and 100 mM G418 sulfate for 10 min at room temperature before setting crystal drops. Equal volumes of preincubated enzyme and well solution (20% polyethylene glycol 3350, 100 mM Hepes, pH 8.0, 10 mM dithiothreitol, 25 mM D-Orn, pH 7.5, 100 mM G418) were used to form the crystal drops. -Butyrolactone, 3% (v/v) final, was added to the drops (final pH = 7.0). The crystals formed overnight at 16 °C under conditions of vapor diffusion. Crystals were cryoprotected in 1.2 x crystal conditions with 20% ethylene glycol and frozen in propane. These conditions differed from those used previously (8) to obtain the putrescine bound structure in which the
-butyrolactone additive was not required, and the well solution contained NaOAc in place of G418 (20% polyethylene glycol 3350, 100 mM Hepes, pH 7.5, 10 mM dithiothreitol, 7.5 mM D-Orn, 200 mM NaOAc).
Data Collection and ProcessingDiffraction data were collected with an R-Axis IV image plate system (MSC, Houston, TX) mounted on a rotating anode and operated at 100 mA and 50 kV, at the University of Texas Southwestern Medical Center. Data were processed with HKL2000 (20). A summary of the statistics for data processing is given in Table I. There was a slight asymmetry in the resolution of the data and also a telescoping effect in the spot shape. After screening many crystals, data adequate for structure determination were collected. Wilson B-values were determined using CCP4 (21).2
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Structure Determination and RefinementInitial phases were calculated by molecular replacement using the program AMoRe (23) and the coordinates of a dimer from the T. brucei ODC/put structure (8) (Protein Data Bank code 1f3t [PDB] ), with putrescine and waters removed, as a search model. The model was built in O version 6.2.2 (24) and refined using CNS (25) utilizing data collected between 35 and 2.45 Å. Non-crystallographic symmetry constraints were kept for residues 38152, 170291, and 317390 for initial rounds of refinement, non-crystallographic symmetry constraints were released for one round, and then restraints were added again for residues 38152, 170238, 247291, 317340, and 350390 for the remainder of the refinement. The peptide torsion angles for 1011 of 1180 non-glycine and non-proline residues fall within the most favored regions, and there are none found in disallowed regions of the Ramachandran plot as determined by the program PROCHECK (26). Water molecules were added using CNS and edited in the program O. Refinement statistics are listed in Table I. The coordinates have been placed in the Protein Data Bank (Protein Data Bank code 1NJJ).
Superposition of Crystal Structures and Determination of r.m.s.d. between Structure CoordinatesC monomers from each of the following T. brucei ODC structures, native ODC (Protein Data Bank code 1QU4
[PDB]
; native_ODC), K69A ODC inactivated with DFMO (Protein Data Bank code 2tod
[PDB]
; K69_ODC/DFMO), and the structure reported in this paper (ODC/G418/D-Orn), were superimposed with the structure of ODC in complex with putrescine (Protein Data Bank code 1f3t
[PDB]
; ODC/put) using Insight II (Accelrys, San Diego, CA). Monomers from each structure were pared down to include only shared residues 3768, 70157, 168296, 312344, 348391, and 402409, and backbone atoms from residues 320340 and 350380 (from the -sheet domain) were superimposed. Superimposed monomers were written out into the same coordinate system using the Biopolymer module of InsightII, and CNS was used to determine the average r.m.s. coordinate difference between structures for residues 45282 (from the
-barrel domain) and for residues in the
-sheet domain (3844, 283342, and 349409), using the CNS rmsd.inp input file.
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RESULTS |
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Crystallization, Data Collection, and Structure DeterminationT. brucei ODC was co-crystallized with D-Orn and G418. The ODC/G418/D-Orn crystal diffracted to 2.45 Å in the space group P21 (a = 67.8 Å, b = 88.5 Å, c = 150.4 Å, = 90.03°) with two homodimers in the asymmetric unit. The final model contains four monomers, 1404 residues, four PLP, four D-Orn, three G418, and 207 water molecules. The R-factor is 26.0% with an Rfree of 28.3% for data between 35- and 2.45-Å resolution (Table I). Representative electron density for the PLP/D-Orn and G418 binding sites is displayed in Fig. 1. Density for most of the structure was unambiguous with the exception that residues at the N terminus (amino acid residues 119), the C terminus (410425), and three surface loops (3136, 158167, and 297311) were disordered. Residues 343348 have poorly defined density as observed previously (7, 8) for all T. brucei ODC structures. Chain specific differences include the following: chain A has an additional six residues (410414) at the C terminus, chain C contains density for additional residues at the N-terminal end (1419) and for residue 392, and chain D has additional density observed for residues 309311 and for the backbone atoms of residues 400 and 401.
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In addition to those regions typically disordered in T. brucei ODC crystals, residues Val-392 to Gln-401, near the active site, were also disordered in the ODC/G418/D-Orn structure (Fig. 2). These residues were well ordered in the previous crystallographic studies of T. brucei ODC with B-factors in the region being comparable with the average B-factor for the overall structure (7, 8). Residues 392401 are in the dimer interface, adjacent to the equivalent loop from the opposing monomer, and contribute residues that form the back of the active site pocket (Fig. 3A). Several of these residues (Phe-397, Asn-398, and Phe-400) have been demonstrated to be important for enzyme activity (17, 27). Although the space group is the same as with previous T. brucei ODC structures (7, 8), the lattice and packing differ. Neither the inhibitor binding site nor the disordered loop appear to be influenced by the lattice.
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The D-Orn Binding SiteThis structure allowed for the orientation of the substrate stereoisomer, D-Orn, to be determined. D-Orn binds ODC with a Kd of 0.27 mM, similar to the binding constant for putrescine (8, 28). During kinetic analysis, D-Orn is not decarboxylated at a detectable rate (kcat/Km < 1 x 106 s1 M1 at 37 °C). However, during the extensive incubation between D-Orn and wild-type T. brucei ODC required for crystallization, D-Orn is converted to putrescine, as observed in the ODC/put structure (8). In contrast, in the ODC/G418/D-Orn structure, density for the carboxylate is observed (Fig. 1A). The D-Orn carboxyl group is positioned on the si face of the PLP cofactor, on the solvent-exposed face of the active site (see Fig. 1A and Fig. 3B). Gly-199, Gly-362, and the His-197 side chains are the only contacts within 4.2 Å of the D-Orn carboxyl group. D-Orn forms an external aldimine with the PLP cofactor, and the -amino group interacts with Asp-361 and Asp-332 (Fig. 3A). The position for the D-Orn
-amino group is similar to that observed for ligands in the putrescine and DFMO complexed ODC structures (7, 8). Lys-69, which forms a Schiff base with PLP in the unliganded structure, has rotated out of the active site to form interactions with Asp-88 and Glu-94, as observed in the structure of T. brucei ODC bound to putrescine (8).
Differences in the active site structure are also observed. In previous structures residues 158165, which are located near the active site entrance, are disordered. In addition to these residues, Leu-166 becomes disordered in this structure, apparently displaced by the carboxylate of D-Orn. Cys-360 is also observed in a different position than for previous ligand-bound structures (e.g. putrescine or DFMO). In the putrescine- and DFMO-bound structures the side chain of Cys-360 is rotated in toward the C carbon of the ligand. In the G418/D-Orn-bound structure, it is rotated out of the active site pocket (Fig. 3), occupying a similar position to that observed in the native, uncomplexed structure (7, 8).
The G418 Binding SiteElectron density for G418 was observed at the domain interface between the N-terminal /
barrel and C-terminal
sheet (Greek key) domain of individual subunits (see Fig. 1B and Fig. 2). This binding site is notably >20 Å from the closest active site. Electron density is interpretable for three of the four monomers (A, B, and C) in the asymmetric unit with the best density observed in the C monomer. No structural differences are observed between this region in monomers A, B, and C compared with monomer D, which could explain why the fourth monomer does not contain G418. Residues within 4.2 Å of G418 include Arg-22, Leu-25, Leu-339, Pro-340, Gln-341, Glu-343, Leu-382, Glu-384, and Asp-385 of the
-sheet domain and Asp-243 of the
/
-barrel domain (Fig. 4). Of these residues only Pro-340, Glu-384, Asp-385, and Asp-243 make H-bonds (3.2 Å or better) with the inhibitor. There are only a few hydrophobic interactions. On the other hand, several negatively charged residues (noted above) may offer electrostatic complementarity to the positively charged G418.
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In an effort to determine whether G418 inhibition results from binding to the site observed in the crystal structure, three residues in the G418 binding site (Asp-243, Leu-339, and Glu-384) were mutated to Ala. Leu-339 and Asp-242 make no observable energetic contribution to G418 binding. E384A ODC exhibited a small but measurable difference in its inhibition by G418 (the IC50 decreased by a factor of 3). The small effect and the unpredictable direction of the effect are consistent with the weak inhibition properties of G418 (data not shown).
Comparative Analysis of T. brucei ODC StructuresThe ODC/G418/D-Orn structure was compared with the native-_ODC, ODC/put, and K69A_ODC/DFMO T. brucei ODC structures. The -sheet domains of the monomers were superimposed, and the average r.m.s.d. of the main-chain atoms was calculated individually for the
/
-barrel domain and also for the superimposed
-sheet domain. The r.m.s.d. between the ODC/G418/D-Orn structure and the putrescine-bound structure is 2-fold greater than observed between the other structures (Table II). A small domain rotation is observed when comparing the ODC/G418/D-Orn structure with the other three structures. The most notable rotation (2-3°) is observed between the ODC/G418/D-Orn and ODC/putrescine structures (see Figs. 4 and 5). This rotation may be the result of inhibitor binding, but differences in crystallization conditions and lattice contacts are also present that may contribute to this observation.
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DISCUSSION |
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The position and orientation of substrate in the ODC active site is constrained both by structure and by chemistry. All three substrate analogs that have been co-crystallized with ODC form a reversible covalent bond (Schiff base or aldimine) with the PLP cofactor. The side-chain amino group (N) of these ligands occupies a well conserved binding site, forming an ion pair with Asp-332 and Asp-361. These interactions constrain the orientation of either D- or L-Orn in the active site of ODC. Further, the role of PLP in ODC reaction chemistry requires the leaving group (carboxylate) to be aligned with the pi orbitals of the conjugated imine bond, perpendicular to the plane of PLP for optimal catalysis (30). Previous analysis of the putrescine and DFMO-bound ODC structures predicted that for the side chain to bind in the extended conformation necessary to form close interactions with these key Asp residues, the ornithine carboxylate must be positioned on the re face of the cofactor for the L-stereoisomer and the si face for the D-isomer (7, 8). The T. brucei ODC/G418/D-Orn structure is the first ODC structure that has been determined bound to a carboxylated substrate analog where this hypothesis could be directly assessed (see Fig. 1A and Fig. 3), and indeed the results confirm the prediction that the carboxyl group of D-Orn is bound on the solvent-exposed si face of PLP.
Thus these data, in combination with the constraints imposed on the side-chain position by the active site structure, strongly suggest that the L-Orn stereoisomer binds with the carboxyl group on the re face of the PLP cofactor, facing the interior of the protein. The mechanism of inactivation of ODC by DFMO further supports this orientation for substrate. Binding of the substrate carboxylate on the re face would position the -methyl group of DFMO near Cys-360, the known nucleophile in the reaction that generates the covalent enzyme-bound species, whereas binding in the opposite orientation would place the reactive group on the other side of the active site from Cys-360 (7). Finally, the structure of diaminopimelate decarboxylase, an enzyme from the same fold class as ODC, has been reported recently (32) in complex with product demonstrating a similar position for the side chain as observed for ODC. These data also support an orientation for the D-stereoisomer substrate that places the carboxylate on the si face of the cofactor.
Amino acid decarboxylases are thought to achieve the largest rate enhancements of any enzyme-catalyzed reaction, increasing the rate by nearly a factor of 1020 (33). For PLP-dependent enzymes a large amount of this rate enhancement is the result of the catalytic power of the cofactor (factor of 1011); however, a significant role for the enzyme in promoting the reaction is clearly required beyond this. The predicted L-CO2 binding site on ODC contains both hydrophobic and negatively charged residues that include Phe-397, Tyr-389, Lys-69 (methylene carbons), and Asp-361. Notably, in both the ligand-bound structures, the N of Lys-69 is turned away from the predicted carboxylate binding site, interacting with Asp-88 and Glu-94. This would be expected to prevent it from forming stabilizing interactions with the substrate carboxylate. Thus, this model for CO2 binding suggests that the binding pocket would enhance decarboxylation by forming more favorable interactions with the more neutral transition state structure (CO2) than the charged ground state structure (CO2). Supporting the hypothesis that the L-CO2 binding site includes Phe-397, steady-state and pre-steady-state analysis of the Phe-397 to Ala mutant ODC demonstrated a reduced rate for the decarboxylation step (27).
The carboxylate binding sites of a number of decarboxylases from diverse structural classes also contain hydrophobic and negatively charged residues. Chalcone synthase-like enzymes have a conserved Phe positioned in the carboxylate binding site that is hypothesized to facilitate decarboxylation (31). Structural analysis of pyruvoyl-dependent histidine decarboxylase (34) demonstrated that the carboxylate binding site is formed by hydrophobic and negatively charged residues. The carboxylate binding site of orotodine-5'-phosphate decarboxylase also contains a negative charge in close proximity of the carboxylate binding site. However controversy over the role of this residue exists. Although it has been suggested that this residue promotes decarboxylation by ground-state destabilization, it appears likely that the major driving force for the reaction is transition state stabilization by other residues in the active site (33).
The position of Cys-360 in the D-Orn complex suggests that mechanism-related conformational changes may also be important to drive the reaction chemistry (Fig. 3A). In this structure, the Cys is rotated out of the active site, similar to native ODC (7, 9, 36). Model building suggests that with L-Orn bound, Cys-360 would also be rotated out of the active site because of steric clashes with the substrate carboxyl group. In contrast, in product complexes the Cys is rotated into the active site (7, 8). This suggests that Cys-360 rotates during the reaction cycle from an external to an internal position, which could be important to prevent proton abstraction prior to decarboxylation. Following decarboxylation, rotation of Cys-360 would position it to serve as a general acid to protonate C, a required step in the reaction. In support, mutation of Cys-360 reduces the rate of the decarboxylation step and causes improper protonation to occur at the C4' position of PLP, leading to product deamination (8).
The G418 binding-site at the junction between the -sheet and
/
-barrel domains (Fig. 2) is consistent with the non-competitive kinetics observed for inhibition of ODC by G418. Two conformational changes are observed in this structure relative to previously published ODC structures. First, superposition of monomers from different structures reveals that the
/
-barrel and
-sheet domains in the ODC/G418/D-Orn structure have undergone a small domain rotation, when compared with previously published T. brucei ODC structures (see Figs. 4 and 5). Second, no defined density is observed in this structure for ten amino acids that had previously been found to be well ordered in all eukaryotic ODC structures published to date. These amino acid residues (392401) form a loop that interacts along the dimer interface with the corresponding loop from the opposing monomer and that contributes residues that form the back of the active site pocket (see Fig. 2 and Fig. 3A). Several of the loop residues have been demonstrated to be essential for enzyme activity, including Phe-397, Asn-398, and Phe-400 (17). Thus the disordering of this loop region upon G418 binding provides a mechanism for enzyme inhibition despite the large distance between the active site and the G418 binding site. These results show that different conformers of ODC are available at low energy and suggest a mechanism by which ODC can be inhibited allosterically by binding ligands to the domain interface.
Mammalian cells produce a protein inhibitor of ODC termed the antizyme (AZ), which is a regulatory protein synthesized in response to high polyamine levels (reviewed in Refs. 35 and 36). AZ binds ODC distant from the active site, inhibiting activity and targeting ODC for degradation. The binding of AZ to ODC increases the reactivity of antibodies raised to sequences within the C terminus (residues 376 to 461) of mouse ODC, suggesting that the C terminus becomes more accessible in the presence of AZ (22). Although AZ does not bind to the same site as G418 (Fig. 2), an interesting question is whether its binding could cause similar structural changes to those observed in the presence of G418, namely the disorder of the 392-401 loop region.
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CONCLUSION |
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FOOTNOTES |
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¶ Recipient of a Burroughs Wellcome Fund Scholar Award in Molecular Parasitology. To whom correspondence should be addressed: Dept. of Pharmacology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9041. Tel.: 214-648-3637; Fax: 214-648-9961; E-mail: margaret.phillips{at}UTSouthwestern.edu.
1 The abbreviations used are: ODC, ornithine decarboxylase; Orn, ornithine; PLP, pyridoxal 5'-phosphate; put, putrescine; AZ, antizyme; DFMO, -difluoromethylornithine; r.m.s.d., root mean square deviation.
2 E. Dodson and P. Wilson. WILSON, a CCP4 supported program. (See Ref. 21.)
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ACKNOWLEDGMENTS |
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REFERENCES |
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