Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity Gernot Rauch 1 , Heidemarie Ehammer 1 , Stephen Bornemann 2 and Peter Macheroux 1 1 Institute of Biochemistry, Graz University of Technology, Austria 2 Department of Biological Chemistry, John Innes Centre, Norwich, UK Chorismate synthase catalyzes the seventh and last step in the shikimate pathway, leading to chorismate, the last common precursor in the biosynthesis of numer- ous aromatic compounds in bacteria, fungi, plants and protozoa. Because of the absence of this pathway in eukaryotic organisms, its enzymes are interesting potential targets for rational drug design [1]. The chorismate 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 the reaction, chorismate synthase activity is based on the supply of reduced FMN, which is bound in the active site of the enzyme [3–5]. Mechanistic studies have indi- cated a functional role of the reduced flavin [6,7] that comprises the transient donation of an electron (or a Keywords enzyme mechanism elimination; flavin; shikimate pathway; site-directed mutagenesis Correspondence P. Macheroux, Institute of Biochemistry, Graz University of Technology, Petersgasse 12 ⁄ II, A-8010 Graz, Austria Fax: +43-316-873 6952 Tel: +43-316-8736450 E-mail: peter.macheroux@tugraz.at (Received 6 December 2007, revised 15 January 2008, accepted 21 January 2008) doi:10.1111/j.1742-4658.2008.06305.x Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic com- pounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)–O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)–O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate 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 the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form 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. Abbreviations EPSP, 5-enolpyruvylshikimate 3-phosphate; NcCS, Neurospora crassa chorismate synthase; wat 1 wat 2 and wat 3, water molecules in chorismate synthase. 1464 FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS charge transfer) to the substrate, prompting cleavage of the C–O bond and thereby facilitating phosphate cleavage. At the end of the catalytic cycle an electron (or negative charge) is redistributed to maintain the reduced form of the flavin cofactor [8–11]. The theoretical and experimental evidence for such a role of the reduced FMN eagerly demanded structural information of the protein. Eventually, the structure determination of Streptococcus pneumoniae chorismate synthase in the presence of oxidized FMN and 5-enol- pyruvylshikimate 3-phosphate (EPSP) provided the first insight into the binding and relative orientation of the cofactor and of the substrate in the active site of the enzyme [10]. Based on this structure we were able to initiate a structure-based mutagenesis study to test mechanistic proposals. In our first study we demon- strated that the invariant histidine residues (His17 and His106) function as general acids in the active site, with His106 protonating the N(1)–C(2)=O locus of reduced flavin whereas His17 appears to be involved in the pro- tonation of the leaving phosphate group [12]. The next target was Asp367, which is in the direct vicinity of the N(5) atom of the isoalloxazine ring system and a likely candidate in a position for the abstraction of hydrogen from the substrate. Single-mutant proteins in which Asp367 has been replaced with alanine or asparagine exhibit a 300- and 600-fold lower activity, respectively, emphasizing the important role of the Asp367 residue as an active-site base. These results provide strong evi- dence that acid–base catalysis is of great importance in the 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 bound EPSP and of the flavin cofactor, revealed two invariant serine residues – Ser16 and Ser127 – interacting with several bound water molecules. As shown in Fig. 1, one water molecule (wat 1) is held by both serine side chains (in the reported structure of S. pneumoniae chorismate synthase, these positions are designated as Ser9 and Ser132, [10]) close to the C(3)–oxygen, while another water molecule (wat 2) is bound between a third water molecule (wat 3) and a C1 carboxyl oxygen of the substrate that is also hydrogen bonded to His106. The third water molecule bridges the first two water molecules. There are both open (Fig. 1A) and closed (Fig. 1B) active-site structures that reveal a tightening up of the site in the latter together with a movement of the His106 side chain away from the fla- vin and towards the substrate [its ring nitrogen atom that 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 and Ser127 residues, we generated two single-mutant pro- teins where the two serine residues were replaced with alanine, producing two single-mutant proteins (Ser16- Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). In this article, we report that the replacement of both invariant serine residues (Ser16AlaSer127Ala double-mutant protein) in the active site of chorismate synthase caused a substantial decrease in activity beyond the detection limit of our assay. In contrast to the single-mutant proteins (Ser16Ala and Ser127Ala) the Ser16AlaSer127Ala dou- ble-mutant protein was not able to form the typical transient intermediate. Based on our results, we pro- pose that Ser16 and Ser127 establish a proton relay system among the isoalloxazine ring, His106 and the EPSP 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. Results Expression and purification of the Ser16Ala and Ser127Ala single-mutant proteins and of the Ser16AlaSer127Ala double-mutant protein The mutant proteins were heterologously expressed in Escherichia coli, strain BL21(DE3)RP at expression levels similar to those of the wild-type protein. The two-step chromatographic procedure developed for the purification of wild-type enzyme yielded similar amounts of the mutant proteins (3 mg of protein per gram 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 all three mutant proteins was comparable to that of the wild-type enzyme. Binding of oxidized FMN to the mutant proteins To characterize the serine mutant proteins in further detail, binding of the oxidized FMN cofactor to the isolated apoproteins was investigated by UV ⁄ visible Scheme 1. Reaction catalyzed by chorismate synthase. G. Rauch et al. Proton relay system in chorismate synthase FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1465 difference absorbance spectroscopy (Fig. 2). The spec- tral changes observed upon binding of oxidized FMN to the serine mutant proteins were identical to those seen with the wild-type enzyme [14]. The dissociation constant for the Ser127Ala mutant protein (Table 1) was similar to that of the wild-type enzyme, whereas the replacement of Ser16 with alanine resulted in a slight increase of the dissociation constant (inset of Fig. 2 and Table 1). Similarly, the Ser16AlaSer127Ala double-mutant protein showed slightly weaker binding of the cofactor (Table 1). Note that for the wild-type E. coli protein, its dissociation constant for flavin decreases by three orders of magnitude when the flavin becomes reduced [15]. Binding of EPSP to the mutant proteins in the presence of oxidized FMN Because the replaced serine residues are located in the direct vicinity of EPSP (Fig. 1A) it is important to ensure that binding of EPSP to the active site is not hampered. Binding of EPSP to the Ser16Ala, Ser127- Ala and Ser16AlaSer127Ala mutant proteins in the presence of oxidized FMN was monitored by UV ⁄ visi- ble spectroscopy. The spectral changes observed upon the binding of EPSP were exploited to determine the dissociation constants for EPSP. The spectral perturba- tions on EPSP binding, and the calculated dissociation constants for the Ser16Ala and the Ser127Ala single- mutant proteins, were found to be similar to those of the wild-type enzyme (Table 1). As shown in Fig. 3, the spectral changes observed when EPSP bound to the double-mutant protein were comparable to those observed with the wild-type protein, whereas the calculated dissociation constant was 10-fold higher than observed with the wild-type enzyme [14]. Note that the K m for EPSP was 2.7 lm with the wild-type enzyme [16] and therefore one order of magnitude lower than the dissociation constant determined in the presence of oxidized flavin. B A Fig. 1. Ser16 and Ser127 residues acting in a proton relay system among His106, FMN and EPSP via a water molecule in the chorismate synthase active site. (A) Stereo- representation of the active site in the open form, where His106 is in a position close to C(2)=O of the flavin. (B) Stereorepresenta- tion of the chorismate synthase active site in the closed form, where His106 makes contact with the O12 of EPSP. The carbon atoms of Ser16, Ser127, His106, FMN and EPSP are colored gray. The red spheres rep- resent water positions. Hydrogen bonds are shown 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 FEBS Fig. 2. Binding of oxidized FMN to the Ser16Ala mutant protein. The plot shows the result of titration of the Ser16Ala mutant protein (23 l M) with oxidized FMN in 50 mM Mops buffer, pH 7.5. Arrows indicate the direction of the spectral changes occurring upon titration with FMN. Difference absor- bance spectra at 0, 9.8, 14.6, 22.3, 41.2 and 78.1 l M oxidized FMN are shown. The inset shows the spectral changes at 379 nm as a function of FMN concentration, revealing a dissociation constant (K d )of60lM. Table 1. Dissociation constant (K d ) values for FMN and EPSP. Ligand K d (lM) Method Wild-type NcCS NcCS Ser16Ala NcCS Ser127Ala NcCS Ser16Ala Ser127Ala FMN 41 ± 5 a 60 ± 7 a 40 ± 9 a 56 b UV ⁄ visible difference spectroscopy EPSP (in the presence of FMN) 17 b 22 b 15 b 155 b UV ⁄ visible spectroscopy a Average of three independent measurements. b Average of two independent measurements. Fig. 3. Binding of EPSP to the Ser16Ala- Ser127Ala double-mutant protein in the presence of oxidized FMN. The course of a titration of the Ser16AlaSer127Ala double- mutant protein with EPSP in 50 m M Mops buffer, pH 7.5 is shown. UV-visible absor- bance spectra of the Ser16AlaSer127Ala double-mutant protein (30 l M) and FMN (25 l M) were recorded at various EPSP con- centrations. The spectra shown are at the following EPSP concentrations: 0, 5.7, 9.9, 21, 42.4, 69.4, 109.3, 148.6, 200 and 250.3 l M. The arrows indicate the direction of the absorbance changes. The inset shows the spectral changes at 397 nm as a function of EPSP concentration, revealing a dissociation constant (K d ) of 155 lM. G. Rauch et al. Proton relay system in chorismate synthase FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1467 Intrinsic FMN:NADPH oxidoreductase activity of the mutant proteins Chorismate synthase from Neurospora crassa has an intrinsic NADPH:FMN oxidoreductase activity that enables the enzyme to generate the reduced FMN cofactor (bifunctionality). The structural basis of this ‘secondary’ catalytic activity is presently not known [4,17]. We investigated the effect of the mutations on the NADPH:FMN oxidoreductase activity of the mutant proteins. From the obtained hyperbolic depen- dency, Michaelis–Menten parameters of 14, 14, 4 and 7 lm were calculated for the wild-type protein and for the Ser16Ala, Ser127Ala and Ser16AlaSer127Ala mutant proteins, respectively. These results demon- strated that none of the amino acid replacements significantly affected the utilization of NADPH as a source of reducing equivalents for activation of the cofactor to its reduced form. Chorismate synthase activity of the serine mutant proteins In order to investigate the influence of the amino acid replacements on the catalytic activity of chorismate syn- thase, we measured the activity of the mutant proteins. An activity assay under aerobic conditions using NADPH as a source of reducing equivalents (15) indi- cated that the amino acid replacements have a large effect 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 the Ser16Ala single-mutant protein and of  12% for the Ser127Ala single-mutant protein compared with that of the wild-type enzyme. However, we were not able to determine any activity for the Ser16AlaSer127Ala double-mutant protein. The precise residual activity of the mutant proteins was then measured using the stopped-flow instrument under anoxic conditions where we used photoreduction to generate the reduced flavin cofactor. In the absence of oxygen, the rate of chorismate formation was six- and 70-fold lower for the Ser127Ala and Ser16Ala mutant proteins, respectively, than for the wild-type enzyme, as shown 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, we performed the same activity assay with a 10-fold higher concentration of the Ser16AlaSer127Ala mutant protein (125 lm instead of 12.5 lm) but again we were unable to detect chorismate formation. The chorismate synthase-catalysed reaction is char- acterized by the occurrence of a transient species with an absorbance maximum at around 390 nm [18]. This species is known to form after the sub- strate binds to the reduced FMN–enzyme binary complex [9] but before EPSP undergoes transforma- tion to the product [18,19]. This species is formed very rapidly (within a few milliseconds) and dis- appears when all substrate has been consumed. The formation of the intermediate by the two serine single-mutant proteins was almost complete within the dead time of the instrument and indistinguishable from that of the wild-type in single-turnover experi- ments (Fig. 4). For the Ser16AlaSer127Ala double- mutant protein we were not able to detect the intermediate. This demonstrates that the Ser16Ala- Ser127Ala double-mutant protein, in contrast to the single-mutant proteins (Ser16Ala and Ser127Ala) is not capable of forming the flavin-derived intermedi- ate. The decay rate of the intermediate in a single- turnover experiment in general reflects the rate of substrate turnover. In the case of the Ser127Ala and Ser16Ala single-mutant proteins, the decay of the transient species was eight- and 140-fold slower, respectively, than that of the wild-type enzyme, in good agreement with the slower rate of substrate turnover (Table 3). In contrast to the wild-type protein, the spectra of the transient flavin species of the two single serine mutant proteins (Ser16Ala and Ser127Ala) have slightly different spectral properties. As shown in Fig. 5, both single-mutant proteins show, in addition to the peak at 390 nm, a broad shoulder in the range of 430–480 nm. Thus, both serine single-mutant pro- teins (Ser16Ala and Ser127Ala) affect the spectral characteristics of the transient flavin intermediate. Sim- ilar spectral changes were observed during turnover with the substrate analogue (6S)-6-fluoro-EPSP [20]. Table 2. Chorismate synthase activity of the serine mutant pro- teins (Ser16Ala, Ser127Ala and Ser16AlaSer127Ala) in comparison with 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. Chorismate synthase activity NcCS Wild-type NcCS Ser16Ala NcCS Ser127Ala NcCS Ser16Ala Ser127Ala k catÆ s )1 0.87 0.012 0.14 Below detection limit % a 100 1.38 16.1 a Chorismate synthase activity compared with wild-type NcCS activity. 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 FEBS Thus, small perturbations in the substrate and its immediate vicinity have similar effects on the flavin environment. Discussion The 1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from EPSP to chorismate by chorismate synthase is still one of the most challeng- ing flavin-dependent reactions. The activity of the chorismate synthase-catalysed reaction is dependent on the supply of reduced FMN, which is bound in the active site of the enzyme [3–5,10]. Several kinetic and mechanistic studies have accumulated substantial evidence for a radical mechanism in which the enzyme-bound reduced FMN facilitates C–O bond cleavage by transient electron donation (or negative charge transfer) to the substrate [6,7]. At the end of the catalytic cycle, an electron (or negative charge) is redistributed to maintain the reduced form of the Fig. 4. Formation of a transient flavin intermediate during substrate turnover with wild-type enzyme (A), the Ser127Ala mutant protein (B) and the Ser16Ala mutant protein (C). The absorbance changes were observed at 390 nm as a function of time in single-turnover experiments under anoxic conditions using stopped-flow spectro- photometry. The formation of the intermediate was obscured by the dead time of the instrument but its exponential decay is clearly visible. Table 3. Decay rates of the transient flavin intermediate. The decay rates for the wild-type NcCS and for the two single-mutant proteins were obtained using stopped-flow spectrophotometry under anaerobic conditions (single turnover). The absorbance changes were observed at 390 nm as a function of time. NcCS Wild-type NcCS Ser16Ala NcCS Ser127Ala NcCS Ser16Ala Ser127Ala Decay rate (s )1 ) 2.4 0.017 0.29 No intermediate detected % a 100 0.7 12 a Chorismate synthase decay rates compared with the wild-type NcCS decay rate. Fig. 5. Observation of the flavin intermediate in the chorismate synthase 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 mutant protein (dashed line). The wild-type (Wt) trace was taken from Kitzing 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. Spectra decayed with time but did not otherwise change. G. Rauch et al. Proton relay system in chorismate synthase FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1469 flavin cofactor [8–11]. The elucidation of the 3D structure of the S. pneumoniae chorismate syn- thase in the presence of oxidized FMN and EPSP (catalytically inactive ternary complex) has provided the first insight into the binding and relative orienta- tion of the cofactor and the substrate in the active site of the enzyme [10]. A structure of the catalytically active ternary complex among enzyme, substrate and reduced FMN would be difficult to obtain because it would turn over to give the product, for which the enzyme has a much poorer affinity. The structure of the active site of chorismate synthase is consistent with the hitherto proposed role of reduced FMN, as out- lined above, and also reveals several invariant amino acid residues in the active site of the enzyme. Based on this structural information we performed our first mutagenesis study where we investigated the role of two conserved histidine residues (His17 and His106), revealing their role as general acid–base catalysts [12]. Recently, we reported experimental evidence that an invariant aspartate residue (Asp367) operates in con- cert with N(5) of the cofactor to bring about the abstraction of the C(6proR) hydrogen of the substrate [13]. In addition to these invariant amino acid residues, the active site of chorismate synthase features two strictly 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 this structure the functional role of the serine residues is not obvious although it was speculated that the side chains help to organize the water molecules in the active site [10]. As shown in Fig. 1, one of these water molecules (wat 1) hydrogen bonds to the C(3)–oxygen of the phosphate group and is held by both serine residues, while another water molecule (wat 2) is positioned between a third water molecule and a C1 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 chorismate synthase, which seems to constitute a proton relay sys- tem among the isoalloxazine ring of FMN, histidine 106 and the EPSP molecule, prompted us to investi- gate the role of Ser16 and Ser127 for the chorismate synthase-catalysed reaction. First, we analyzed the ability of the mutant proteins to bind cofactor (Fig. 2) and substrate (Fig. 3). All three serine mutant proteins were able to bind oxidized FMN with dissociation constants comparable to that of the wild-type enzyme but we observed a significant difference in the ability to bind EPSP, as expected. While the serine single-mutant proteins (Ser16Ala and Ser127Ala) have EPSP dissociation constants similar to 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 Ser127 residues stabilize a water molecule (wat 1), which forms 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 that the entire hydrogen bonding network is disrupted in the double-mutant protein, as indicated by the 10-fold higher dissociation constant for EPSP binding (Fig. 3 and 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 by factors of 6 and 70 for the Ser127Ala and the Ser16- Ala mutant proteins, respectively (Table 2). This is also probably a result of the loss of appropriately ordered water within the EPSP-binding site. It is possi- ble that the Ser16Ala mutation is more disruptive because it might also affect the orientation of its neighbour, His17, which normally hydrogen bonds to the phosphate-leaving group. Most importantly, the Ser16AlaSer127Ala double-mutant protein is devoid of any detectable catalytic activity, indicating that replacement of both serine residues produces a syner- gistic effect. This result is consistent with the inability of the double-mutant protein to form the transient flavin intermediate, which normally forms before any bond-breaking steps occur [18,19]. Taken together, our results suggest that the absence of one serine residue leads to a partial disruption of the water structure (‘conserved water molecules’), whereas the absence of both serine residues generates an environment that abrogates 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 the isoalloxazine ring to the imidazole ring of His106, then shuttled to the phosphate ester oxygen atom of the phosphate-leaving group, a process mediated by wat 1, wat 2, wat 3 and the carboxyl group of EPSP, which serve as a proton translocation system in the active site (see Fig. 1B and Scheme 2). Interestingly, the proton on the N(1)–C(2)=O locus is likely to have originally come from His106. The transient flavin intermediate is thought to be the result of the protonation of anionic reduced flavin on binding of EPSP to give neutral reduced flavin [9], and the associated general acid is thought to be His106 [10]. It is therefore possible that disruption of the proposed proton relay system affects not only phosphate cleavage, but also this first flavin- protonation step, by affecting the initial protonation state 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 FEBS Such a proton relay system is attractive for several reasons. The enzyme initiates catalysis by ‘separation’ of an electron and a proton, both of which are derived from the reduced protonated flavin in a proton-cou- pled electron transfer step. The electron is donated to the substrate in order to facilitate C–O bond-breakage. Phosphate dianions are poor leaving groups, and although interactions with His10, Arg49 and Arg337 facilitate the neutralization of the negative charge on the phosphate group of the substrate, a mechanism for lowering the incipient negative charge on the oxygen of the CO bond being cleaved would be expected. Upon product dissociation, this internal proton relay system can be reloaded, leading to protonation of His106, 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 has some conformational flexibility allowing it to assume different positions in the active site [10]. In the so-called open conformation (Fig. 1A), His106 assumes a 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 the substrate and makes contact with an oxygen atom (O12) of the substrate’s carboxylate group, which, in turn, is in hydrogen bond distance to wat 2. Hence, it appears that His106 and the substrate’s carboxylate group function as a gate, controlling proton transfer in the enzyme active site during catalysis. Furthermore, the tightening of the active site in the closed structure is required for a hydrogen bond to form between wat 1 and wat 3, adding another element to such a gating mechanism. In summary, our data provide evi- dence that the two invariant serine residues are required to organize a chain of water molecules in the active site of chorismate synthase, which form a pro- ton relay system among the isoalloxazine ring of FMN, His106 and substrate. This proton relay system is essential for catalysis and is probably synchronized with the electron transfer process to the substrate, emphasizing the unique character of the chorismate synthase reaction. Experimental procedures Reagents All chemicals were of the highest grade available and obtained from Sigma or Fluka (Buchs, Switzerland). DEAE Sephacel was from Amersham Biosciences, and cellulose phosphate (P11) was from Whatman (Kent, UK). DNA restriction and modification enzymes were obtained from Fermentas GmbH or from New England Biolabs (Beverly, MA, USA). Plasmid DNA preparation was performed using the Nucleobond AX plasmid preparation kit (Mache- rey-Nagel GmbH & Co. KG, Germany). PCR primers were purchased from VBC-Genomics (Vienna, Austria). EPSP was synthesized from shikimate-3-phosphate using recombi- nant E. coli EPSP synthase and purified by HPLC. Site-directed mutagenesis Basic molecular biology manipulations were performed using standard techniques [21]. Amino acid replacements were performed using the QuikChange site-directed muta- genesis kit from Stratagene (La Jolla, CA, USA). The construct pET21a–N. crassa chorismate synthase (pET21a– NcCS) served as the template. The following oligonucleo- tides containing the appropriate codon exchange were used for the procedure (the changed codons are underlined and nucleotides exchanged are in bold): S16A, forward pri- mer, 5¢-CGACCTATGGCGAG GCGCACTGCAAGTCG- 3¢, and reverse primer, 5¢-CGACTTGCAGTG CGCCTCGC CATAGGTCG-3¢; S127A, forward primer, 5¢-GCGGCCG CTCT GCCGCCCGCGAGACC-3¢, and reverse primer, 5¢-GGTCTCGCGGGC GGCAGAGCGGCCGC-3¢. For the S16AS127A mutein, the construct pET21a–NcCS-S16A served as the template and the primer for the S127A replace- CO 2 – OH OO – O 2 C H 3 6 N H N NH H N O O 1 5 N H N NH N O O Electron transfer H 2 C N NH His106 H 2 C N H NH His106 P O O O NH CH C H 2 C O H O O H H Ser127 O H H NH CH C H 2 C O HO Ser16 wat1 wat2 Scheme 2. Proposed proton relay system. G. Rauch et al. Proton relay system in chorismate synthase FEBS Journal 275 (2008) 1464–1473 ª 2008 The Authors Journal compilation ª 2008 FEBS 1471 ment was used in the procedure. All manipulations were performed following the manufacturer’s instructions. The mutations were verified by DNA sequencing (MWG-Biotech AG, Germany). Production and purification of NcCS The Ser16Ala, Ser127Ala and Ser16AlaSer127Ala mutant proteins were produced and purified as described for the wild-type enzyme [14]. Concentration of the purified mutant proteins was carried out using Ultracel YM-10 Centriprep concentrators (Amicon Bioseparations, Bedford, MA, USA). UV-visible absorbance spectrophotometry Absorbance spectra for EPSP titration experiments were recorded using a Specord 210 spectrophotometer equipped with 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 absorbance spectrophotometry Binding of oxidized FMN to NcCS can be directly moni- tored by difference UV ⁄ visible spectrophotometry. For this experiment, 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 filled with enzyme solution, whereas the second chamber was filled with the same volume of buffer. The titration was per- formed by successive additions of the same volume of an FMN solution to the enzyme solution of the sample cuvette and to the buffer compartment of the reference cuvette. To compensate for the dilution of the enzyme solution in the reference cuvette, the same volume of buffer was added to the enzyme solution in the reference cuvette. The observed spectral changes on binding of oxidized FMN to the enzyme were exploited to determine the dissociation con- stants for oxidized FMN. Activity assay under aerobic conditions Chorismate synthase activity was determined by measuring chorismate formation at 281 nm. The reactions were started by the addition of 30 lm EPSP to a mixture of 100 lm NADPH, 25 lm FMN and 4 lm of either the wild-type NcCS or the mutant proteins. Reactions were carried out in 50 mm Mops, pH 7.5, at 25 °C [14]. Stopped-flow spectrophotometry Single-turnover and multiple-turnover experiments were carried out using a Hi-Tech Scientific SF-61 stopped-flow spectrophotometer (Salisbury, UK) at 25 °C. The dead time of the instrument was measured to be 4.1 ± 0.3 ms [22]. The stopped-flow observation cell had a 1.0 cm path length. Enzyme and substrate solutions were made anaerobic by exchanging the dissolved oxygen with argon by several cycles of evacuation and flushing. Anaerobic substrate solution was rapidly mixed with anaerobic enzyme solution in the spectro- photometer. Both solutions contained FMNH 2 that was reduced using photoirradiation, in the presence of potassium oxalate, prior to mixing. After mixing, the anaerobic reaction 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 proteins or 12.5 lm of the Ser16Ala and Ser16AlaSer127Ala mutant proteins). Chorismate formation was monitored at 275 nm under anaerobic conditions. For determination of the decay rates of the transient fla- vin intermediate for the wild-type and mutant proteins, the absorbance changes were observed at 390 nm, as a function of time, in single-turnover experiments with 20 lm enzyme and 15 lm substrate. Kinetic data were fitted using the Hi- Tech Scientific kinetasyst 3.14 software. Spectra of reaction intermediates Absorbance spectra were recorded with a Hewlett-Packard photodiode 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 anaerobic by exchanging the dissolved oxygen by several cycles of evacuation and flushing with argon. The anaerobic enzyme solution containing FMNH 2 was reduced using photoirra- diation in the presence of potassium oxalate before mixing with 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 recorded between 300 and 600 nm. Each blank was obtained from control spectra in the presence of fully reduced FMN, but in the absence of substrate. Acknowledgements This work was supported, in part, by the Fonds zur Fo ¨ rderung der wissenschaftlichen Forschung (FWF) through grant P17471 to PM. References 1 Coggins JR, Abell C, Evans LB, Frederickson M, Rob- inson DA, Roszak AW & Lapthorn AJ (2003) Experi- ences with the shikimate-pathway enzymes as targets for rational drug design. Biochem Soc Transactions 31, 548–552. Proton relay system in chorismate synthase G. 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