Crystal Structure of Escherichia coli Ketopantoate Reductasein a Ternary Complex with NADP� and Pantoate BoundSUBSTRATE RECOGNITION, CONFORMATIONAL CHANGE, AND COOPERATIVITY*□S
Received for publication, December 5, 2006, and in revised form, January 4, 2007 Published, JBC Papers in Press, January 16, 2007, DOI 10.1074/jbc.M611171200
Alessio Ciulli‡, Dimitri Y. Chirgadze§, Alison G. Smith¶, Tom L. Blundell§, and Chris Abell‡1
From the ‡University Chemical Laboratory, Lensfield Road, Cambridge CB2 1EW, United Kingdom, §Department of Biochemistry,University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, United Kingdom, and ¶Department of Plant Sciences,University of Cambridge, Downing Street, Cambridge CB2 3EA, United Kingdom
Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes theNADPH-dependent reduction of ketopantoate to pantoate,an essential step for the biosynthesis of pantothenate (vita-min B5). Inhibitors of the enzymes of this pathway have beenproposed as potential antibiotics or herbicides. Here we pres-ent the crystal structure of Escherichia coli KPR in a precata-lytic ternary complex with NADP� and pantoate bound,solved to 2.3 A of resolution. The asymmetric unit containstwo protein molecules, each in a ternary complex; however,one is in a more closed conformation than the other. A hingebending between the N- and C-terminal domains is observed,which triggers the switch of the essential Lys176 to form a keyhydrogen bond with the C2 hydroxyl of pantoate. Pantoateforms additional interactions with conserved residues Ser244,Asn98, and Asn180 and with two conservatively varied resi-dues, Asn194 and Asn241. The steady-state kinetics of activesite mutants R31A, K72A, N98A, K176A, S244A, and E256Aimplicate Asn98 as well as Lys176 and Glu256 in the catalyticmechanism. Isothermal titration calorimetry studies withthese mutants further demonstrate the importance of Ser244for substrate binding and of Arg31 and Lys72 for cofactorbinding. Further calorimetric studies show that KPR discrim-inates binding of ketopantoate against pantoate only withNADPH bound. This work provides insights into the roles ofactive site residues and conformational changes in substraterecognition and catalysis, leading to the proposal of a detailedmolecular mechanism for KPR activity.
Pantothenate (vitamin B5) is the precursor of the 4�-phos-phopantetheine moiety of coenzyme A and acyl carrier pro-teins, which play an important role inmetabolism and fatty acidbiosynthesis (1–3). The biosynthetic pathway for pantothenatehas been elucidated in Escherichia coli and other bacteria and is
composed of four enzymes. The first two convert �-ke-toisovalerate to pantoate, then in a separate branch L-aspartateis decarboxylated to produce �-alanine. Finally, pantoate and�-alanine are condensed together to form pantothenate (4, 5).The pathway is similar in plants and fungi, although they appearto use a different route to �-alanine (6). Bioinformatics analysishas identified the pantothenate pathway as a potential antimi-crobial target (7). This is supported by recent genetic studieswhich show that a pantothenate auxotroph of Mycobacteriumtuberculosis fails to establish chronic infections in mice (8).Ketopantoate reductase (KPR,2 EC 1.1.1.169), encoded by
the panE gene, is the second enzyme in the pathway and cata-lyzes the NADPH-dependent reduction of ketopantoate topantoate. Previous biochemical studies on the Escherichia colienzyme established that hydride transfer is stereospecific fromthe pro-S proton of NADPH to the si face of ketopantoate(Scheme 1A), and the reaction equilibrium favors NADP� andpantoate formation (9, 10). Steady-state kinetic and inhibitionanalysis are consistent with a sequential ordered bi:bi kineticmechanism (Scheme 1B) in which NADPH binding is followedby ketopantoate binding, and then pantoate release precedesNADP� release (10). The pHdependence of catalysis is consist-ent with the involvement of a general acid/base in the catalyticmechanism (9, 10). Site-directedmutagenesis implicated Lys176and Glu256 as important for catalysis (9).The crystal structure of the apoenzymewas solved byMatak-
Vinkovic et al. (11) at 1.7 Šof resolution. KPR belongs to the6-phosphogluconate dehydrogenase superfamily in the SCOPdata base (11, 12). Among other enzymes in this superfamily areacetohydroxy acid isomeroreductase (13), short chain L-3-hy-droxyacyl-CoA dehydrogenase (14), �1-pyrroline-5-carboxy-late reductase (15), and prephenate dehydrogenase (16). Thesecondary structure of KPR comprises 13 �-helices and 11�-strands. The enzyme is monomeric with a molecular mass of34 kDa and is composed of a coenzyme binding domain and asubstrate binding domain separated by a large cleft. The N-ter-minal domain has an �� Rossmann-type fold featured in manynucleotide-binding proteins, with a glycine-rich region(7GCGALG12) for coenzyme recognition (17). The C-terminalsubstrate binding domain is composed of 8 �-helices and has acore of two long antiparallel helices, which is a common motifwithin the superfamily.
* This work was supported by the Biotechnology and Biological SciencesResearch Council. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.
□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1, Tables S1 and S2, and Movies S1 and S2.
The atomic coordinates and structure factors (code 2OFP) have been deposited inthe Protein Data Bank, Research Collaboratory for Structural Bioinformatics,Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed. Tel.: 44-1223-336405; Fax:44-1223-336362; E-mail: [email protected].
2 The abbreviations used are: KPR, ketopantoate reductase; ITC, isothermaltitration calorimetry; r.m.s.d., root-mean-square deviation; WT, wild-type.
More recently, we solved the crystal structure of KPR in com-plex with NADP� to 2.1 Å of resolution (18). Surprisingly, itwas found to be in the same open conformation as the structureof apoenzyme. The cofactor is bound in an extended conforma-tion encompassing the active site cleft, with the nicotinamidering adopting a syn conformation. Analysis of this structure ledto the proposal that Lys176 could act as the general acid/base forcatalysis. This required a significant change in the conforma-tion of the lysine to switch to an “active” state where it canhydrogen bond to the substrate. However, there was no directstructural evidence for this conformational change of Lys176.Elucidation of the atomic details of substrate binding and of thecatalytic mechanism required crystal structures of the ternarycomplex, which has yet been unsuccessful (18, 19).Here we present the three-dimensional structure of E. coli
KPR in a ternary complex with NADP� and pantoate, solved to2.3 Å of resolution. Two protein molecules were found in theasymmetric unit, each with NADP� and pantoate bound. Onecomplex is in an open form, and the other is in a closed form,demonstrating that a hinge bending domain closure occurs.Calorimetric and kinetic studies on ternary complexes andengineeredmutants provide additional insights into themolec-ular details of catalysis.
EXPERIMENTAL PROCEDURES
Materials—All chemicals were purchased from SigmaAldrich unless otherwise stated. Solutions for crystallographywere fromQiagenNextal. Ketopantoate andpantoatewere pre-pared by hydrolysis of their respective lactones with NaOH asdescribed elsewhere (20).Protein Expression and Purification—Wild-type (WT) KPR
and site-directed mutants of KPR were expressed from apRSETA plasmid, which adds 17 amino acid residues (MRG-SHHHHHHGLVPRGS) to the N terminus of the recombinantprotein, including a His6 tag. Expression, single-step affinitypurification, and characterization of His6-KPR proteins wereconducted as previously described (21). Purified proteins werebuffer-exchanged in 50–100 mM HEPES-HCl, pH 7.6, using aHiPrep 26/10 Desalting column (GE Healthcare) and used forcalorimetric and kinetic studies. For crystallization studies,
the WT protein was further puri-fied by size exclusion chromatog-raphy using a HiLoad 26/60 Super-Dex 200 gel filtration column (GEHealthcare). The column waseluted using 50 mM HEPES-HCl,pH 7.6, at a flow rate of 1.0 mlmin�1. His6-KPR runs as a mono-mer in gel filtration (apparentmolecular mass of �32 kDa). Pro-tein concentration was deter-mined by A280, with an extinctionco-efficient of 62,650 M�1 cm�1
obtained from amino acid analysis(Protein and Nucleic Acid Chem-istry Facility, Cambridge). Theintegrity and purity (�95%) ofprotein samples were determined
by SDS-PAGE and electrospray mass spectrometry.Crystallization of His6-KPR—Crystal trials were carried out
using vapor diffusion by the hanging drop vapor-diffusion tech-nique using a protein concentration of 15–30 mg/ml. Beforecrystallization KPR samples were incubated at 20 °C with 2 mM
NADP� and 10 mM pantoate for 10 min. Single rhombohe-drally shaped crystals of KPR were obtained in drops made of 2�l of protein solution mixed with an equal volume of well solu-tion composed of 35% v/v dioxane. To facilitate data collectionat 100 K, these crystals were cryo-protected using the well solu-tion in the presence of 20% v/v 2-methyl-2,4-pentanediol.X-ray Data Collection, Structure Determination, and
Refinement—X-ray data to 2.3 Å of resolution were collected atEuropean Synchrotron Radiation Facility synchrotron, beamstation ID14.4 (Grenoble, France). The diffraction data statis-tics are shown in Table 1. The data were scaled, merged, andreduced using HKL suite (22). The crystal belongs to the prim-itive tetragonal lattice, space groupP41212,with cell parametersa � b � 101.7 Å and c � 171.2 Å. Analysis of the Matthewscoefficient indicated the presence of two molecules in theasymmetric unit of the crystal. This corresponds to a solventcontent of 58%. Calculation of the self-rotation functionshowed the presence of a non-crystallographic 2-fold axis sug-gesting a dimeric arrangement of the two molecules of KPR.The structure was solved by molecular replacement usingAMoRe from the CCP4 suite (23–25). First, the KPR�NADP�
complex (holoKPR, PDB code 1YJQ (18)) was used as themolecular replacement search probe, and one clear solution(monomerA)was found.However, the second solutionwas notpresent in the top 10 peaks of the rotation function. The secondmolecule (monomer B) was subsequently found using molecu-lar replacement with apoKPR (PDB code 1KS9 (11)) as thesearch probe. Before calculation, the probes were edited byremoving all non-protein atoms. The apo- and holoproteinprobeswere placed in the crystal cell according to the rotationaland translational parameters obtained. The resulting correla-tion coefficient and Rcryst between observed and calculatedstructure factor amplitudes were 54.8 and 49.2%, respectively.The non-crystallographic 2-fold relationship between the two
SCHEME 1. A, ketopantoate is reduced by NADPH, with relevant atoms numbered. The pro-S hydride of NADPHis transferred to the si face of ketopantoate. B, sequential ordered bi:bi kinetic mechanism.
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KPR molecules in the asymmetric unit was consistent with theresults of the self-rotation function.Aftermolecular replacement, rigid body refinement was per-
formed by splitting each of KPR monomers into two separaterigid bodies corresponding to the N- and C-terminal domains(residues 1–169 and 170–291, respectively). Simulated anneal-ing was carried out using CNS (26), improving the Rcryst andRfree factors to 27.8 and 32.3%, respectively, and initial electrondensity maps were produced. The structure was refined usingsuccessive rounds of manual rebuilding in Coot version 0.0.33(27) and maximum likelihood refinement using Refmac 5 fromthe CCP4 suite (25, 28) until no unexplained electron densityremained, and the Rcryst and Rfree values converged at 15.6 and21.5%, respectively (Table 1). The final structure includes twomolecules of KPR (monomer A, residues 0–292; monomer B,residues 0–293), each in complexwith onemolecule ofNADP�
and one molecule of pantoate. In addition, four molecules ofdioxane and one molecule of acetate have been included in themodeled solvent. Coordinates for the non-protein moleculeswere obtained from theHiCUP data base (29), and some refine-ment libraries were obtained using monomer library Sketcher(CCP4). The following residues were built with double confor-mations: Arg170, Glu209, and Glu240 in monomer A; Tyr36,
Ser77, Arg170,Glu172,Gln206,Glu209,Glu216, andArg217 inmon-omer B. None of the above residues is near the active site.Site-directed Mutagenesis—Site-directed mutants of KPR
corresponding to R31A, K72A, N98A, K176A, S244A, andE256A were made with the QuikChange site-directedmutagenesis kit (Stratagene) according to the manufacturer’sinstructions using pRSETA-panE as a template. The primersused were from Sigma Genosys and are listed in supplementalTable S1. Eachmutationwas verified byDNA sequencing (LarkTechnologies), and the molecular masses of the recombinantHis-tagged mutant enzymes were determined using electro-spray mass spectrometry.UV-based Kinetic Analysis—Assays were carried out on a
Biotek Powerwave XS plate reader equipped with KC4 Version3.2 Biotek instrument software using a 96-well plate with a totalvolume of 0.2 ml in each well. Enzyme activity was assayed at25 °C bymonitoring the decrease in absorbance at 340 nm overtime due to the enzyme-catalyzed oxidation of NADPH toNADP� (�340 nm for NADPH � 6220 M�1 cm�1). A typicalreaction forWTKPRcontained 100mMHEPES-HCl buffer, pH7.6, 2–100 �M NADPH, 0.02–1 mM ketopantoate, and 1–5 nMenzyme. The concentration of active WT and mutant forms ofKPR was accurately determined by performing ITC titrationswith the cofactor (see below). The reaction was initiated byaddition of ketopantoate. Measurements were obtained at leastin duplicate. Initial rates were obtained from the data corre-sponding to the conversion of the first 10% of substrate. Datawere fitted to the Michaelis-Menten rate equation using theGraFit software (Version 5.0.6, Erithacus Software Ltd).Isothermal Titration Calorimetry—Titrations were per-
formed at 27 °C on OMEGA and VP-ITC isothermal titrationcalorimeters (Microcal, Inc.). Thermodynamic analysis ofbinary complexes were performed as previously described (18,21). For thermodynamic analysis of ternary complexes, the sec-ond ligand was titrated to the binary complex formed aftercompletion of titrations of KPR with the first ligand. To ensurenear saturation of the enzyme, the final concentration of thefirst ligand was �10�Kd in the case of ketopantoate and pan-toate and 2–3� the enzyme concentration in the case ofNADPH and NADP�. Concentrations of cofactors were meas-ured by UV-visible spectrophotometry using the extinctioncoefficients 6,220 M�1 cm�1 at 340 nm for NADPH (30) and18,000 M�1 cm�1 at 260 nm for NADP� (31). The heat changeaccompanying the titration was recorded as differential powerby the instrument and determined by integration of each peakobtained. Titrations of ligand to buffer were performed to allowfor baseline corrections. The corrected heat change was thenfitted using nonlinear least-squares minimization to obtain thedissociation constants,Kd, the enthalpy of binding,�H, and thestoichiometry, n. A stoichiometry of 1, determined from titra-tions with NADP(H) under high c values conditions, was fixedduring curve-fitting of data obtained for ketopantoate and pan-toate under low c values (21).
RESULTS
Previous attempts to obtain ternary complexes of KPR weremade with the aim of forming “dead-end” complexes by co-crystallization with either NADP� and ketopantoate (18) or
TABLE 1Crystallographic data collection and refinement statisticsESRF, European Synchrotron Radiation Facility.
Data collectionX-ray source ESRF, ID14.4Space group P41212Cell parameters, Å (� � � � � � 90°)a � b 101.7c 171.2
Wavelength, Å 0.977Resolution range, Å (outer shell) 50.0-2.30 (2.35-2.30)No. of unique reflections 40,629Multiplicity 9.3Rmerge, % (outer shell) 7.3 (42.5)Average I/�(I) 12.8% Reflections with I/�(I) � 3, (outer shell) 86.6 (64.5)Completeness, % (outer shell) 99.9 (100)Mosaicity, ° 0.33Wilson B, Å2 38.8
RefinementResolution range, Å 43.9-2.3Rcryst,a % 15.6Rfree,b % 21.5Number of reflectionsWorking set 36,501Test set 2,032
Number of protein residues 587Water molecules 508Dioxane 4Acetate 1NADP� (NAP) 2Pantoate (PAF) 2
r.m.s.d. bonds,c Å 0.014r.m.s.d. angles,c ° 1.45Overall mean B,c Å2 36.3
aRcryst � �Fobs� � �Fcalc�/�Fobs�, and Fobs and Fcalc are observed and calculatedstructure factor amplitudes.
b Rfree as for Rcryst using a random subset of the data excluded from the refinement.c Estimated coordinate error based on the free R-value as calculated by Refmac 5.d Calculated with Procheck (46).
Structure of KPR Ternary Complexes
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with NADPH and pantoate (19). In both cases crystals wereobtained at pH 4.5; however, analyses of the crystal structuresrevealed no evidence of substrate or product bound. In the firststructure, the binary complex with NADP� alone was obtained(18). Binding of ketopantoate was not observed due to its lowaffinity for the KPR�NADP� complex under the experimentalconditions of crystallization. In the second structure the lowpHof crystallization resulted in the chemical degradation of NADPHto 2�-phospho-ADP-ribose (19). Electron density correspondingto 2�-phospho-ADP-ribose was observed in the active site,whereas no bound pantoate could be observed. These resultsclearly indicated that crystallization of the enzyme at low pH wasnot suitable for obtaining ternary complexes. Therefore, the for-mation of ternary complexes at the more physiologically relevantpH range of 7.0–8.0 was investigated.Kinetic andThermodynamic Studies of Ternary Complexes—
Kinetic analysis of the KPR-catalyzed reaction provided inter-esting results. At pH 7.5, the enzyme rapidly turns over in theforward direction using NADPH and ketopantoate as sub-strates, with kinetic parameters kcat � 25 s�1,Km(NADPH)� 7
�M, and Km (ketopantoate) � 30 �M (21) (see also Table 4). Incontrast, it was not possible to detect the reverse reaction at pH7.5, even using up to millimolar concentrations of NADP� andpantoate and 20 �M enzyme. The failure to detect any catalyticactivity at pH 7.5 using NADP� and pantoate as substrates isconsistent with the large apparent equilibrium constant,
Keq �NADP��pantoate�
NADPH�ketopantoate�� 676 (Eq. 1)
measured by Zheng and Blanchard under identical conditions(10). Further evidence for the stability of theMichaelis-Mententernary complex KPR�NADP��pantoate at pH 7.5 came fromNMR spectroscopy. Simultaneous binding of NADP� and pan-toate to KPR was detected in a 1HWaterLOGSY NMR experi-ment (18, 32), and no formation of NADPH or ketopantoatewas observed (data not shown), suggesting the ternary complexcould be formed under thermodynamic equilibrium.The formation of ternary complexes of KPR with coenzyme
and substratemolecules was investigated by ITC at 27 °C. Fig. 1
FIGURE 1. Thermodynamic analyses of formation of binary and ternary complexes of E. coli KPR with NADP� and pantoate by ITC. Integrated data, correctedfor the heat of dilution, are shown from titrations at 27 °C of 50 �M His6-KPR with 8 mM pantoate (A) and with 0.7 mM NADP� (B). The resulting binary complexes werefurther titrated with 0.7 mM NADP� (C) and with 8 mM pantoate (D). The line represents the least-squares fit to the single-site binding model by the ORIGIN program.
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shows a thermodynamic cycle for the formation of the ternarycomplex KPR�NADP��pantoate and the corresponding ITCbinding curves to form binary (Fig. 1,A and B) and ternary (Fig.1, C and D) complexes. The Kd of NADP� and pantoate fromthe binary complexes were 6 and 270 �M (Table 2). The aboveKd values are similar to those from the ternary complex (Kd of 9and 160 �M), thus excluding cooperativity between NADP�
and pantoate binding. However, the relative enthalpic andentropic terms changed significantly between binary and ter-nary complexes. For both pantoate and NADP�, formation ofthe binary complex is exothermic (�H of �8.1 and �3.2 kcal/mol; see also Fig. 1, A and B), whereas formation of the ternarycomplex is endothermic (�H of 1.8 and 6.3 kcal/mol) anddriven by favorable �T�S. Such large and compensating
changes in �H and �T�S (�10kcal/mol for each ligand) are oftenassociated with protein conforma-tional changes or protonationevents coupled to the formation ofthe protein-ligand complex (33).Similarly, compensating enthalpicand entropic changeswere observedfor the formation of the dead-endKPR�NADP��ketopantoate ternarycomplex relative to the binary com-plexes, with no significant changesin Kd (Table 2). However, analysis ofternary complexes with NADPHshowed evidence of cooperativitybetweencofactorandsubstrate.Bind-ing of NADPH decreases the affinityof pantoate for the enzyme by�4-fold (Table 2). In contrast, keto-pantoate binding to theKPR�NADPH(Km� 30�M) is significantly strongerthan that to apoKPR (Kd � 5 mM),suggesting that it is enhanced by thepresence of NADPH.Crystallization and Structure
Determination—Based on the re-sults of the kinetic and thermody-namic studies of ternary complexes,we attempted to co-crystallize KPR
in the presence of saturating amounts of NADP� and pantoateat pH 7.5. As a result of a sparse matrix screening of a solutioncontaining 0.5–1mMHis6-KPR, 2mMNADP�, 10mMpantoatein 50 mM HEPES-HCl, pH 7.5, single rhombohedrally shapedcrystals were identified in 35% v/v dioxane (solution 51 of TheClassics Suite, Nextal Qiagen). Attempts to crystallize apoKPRor binary complexes of KPR under identical conditions wereunsuccessful. Larger crystals were obtained from drops set upby mixing 2 �l of protein solution with 2 �l of this solution.The crystals diffracted to 2.3 Å of resolution. The crystalstructure of the KPR�NADP��pantoate complex was solvedusing molecular replacement in AMoRe (23, 24). Two mol-ecules of KPR were found in the asymmetric unit of the crys-
FIGURE 2. The structure of the KPR�NADP��pantoate ternary complex. The two monomers present in theasymmetric unit are related by a non-crystallographic 2-fold axis. Monomer A (shown in green, right) is in anopen form, whereas monomer B (shown in cyan, left) is in a closed form. The secondary structure elements arelabeled in monomer B. NADP� and pantoate are shown as sticks (yellow, carbon; blue, nitrogen; red, oxygen;orange, phosphorus). Figs. 2, 3, 4, and 6 were generated and rendered using Pymol Version 0.99 (45).
TABLE 2Thermodynamic parameters of cofactor and substrate binding to form binary and ternary complexes with E. coli KPRAll ITC titrations were performed at 27 °C in 100 mM HEPES HCl, pH 7.6. Errors quoted are those returned by Origin on the curve fitting.
Ligand in syringe Protein Kd �G �H �T�S�M kcal/mol kcal/mol kcal/mol
tal (Fig. 2, see “Experimental Procedures” for details). The2Fo � Fc and Fo � Fc electron density maps clearly showedthe presence of NADP� and pantoate in the active site cleftof both protomers (Fig. 3).Overall Crystal Structure—The two molecules of KPR are
arranged as a dimer and are related to each other by a non-crystallographic 2-fold axis (Fig. 2). The dimeric interface com-prises �5 and �11 at the end of the N-terminal domain and �9of theC-terminal domain. This dimeric arrangement is likely tobe due to crystallographic packing, as the biologically func-tional state ofKPR ismonomeric (10), and the asymmetric unitsof all previous crystal structures consist of one protein mono-mer (11, 18, 19). One complex (monomer B) is in a more closedform than the other (monomer A). Analysis of main chainatoms r.m.s.d. upon superposition over the N- and C-terminaldomain is consistent with a movement between the twodomains (see supplemental Table S2). Closer inspection revealsthe conformational change is a hinge bending, i.e. a motionperpendicular to the plane of the domain interface (see supple-mental Movie S1).Hinge Bending; Comparison with Apo- and HoloKPR—Co-
factor-induced hinge bending domain closure has beenobserved for several dehydrogenases, including alcohol dehy-drogenase (34), formate dehydrogenase (35), glutamate dehy-drogenase (36), diaminopimelate dehydrogenase (37), and
aspartate-�-semialdehyde dehydrogenase (38). It was, there-fore, surprising that the crystal structure of the KPR�NADP�
complex compared with apoKPR revealed no evidence of ahinge bending induced by the cofactor (18). Fig. 4 shows asuperposition over the main chain atoms of the N-terminaldomain and conformational changes of the protein backbone inthe C-terminal domain between apo- and holoKPR (Fig. 4A),holo- andmonomerA (Fig. 4B), andmonomerA andmonomerB (Fig. 4C). The pairwise r.m.s.d. values of the C-terminaldomain main chain atoms are 1.56 Å (apo/holo), 1.85 Å (holo/monomerA), and 3.45Å (monomerA/monomerB), suggestingthe conformational change is more pronounced along the reac-tion coordinates (Table 3).An analysis of the conformational changes between these
pairs of structure using the program DynDom (39) reveals theC-terminal domain motions occur around three different rota-tion axes (see the gray arrows in Fig. 4). Apo- and holoKPR areboth in an open form and are closer to monomer A than tomonomer B (see Fig. 4 and Table 3). No hinge bending domainclosure is identified in either apo/holo or holo/monomer A. Ineach case the rotation axis has a component perpendicular tothe active site cleft (see Fig. 4,A and B), indicating that a degreeof shear-like domainmotion occurs. In contrast, hinge bendingis observed between the two KPR�NADP��pantoate ternarycomplexes (Fig. 4C). The interdomain region formed by resi-
FIGURE 3. Crossed-eye stereo view of the active site cleft of the KPR�NADP��pantoate complex. Crystal structure showing the binding of NADP� andpantoate in the KPR active site at 2.3 Šresolution for the open (A) and the closed (B) ternary complexes. The ligands are shown as sticks with yellow carbons, withthe final 2Fo � Fc electron density identifying the presence of the ligands superimposed in gray and contoured at 1.5�. The electron density is also shown forLys176. Key protein residues are shown with carbon atoms in green (A) and cyan (B), nitrogen in blue, oxygen in red, and phosphorus in orange. Hydrogen bondsare indicated with magenta dashed lines.
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dues 169–176 (shown in orange in Fig. 4C) acts a mechanicalhinge (39). A 14° rotation around a hinge axis parallel to theactive site cleft triggers the domain closure to produce the cat-alytic ternary complex (see supplemental Movie S1).Cofactor Binding—The cofactor binds in an extended con-
formation at the active site cleft in both ternary complexes(Figs. 2 and 3). The non-covalent interactions with the proteinresidues are shown schematically in Fig. 5. The main interac-tions of NADP� inmonomer A are similar to those observed inthe structure of holoKPR by Lobley et al. (18). The 2�-phos-phate group makes two hydrogen bonds with the guanidiniumpart of the Arg31 side chain; the adenine group sits in a hydro-phobic pocket defined by the aliphatic parts of the side chains ofArg31, Leu6, Leu71, and Gln75; the pyrophosphate group ishydrogen-bonded to the backbone amides of Ala10 and Leu11from the glycine-rich region; the nicotinamide-ribose 2�- and3�-hydroxyls form hydrogen bonds with the carboxylate groupof Glu256 and with the backbone amide of Asn98; the nicotina-mide groupmakes hydrophobic contacts with the side chains ofLeu11 and Thr118; and the nicotinamide carboxyl and amidegroups form hydrogen bonds with the backbone amide andcarbonyl of Ala122.
The binding mode of NADP� in monomer B is nearly iden-tical to that in the open complex, the only difference being a
subtle movement of the 2�-phosphate moiety (indicated by anarrow in Fig. 4C). However, several interactions with the pro-tein differ substantially due tomovements of active site residuesduring the hinge bending (see Figs. 3 and 5). First, the side chainof Arg253 swings by�8 Å to form two hydrogen bonds with the2�-phosphate of the cofactor, leaving only one hydrogen bondfrom Arg31. Second, rotation of the side chain of Asn98 bringsits amide group into hydrogen-bond contact with the nicotin-amide ribose 2�-hydroxyl. Third, the domain closure brings the�9-loop–�10 motif on top of the C-terminal domain towardthe reaction center, allowingAsn241 to formahydrogen bond tothe nicotinamide carbonyl oxygen as well as to the C4 hydroxylof pantoate. Finally, a subtle movement of the side chain ofLys72 allows hydrogen bonding with the pyrophosphate ofNADP�.Substrate Binding—The ternary complex crystal structures
here reported provide the first evidence of substrate binding toKPR. The hinge bending brings the C2 carbon of pantoate�3.3Å from the C4 carbon of NADP� (Fig. 4C). This distance isoptimal for hydride transfer (40), suggesting monomer B is theprecatalytic ternary complex. Significantly, the C2 hydroxyl ofpantoate forms a hydrogen bond with the side chain amine ofLys176 at 2.7 Å of distance (Figs. 3B and 6A). This is the firstdirect evidence of this interaction, and of the “active state” con-formation of Lys176 previously proposed by Lobley et al. (18).The side chain amine of Lys176 forms additional hydrogenbonds with the side chain carbonyl of Asn98 and main chaincarbonyl of Thr118 (Fig. 6A). In monomer B, pantoate is fullyenclosed between the two domains in such a way as to excludeaccess by the solvent. The carboxylate group forms hydrogenbonds with the side chain hydroxyl and the backbone amide ofSer244 and with the side chain amide of Asn98. The C2 hydroxylforms an additional hydrogen bondwith the side chain amide ofAsn180. The C4 hydroxyl makes two hydrogen bonds with theside chain amides of Asn194 and Asn241. Finally, the C3 dimeth-
FIGURE 4. Conformational changes of KPR during catalysis. Superposition of apo/holo (A), holo/monomer A (B), and monomer A/monomer B (C) are shownaccording to the backbone atoms of the N-terminal domain (residues 1–169). The rotation axes for the C-terminal domain are shown as gray arrows. ApoKPR(11) is colored in purple, the KPR�NADP� binary complex (18) is in yellow, and the KPR�NADP��pantoate ternary complexes are in green (monomer A) and cyan(monomer B). NADP� and pantoate molecules are shown as sticks, with carbon atoms colored according to their respective structures.
TABLE 3Root mean square deviations from superposition of KPR structures
Superposition on mainchain atoms of residues
1–169
r.m.s.d. (main chain atoms, Å)
HoloKPR Monomer A Monomer B
N-terminal domain only (residues 1–169)ApoKPR 0.58 0.61 0.61HoloKPR 0.47 0.48Monomer A 0.23
C-terminal domain only (residues 170–292)ApoKPR 1.56 2.63 5.54HoloKPR 1.85 5.01Monomer A 3.45
Structure of KPR Ternary Complexes
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yl group sits in a hydrophobic pocket defined by the side chainsof Thr119, Val179, Ile183, Val234, and Thr238.In monomer A pantoate is more than 6.5 Å away from
NADP�, with water molecules filling the gap left in the activesite. Throughout the closed-open conformational change, thecarboxylate group of pantoate remains anchored to Ser244 andAsn98, and the C2 and C4 hydroxyls maintain their hydrogenbonds with Asn180 and Asn194, respectively (Fig. 6B). Thehydrogen bond to Asn180 appears to be important to stabilizethe C2 hydroxyl of pantoate. This interaction may account forthe enzyme selectivity for pantoate over ketopantoate (20-foldwith apo-KPR and 80-fold with KPR�NADP�). The hydrogenbondwith Lys176 is crucially lost, as its side chain switches awayfrom the substrate to form intramolecular hydrogen bondswiththe Glu172 carboxylate and with the Tyr230 hydroxyl (notshown). A similar “resting state” of the protonated Lys176 hadbeen previously observed in the crystal structure of the
KPR�NADP� complex (18). BecauseLys176 exhibited a pK value of 7.8 inthe KPR�NADP��pantoate complex(10), it is possible that under thecrystallization conditions (pH �7.5) the side chain amine of Lys176 ispartly deprotonated, resulting in theactive state observed in the closedternary complex. It is interesting tonote that Lys176 adopted a similarorientation in the apo structure,obtained at pH 9.4 (11). A hydrogenbond between the C4 hydroxyl andAsn241 is also lost during the open-ing of the hinge. Subtle rotationsaround the carbon backbone of pan-toate bring its C3 dimethyl groupcloser to the C-terminal domain, tomake hydrophobic contacts withVal179, Ile183, and Val234.Steady-state Kinetics and Binding
Thermodynamics of Active SiteMutants—The kinetic parametersfor ketopantoate reduction byR31A, K72A, N98A, K176A, S244A,and E256A mutants of KPR weremeasured and compared with WT(Table 4). Sequence alignment of 19bacterial ketopantoate reductasesrevealed that Lys72, Asn98, Lys176,Ser244, and Glu256 are strictly con-served, and Arg31 is conservativelyvaried (see supplemental Fig. S1).The thermodynamic parametersof cofactor and substrate bindingto form binary complexes withthese mutants of KPR were alsodetermined by ITC (Table 5). Theeffects of the mutations on forma-tion of ternary complexes were notinvestigated due to the absence of
cooperativity between substrates and NADP�.The K176A mutant exhibited the lowest activity among all
themutants analyzed, showing a 1670-fold decrease in kcat. TheN98A and E256Amutations decreased kcat by 10-fold. All threemutants also exhibited large decreases in kcat/Km for ketopan-toate. In contrast, the other mutants showed kcat values within2-fold of the WT enzyme (Table 4). These kinetic results areconsistent with previous results of in vivo studies, whichshowed that N98A, K176A, and E256Awere unable to comple-ment a pantoate auxotroph of Salmonella typhimurium (18).The K176A mutation increased the Kd of the substrates by�4-fold and decreased the Kd of the coenzymes, albeit moresignificantly for NADPH than NADP� (Table 5). Both N98Aand E256A mutations affected the thermodynamics of coen-zyme binding, leading to larger favorable �H and less favorable�T�S relative to WT (Table 5). Interestingly, the N98A muta-tion exhibited a 6-fold increase in binding affinity for both
FIGURE 5. Schematic diagram showing non-covalent interactions between NADP� and the binding siteresidues. Binding interactions of NADP� (in bold) with the open (A) and closed (B) ternary complexes. Hydro-gen bonds are indicated with dashed lines, and their distances are indicated in Å. Residues involved in hydro-phobic contacts are surrounded by dashed lines.
Structure of KPR Ternary Complexes
8494 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282 • NUMBER 11 • MARCH 16, 2007
NADPH and NADP�. No heat effect was observed by ITCbetweenN98A or E256A and ketopantoate or pantoate (at con-centrations up to 50 mM).The S244A mutant showed comparable kcat to WT enzyme.
However, the Km value for ketopantoate increased to 7 mM,resulting in a 380-fold decrease in kcat/Km (Table 4). Theseresults suggest that the S244A mutation destabilizes both theground state and the transition state to a comparable extent.The Kd for ketopantoate and pantoate to S244A increased by�20-fold relative toWT, consistent with interactions of Ser244with the substrate carboxylate group (Fig. 6). The thermody-namic parameters for cofactor binding were not significantlychanged upon mutation of Ser244.The R31A and K72A mutants exhibited slightly higher kcat
thanWT but increased the Km for NADPH by 4 and �10-fold,respectively (Table 4). Further evidence for the involvement ofArg31 and Lys72 in cofactor recognition came from ITC. AscomparedwithWTenzyme, theKd forNADPHandNADP� toK72A are increased 18- and 32-fold, respectively (Table 5). TheR31Amutation increased theKd for NADP� by 14-fold but didnot significantly increase theKd forNADPH.TheK72Amutantexhibited a �60-fold increase in Km for ketopantoate. This wassurprising since Lys72 is more than 7 Å away from pantoate inthe crystal structure (Fig. 6). Furthermore, the calorimetricstudies showed no significant changes in substrate binding toK72A as compared with WT (Table 5).
DISCUSSION
Previous mutagenesis studies (9) and analysis of the crystalstructure of the KPR�NADP� complex (18) have led to the pro-posal that Lys176 acts as the general acid in the physiologicallyimportant direction of ketopantoate reduction by donating aproton to the developing alkoxide on ketopantoate. The crystalstructure of the ternary complex KPR�NADP��pantoate in itsclosed form provides the first evidence of a hydrogen bondbetween Lys176 and the C2 oxygen of product pantoate,observed at 2.7 Šdistance.A sequential ordered bi:bi kinetic mechanism (Scheme 1B)
for KPR activity was proposed by Zheng and Blanchard (10)based on product inhibition analyses. Sequential orderedmechanisms often occur in reactions catalyzed by dehydroge-nases, with the coenzyme binding first (41). This has beenexplained by a conformational change upon binding of the nic-otinamide adenine dinucleotide that increases the affinity ofthe enzyme for the second substrate (42). Our ITC results showthat the binding of ketopantoate is enhanced by more than 2
FIGURE 6. Detailed view of the substrate binding site. Non-covalent inter-actions of pantoate (shown as sticks, with yellow carbons) with active siteresidues in the closed (cyan) (A) and open (green) (B) ternary complexes.NADP� is shown as lines. Hydrogen bonds are indicated with magenta dashedlines, and their distances are indicated in Å.
TABLE 4Steady-state kinetic parameters of WT and mutant E. coli KPRKinetic parameters were determined at 25 °C in 100 mM HEPES-HCl buffer, pH 7.6.
KPR kcat Vrel
NADPH KetopantoateKm at Ketopantoate V/Krel Km at NADPH V/Krel
orders of magnitude in the presence of NADPH, consistentwith the compulsory ordered mechanism. The binding ofNADPH concomitantly protects the enzyme from pantoateinhibition, presumably due to a steric repulsion between thehydrogens present in each reduced center. This selectivity forketopantoate over pantoate is only observed with theKPR�NADPH complex. In contrast, the selectivity is reversedwith the KPR�NADP� complex or the apoenzyme, which areboth in a more open conformation. We speculate that theKPR�NADPH complex must be in a more closed conformationthan KPR�NADP�. This conclusion is consistent with (a) thedifferences in substrate binding cooperativity between the twoholoenzymes, (b) the different effects of R31A and K176A oncoenzyme binding, and (c) the different�G,�S, and heat capac-ity �Cp changes associated with coenzyme binding (18).
Previouskinetic studies suggest thathydride transfer isnot rate-limiting in catalysis (9, 10, 43). Here we show that the mutantsR31A andK72A bindNADP� less well but have an increased kcat.Conversely, theN98Amutant bindsNADP�more tightly andhasa decreased kcat. These results are consistent with dissociation ofNADP� being partially rate-limiting for catalysis.The two crystal structures of ternary complexes reported
here and the previously reported structures provide snapshotsof the enzyme in action (see supplemental Movie S2). Thesestructures and additional kinetic and thermodynamic studies
with active site mutants allow us topropose a detailed catalytic mecha-nism for E. coli ketopantoate reduc-tase (Scheme 2). The initial bindingof NADPH with recognition of its2�-phosphate and pyrophosphategroups by the side chains of Arg31and the conserved Lys72 inducesconformational changes to providean active site environment thatfavors ketopantoate binding anddiscriminates against pantoatebinding. An extended hydrogen
bonding network provided by conserved residues Glu256 andAsn98 orientates the ribose-nicotinamide moiety of the cofac-tor in the conformation required for productive substrate bind-ing and optimal hydride transfer. A significant hinge bendingencloses the active site around ketopantoate to provide a sol-vent-inaccessible environment in which catalysis occurs. Afterbinding of ketopantoate, the C4 pro-S hydride of NADPH istransferred to the si face of ketopantoate, with protonation ofthe developing alkoxide by Lys176 in its active state (Scheme 2,left). The substrate is locked during each step of the reaction viahydrogen bonds of its carboxylate group to the conservedSer244. Other binding interactions are provided by the sidechains of four asparagine residues, Asn98, Asn180, Asn194, andAsn241. In addition to binding the substrate, Asn98 plays a cen-tral role in the catalytic mechanism by stabilizing the activeconformation of Lys176 and by promoting the dissociation ofNADP�. The opening of the hinge subsequently allows the rep-rotonation of Lys176 from solvent and its return to the restingstate (Scheme 2, right). The loss of hydrogen bonds from Lys176and Asn241 facilitates the release of pantoate from the openternary complex. The subsequent rate-limiting release ofNADP� ends the catalytic cycle.Conclusions—Ketopantoate reductase catalyzes an essential
step in the biosynthesis of pantothenate. This paper reports thecrystal structure of the enzyme captured in a precatalytic ter-
SCHEME 2. Proposed catalytic mechanism of E. coli ketopantoate reductase.
TABLE 5Thermodynamic parameters for cofactor and substrate binding with WT and mutant forms of KPRAll ITC titrations were run at 27 °C in 50 mMHEPES-HCl, pH 7.6. Errors quoted are S.D. of the mean parameter values from at least duplicate titrations. Otherwise, errorsare those returned by Origin on the curve fitting. ND, not determined.
nary complexwithNADP� and pantoate bound, giving the firstdirect evidence of a hinge bending domain closure in KPR andof a hydrogen bond between Lys176 and the substrate. Thestructure and thermodynamics of ternary complexes provideinsights into the interplay of conformational changes and coop-erativity in the sequential ordered mechanism. Additionalkinetic and calorimetric studies with site-directed mutantshave elucidated the roles of active site residues Arg31, Lys72,Asn98, Lys176, Ser244, and Glu256 in substrate recognition andcatalysis. Our crystal structure has highlighted the importanceof other residues, including Asn180, Asn194, Asn241, and Arg253,which will be the subject of future mutagenesis studies. Finally,the crystal structure of the closed ternary complex will be auseful template for designing inhibitors that mimic the struc-ture of the transition state during hydride transfer. The rele-vance of ligand-induced domainmovements in drug design hasbeen recently stressed (44) and may prove useful in designinginhibitors against KPR.
Acknowledgment—We thank Sarah L. Maslen for assistance withmass spectrometry of the KPR mutants.
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