Báo cáo khoa học: Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity docx

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Replacement of two invariant serine residues inchorismate synthase provides evidence that a proton relaysystem is essential for intermediate formation andcatalytic activityGernot Rauch1, Heidemarie Ehammer1, Stephen Bornemann2and Peter Macheroux11 Institute of Biochemistry, Graz University of Technology, Austria2 Department of Biological Chemistry, John Innes Centre, Norwich, UKChorismate synthase catalyzes the seventh and last stepin the shikimate pathway, leading to chorismate, thelast common precursor in the biosynthesis of numer-ous aromatic compounds in bacteria, fungi, plants andprotozoa. Because of the absence of this pathway ineukaryotic organisms, its enzymes are interestingpotential targets for rational drug design [1]. Thechorismate synthase reaction involves an anti-1,4-elimi-nation of the 3-phosphate and the C(6proR) hydrogen,as shown in Scheme 1 [2,3].Although no net redox change occurs during thereaction, chorismate synthase activity is based on thesupply of reduced FMN, which is bound in the activesite of the enzyme [3–5]. Mechanistic studies have indi-cated a functional role of the reduced flavin [6,7] thatcomprises the transient donation of an electron (or aKeywordsenzyme mechanism elimination; flavin;shikimate pathway; site-directedmutagenesisCorrespondenceP. Macheroux, Institute of Biochemistry,Graz University of Technology,Petersgasse 12 ⁄ II, A-8010 Graz, AustriaFax: +43-316-873 6952Tel: +43-316-8736450E-mail: peter.macheroux@tugraz.at(Received 6 December 2007, revised 15January 2008, accepted 21 January 2008)doi:10.1111/j.1742-4658.2008.06305.xChorismate synthase is the last enzyme of the common shikimate pathway,which catalyzes the anti-1,4-elimination of the 3-phosphate group and theC-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) togenerate chorismate, a precursor for the biosynthesis of aromatic com-pounds. Enzyme activity relies on reduced FMN, which is thought todonate an electron transiently to the substrate, facilitating C(3)–O bondbreakage. The crystal structure of the enzyme with bound EPSP and theflavin cofactor highlighted two invariant serine residues interacting with abound water molecule that is close to the C(3)–O of EPSP. In this articlewe present the results of a mutagenesis study where we replaced the twoinvariant serine residues at positions 16 and 127 of the Neurospora crassachorismate synthase with alanine, producing two single-mutant proteins(Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16Ala-Ser127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively,than that of the wild-type protein. No residual activity was detected for theSer16AlaSer127Ala double-mutant protein, and formation of the typicaltransient intermediate, characteristic for the chorismate synthase-catalysedreaction, was not observed, in contrast to the single-mutant proteins. Onthe basis of the structure of the enzyme, we propose that Ser16 and Ser127form part of a proton relay system among the isoalloxazine ring of FMN,histidine 106 and the phosphate group of EPSP that is essential for the for-mation of the transient intermediate and for substrate turnover.AbbreviationsEPSP, 5-enolpyruvylshikimate 3-phosphate; NcCS, Neurospora crassa chorismate synthase; wat 1 wat 2 and wat 3, water molecules inchorismate synthase.1464 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBScharge transfer) to the substrate, prompting cleavageof the C–O bond and thereby facilitating phosphatecleavage. At the end of the catalytic cycle an electron(or negative charge) is redistributed to maintain thereduced form of the flavin cofactor [8–11].The theoretical and experimental evidence for such arole of the reduced FMN eagerly demanded structuralinformation of the protein. Eventually, the structuredetermination of Streptococcus pneumoniae chorismatesynthase in the presence of oxidized FMN and 5-enol-pyruvylshikimate 3-phosphate (EPSP) provided the firstinsight into the binding and relative orientation of thecofactor and of the substrate in the active site of theenzyme [10]. Based on this structure we were able toinitiate a structure-based mutagenesis study to testmechanistic proposals. In our first study we demon-strated that the invariant histidine residues (His17 andHis106) function as general acids in the active site, withHis106 protonating the N(1)–C(2)=O locus of reducedflavin whereas His17 appears to be involved in the pro-tonation of the leaving phosphate group [12]. The nexttarget was Asp367, which is in the direct vicinity of theN(5) atom of the isoalloxazine ring system and a likelycandidate in a position for the abstraction of hydrogenfrom the substrate. Single-mutant proteins in whichAsp367 has been replaced with alanine or asparagineexhibit a 300- and 600-fold lower activity, respectively,emphasizing the important role of the Asp367 residueas an active-site base. These results provide strong evi-dence that acid–base catalysis is of great importance inthe chorismate synthase reaction [13].Multiple sequence alignments of chorismate synthas-es from bacterial, fungal, plant and protozoan origin,of the crystal structure of the enzyme with boundEPSP and of the flavin cofactor, revealed two invariantserine residues – Ser16 and Ser127 – interacting withseveral bound water molecules. As shown in Fig. 1,one water molecule (wat 1) is held by both serine sidechains (in the reported structure of S. pneumoniaechorismate synthase, these positions are designated asSer9 and Ser132, [10]) close to the C(3)–oxygen, whileanother water molecule (wat 2) is bound between athird water molecule (wat 3) and a C1 carboxyl oxygenof the substrate that is also hydrogen bonded toHis106. The third water molecule bridges the first twowater molecules. There are both open (Fig. 1A) andclosed (Fig. 1B) active-site structures that reveal atightening up of the site in the latter together with amovement of the His106 side chain away from the fla-vin and towards the substrate [its ring nitrogen atomthat participates in hydrogen bonding moves 1.3 A˚away from the C(2=O) oxygen of the flavin ring sys-tem and 1.2 A˚closer to the oxygen atom of the car-boxyl group of EPSP].In order to probe the pertinent role of the Ser16 andSer127 residues, we generated two single-mutant pro-teins where the two serine residues were replaced withalanine, producing two single-mutant proteins (Ser16-Ala and Ser127Ala) and a double-mutant protein(Ser16AlaSer127Ala). In this article, we report thatthe replacement of both invariant serine residues(Ser16AlaSer127Ala double-mutant protein) in theactive site of chorismate synthase caused a substantialdecrease in activity beyond the detection limit of ourassay. In contrast to the single-mutant proteins(Ser16Ala and Ser127Ala) the Ser16AlaSer127Ala dou-ble-mutant protein was not able to form the typicaltransient intermediate. Based on our results, we pro-pose that Ser16 and Ser127 establish a proton relaysystem among the isoalloxazine ring, His106 and theEPSP molecule that delivers protons via water mole-cules either to His106, protonating the flavin at N(1)position, or to the C(3)–oxygen of the phosphate-leaving group to facilitate C–O bond breakage.ResultsExpression and purification of the Ser16Ala andSer127Ala single-mutant proteins and of theSer16AlaSer127Ala double-mutant proteinThe mutant proteins were heterologously expressedin Escherichia coli, strain BL21(DE3)RP at expressionlevels similar to those of the wild-type protein. Thetwo-step chromatographic procedure developed forthe purification of wild-type enzyme yielded similaramounts of the mutant proteins (3 mg of protein pergram of wet cell paste) [14]. Because of the weak bind-ing of (oxidized) FMN, all mutant proteins were iso-lated in their apo-form. The apparent stability of allthree mutant proteins was comparable to that of thewild-type enzyme.Binding of oxidized FMN to the mutant proteinsTo characterize the serine mutant proteins in furtherdetail, binding of the oxidized FMN cofactor to theisolated apoproteins was investigated by UV ⁄ visibleScheme 1. Reaction catalyzed by chorismate synthase.G. Rauch et al. Proton relay system in chorismate synthaseFEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1465difference absorbance spectroscopy (Fig. 2). The spec-tral changes observed upon binding of oxidized FMNto the serine mutant proteins were identical to thoseseen with the wild-type enzyme [14]. The dissociationconstant for the Ser127Ala mutant protein (Table 1)was similar to that of the wild-type enzyme, whereasthe replacement of Ser16 with alanine resulted in aslight increase of the dissociation constant (inset ofFig. 2 and Table 1). Similarly, the Ser16AlaSer127Aladouble-mutant protein showed slightly weaker bindingof the cofactor (Table 1). Note that for the wild-typeE. coli protein, its dissociation constant for flavindecreases by three orders of magnitude when the flavinbecomes reduced [15].Binding of EPSP to the mutant proteins in thepresence of oxidized FMNBecause the replaced serine residues are located in thedirect vicinity of EPSP (Fig. 1A) it is important toensure that binding of EPSP to the active site is nothampered. Binding of EPSP to the Ser16Ala, Ser127-Ala and Ser16AlaSer127Ala mutant proteins in thepresence of oxidized FMN was monitored by UV ⁄ visi-ble spectroscopy. The spectral changes observed uponthe binding of EPSP were exploited to determine thedissociation constants for EPSP. The spectral perturba-tions on EPSP binding, and the calculated dissociationconstants for the Ser16Ala and the Ser127Ala single-mutant proteins, were found to be similar to those ofthe wild-type enzyme (Table 1). As shown in Fig. 3,the spectral changes observed when EPSP boundto the double-mutant protein were comparable tothose observed with the wild-type protein, whereas thecalculated dissociation constant was 10-fold higherthan observed with the wild-type enzyme [14]. Notethat the Kmfor EPSP was 2.7 lm with the wild-typeenzyme [16] and therefore one order of magnitudelower than the dissociation constant determined in thepresence of oxidized flavin.BAFig. 1. Ser16 and Ser127 residues acting ina proton relay system among His106, FMNand EPSP via a water molecule in thechorismate synthase active site. (A) Stereo-representation of the active site in the openform, where His106 is in a position close toC(2)=O of the flavin. (B) Stereorepresenta-tion of the chorismate synthase active sitein the closed form, where His106 makescontact with the O12 of EPSP. The carbonatoms of Ser16, Ser127, His106, FMN andEPSP are colored gray. The red spheres rep-resent water positions. Hydrogen bonds areshown as dashed lines. wat 1, wat 2,wat 3, are different water molecules.Proton relay system in chorismate synthase G. Rauch et al.1466 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBSFig. 2. Binding of oxidized FMN to theSer16Ala mutant protein. The plot showsthe result of titration of the Ser16Ala mutantprotein (23 lM) with oxidized FMN in 50 mMMops buffer, pH 7.5. Arrows indicate thedirection of the spectral changes occurringupon titration with FMN. Difference absor-bance spectra at 0, 9.8, 14.6, 22.3, 41.2 and78.1 lM oxidized FMN are shown. The insetshows the spectral changes at 379 nm as afunction of FMN concentration, revealing adissociation constant (Kd)of60lM.Table 1. Dissociation constant (Kd) values for FMN and EPSP.LigandKd(lM)MethodWild-typeNcCSNcCSSer16AlaNcCSSer127AlaNcCS Ser16AlaSer127AlaFMN 41 ± 5a60 ± 7a40 ± 9a56bUV ⁄ visible difference spectroscopyEPSP(in the presence of FMN)17b22b15b155bUV ⁄ visible spectroscopyaAverage of three independent measurements.bAverage of two independent measurements.Fig. 3. Binding of EPSP to the Ser16Ala-Ser127Ala double-mutant protein in thepresence of oxidized FMN. The course of atitration of the Ser16AlaSer127Ala double-mutant protein with EPSP in 50 mM Mopsbuffer, pH 7.5 is shown. UV-visible absor-bance spectra of the Ser16AlaSer127Aladouble-mutant protein (30 lM) and FMN(25 lM) were recorded at various EPSP con-centrations. The spectra shown are at thefollowing EPSP concentrations: 0, 5.7, 9.9,21, 42.4, 69.4, 109.3, 148.6, 200 and250.3 lM. The arrows indicate the directionof the absorbance changes. The insetshows the spectral changes at 397 nm as afunction of EPSP concentration, revealing adissociation constant (Kd) of 155 lM.G. Rauch et al. Proton relay system in chorismate synthaseFEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1467Intrinsic FMN:NADPH oxidoreductase activityof the mutant proteinsChorismate synthase from Neurospora crassa has anintrinsic NADPH:FMN oxidoreductase activity thatenables the enzyme to generate the reduced FMNcofactor (bifunctionality). The structural basis of this‘secondary’ catalytic activity is presently not known[4,17]. We investigated the effect of the mutationson the NADPH:FMN oxidoreductase activity of themutant proteins. From the obtained hyperbolic depen-dency, Michaelis–Menten parameters of 14, 14, 4 and7 lm were calculated for the wild-type protein and forthe Ser16Ala, Ser127Ala and Ser16AlaSer127Alamutant proteins, respectively. These results demon-strated that none of the amino acid replacementssignificantly affected the utilization of NADPH as asource of reducing equivalents for activation of thecofactor to its reduced form.Chorismate synthase activity of the serinemutant proteinsIn order to investigate the influence of the amino acidreplacements on the catalytic activity of chorismate syn-thase, we measured the activity of the mutant proteins.An activity assay under aerobic conditions usingNADPH as a source of reducing equivalents (15) indi-cated that the amino acid replacements have a largeeffect on the activity of the mutant proteins in com-parison to the wild-type enzyme. Under these condi-tions, we measured a residual activity of  2% for theSer16Ala single-mutant protein and of  12% for theSer127Ala single-mutant protein compared with thatof the wild-type enzyme. However, we were not ableto determine any activity for the Ser16AlaSer127Aladouble-mutant protein.The precise residual activity of the mutant proteinswas then measured using the stopped-flow instrumentunder anoxic conditions where we used photoreductionto generate the reduced flavin cofactor. In the absenceof oxygen, the rate of chorismate formation was six- and70-fold lower for the Ser127Ala and Ser16Ala mutantproteins, respectively, than for the wild-type enzyme, asshown in Table 2. The residual activity of the Ser16Ala-Ser127Ala double-mutant protein was below the detec-tion limit of the instrument (Table 2). Therefore, weperformed the same activity assay with a 10-fold higherconcentration of the Ser16AlaSer127Ala mutant protein(125 lm instead of 12.5 lm) but again we were unableto detect chorismate formation.The chorismate synthase-catalysed reaction is char-acterized by the occurrence of a transient specieswith an absorbance maximum at around 390 nm[18]. This species is known to form after the sub-strate binds to the reduced FMN–enzyme binarycomplex [9] but before EPSP undergoes transforma-tion to the product [18,19]. This species is formedvery rapidly (within a few milliseconds) and dis-appears when all substrate has been consumed. Theformation of the intermediate by the two serinesingle-mutant proteins was almost complete withinthe dead time of the instrument and indistinguishablefrom that of the wild-type in single-turnover experi-ments (Fig. 4). For the Ser16AlaSer127Ala double-mutant protein we were not able to detect theintermediate. This demonstrates that the Ser16Ala-Ser127Ala double-mutant protein, in contrast to thesingle-mutant proteins (Ser16Ala and Ser127Ala) isnot capable of forming the flavin-derived intermedi-ate. The decay rate of the intermediate in a single-turnover experiment in general reflects the rate ofsubstrate turnover. In the case of the Ser127Ala andSer16Ala single-mutant proteins, the decay of thetransient species was eight- and 140-fold slower,respectively, than that of the wild-type enzyme, ingood agreement with the slower rate of substrateturnover (Table 3).In contrast to the wild-type protein, the spectra ofthe transient flavin species of the two single serinemutant proteins (Ser16Ala and Ser127Ala) haveslightly different spectral properties. As shown inFig. 5, both single-mutant proteins show, in additionto the peak at 390 nm, a broad shoulder in the rangeof 430–480 nm. Thus, both serine single-mutant pro-teins (Ser16Ala and Ser127Ala) affect the spectralcharacteristics of the transient flavin intermediate. Sim-ilar spectral changes were observed during turnoverwith the substrate analogue (6S)-6-fluoro-EPSP [20].Table 2. Chorismate synthase activity of the serine mutant pro-teins (Ser16Ala, Ser127Ala and Ser16AlaSer127Ala) in comparisonwith the activity of the wild-type enzyme. The formation of choris-mate was monitored at 275 nm using stopped-flow spectrophoto-metry under anaerobic conditions.ChorismatesynthaseactivityNcCSWild-typeNcCSSer16AlaNcCSSer127AlaNcCSSer16AlaSer127AlakcatÆs)10.87 0.012 0.14 Belowdetectionlimit%a100 1.38 16.1aChorismate synthase activity compared with wild-type NcCSactivity.Proton relay system in chorismate synthase G. Rauch et al.1468 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBSThus, small perturbations in the substrate and itsimmediate vicinity have similar effects on the flavinenvironment.DiscussionThe 1,4-elimination of the 3-phosphate group andthe C-(6proR) hydrogen from EPSP to chorismate bychorismate synthase is still one of the most challeng-ing flavin-dependent reactions. The activity of thechorismate synthase-catalysed reaction is dependenton the supply of reduced FMN, which is bound inthe active site of the enzyme [3–5,10]. Several kineticand mechanistic studies have accumulated substantialevidence for a radical mechanism in which theenzyme-bound reduced FMN facilitates C–O bondcleavage by transient electron donation (or negativecharge transfer) to the substrate [6,7]. At the end ofthe catalytic cycle, an electron (or negative charge) isredistributed to maintain the reduced form of theFig. 4. Formation of a transient flavin intermediate during substrateturnover with wild-type enzyme (A), the Ser127Ala mutant protein(B) and the Ser16Ala mutant protein (C). The absorbance changeswere observed at 390 nm as a function of time in single-turnoverexperiments under anoxic conditions using stopped-flow spectro-photometry. The formation of the intermediate was obscured bythe dead time of the instrument but its exponential decay is clearlyvisible.Table 3. Decay rates of the transient flavin intermediate. Thedecay rates for the wild-type NcCS and for the two single-mutantproteins were obtained using stopped-flow spectrophotometryunder anaerobic conditions (single turnover). The absorbancechanges were observed at 390 nm as a function of time.NcCSWild-typeNcCSSer16AlaNcCSSer127AlaNcCS Ser16AlaSer127AlaDecayrate (s)1)2.4 0.017 0.29 No intermediatedetected%a100 0.7 12aChorismate synthase decay rates compared with the wild-typeNcCS decay rate.Fig. 5. Observation of the flavin intermediate in the chorismatesynthase reaction. Comparison of the difference absorbance spec-tra formed during the reaction with wild-type enzyme (solid line),the Ser16Ala mutant protein (dotted line) and the Ser127Ala mutantprotein (dashed line). The wild-type (Wt) trace was taken fromKitzing et al. [12]. The spectra were obtained during a multiple-turnover experiment, and those obtained with the highest ampli-tude within the first few seconds after mixing are shown. Spectradecayed with time but did not otherwise change.G. Rauch et al. Proton relay system in chorismate synthaseFEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1469flavin cofactor [8–11]. The elucidation of the3D structure of the S. pneumoniae chorismate syn-thase in the presence of oxidized FMN and EPSP(catalytically inactive ternary complex) has providedthe first insight into the binding and relative orienta-tion of the cofactor and the substrate in the active siteof the enzyme [10]. A structure of the catalyticallyactive ternary complex among enzyme, substrate andreduced FMN would be difficult to obtain because itwould turn over to give the product, for which theenzyme has a much poorer affinity. The structure ofthe active site of chorismate synthase is consistent withthe hitherto proposed role of reduced FMN, as out-lined above, and also reveals several invariant aminoacid residues in the active site of the enzyme. Based onthis structural information we performed our firstmutagenesis study where we investigated the role oftwo conserved histidine residues (His17 and His106),revealing their role as general acid–base catalysts [12].Recently, we reported experimental evidence that aninvariant aspartate residue (Asp367) operates in con-cert with N(5) of the cofactor to bring about theabstraction of the C(6proR) hydrogen of the substrate[13]. In addition to these invariant amino acid residues,the active site of chorismate synthase features twostrictly conserved (99.5% of 400 sequences aligned)serine residues located near the substrate on the oppo-site site of the isoalloxazine ring (Fig. 1A). From thisstructure the functional role of the serine residues isnot obvious although it was speculated that the sidechains help to organize the water molecules in theactive site [10]. As shown in Fig. 1, one of these watermolecules (wat 1) hydrogen bonds to the C(3)–oxygenof the phosphate group and is held by both serineresidues, while another water molecule (wat 2) ispositioned between a third water molecule and aC1 carboxyl oxygen of the substrate that is also hydro-gen bonded to His106. The third water molecule(wat 3) links the first two water molecules. This inter-esting configuration in the active site of chorismatesynthase, which seems to constitute a proton relay sys-tem among the isoalloxazine ring of FMN, histidine106 and the EPSP molecule, prompted us to investi-gate the role of Ser16 and Ser127 for the chorismatesynthase-catalysed reaction.First, we analyzed the ability of the mutant proteinsto bind cofactor (Fig. 2) and substrate (Fig. 3). Allthree serine mutant proteins were able to bind oxidizedFMN with dissociation constants comparable to thatof the wild-type enzyme but we observed a significantdifference in the ability to bind EPSP, as expected.While the serine single-mutant proteins (Ser16Ala andSer127Ala) have EPSP dissociation constants similarto those of the wild-type enzyme, the dissociation con-stant for the Ser16AlaSer127Ala double-mutant pro-tein was 10-fold higher than for the wild-type enzyme(Table 1). As shown in Fig. 1A, the Ser16 and Ser127residues stabilize a water molecule (wat 1), whichforms a hydrogen bond to the C(3)-oxygen of EPSP.Moreover, wat 2 forms a hydrogen bond to the car-boxylate group of EPSP. Therefore, we conclude thatthe entire hydrogen bonding network is disrupted inthe double-mutant protein, as indicated by the 10-foldhigher dissociation constant for EPSP binding (Fig. 3and Table 1).Next, we studied the effects on the catalytic proper-ties of our mutant proteins. Both single-mutant pro-teins showed a modest to strong decrease in activity byfactors of 6 and 70 for the Ser127Ala and the Ser16-Ala mutant proteins, respectively (Table 2). This isalso probably a result of the loss of appropriatelyordered water within the EPSP-binding site. It is possi-ble that the Ser16Ala mutation is more disruptivebecause it might also affect the orientation of itsneighbour, His17, which normally hydrogen bonds tothe phosphate-leaving group. Most importantly, theSer16AlaSer127Ala double-mutant protein is devoidof any detectable catalytic activity, indicating thatreplacement of both serine residues produces a syner-gistic effect. This result is consistent with the inabilityof the double-mutant protein to form the transientflavin intermediate, which normally forms before anybond-breaking steps occur [18,19]. Taken together, ourresults suggest that the absence of one serine residueleads to a partial disruption of the water structure(‘conserved water molecules’), whereas the absence ofboth serine residues generates an environment thatabrogates the required water structure.Therefore, we propose a mechanism in which a pro-ton is relocated from the N(1)–C(2)=O locus of theisoalloxazine ring to the imidazole ring of His106, thenshuttled to the phosphate ester oxygen atom of thephosphate-leaving group, a process mediated by wat 1,wat 2, wat 3 and the carboxyl group of EPSP, whichserve as a proton translocation system in the active site(see Fig. 1B and Scheme 2). Interestingly, the protonon the N(1)–C(2)=O locus is likely to have originallycome from His106. The transient flavin intermediate isthought to be the result of the protonation of anionicreduced flavin on binding of EPSP to give neutralreduced flavin [9], and the associated general acid isthought to be His106 [10]. It is therefore possible thatdisruption of the proposed proton relay system affectsnot only phosphate cleavage, but also this first flavin-protonation step, by affecting the initial protonationstate of His106.Proton relay system in chorismate synthase G. Rauch et al.1470 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBSSuch a proton relay system is attractive for severalreasons. The enzyme initiates catalysis by ‘separation’of an electron and a proton, both of which are derivedfrom the reduced protonated flavin in a proton-cou-pled electron transfer step. The electron is donated tothe substrate in order to facilitate C–O bond-breakage.Phosphate dianions are poor leaving groups, andalthough interactions with His10, Arg49 and Arg337facilitate the neutralization of the negative charge onthe phosphate group of the substrate, a mechanism forlowering the incipient negative charge on the oxygenof the CO bond being cleaved would be expected.Upon product dissociation, this internal proton relaysystem can be reloaded, leading to protonation ofHis106, such that the enzyme is ready for the next sub-strate turnover.In this mechanism, His106 plays a central role,supported by structural evidence that this residue hassome conformational flexibility allowing it to assumedifferent positions in the active site [10]. In theso-called open conformation (Fig. 1A), His106 assumesa position close to the C(2)=O position of the isoallox-azine ring system, whereas in the closed conformation(Fig. 1B), it moves away from the flavin towards thesubstrate and makes contact with an oxygen atom(O12) of the substrate’s carboxylate group, which, inturn, is in hydrogen bond distance to wat 2. Hence, itappears that His106 and the substrate’s carboxylategroup function as a gate, controlling proton transfer inthe enzyme active site during catalysis. Furthermore,the tightening of the active site in the closed structureis required for a hydrogen bond to form betweenwat 1 and wat 3, adding another element to such agating mechanism. In summary, our data provide evi-dence that the two invariant serine residues arerequired to organize a chain of water molecules in theactive site of chorismate synthase, which form a pro-ton relay system among the isoalloxazine ring ofFMN, His106 and substrate. This proton relay systemis essential for catalysis and is probably synchronizedwith the electron transfer process to the substrate,emphasizing the unique character of the chorismatesynthase reaction.Experimental proceduresReagentsAll chemicals were of the highest grade available andobtained from Sigma or Fluka (Buchs, Switzerland). DEAESephacel was from Amersham Biosciences, and cellulosephosphate (P11) was from Whatman (Kent, UK). DNArestriction and modification enzymes were obtained fromFermentas GmbH or from New England Biolabs (Beverly,MA, USA). Plasmid DNA preparation was performedusing the Nucleobond AX plasmid preparation kit (Mache-rey-Nagel GmbH & Co. KG, Germany). PCR primers werepurchased from VBC-Genomics (Vienna, Austria). EPSPwas synthesized from shikimate-3-phosphate using recombi-nant E. coli EPSP synthase and purified by HPLC.Site-directed mutagenesisBasic molecular biology manipulations were performedusing standard techniques [21]. Amino acid replacementswere performed using the QuikChange site-directed muta-genesis kit from Stratagene (La Jolla, CA, USA). Theconstruct pET21a–N. crassa chorismate synthase (pET21a–NcCS) served as the template. The following oligonucleo-tides containing the appropriate codon exchange wereused for the procedure (the changed codons are underlinedand nucleotides exchanged are in bold): S16A, forward pri-mer, 5¢-CGACCTATGGCGAGGCGCACTGCAAGTCG-3¢, and reverse primer, 5¢-CGACTTGCAGTGCGCCTCGCCATAGGTCG-3¢; S127A, forward primer, 5¢-GCGGCCGCTCTGCCGCCCGCGAGACC-3¢, and reverse primer,5¢-GGTCTCGCGGGCGGCAGAGCGGCCGC-3¢. For theS16AS127A mutein, the construct pET21a–NcCS-S16Aserved as the template and the primer for the S127A replace-CO2–OHOO–O2CH36NHNNHHNOO15NHNNHNOOElectrontransferH2CNNHHis106H2CNHNHHis106POOONHCHCH2COHOOHHSer127OHHNHCHCH2COHOSer16wat1wat2Scheme 2. Proposed proton relay system.G. Rauch et al. Proton relay system in chorismate synthaseFEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1471ment was used in the procedure. All manipulations wereperformed following the manufacturer’s instructions. Themutations were verified by DNA sequencing (MWG-BiotechAG, Germany).Production and purification of NcCSThe Ser16Ala, Ser127Ala and Ser16AlaSer127Ala mutantproteins were produced and purified as described for thewild-type enzyme [14]. Concentration of the purified mutantproteins was carried out using Ultracel YM-10 Centriprepconcentrators (Amicon Bioseparations, Bedford, MA, USA).UV-visible absorbance spectrophotometryAbsorbance spectra for EPSP titration experiments wererecorded using a Specord 210 spectrophotometer equippedwith a thermostated cell holder (Analytik Jena, Germany).All experiments were performed in 50 mm Mops buffer,pH 7.5, at 25 °C.UV ⁄ visible difference absorbancespectrophotometryBinding of oxidized FMN to NcCS can be directly moni-tored by difference UV ⁄ visible spectrophotometry. For thisexperiment, tandem cuvettes (Hellma GmbH & Co. KG,Germany) were used. Initially, the first chamber in the opti-cal path of the sample and the reference cuvette was filledwith enzyme solution, whereas the second chamber wasfilled with the same volume of buffer. The titration was per-formed by successive additions of the same volume of anFMN solution to the enzyme solution of the sample cuvetteand to the buffer compartment of the reference cuvette. Tocompensate for the dilution of the enzyme solution in thereference cuvette, the same volume of buffer was added tothe enzyme solution in the reference cuvette. The observedspectral changes on binding of oxidized FMN to theenzyme were exploited to determine the dissociation con-stants for oxidized FMN.Activity assay under aerobic conditionsChorismate synthase activity was determined by measuringchorismate formation at 281 nm. The reactions were startedby the addition of 30 lm EPSP to a mixture of 100 lmNADPH, 25 lm FMN and 4 lm of either the wild-typeNcCS or the mutant proteins. Reactions were carried out in50 mm Mops, pH 7.5, at 25 °C [14].Stopped-flow spectrophotometrySingle-turnover and multiple-turnover experiments werecarried out using a Hi-Tech Scientific SF-61 stopped-flowspectrophotometer (Salisbury, UK) at 25 °C. The dead timeof the instrument was measured to be 4.1 ± 0.3 ms [22]. Thestopped-flow observation cell had a 1.0 cm path length.Enzyme and substrate solutions were made anaerobic byexchanging the dissolved oxygen with argon by several cyclesof evacuation and flushing. Anaerobic substrate solution wasrapidly mixed with anaerobic enzyme solution in the spectro-photometer. Both solutions contained FMNH2that wasreduced using photoirradiation, in the presence of potassiumoxalate, prior to mixing. After mixing, the anaerobicreaction mixture contained reduced FMN (80 lm), EPSP(100 lm), oxalate (1 mm), 50 mm Mops, pH 7.5, and enzyme(1.25 lm for the wild-type and Ser127Ala mutant proteinsor 12.5 lm of the Ser16Ala and Ser16AlaSer127Ala mutantproteins). Chorismate formation was monitored at 275 nmunder anaerobic conditions.For determination of the decay rates of the transient fla-vin intermediate for the wild-type and mutant proteins, theabsorbance changes were observed at 390 nm, as a functionof time, in single-turnover experiments with 20 lm enzymeand 15 lm substrate. Kinetic data were fitted using the Hi-Tech Scientific kinetasyst 3.14 software.Spectra of reaction intermediatesAbsorbance spectra were recorded with a Hewlett-Packardphotodiode array instrument (model HP8452) using a cuv-ette with a side arm. The enzyme solutions and the sub-strate in the side arm of the cuvette were made anaerobicby exchanging the dissolved oxygen by several cycles ofevacuation and flushing with argon. The anaerobic enzymesolution containing FMNH2was reduced using photoirra-diation in the presence of potassium oxalate before mixingwith the substrate and recording the spectra. After mixing,the anaerobic reaction mixture contained enzyme (40 lm),reduced FMN (80 lm), EPSP (80 lm) and oxalate (1 mm),in 50 m m Mops, pH 7.5. Absorbance spectra were recordedbetween 300 and 600 nm. Each blank was obtained fromcontrol spectra in the presence of fully reduced FMN, butin the absence of substrate.AcknowledgementsThis work was supported, in part, by the Fonds zurFo¨rderung der wissenschaftlichen Forschung (FWF)through grant P17471 to PM.References1 Coggins JR, Abell C, Evans LB, Frederickson M, Rob-inson DA, Roszak AW & Lapthorn AJ (2003) Experi-ences with the shikimate-pathway enzymes as targetsfor rational drug design. Biochem Soc Transactions 31,548–552.Proton relay system in chorismate synthase G. Rauch et al.1472 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS2 Floss HG, Onderka DK & Carroll M (1972) Stereo-chemistry of the 3-deoxy-d-arabino-heptulosonate7-phosphate synthetase reaction and the chorismatesynthetase reaction. 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Planta 207 , 325–334.8 Dmitrenko O, Wood HB, Bach RD & Ganem B (2001)A theoretical study of the chorismate synthase reaction.Organic Letters 3, 4137–4140.9 Macheroux P, Bornemann S, Ghisla S & ThorneleyRNF (1996) Studies with flavin analogs provide evi-dence that a protonated, reduced FMN is the substrateinduced transient intermediate in the chorismatesynthase reaction. J Biol Chem 271, 25850–25858.10 Maclean J & Ali S (2003) The structure of chorismatesynthase reveals a novel flavin-binding site fundamentalto a unique chemical reaction. Structure 11, 1499–1511.11 Osborne A, Thorneley RNF, Abell C & Bornemann S(2000) Studies with substrate and cofactor analoguesprovide evidence for a radical mechanism in the choris-mate synthase reaction. J Biol Chem 275, 35825–35830.12 Kitzing K, Auweter S, Amrhein N & Macheroux P(2004) Mechanism of chorismate synthase. Role of thetwo invariant histidine residues in the active site. 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J Am Chem Soc 112, 8581–8583.17 Henstrand JM, Amrhein N & Schmid J (1995) Cloningand characterization of a heterologously expressedbifunctional chorismate synthase ⁄ flavin reductase fromNeurospora crassa. J Biol Chem 270, 20447–20452.18 Bornemann S, Lowe DJ & Thorneley RNF (1996)The transient kinetics of Escherichia coli chorismatesynthase: substrate consumption, product formation,phosphate dissociation and characterisation of a flavinintermediate. Biochemistry 35, 9907–9916.19 Hawkes TR, Lewis T, Coggins JR, Mousdale DM,Lowe DJ & Thorneley RNF (1990) Chorismatesynthase: pre-steady-state kinetics of phosphate releasefrom 5-enolpyruvylshikimate 3-phosphate. Biochem J265, 899–902.20 Bornemann S, Ramjee MK, Balasubramanian S,Abell C, Coggins JR, Lowe DJ & Thorneley RNF(1995) Escherichia coli chorismate synthase catalyzesthe conversion of (6S)-6-fluoro-5-shikimate-3-phosphate to 6-fluorochorismate. J Biol Chem 270,22811–22815.21 Sambrook J, Fritsch EF & Maniatis T (1989)Molecular Cloning: A Laboratory Manual, 2nd edn.Cold Spring Harbor Laboratory, Cold Spring Harbor,NY.22 Ramjee MK (1992) The characterization of andmechanistic studies on Escherichia coli chorismatesynthase. In The Characterization of and MechanisticStudies on Escherichia coli Chorismate Synthase.PhD Thesis, University of Sussex, Brighton, UK.G. Rauch et al. Proton relay system in chorismate synthaseFEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1473 . Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation. replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant
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