Biochemistry 2009, 48, 8879–8890 8879 DOI: 10.1021/bi901046x
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The Mechanism of the Reaction Catalyzed by Uronate Isomerase Illustrates How an Isomerase May Have Evolved from a Hydrolase within the Amidohydrolase Superfamily† )
Tinh T. Nguyen,§ Alexander A. Fedorov, LaKenya Williams,§ Elena V. Fedorov, Yingchun Li,§ Chengfu Xu,§ Steven C. Almo,*, and Frank M. Raushel*,§ §
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Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77842-3012, and Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Received June 20, 2009; Revised Manuscript Received August 10, 2009 ABSTRACT: Uronate isomerase (URI) catalyzes the reversible isomerization of D-glucuronate to D-fructuronate and of D-galacturonate to D-tagaturonate. URI is a member of the amidohydrolase superfamily (AHS), a highly divergent group of enzymes that catalyze primarily hydrolytic reactions. The chemical mechanism and active site structure of URI were investigated in an attempt to improve our understanding of how an active site template that apparently evolved to catalyze hydrolytic reactions has been reforged to catalyze an isomerization reaction. The pH-rate profiles for kcat and kcat/Km for URI from Escherichia coli are bellshaped and indicate that one group must be unprotonated and another residue must be protonated for catalytic activity. Primary isotope effects on the kinetic constants with [2-2H]-D-glucuronate and the effects of changes in solvent viscosity are consistent with product release being the rate-limiting step. The X-ray structure of Bh0493, a URI from Bacillus halodurans, was determined in the presence of the substrate D-glucuronate. The bound complex showed that the mononuclear metal center in the active site is ligated to the C-6 carboxylate and the C-5 hydroxyl group of the substrate. This hydroxyl group is also hydrogen bonded to Asp-355 in the same orientation as the hydroxide or water is bound in those members of the AHS that catalyze hydrolytic reactions. In addition, the C-2 and C-3 hydroxyl groups of the substrate are hydrogen bonded to Arg-357 and the carbonyl group at C-1 is hydrogen bonded to Tyr-50. A chemical mechanism is proposed that utilizes a proton transfer from C-2 of D-glucuronate to C-1 that is initiated by the combined actions of Asp-355 from the end of β-strand 8 and the C-5 hydroxyl of the substrate that is bound to the metal ion. The formation of the proposed cis-enediol intermediate is further facilitated by the shuttling of the proton between the C-2 and C-1 oxygens by the conserved Tyr-50 and/or Arg-355.
Uronate isomerase (URI)1 catalyzes the first step in the pathway for the metabolism of D-glucuronate and D-galacturonate. In this transformation, D-glucuronate and D-galacturonate are initially isomerized into their corresponding keto products, D-fructuronate and D-tagaturonate, respectively (1). D-Fructuronate and D-tagaturonate are then reduced to D-mannonate and D-altronate, respectively, by mannonate and altronate dehydrogenase in the presence of NADH (2). The pathways converge through a dehydration reaction in which mannonate dehydrase and altronate dehydrase convert mannonate and altronate to 2-keto-3-deoxy-D-gluconic acid (KDG). This product is then phosphorylated by the enzyme ketodeoxygluconic acid kinase with ATP to form 2-keto-3-deoxy-6-phosphogluconic acid (KDG-6-P). In the final step of this pathway, 2-keto-3-deoxy6-phosphogluconic acid is cleaved by an aldolase to yield pyruvate and D-glyceraldehyde 3-phosphate, which enter the This work was supported in part by the National Institutes of Health (Grant GM71790) and the Robert A. Welch Foundation (A-840). *To whom correspondence should be addressed. F.M.R.: telephone, (979) 845-3373; fax, (979) 845-9452; e-mail,
[email protected]. S.C.A.: telephone, (718) 430-2746; fax, (718) 430-8565; e-mail, almo@ aecom.yu.edu. 1 Abbreviations: URI, uronate isomerase; KDG, 2-keto-3-deoxy-Dgluconic acid; KDG-6-P, 2-keto-3-deoxy-6-phospho-D-gluconic acid; MDH, mannonate dehydrogenase; ICP-MS, inductively coupled plasma mass spectrometry; PDB, Protein Data Bank; rmsd, root-meansquare deviation.
citric acid cycle and glycolysis. The entire pathway is summarized in Scheme 1 (1, 2). We have demonstrated that uronate isomerase is a member of the amidohydrolase superfamily of enzymes based on sequence alignments and three-dimensional structural comparisons (3). The majority of the functionally characterized members of the amidohydrolase superfamily catalyze the hydrolysis of amide or ester bonds to carbon or phosphorus centers (4, 5). Well-characterized examples include dihydroorotase (6), urease (7), and phosphotriesterase (8). Members of this superfamily also catalyze the deamination of many nucleotides, including adenosine (9), cytosine (10), and guanine (11). The active sites of these enzymes generally contain a mononuclear or binuclear metal center that is perched at the C-terminal end of the β-barrel within a (β/R)8 structural fold. The most highly conserved residues in the AHS include two histidines from β-strand 1, histidines after the ends of β-stands 5 and 6, and an aspartic acid from β-strand 8. Since URI catalyzes an isomerization of an aldose sugar to the corresponding ketose product, this enzyme is one of the most divergent members of the amidohydrolase superfamily. The mechanistic details of this transformation are therefore of significant interest for an improved understanding of how an active site that originally evolved to catalyze hydrolytic reactions has been reforged to undergo an isomerization reaction.
r 2009 American Chemical Society
Published on Web 08/14/2009
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pubs.acs.org/Biochemistry
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Nguyen et al.
Scheme 1
Scheme 2
We have previously demonstrated that the hydrogen originally at C-2 of D-glucuronate is ultimately found at the proR position at C-1 of D-fructuronate and that this hydrogen slowly exchanges with solvent (12). These results are consistent with a proton transfer mechanism with a cis-enediol intermediate. The general mechanism, shown in Scheme 2, indicates a requirement for at least two residues that participate in the transformation of D-glucuronate into D-fructuronate. A general base (:B1) abstracts the proton from C-2 of Dglucuronate, and a general acid (H:B2) facilitates the transfer of a proton to the carbonyl oxygen at C-1 to produce the cis-enediol intermediate. In the subsequent step, the ketose product is generated by the transfer of a proton from the hydroxyl group at C-2 of the proposed intermediate and protonation of C-1 by H:B1. For compounds such as D-glucuronate, the enzymatic transformation is made more complicated by the fact that in solution the substrate exists almost entirely as a mixture of two anomeric cyclic hemiacetals. This paper focuses on a determination of the chemical mechanism for the isomerization reaction catalyzed by URI from Escherichia coli. The rate-limiting steps have been interrogated by measuring the primary kinetic isotope effects with [2-2H]-D-glucuronate and solvent isotope effects with D2O for the wild-type and mutant enzymes. The rate limitation imposed by product release has been examined using solvent viscosity effects. The identity of the residues involved in the proton transfer events has been probed by pH-rate profiles and characterization of the kinetic constants for mutant enzymes. These approaches have been augmented by the determination of the X-ray structure of a uronate isomerase from Bacillus halodurans (Bh0493) in the presence of D-glucuronate, D-fructuronate, and two mimics of the cisenediol intermediate.
Scheme 3
MATERIALS AND METHODS Materials. D-Glucuronic acid (I), NADH, buffers, and all other chemicals were purchased from Sigma-Aldrich or Acros, unless otherwise stated. D-Arabinaric acid (III) and the monohydroxamate derivative of this compound (II) were synthesized as previously described (12). 2,6-Anhydro-L-gulonic acid (IV) was synthesized starting from L-xylose (13, 14). The structures of these compounds are presented in Scheme 3. Oligonucleotide syntheses and DNA sequencing were performed by the Gene Technologies Lab of Texas A&M University. Metal analyses were conducted using inductively coupled plasma mass spectrometry (ICP-MS) as previously described (12). Site-Directed Mutagenesis. Site-directed mutagenesis of URI was performed using the QuikChange mutagenesis kit from Stratagene. The following mutants were obtained by this method: H33N, H33A, H35N, H35A, H59N, H59A, Y60F, Y60A, R186K, R186M, D238N, H297N, R302K, R302M, H297A, W381F, W381A, D412N, D412A, R414K, and R414M. The mutations were confirmed by DNA sequencing of the modified plasmids. Protein Expression and Purification. The uxaC gene encoding uronate isomerase in E. coli was cloned into the pET28 expression vector. The protein was expressed in E. coli strain BL21(DE3) and purified as previously described (12). The enzymes contained up to 1 equiv of zinc (depending on the mutant) as measured by ICP-MS. Enzyme Assays. The conversion of D-glucuronate to D-fructuronate by URI was coupled to the reduction of D-fructuronate with NADH by mannonate dehydrogenase (MDH) as previously described (12). The assays were monitored spectrophotometrically by following the decrease in absorbance at 340 nm. The standard assay conditions included 50 mM HEPES (pH 8.0), varying
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Table 1: Data Collection and Refinement Statistics for Crystals of the Complexes of Uronate Isomerase from B. halodurans with Various Ligands URI 3
D-arabinarate
URI 3
URI 3 arabinohydroxamate
D-arabinarate
URI 3
D-glucuronate
URI 3
D-fructuronate
Data Collection space group no. of molecules in the asymmetric unit cell dimensions a, b, c (A˚) β (deg) resolution (A˚) no. of unique reflections Rmerge I/σI completeness (%)
R32 2
C2 12
R32 2
C2 12
P4122 3
149.35, 149.35, 254.16
274.82, 156.52, 185.96 116.20 2.2 352288 0.065 19.3 98.8
149.30, 149.30, 254.02
84.35, 99.14, 126.31
2.2 55174 0.071 20.6 99.8
274.09, 156.88, 185.21 115.78 2.1 404122 0.074 15.2 98.7
1.9 128635 0.086 17.1 93.9
25.0-2.2 0.204 0.226 6744 312 0.007 1.3 arabinohydroxamate 26 4Zn2þ, 2CO32-, 2Cl3HK8
25.0-2.1 0.227 0.258 40860 1824 0.006 1.3 D-glucuronate 156 12Zn2þ, 12CO32-, 4Cl3HK9
25.0-1.9 0.213 0.241 10164 572 0.006 1.3 D-fructuronate 39 4Zn2þ, 3CO32-, 1Cl3HKA
2.2 55294 0.058 27.1 99.7
Refinement resolution (A˚) Rcryst Rfree no. of protein atoms no. of waters rmsd for bond lengths (A˚) rmsd for bond angles (deg) bound inhibitor no. of inhibitor atoms bound ions PDB entry
25.0-2.2 0.218 0.238 6766 259 0.007 1.3 D-arabinarate 24 4Zn2þ, 2CO32-, 2Cl3HK5
25.0-2.2 0.213 0.245 40860 1880 0.006 1.3 D-arabinarate 144 12Zn2þ, 12CO32-, 4Naþ, 4Cl3HK7
concentrations of D-glucuronate, 0.2 mM NADH, excess MDH, and URI in a final volume of 250 μL. The pH dependence of the kinetic parameters, kcat and kcat/Km, was measured over the pH range of 5.3-10.3 at 0.20 pH intervals. The buffers used for the pH-rate profiles were MES, PIPES, HEPES, CHES, and CAPS. The pH values were recorded after the completion of the assays. The effects of solvent viscosity on the kinetic constants were determined at pH 8.0 using sucrose as the microviscogen at 25 °C. The concentrations of sucrose were 0, 10, 14, 20, 24, and 32% (w/w), and the corresponding relative viscosities were 1, 1.3, 1.5, 1.9, 2.2, and 3.2 (15, 16). The solvent isotope effects on the kinetic parameters for URI and two mutant enzymes (D412N and R414M) were measured in 99% D2O at pD 8.4. The primary deuterium kinetic isotope effects were obtained by direct comparison of the kinetic constants at pH 8.0 for [2-1H]-D-glucuronate and [2-2H]-D-glucuronate. Preparation of [2-2H]Glucuronic Acid. [2-2H]-D-Glucuronate was prepared from [2-2H]-D-glucose in three steps. The [2-2H]-D-glucose was refluxed in methanol in the presence of dry Dowex-50(Hþ) for 12 h to form a mixture of R- and β-methyl [2-2H]-D-glucopyranoside (17). The solvent was removed under reduced pressure to yield crystals of the pure R-anomer. Methyl [2-2H]-R-D-glucopyranoside was quantitatively oxidized at C-6 using a 2,2,6,6-tetramethyl-1-piperidinyloxy/sodium bromide/ sodium hypochlorite mixture at pH 10, to form methyl [2-2H]R-D-glucuronopyranoside (18, 19). The product was washed with methanol and purified using a column of DEAE-Sephadex with a gradient of sodium bicarbonate. The fractions containing the desired product were evaporated to dryness. The methyl [2-2H]D-glucuronopyranoside was demethylated with concentrated HCl at 4 °C and the pH adjusted to ∼10 with sodium hydroxide. The concentration of [2-2H]-D-glucuronate was determined
enzymatically and was produced in an overall yield of 46%. The products from each step in the synthesis were characterized by 1H and 13C NMR and mass spectrometry. Data Analysis. The kinetic parameters, kcat and kcat/Km, for uronate isomerase with D-glucuronate as the substrate were determined by fitting the initial velocity data to eq 1 v=Et ¼ ðkcat ½AÞ=ðKa þ ½AÞ
ð1Þ
where v is the initial velocity, Et is the total enzyme concentration, kcat is the turnover number, [A] is the substrate concentration, and Km is the Michaelis constant. The profiles for the variation of kcat or kcat/Km with pH were fit to eq 2 log y ¼ log½c=ð1 þ H=Ka þ Kb =HÞ
ð2Þ
where c is the pH-independent value of y, Ka and Kb are the dissociation constants of the ionizable groups, and H is the proton concentration. The competitive inhibition patterns were fit to eq 3 v=Et ¼ ðkcat ½AÞ=½Ka ð1 þ I=Kis Þ þ ½A
ð3Þ
where Kis is the slope inhibition constant and I is the concentration of the inhibitor. Crystallization and Data Collection. Five different crystalline complexes (Table 1) were grown by the hanging drop method at room temperature for Bh0493 from B. halodurans: (a) complex with D-arabinarate, crystal form 1; (b) complex with D-arabinarate, crystal form 2; (c) complex with arabinohydroxamate; (d) complex with D-glucuronate; and (e) complex with D-fructuronate. The initial protein solution for all five crystallizations contained Bh0493 (16 mg/mL) in 10 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM methionine, 10% glycerol, 1.0 mM DTT,
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0.2 mM ZnCl2, and the corresponding substrate or inhibitor at 40 mM. The crystallization conditions were as follows. For the Bh0493 3 D-arabinarate complex (form 1), the protein solution contained 40 mM D-arabinarate and the precipitant contained 25% PEG 3350, 0.1 M Tris (pH 8.5), and 0.2 M NaCl. Crystals appeared in 4-5 days and exhibited diffraction consistent with space group R32, with two copies of the complex per asymmetric unit. For the Bh0493 3 D-arabinarate complex (form 2), the protein solution contained 40 mM D-arabinarate and the precipitant contained 20% PEG 3350 and 0.2 M sodium citrate (pH 6.0). Crystals appeared in 4 days and exhibited a diffraction pattern consistent with space group C2, with 12 copies of the complex per asymmetric unit. For the Bh0493 3 arabinohydroxamate complex, the protein solution contained 40 mM arabinohydroxamate and the precipitant contained 25% PEG 3350, 0.1 M Tris (pH 8.5), and 0.2 M NaCl. Crystals appeared in 2 days and exhibited a diffraction pattern consistent with space group R32, with two copies of the complex per asymmetric unit. For the Bh0493 3 D-glucuronate complex, the protein solution contained 40 mM D-glucuronate and the precipitant contained 20% PEG 3350 and 0.2 M ammonium citrate (pH 6.0). For this complex, the protein solution was incubated on ice with D-glucuronate for 2 h before crystallization. The crystals appeared in 9 days and exhibited diffraction consistent with space group C2, with 12 copies of the complex per asymmetric unit. For the Bh0493 3 D-fructuronate complex, the protein solution was incubated on ice for ∼2 months with 40 mM D-glucuronate before crystallization. The precipitant contained 25% PEG 3350, 0.1 M Tris (pH 8.5), and 0.2 M NaCl. Crystals appeared in 2 weeks and exhibited diffraction consistent with space group P4122, with three copies of the complex per asymmetric unit. Prior to data collection, the crystals of all Bh0493 complexes (Table 1) were transferred to cryoprotectant solutions composed of their mother liquids and 20% glycerol and flash-cooled in a nitrogen stream. All data sets were collected at the NSLS X4A beamline (Brookhaven National Laboratory) on an ADSC CCD detector (Table 1). Diffraction intensities were integrated and scaled with DENZO and SCALEPACK (20). The data collection statistics are given in Table 1. Structure Determination and Model Refinement. All five URI structures (Table 1) were determined by molecular replacement with the fully automated molecular replacement pipeline BALBES (21), using only input diffraction and sequence data. The native uronate isomerase from B. halodurans (PDB entry 2Q08) was used by BALBES as a template in all five structure determinations. Partially refined structures of all URI crystal forms (Table 1) were output from BALBES without manual intervention. Several iterative cycles of refinement were performed for each crystal form, including manual model rebuilding with TOM (22), refinement with CNS (23), automatic model rebuilding with ARP (24), and solvent building with the CCP4 suite (25). The rhombohedral crystal form of the Bh0493 3 D-arabinarate complex contains two copies of the complex in the asymmetric unit of the cell; the monoclinic crystal form of the same complex contains 12 copies of the complex in the asymmetric unit packed as four trimers. The first residue and last 13 residues of every molecule are disordered in the first crystal form of this complex. The four N-terminal residues and last 14 residues are disordered in every molecule of the second crystal form of the Bh0493 3 D-arabinarate complex. The disordered residues are not included in the final models. The asymmetric unit of the
Nguyen et al.
FIGURE 1: pH-rate profile for the wild-type uronate isomerase from E. coli containing 1 equiv of zinc. The data were fit to eq 2: (A) plot of log kcat vs pH and (B) plot of log kcat/Km vs pH.
Bh0493 3 arabinohydroxamate crystalline complex contains two molecules of the complex, where the first residue and the last 14 residues of every molecule are disordered. The asymmetric unit of the Bh0493 3 D-glucuronate crystalline complex contains 12 copies of the complex packed as four trimers. The first residue and the last 13 residues are disordered in every molecule of this complex. The asymmetric unit of the Bh0493 3 D-fructuronate crystalline complex contains one trimer of the complex. The first two residues and last 14 residues are disordered in every molecule and are not included in the final models. This complex was produced by a long incubation and subsequent cocrystallization of Bh0493 with D-glucuronate, but the electron density of the bound inhibitor can be interpreted only as the product, Dfructuronate. The Zn2þ ions bound in the active sites were clearly visible in every molecule of every URI complex listed in Table 1. Additional ions (Naþ, Zn2þ, and Cl-) located on the local 3-fold axis of every Bh0493 trimer also exhibited good density in all five URI crystalline complexes. Final crystallographic refinement statistics for all of the URI complexes are provided in Table 1. RESULTS Requirement for a Divalent Cation. The importance of a metal ion for the catalytic activity of uronate isomerase was reinvestigated. The apoenzyme was prepared and subsequently tested for enzymatic activity using D-glucuronate as the substrate. The wild-type URI from E. coli was found to contain 0.9 equiv of zinc after purification. This protein (3 mL) at a concentration of 3.0 mg/mL was dialyzed against 1 L of dialysis buffer containing 20 mM dipicolinate in 50 mM MES (pH 6.0). The buffer was changed three times over the course of 48 h, and then the catalytic activity and metal content of the enzyme were determined. The
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Table 2: Kinetic Parameters and Metal Content of Mutants of URI from E. colia enzyme
kcat (s-1)
Km (mM)
kcat/Km (M-1 s-1)
URI/Zn
wild type H33N (H26)b H33A H35N (H28) H35A H59N (H49) H59A Y60F (Y50) Y60A R186K (R170) R186M D238N H297N (M258) H297A R302K (K303) R302M W381F (W325) W381A D412N (D355) D412A R414K (R357) R414M
196 ( 6 2.1 ( 0.1 0.60 ( 0.01 4.0 ( 0.2 0.70 ( 0.04 15 ( 1 0.60 ( 0.01 21.7 ( 0.1 13.9 ( 0.1 54 ( 2 4.7 ( 0.1 60 ( 1 30 ( 2 10 ( 1 160 ( 4 180 ( 9 16 ( 1 250 ( 6 0.60 ( 0.01 (9.0 ( 0.3) 10-3 5.8 ( 0.1 0.70 ( 0.01
0.50 ( 0.05 (5.0 ( 0.4) 10-2 0.20 ( 0.01 9.4 ( 1.1 39 ( 5 0.70 ( 0.04 0.70 ( 0.07 0.16 ( 0.01 0.21 ( 0.01 2.6 ( 0.2 38 ( 3 1.3 ( 0.1 56 ( 5 (2.2 ( 0.3) 102 2.5 ( 0.2 (2.0 ( 0.3) 102 1.7 ( 0.1 21 ( 2 1.00 ( 0.04 0.40 ( 0.05 0.82 ( 0.02 1.4 ( 0.1
(4.0 ( 0.4) 105 (4.7 ( 0.4) 104 (3.0 ( 0.2) 103 (4.3 ( 0.5) 102 18 ( 2 (2.1 ( 0.1) 104 (8.3 ( 0.1 102 (1.4 ( 0.1) 105 (6.6 ( 0.3) 104 (2.1 ( 0.2) 104 (1.3 ( 0.1) 102 (4.6 ( 0.1) 104 (5.0 ( 0.5) 102 43 ( 7 (6.3 ( 0.5) 104 (8.8 ( 1.3) 102 (9.5 ( 0.4) 103 (1.2 ( 0.1) 104 (6.0 ( 0.3) 102 21 ( 3 (7.1 ( 0.2) 103 (5.4 ( 0.2) 102
0.90 0.07 0.20
