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Kinetic and Structural Analysis of α-D-Glucose-1-PhosphateCytidylyltransferase from Salmonella typhi

Nicole M. Koropatkin, W. Wallace Cleland⊥, and Hazel M. Holden⊥

Department of Biochemistry, University of Wisconsin, Madison, WI 53706

Running Title: structural and kinetic analysis of cytidylyltransferase

⊥To whom correspondence should be addressed:EMAIL: Hazel_Holden@biochem.wisc.edu or Cleland@biochem.wisc.eduPHONE: 608-262-4988. FAX: 608-262-1319.

This research was supported by grants from the NIH (DK47814 to H. M. H. and GM18938 to W.W. C.)

Abbreviations: CMP, cytidine 5'-monophosphate; CDP, cytidine 5'-diphosphate; CTP, cytidine5'-triphosphate; NMP, nucleotide 5'-monophosphate; NDP, nucleotide 5'-diphosphate

Tyvelose is a 3,6-dideoxyhexosefound in the O-antigen of the surfacelipopolysaccharides of somepathogenic bacteria. It is synthesizedvia a complex biochemical pathwaythat is initiated by the formation ofCDP-D-glucose. The production ofthis ligand is catalyzed by the enzymeglucose-1-phosphatecytidylyltransferase, which utilizes α-D-glucose-1-phosphate and MgCTP assubstrates. Previous x-raycrystallographic investigations havedemonstrated that the Salmonellatyphi enzyme complexed with theproduct CDP-glucose is a fullyintegrated hexamer displaying 32point group symmetry. The bindingpocket for CDP-glucose is sharedbetween two subunits. Here wedescribe both a detailed kineticanalysis of the cytidylyltransferaseand a structural investigation of theenzyme complexed with MgCTP.These data demonstrate that thereaction catalyzed by thecytidylyltransferase proceeds via asequential rather than a bi bi ping

pong mechanism as was previouslyreported. Additionally, the enzymeutilizes both CTP and UTP equallywell as substrates. The structure ofthe enzyme with bound MgCTPreveals that the binding pocket for thenucleotide is contained within onesubunit rather than shared betweentwo. Key side chains involved innucleotide binding include Thr 14,Arg 15, Lys 25, and Arg 111. In theprevious structure of the enzymecomplexed with CDP-glucose, thoseresidues defined by Thr 14 to Ile 21were disordered. The kinetic and x-ray crystallographic data presentedhere support a mechanism for thisenzyme which is similar to thatreported for the glucose-1-phosphatethymidylyltransferases.

O-antigens are glycan side chains ofbacterial surface lipopolysaccharidesthat play a role in pathogenicity and arerecognized by the host’s immunologicaldefense system (1). Tyvelose, as shownin Scheme 1, is a 3,6-dideoxyhexose thatoccurs in the O-antigens of some types

JBC Papers in Press. Published on January 20, 2005 as Manuscript M414111200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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of Gram-negative bacteria such asYersinia pseudotuberculosis IVA andSalmonella typhi (2). It is produced viaa complex biochemical pathway thatemploys α-D-glucose-1-phosphate as thestarting ligand. The first committed stepin the pathway, as shown in Scheme 1, isthe transfer of a CMP moiety from CTPto glucose-1-phosphate. This reaction iscatalyzed by α -D-glucose-1-phosphatecytidylyltransferase which was firstpurified in the early 1960s from variousbacterial species (3-7). These initialreports established the kineticparameters of the enzyme and were ingeneral agreement despite the varyingsources of protein. All of the studiesd e m o n s t r a t e d t h a t t h ecytidylyltransferase has a strictpreference for CTP, with only traceactivity in the presence of dCTP andUTP, and no measurable activity withany other NTP or dNTP (4, 6, 7). Theenzyme was shown to have maximalactivity at pH ~8 and to display feedbackinhibition by CDP-paratose, CDP-tyvelose, and other intermediates in theproduction of CDP-tyvelose (4-7).Additionally, the type of inhibitionobserved was consistent with multiplebinding sites for the inhibitor. The over-all reaction was shown to be readilyreversible with an equilibrium constantin the direction of CDP-glucoseformation of 0.57 to 1.0 (4, 6). Theseinitial reports also indicated that thequaternary structure of the enzyme wasmonomeric with an overall molecularweight of 110,000 (7).

Following these earlier studies,highly purified samples of α-D-glucose-1-phosphate cytidylyltransferase weresubsequently obtained from Salmonellae n t e r i c a s t r a in LT2 , Y .pseudotuberculosis, and Streptomycesglaucescens GLA.0 (8-10). In contrast,

these investigations indicated thequaternary structure of the enzyme to betetrameric rather than monomeric with asubunit molecular weight of ~30,000 (9).A detailed kinetic analysis of thecytidylyltransferase from S. entericasuggested that its catalytic mechanismproceeds via a bi bi ping-pong reaction(8). In such a mechanism, CTP bindsfirst to the enzyme followed by an attackof an enzymatic base on the α -phosphorus of the nucleotide, resultingin the formation of an enzyme-CMPcovalent intermediate and the release ofpyrophosphate. Glucose-1-phosphatesubsequently binds and conducts anucleophilic attack on the phosphorus ofthe enzyme-CMP intermediate resultingin CDP-glucose formation. Strikingly,this mode of catalysis is in sharp contrastto the sequential mechanism reported forthe related glucose-1-phosphatethymidylyltransferases (11, 12).

Recently, the x-ray crystallographicstructure of the S. typhi glucose-1-phosphate cy t idy ly l t ransferasecomplexed with CDP-glucose wasdetermined in this laboratory (13). Theenzyme crystallized in the space groupP6322 with one subunit in theasymmetric unit. Contrary to previousreports, the crystallographic data clearlydemonstrated the enzyme to be a fullyintegrated hexamer displaying 32 pointgroup symmetry. Subsequent analyticalul t racentr i fugat ion experimentsconfirmed the hexameric nature of thecytidylyltransferase (13). A view of onehalf of the hexamer is shown in Figure 1.Note that each active site is formed byresidues contributed from two subunits,specifically those related by thecrystallographic dyads. Each subunitdisplays a “bird-like” appearance withthe “body” dominated by a seven-stranded mixed β-sheet and the two

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“wings” formed by β-hairpin motifs.Here we present both a detailed kineticanalysis of the enzyme and the three-dimensional structure of the proteincomplexed with CTP. Thesebiochemical and x-ray crystallographicdata indicate that the reaction proceedsvia a sequential mechanism, that theenzyme can utilize both UTP and CTP,that the binding site for CTP is containedwithin a single subunit of the hexamer,and that the “wings” of each subunitadopt different orientations when CTPversus CDP-glucose is bound in theactive site.

EXPERIMENTAL PROCEDURES

Protein Purification. The S. typhiglucose-1-phosphate cytidylyltransferasewas purified according to previouslypublished procedures (13). Allchemicals and enzymes were purchasedfrom Sigma. The concentrations ofCTP, CDP-glucose, UTP, and UDP-glucose were determined by UVspectrophotometry assuming extinctioncoefficients at 271 nm of 9.3 mM-1 cm-1

for CTP and CDP-glucose and 10.3 mM-

1 cm-1 for UTP and UDP-glucose.Enzymatic Assays for the Forward

and Reverse Reactions. The activity ofglucose-1-phosphate cytidylyltransferasein the forward reaction was followed bytwo spectrophotometric methods.Absorbance readings were taken with aBeckman DU 640B spectrophotometerequipped with a Peltier temperaturecontrolled 6-cell auto sampler. Thes u b s t r a t e s p e c i f i c i t y o fcytidylyltransferase was assessed in acoupled assay in which the production ofpyrophosphate was linked to theoxidation of NADH. These assays wereperformed in a discontinuous mannerutilizing the pyrophosphate detection kit

from Sigma (product P-7275). Thepyrophosphate detection kit containedthe coupling enzymes pyrophosphate-dependent phosphofructokinase,aldolase, triosephosphate isomerase, andglycerophosphate dehydrogenase, aswell as the appropriate substrates. Forthis set of experiments, thepyrophosphate reagent was prepared byresuspending one vial of the lyophilizedreagent mixture in 2 mls of sterile water,rather than the recommended 4 mls. Theassay was initiated by the addition ofcytidylyltransferase (18 µls, 0.25 µg/ml)to 3 mls of solution containing 1 - 2 mMNTP, 3 - 4 mM MgCl2, 1 - 2 mM ofvarious sugar-1-phosphates, and 25 mMHEPPS (pH 8.0). At various times (0min to 24 hours), 800 µls were removedand quenched by the addition of 40 µlsof 2 M HCl and 20 µls of CCl4 followedby 30 s of vortexing. These aliquotswere subsequently centrifuged for oneminute and 800 µls were withdrawn andtransferred to a microfuge tubecontaining 35 µls of 2 M NaOH in orderto neutralize the pH. To this solution,165 µ ls of the pyrophosphate reagentwere added and the reaction wasincubated at 30 °C for 20 minutes. Theamount of pyrophosphate produced overtime was assessed by the decrease inabsorbance at 340 nm according to themanufacturer’s instructions. Negativecontrols were performed in whichcytidylyltransferase, CTP, or glucose-1-phosphate was omitted from thereaction.

The kinetic constants for thecytidylyltransferase forward reactionwere assessed via a continuous coupledassay for the detection of pyrophosphate.Unfortunately, an unidentifiedcontaminant in the Sigma pyrophosphatedetection kit acted as a substrate for thecytidylyltransferase reaction when only

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CTP or UTP were added to the reaction.Therefore, this kit could not be used toperform a continuous assay. Instead, theindividual reagents of the kit werepurchased from Sigma and thepyrophosphate reagent was reconstitutedfrom the individual components. Each350 µ l reaction contained 14.4 mMimidazole (pH 7.4), 1.6 mM citrate, 32µM EDTA, 64 µM MnCl2, 6.4 µ MCoCl2, 256 µ M β -NADH, 3.8 mMfructose-6-phosphate , 0 .06 Upyrophosphate-dependentphosphof ruc tok inase , 0 .6 Uglycerophosphate dehydrogenase, 5.6 Utriosephosphate isomerase, 0.84 Ualdolase, 0.5 mgs of bovine serumalbumin, and 0.35 µ gs ofcytidylyltransferase, and varyingamounts of CTP with MgCl2, andglucose-1-phosphate. CTP was variedfrom 50 µM to 1 mM at fixed levels ofglucose-1-phosphate in the range of 25µM to 1 mM. The MgCl2 concentrationwas fixed at 2.5 mM above theconcentration of CTP in each reaction.40 µls of the variable substrate, glucose-1-phosphate or CTP and MgCl2, weretransferred to a quartz cuvette and thereaction was initiated by the addition of310 µ ls containing the remainingreaction components. The reaction wasquickly mixed and the absorbance at 340nm was monitored every 15 secondsover 7 minutes. During this time, thereactions were kept at a constanttemperature of 26 °C. The continuousassay was also performed varying α-D-xylose-1-phosphate while keeping theconcentration of CTP fixed at 1 mM.Similarly, the concentrations of dCTP orUTP were varied while maintaining theconcentration of glucose-1-phosphate at1 mM. In the assays involving dCTPand xylose-1-phosphate, 20x theconcentration of cytidylyltransferase was

required to achieve an observablereaction velocity. In order to comparereaction velocities, the rate of thereaction with CTP and glucose-1-phosphate at this higher enzymeconcentration was also measured.Control experiments for the continuousassay were performed in whichcytidylyl transferase, glucose-1-phosphate, or NTP were omitted fromthe assay to ensure that pyrophosphateproduction did not occur unless both ofthe substrates and cytidylyltransferasewere present.

The products of the reaction withCTP, UTP, glucose-1-phosphate andxylose-1-phosphate were identified viaHPLC analysis. HPLC analysis of thereaction products was performed on anÄKTA HPLC (Amersham-Pharmacia)equipped with a 1 ml Resource Qcolumn using two different sets ofconditions. The reaction products wereanalyzed at pH 4.0 with a gradient of20 mM to 500 mM ammonium acetateover 20 mls and at pH 8.5 with agradient of 20 mM to 500 mMammonium carbonate over 20 mls. Theproducts of the reaction were identifiedby comparing the chromatograms tothose of authentic samples of CMP,UMP, CDP, UDP, CDP-glucose, UDP-glucose, CTP or UTP.

The kinetic constants for the reverse(NTP synthesis) reaction weredetermined via a discontinuous HPLCassay. Each 1 - 5 ml reaction contained1 µg/ml of cytidylyltransferase and 25mM HEPPS (pH 8.0) while theconcentrations of CDP-glucose andsodium pyrophosphate or UDP-glucoseand sodium pyrophosphate were variedover the range of 20 µM to 10 mM. Theconcentration of MgCl2 was fixed at 2mM above the concentration ofpyrophosphate. Reaction aliquots were

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removed at various times and quenchedwith HCl and carbon tetrachloride asdescribed for the discontinuous coupledassay. One ml samples of the reactionwere injected onto a 1 ml Resource Qcolumn pre-equilibrated with 20 mMammonium acetate (pH 4.0). The NTPproduct was separated from the NDP-glucose substrate with a linear gradientfrom 0 mM to 300 mM LiCl over 20mls. The elution profile was monitoredby UV absorbance at 271 nm. Theretention volumes for CDP-glucose,CTP, UDP-glucose, and UTP were 9.2mls, 13.4 mls, 9.7 mls, and 15.3 mlsrespectively. The area under the elutionpeak for UTP and CTP was calculatedand compared to a standard curverelating the area under the elution peakto the µmoles of NTP.

Measurement of the EquilibriumConstant and Divalent CationDependence. The equilibrium constantand Mg2+ dependence for thecytidylyltransferase reaction wereassessed via an HPLC based assay todetect the relative proportion of CDP-glucose to CTP and vice versa. For themeasurement of the equilibriumconstant, the standard reaction contained1 mM each of sodium pyrophosphateand CDP-glucose or 1 mM each of CTPand glucose-1-phosphate, 3 mM MgCl2,and 25 mM HEPPS (pH 8.0). Each 1 mlreaction contained 2.5 µ gs ofcytidylyltransferase. To determine thedependence of the cytidylyltransferasereaction on the concentration of Mg2+,CTP and glucose-1-phosphate werefixed at 0.2 mM and 1 mM, respectively,while the concentration of MgCl2 wasvaried from 0.7 mM to 5.2 mM. These 1ml reactions contained 1.0 µ g ofcytidylyltransferase. The reactionproducts were separated on a 1 mlResource Q column with a linear

gradient of 20 mM to 250 mMammonium carbonate (pH 8.5) over 20mls. The elution profile was monitoredby UV absorbance at 271 nm and thearea under the peaks was used todetermine the proportion of CDP-glucose to CTP.

Kinetic Data Analysis. The kineticconstants for the forward reaction wereassessed using the data obtained fromthe continuous spectrophotometric assay.Before the final data analysis, the initialvelocity pattern was determined byplotting each data set to the equation:

The double reciprocal plot clearlyshowed a series of intersecting linessuggesting a sequential mechanism(Figure 2). Therefore, all 60 data pointsfrom the continuous assay with CTP andglucose-1-phosphate were fitted toEquation 2:

The data were fitted with the programSEQUENO in order to calculate V, Ka,Kb, Kia, and Kib from K ib = (K ia Kb)/Ka

where Kia and K ib are the dissociationconstants for binary EA and EBcomplexes, Ka and K b are Michaelisconstants for A and B, and V is themaximum velocity (14). In thisequation, A is the concentration of CTP,while B is the concentration of glucose-1-phosphate.

The kinetic parameters for the backreaction and for the alternate substratesfor the forward reaction were obtained

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by fitting the data to the Michaelis-Menten equation (3) of the form:

Crystallization of Glucose-1-Phosphate Cytidylyltransferase. Prior tocrystallization, the protein wasconcentrated to 9.5 mg/ml. CTP andMgCl2 were added to f inalconcentrations of 4 mM and 8 mM,respectively. Crystals were grown bythe hanging drop method of vapordiffusion against a precipitant solutioncontaining 24 - 28 % poly(ethyleneglycol) 400, 100 mM MgCl2, 100 mMHEPES (pH 7.0). The crystals belongedto the space group P6322 with unit celldimensions of a = b = 84.2, c = 157.4and one subunit per asymmetric unit.

X-ray Data Collection and StructuralAnalysis. Prior to x-ray data collection,the crystals were serially transferredfrom the hanging drop experiments to acryoprotectant solution containing 30 %poly(ethylene glycol) 400, 10 %ethylene glycol, 400 mM NaCl, 100 mMMgCl2, 4 mM CTP, and 100 mMHEPES (pH 7.0), flash-cooled to -150 ºCin a stream of nitrogen vapor, and storedunder liquid nitrogen until synchrotronbeam time became available. X-ray datafrom a single crystal were collected on aCCD detector at the COM-CAT 32-IDbeamline (Advanced Photon Source,Argonne National Laboratory, Argonne,IL). All x-ray data were processed withHKL2000 and sca led wi thSCALEPACK (15). Relevant x-ray datacollection statistics are presented inTable I.

The structure of S. typhi glucose-1-phosphate cy t idy ly l t ransferasecomplexed with CTP was solved viamolecular replacement with the program

AMoRe and employing as a searchmodel the previously determinedstructure of the enzyme with boundCDP-glucose (PDB 1TZF) (16).Alternate cycles of manual modelbuilding with the graphics programTURBO (17) and least-squaresrefinement with TNT (18) reduced theR-factor to 20.2 % for all measured x-raydata from 25 to 2.5 Å. The polypeptidechain extends from Ala 2 - Ala 162, Gly164 - Gln 177, and Lys 181 - Glu 259.There is a metal coordinated to His 79,which is most likely a nickel ionresulting from the Ni-NTA columnchromatography purification. Note thatthe His6 tag at the N-terminus wasremoved from the protein prior to thekinetic and structural analyses. Relevantleast-squares refinement statistics arepresented in Table II. X-ray coordinateshave been deposited in the Protein DataBank (1WVC).

RESULTS AND DISCUSSION

In order to determine the substratespecificity for cytidylyltransferase,pyrophosphate production was assessedutilizing the pyrophosphate detection kitmarketed by Sigma. The controlexperiments, however, indicated thatpyrophosphate was produced even whenonly CTP and cytidylyltransferase wereadded to the coupling enzymes. Theproducts from the reaction containingCTP, coupl ing reagent , andcytidylyltransferase were verified byHPLC and indicated that in the absenceof glucose-1-phosphate, a compoundconsistent with the HPLC profile ofCDP-glucose was produced. In addition,electro-spray ionization (ESI) massspectrometry on the product of thisreaction showed that it had the samemolecular weight as CDP-glucose. It

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should be noted that CMP was notdetected as a product of this reaction norwas it detected in prolonged incubationsof cytidylyltransferase, CTP, and MgCl2.Since the pyrophosphate reagent in thedetection kit appeared contaminated witha substrate for the cytidylyltransferasereaction, this reagent could only beutilized to perform a discontinuous assayas described in the ExperimentalProcedures. In this set of assays, CTP,dCTP, UTP, dUTP, ATP, and GTP weretested for activity with glucose-1-phosphate. Similarly, D-glucose-1-phosphate, D-xylose-1-phosphate, D-mannose-1-phosphate, D-galactose-1-phosphate, D-fructose-6-phosphate, andD-glucose-6-phosphate were tested assubstrates with CTP. Only reactionscontaining CTP, dCTP, UTP, glucose-1-phosphate, and xylose-1-phosphateproduced detectable levels ofpyrophosphate. The kinetic parametersof the cytidylyltransferase reaction withthese substrates were further assessed ina continuous assay system.

To confirm the identity of theproducts from the cytidylyltransferasereaction in the forward direction,samples of the reaction with CTP, dCTP,UTP, glucose-1-phosphate, and xylose-1-phosphate were analyzed by HPLC.The substrate and product peaks fromthe reaction were identified bycomparing the retention volumes tothose of known samples of CTP, CDP,CMP, CDP-glucose, UTP, UDP, UMP,and UDP-glucose at both pH 8.5 and pH4.0. In all reactions, the only productidentified was the NDP-sugar, with noconcomitant formation of NMP. Inaddition, prolonged incubation ofcytidylyltransferase with CTP or UTPdid not result in CMP or UMPformation, confirming that the enzymedoes not perform a pyrophorylysis side

reaction. Thus it can be concluded thatthe measurement of pyrophosphate is anappropriate method to determine thekinetics of product formation.

The kinetic constants for the forwardcytidylyltransferase reaction weredetermined using a continuous assaycoupl ing the product ion ofpyrophosphate to the oxidation ofNADH. In order to establish whetherthe cytidylyltransferase reaction followsa sequential or ping-pong mechanism,the glucose-1-phosphate concentrationwas varied at several fixedconcentrations of CTP while measuringthe initial velocity of the reaction.Double reciprocal plots of the initialvelocity with respect to substrateconcentration resulted in a series of linesthat intersected in the lower left quadrantas shown in Figure 2. The initialvelocity pattern is consistent with as e q u e n t i a l m e c h a n i s m f o rcytidylyltransferase. This is in sharpcontrast to that previously reported forthe Salmonella glucose-1-phosphatecytidylyltransferase which suggested aping-pong mechanism on the basis ofdouble reciprocal plots of the initialvelocity pattern (8). The initial velocitydata in the present investigation werefitted to the rate equation for a sequentialmechanism using the least-squaresfitting program SEQUENO (14). Thedata fit the rate equation for a sequentialmechanism well with σ = 0.18 µM/minfor the initial velocity values. Theresults from this analysis are reported inTable III. The Km for CTP (Ka) is 148 ±7 µ M and the Km for glucose-1-phosphate (Kb) is 52 ± 2 µM. The Vmax

for the forward reaction with CTP andglucose-1-phosphate is 10.4 ± 0.2µM/min. The dissociation constants (Ki)of CTP and glucose-1-phosphate are 35± 8 µM and 12 ± 3 µM, respectively.

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The fact that the dissociation constantsare lower than the Km values for CTPand glucose-1-phosphate argues that thesubstrates bind in an anti-synergisticfashion in the forward reaction.

The continuous assay was alsoutilized to determine the kineticparameters from the reaction with UTPand glucose-1-phosphate, dCTP andglucose-1-phosphate, and CTP andxylose-1-phosphate. The Km for UTP is159 ± 8 µM and the Vmax of the reactionis 15.4 ± 0.3 µM/min. The ratio of theVmax of the reaction with UTP versusCTP is 1.5 ± 0.04 suggesting that therates of the reaction with UTP or CTPare essentially the same. These dataindicate that the enzyme appears toutilize CTP and UTP equally well, afinding which has not been reported forany other hexose-1-phosphatec y t i d y l y l t r a n s f e r a s e o ruridylyltransferase. However, the rate ofthe reaction is severely decreased whenCTP is replaced with dCTP. The Km fordCTP is 3.0 ± 0.5 mM which is 20xgreater than the Km for CTP and the rateof the reaction is 30 % that of thereaction with CTP. Of the hexose-1-phosphates tested, only xylose-1-phosphate acted as a substrate in theforward reaction. The Km for xylose-1-phosphate is 3.6 ± 0.3 mM, which is 72xgreater than the Km for glucose-1-phosphate. The rate of the reaction withxylose-1-phosphate, however, is ~40 %faster than the reaction with glucose-1-phosphate. This rate increase could beexplained if the product CDP-xylose hasa low affinity for the enzyme in whichcase the product release step for CDP-xylose would be much faster than thatfor CDP-glucose. This would give ahigher apparent velocity for the reactionof CTP and xylose-1-phosphate.

The equilibrium constant forcytidylyltransferase was analyzed bymeasuring the proportion of substrateversus product via HPLC analysis,obtained from both the forward andreverse reactions. These data indicate aKeq of 0.27 with the reaction having aslight preference for CTP synthesis.There is some indication in the kineticdata that a second Mg+2 ion is requiredin addition to that forming the Mg+2-CTP complex. Thus a 2 mM excess ofMg+2 over Mg2+-CTP was needed forfull activity although a 0.5 mM excessshould have been sufficient to keepMgCTP in that form. The dissociationconstant of the second metal ion ispresumably less than 1 mM.

Kinetic parameters for the backreaction (NTP synthesis) indicate thatthe enzyme has a clear preference forCDP-glucose over UDP-glucose asshown in Table IV. The Km for CDP-glucose is 11 ± 2 µM while the Km forUDP-glucose is 2535 ± 240 µ M.However, the Vmax for both substrates isnearly the same at approximately 8µM/min. The Km for pyrophosphate is67 ± 8 µM.

The dramatic difference in the Km forCDP-glucose versus UDP-glucose but anearly identical Km for CTP and UTPmay be explained if the binding of thetriphosphate nucleotide is driven by theaff ini ty of the enzyme forpyrophosphate. Indeed, the K m forpyrophosphate is less than the Km forCTP or UTP (158 and 149 µ Mrespectively). The relatively low affinityof the enzyme for UDP-glucose suggeststhat the rate of dissociation of the UDP-glucose product is likely not rate limitingand thus explains the slightly elevatedVmax for the forward reaction with UTPversus CTP. The results from the backreaction are somewhat more difficult to

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expla in . Addi t iona l x - raycrystallographic and kinetic data,including the determination of the Km forglucose-1-phosphate and pyrophosphatein the presence of UTP and UDP-glucose, respectively, will be required tofully explain the current observations.

Electron density corresponding to thebound nucleotide, MgCTP, is shown inFigure 3. As can be seen, the cytosinering is in the anti-conformation while theribose adopts the C2'-endo pucker. Atthis resolution it is not possible tocompletely define the coordinationgeometry about the Mg+2 ion. However,it is apparent from this structure that theMg+2 ion sits within 2.5 Å and 3.1 Å ofthe α-phosphoryl oxygens (Figure 4a).Additionally, the side chains of Asp 131and Asp 236 lie within coordinationdistance of the magnesium (2.0 Å and2.1 Å, respectively). There is a secondpeak of electron density positioned nearthe α,β-bridging oxygen (2.4 Å) and a γ-phosphoryl oxygen (2.8 Å) of thenucleotide. This has been tentativelymodeled as a magnesium ion.

A close-up view of the active sitewith bound MgCTP is shown in Figure4a. The backbone amide nitrogen of Gly10, the carbonyl oxygen of Ser 106, andthe guanidinium group of Arg 111 all liewithin hydrogen bonding distance to thecarbonyl oxygen, the amino group, andthe ring nitrogen, respectively, of thecytosine base. The 2-hydroxyl group ofthe nucleotide ribose sits within ~2.9 Åof the backbone amide nitrogen of Gly11 and a water molecule whereas the 3-hydroxyl group lies within 2.9 Å and2.7 Å of the backbone nitrogen of Gly130 and the carbonyl oxygen of Leu 8,respectively. In the previouslydetermined structure of the enzymecomplexed with its product, CDP-glucose, the loop defined by Thr 14 to

Ile 21 was disordered (13). Strikingly,in the presence of CTP, the loopbecomes ordered and both Oγ of Thr 14and Nε of Arg 15 participate in hydrogenbonding interactions with γ-phosphoryloxygens (Figure 4a). Note that the sidechain of Lys 25 projects towards the α-phosphoryl group of CTP with its ε-nitrogen sitting at 2.4 Å from aphosphoryl oxygen.

Shown in Figure 4b is a superpositionof the subunits with either bound CDP-glucose or CTP. The polypeptide chainsfor these two models correspond with aroot-mean-square deviation of 0.45 Å for203 structurally equivalent α-carbons.The most dramatic structural changesthat occur upon binding either CTP orCDP-glucose are limited primarily to theβ-hairpin regions labeled β3, β4 and β9,β10 in Figure 4b. All of the residuesinvolved in anchoring CTP to the proteinare contained within the same subunit ofthe hexamer. This is in marked contrastto that observed in the model of theenzyme with bound product, CDP-glucose, whereby part of the bindingpocket is provided by a second subunitin the hexamer. Specifically, Glu 178and Lys 179 from a neighboringmolecule form electrostatic interactionswith CDP-glucose (13). These residuesare disordered in the absence of CDP-glucose (Figure 4b).

An overlay of the CTP and CDP-glucose ligands, when bound in theactive site of the cytidylyltransferase, isgiven in Figure 4c. As can be seen, thetwo ligands adopt similar positions withrespect to their base and ribosylmoiet ies . Quite remarkableconformational differences, however,occur beginning at their α-phosphorusatoms. Specifically, the torsional angledefined by the bond between O5 of theribosyl group and the α -phosphorus

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atom differs by ~115o in these twomodels. As a result the glucosyl groupin CDP-glucose abuts the side chain ofTyr 129 whereas the γ-phosphoryl groupof CTP sits near Thr 14 and Arg 15(Figure 4c).

These two structures further argueagainst a bi bi ping-pong mechanism inthat the only potential base near the α-phosphorus is Lys 25. Strikingly, the β-phosphorus of the CDP-glucose ligand islocated at ~3.4 Å from the α-phosphorusof CTP in the overlay presented inFigure 4c. Assuming that the binding ofthe glucose moiety in CDP-glucosemimics what would occur for thesubstrate, glucose-1-phosphate, it is easyto envisage a catalytic mechanism forthe cytidylyltransferase that proceeds viadirect nucleophilic attack of the glucose-1-phosphate phosphoryl oxygen on theα-phosphorus of CTP. In conclusion,from both the structural and kinetic datapresented here it can be concluded thatthe cytidylyltransferase reaction issequential and similar to that reportedf o r t h e glucose-1-phosphatethymidylyltransferases (11, 12).

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12. Barton, W. A., Lesniak, J.,Biggins, J. B., Jeffrey, P. D.,Jiang, J., Rajashankar, K. R.,Thorson, J. S., and Nikolov, D.B. (2001) Nat Struct Biol 8, 545-551

13. Koropatkin, N. M., and Holden,H. M. (2004) J Biol Chem 279,44023-44029

14. Cleland, W. W. (1979) MethodsEnzymol 63, 103-138

15. Otwinowski, Z., and Minor, W.(1997) Methods Enzymol 276,307-326

16. Navaza, J. (1987) ActaCrystallogr Sect A 43, 645-653

17. Roussel, A., and Cambillau, C.(1998) Silicon GraphicsGeometry Partners Directory(Graphics, S., ed) pp. 77-79,Silicon Graphics, MountainView, CA

18. Tronrud, D. E., Ten Eyck, L. F.,and Matthews, B. W. (1987) ActaCrystallogr Sect A 43, 489-501

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FIGURE LEGENDS

Fig. 1. Ribbon representation of glucose-1-phosphate cytidylyltransferase from S. typhi.The cytidylyltransferase from S. typhi is a hexamer displaying 32 symmetry. For the sakeof clarity, only half of the hexamer is shown. The active sites are indicated by thepositions of the CDP-glucose ligands, which are displayed in ball-and-stickrepresentations.

Fig. 2. Double reciprocal plots of initial rates of formation of pyrophosphate. In thisplot, the concentration of CTP is varied at several fixed concentrations of glucose-1-phosphate equal to 25 µM (open circles), 33 µM (open squares), 50 µM (filled squares),0.1 mM (filled circles), and 1 mM (filled triangles). Initial velocities (v) are expressed asmM/min of pyrophosphate.

Fig. 3. Representative electron density. Shown is the electron density corresponding tothe nucleotide. The map was calculated with coefficients of the form (Fo - Fc), where Fowas the native structure factor amplitude and Fc was the calculated structure factor

amplitude from the model lacking coordinates for the MgCTP ligand. The map wascontoured at 3 σ. There were two magnesium ions modeled into the electron density andthey are represented by the green spheres.

Fig. 4. Structure of the cytidylyltransferase with bound CTP. A close-up view of theactive site, within ~3.2 Å, is shown in (a). The green spheres represent boundmagnesium ions. The dashed green lines indicate possible ligand coordination to thesecations. At a resolution of 2.5 Å it is not possible to completely define the coordinationspheres of the magnesiums. Ordered water molecules are depicted as red spheres.Possible hydrogen bonds (within 3.2 Å) between CTP and the protein are indicated by thedashed lines. A superposition of the α-carbon traces for the models of thecytidylyltransferase with either bound CDP-glucose (yellow) or CTP (blue) is depicted in(b). Note that the β-hairpin motif labeled β9, β10 in (b) forms part of the active site in aneighboring molecule in the hexamer. The manner in which the enzyme accommodateseither CTP or the product, CDP-glucose, is depicted in (c).

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Table I. X-ray Data Collection Statistics.

Wavelength (Å) 0.95730

Resolution Limits (Å) 50 - 2.52.59 – 2.5b

No. independentreflections

117801064

Completeness (%) 97.490.3

Redundancy 9.55.3

Avg I / Avg σ(I) 26.43.1

Rsyma (%) 6.2

27.4

aRsym = (Σ|I - I |ΣI) x 100. bStatistics for the highest resolution bin.

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Table II. Least-Squares Refinement Statistics.

Resolution Limits (Å) 25.0 – 2.5aR-factor (overall) %/no.reflections

20.2 (11746)

R-factor (working) %/no.reflections

19.9 (10623)

R-factor (free) %/no.reflections

27.2 (1123)

No. Protein Atoms 1996No. Hetero-atoms 123Average B values (Å2)Protein Atoms 48.7CTP 42.8solvents 52.2Weighted RMS Deviationsfrom IdealityBond Lengths (Å) 0.010Bond Angles (deg) 2.2Trigonal Planes (Å) 0.004General Planes (Å) 0.006bTorsional Angles (deg) 20.0aR-factor = (Σ|Fo - Fc| / Σ|Fo|) x 100 where Fo is the observed structure-factor amplitude

and Fc is the calculated structure-factor amplitude.bThe torsional angles were not restrained during the refinement.

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Table III. Kinetic Parameters for the Forward Reaction with CTP and Glucose-1-Phosphate.

Vmax 10.4 (± 0.2) µM/minKA 148 (± 7) µMKB 52 (± 2) µMKia 35 (± 8) µMKib 12 (± 3) µMThese parameters were generated from a least-squares fit of the data to the rate equationfor a sequential mechanism, assuming A = CTP and B = glucose-1-phosphate. The fitgave σ = 0.18 µM/min.

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Table IV. Kinetic Parameters of the Reverse Reaction (NTP synthesis).

Substrate Km (µM) Vmax (µM/min)CDP-glucose 11 (± 2) 8.0 (± 0.2)pyrophosphate 67 (± 8) 8.3 (± 0.3)UDP-glucose 2535 (± 240) 7.6 (± 0.3)These parameters were calculated by fitting the data to the Michaelis-Menten equation.

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Nicole M. Koropatkin, W. Wallace Cleland and Hazel M. Holdenfrom Salmonella typhi

Kinetic and structural analysis of alpha-D-glucose-1-phosphate cytidylyltransferase

published online January 5, 2005 originally published online January 5, 2005J. Biol. Chem. 

  10.1074/jbc.M414111200Access the most updated version of this article at doi:

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Supplemental material:

  http://www.jbc.org/content/suppl/2005/01/12/M414111200.DC1

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