The Catalytic Mechanism of Indole-3-glycerol Phosphate Synthase: Crystal Structures of Complexes of the Enzyme from Sulfolobus solfataricus with Substrate Analogue, Substrate, and Product

doi:10.1016/S0022-2836(02)00378-9 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 319, 757–766

The Catalytic Mechanism of Indole-3-glycerol Phosphate Synthase: Crystal Structures of Complexes of the Enzyme from Sulfolobus solfataricus with Substrate Analogue, Substrate, and Product M. Hennig1*, B.D. Darimont2, J.N. Jansonius1 and K. Kirschner2 1

Division Structural Biology Biozentrum, University of Basel, Klingelbergstr. 70 CH-4056 Basel, Switzerland 2 Division Biophysical Chemistry, Biozentrum University of Basel Klingelbergstr. 70, CH-4056 Basel, Switzerland

Indoleglycerol phosphate synthase catalyzes the ring closure of an N-alkylated anthranilate to a 3-alkyl indole derivative, a reaction requiring Lewis acid catalysis in vitro. Here, we investigated the enzymatic reaction mechanism through X-ray crystallography of complexes of the hyperthermostable enzyme from Sulfolobus solfataricus with the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate, a substrate analogue and the product indole-3-glycerol phosphate. The substrate and the substrate analogue are bound to the active site in a similar, extended conformation between the previously identified phosphate binding site and a hydrophobic pocket for the anthranilate moiety. This binding mode is unproductive, because the carbon atoms that are to be joined are too far apart. The indole ring of the bound product resides in a second hydrophobic pocket adjacent to that of the anthranilate moiety of the substrate. Although the hydrophobic moiety of the substrate moves during catalysis from one hydrophobic pocket to the other, the triosephosphate moiety remains rigidly bound to the same set of hydrogen-bonding residues. Simultaneously, the catalytically important residues Lys53, Lys110 and Glu159 maintain favourable distances to the atoms of the ligand undergoing covalent changes. On the basis of these data, the structures of two putative catalytic intermediates were modelled into the active site. This new structural information and the modelling studies provide further insight into the mechanism of enzyme-catalyzed indole synthesis. The charged 1-amino group of Lys110 is the general acid, and the carboxylate group of Glu159 is the general base. Lys53 guides the substrate undergoing conformational transitions during catalysis, by forming a salt-bridge to the carboxylate group of its anthranilate moiety. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: ligand complexes; X-ray structural analysis; (ba)8-barrel; catalytic mechanism; tryptophan biosynthesis

Introduction Present addresses: M. Hennig, Hoffmann-La Roche AG, Pharma Research Discovery Technologies, X-ray Crystallography 65/308, CH-4070 Basel, Switzerland; B. D. Darimont, Institute of Molecular Biology, 1229, University of Oregon, Eugene, OR 97403-1229, USA Abbreviations used: CdRP, 1-(o-carboxyphenylamino)1-deoxyribulose 5-phosphate; eIGPS, IGP synthase from Escherichia coli; ePRAI, phosphoribosyl-anthranilate isomerase from E. coli; IGP, indole-3-glycerol phosphate; rCdRP, reduced CdRP, 1-(o-carboxyphenylamino)-1deoxyribityl 5-phosphate; sIGPS, IGP synthase from Sulfolobus solfataricus; tIGPS, IGP synthase from Thermotoga maritima. E-mail address of the corresponding author: [email protected]

Indole-3-glycerol phosphate synthase (IGPS) catalyzes the fifth reaction in the pathway of biosynthesis of tryptophan, in which the substrate 1-(o-carboxyphenylamino) 1-deoxyribulose 5-phosphate (CdRP, Figure 1(a)) undergoes a ring closure reaction to the product indole-3-glycerol phosphate (IGP, Figure 1(d)). The enzyme-catalyzed synthesis of the indole ring was proposed by Parry1 to occur by means of the intermediates I1 and I2 (Figure 1(b) and (c)), in analogy to the Bischler synthesis of indoles from a-arylamino ketones.2 The chemical reaction presumably consists of a sequence of condensation, decarboxylation and

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

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Figure 1. The mechanism of the indoleglycerol phosphate synthase reaction as proposed by Parry.1 CdRP (a) is converted via the intermediates (b) I1 and (c) I2 to (d) IGP. The substrate analogue rCdRP carries a secondary OH-group at the C20 atom of (a) CdRP. See the text for details.

dehydration, and requires heating with Lewis acids. It is practically irreversible, due to both the formation of the pyrrole ring of the indole and the release of CO2. At neutral pH, the biochemical reaction does not proceed spontaneously at a measurable rate. Yanofsky3 showed that 4-methylanthranilate is converted to 6-methyl IGPS, proving that the C2 atom of the ribulose moiety of CdRP condenses with the C1 atom of the anthranilate moiety. Moreover, the carboxyl group of CdRP must play an essential, as yet unknown, role in the bond-forming process, because 1-(phenylamino)

Catalytic Mechanism of IGPS

1-deoxyribulose 5-phosphate is not converted to IGP by IGPS.4,5 IGPS is active as a separate monomeric enzyme in most microorganisms,6 but it is occasionally part of a bi- or multifunctional enzyme, being fused to one or more of the other enzymes of the tryptophan biosynthesis pathway. The crystal structures of the bifunctional enzyme IGPS/phosphoribosyl anthranilate isomerase from Escherichia coli (eIGPS:ePRAI)7,8 as well as that of the monofunctional IGPS from the hyperthermophile Sulfolobus solfataricus (sIGPS)9,10 have been ˚ resolution. In both cases, IGPS determined at 2 A has the (ba)8-barrel fold, with an N-terminal extension of about 45 residues (Figure 2). Within the first 20 residues, an additional helix a0 is located at the C-terminal face of the central parallel b-barrel. It forms one wall of the active site, which is identified by a molecule of the bound product IGP (Figure 2) as will be described for the first time in this work. On the basis of mutational analyses of eIGPS,11,12 residues K53, K110, E159 and N180 (numbering of sIGPS) were identified as essential, and both E51 and R182 as important for catalysis. The Ca atoms of these residues are labelled in Figure 2. The purpose of the present study is to improve the understanding of the catalytic mechanism of IGPS by correlating new structural data on ligand complexes of sIGPS with previous enzyme kinetic and mutational data on eIGPS. sIGPS is far more thermostable than eIGPS,11 but catalytically much less active at low temperatures.9,13,14 This work reports the crystal structures of sIGPS complexed with the substrate analogue rCdRP, with the product IGP, and also with the substrate CdRP. These structures identify K110 as the general acid and E159 as the general base in the catalytic mechanism, and clarify the so far unknown roles of E51, K53, N180, and R182.

Figure 2. Stereo view of the complex of sIGPS with IGP bound to the active site. The ribbon drawing gives the N-terminal extension in dark blue, and the subsequent eight canonical ba-modules in rainbow colours. The positions of the Ca atoms of the catalytically important residues E51, K53, K110, E159, N180 and R182 are marked as labelled spheres. The Figure was produced with MOLSCRIPT.30

759

Catalytic Mechanism of IGPS

Table 1. Statistics for the X-ray data collection and refinement sIGPS:CdRP Space group ˚) Cell dimensions (A Temperature (K) X-ray source Detector ˚) Resolution range (A Measured reflections Unique reflections Completeness (%) Rsym (%)a

sIGPS:rCdRP

P21 21 21 P21 21 21 a ¼ 58:2 a ¼ 57:8 b ¼ 73:9 b ¼ 73:7 c ¼ 104:5 c ¼ 104:4 273 293 Rotating anode generator (50 mA, 40 kV) Mar-research image plate area detector (300 mm) 15.0–2.40 15.0–2.05 60,396 114,105 17,120 27,283 94.6 95.7 10.6 7.7

No. protein atoms 2003 No. water molecules 207 No. ligand atoms 23 ˚) r.m.s. distances (A 0.012 r.m.s. bond angles (8) 1.9 (%)b 25.2 Rstart-refinement factor end-refinement (%) 19.7 Rwork (5% of data, %) 24.7 Rend-refinement free ˚ 2) 24.2 Mean B-factor, protein (A ˚ 2) Mean B-factor, ligand (A 39.8 ˚ 2) 43.6 Mean B-factor, solvent (A  P P P P  a Rsym ¼ hkl i IðhklÞi 2 kIðhklÞl= hkl i IðhklÞi :  P P  b Rfactor ¼ hkl Fo ðhklÞ 2 Fc ðhklÞ= hkl Fo ðhklÞ:

Results and Discussion Crystallization and structure determination of the enzyme– ligand complexes Crystals of sIGPS were grown under similar conditions as used for the enzyme liganded with phosphate,9 that is, in 0.05 M potassium phosphate buffer containing 1.3 M ammonium sulfate (see Materials and Methods). Ligand concentrations were adjusted to saturate the active site of sIGPS, by competing efficiently with the concentrations of phosphate and sulfate in the crystallization buffer. Chemical reduction of CdRP (Figure 1(a)) generates the non-reactive substrate analogue rCdRP,15 in which the keto group at C20 is converted to a diastereomeric hydroxyl group, probably without enantiomeric selectivity. rCdRP is a potent competitive inhibitor of both and sIGPS eIGPS (KdrCdRP ¼ 0:25 mM16,17) rCdRP ¼ 0:014 mM; A. Merz & K.K., unpublished (Kd results). Crystallizing sIGPS at pH 5.0 and 4 8C in the presence of rCdRP yielded directly the rCdRP complex. Crystals of sIGPS were soaked with IGP at pH 6.0 and 25 8C to obtain the sIGPS:IGP complex. Because the catalytic turnover number of sIGPS is only 0.03 s21 at 37 8C,18 the structure of the enzyme– substrate complex could be obtained with crystals of sIGPS grown at pH 6.5 and 25 8C, which were briefly soaked in solutions containing CdRP, followed by chilling to 0 8C. The crystal structures of the three sIGPS:ligand complexes were determined and the final atomic models always include all 248 amino acid residues as well as the respective ligand. They all had the

2003 253 23 0.013 1.9 34.4 15.2 22.5 27.8 26.3 52.6

sIGPS:IGP P21 21 21 a ¼ 57:9 b ¼ 73:8 c ¼ 104:2 293 15.0– 2.00 141,730 28,014 91.1 6.3 2003 242 19 0.012 1.7 24.8 15.9 21.2 29.6 37.5 53.8

same space group and very similar cell dimensions (Table 1) as the enzyme liganded with phosphate,9 and their overall three-dimensional structures were essentially identical. Moreover, the superposition of the active-site structures of eIGPS and sIGPS revealed that the locations and the conformations of the side-chains of the six catalytically important residues are very similar (data not shown). Table 2 presents the catalytic and the hydrophobic residues that contact the active-site ligands, as well as their distribution over four distinct regions of the active site. The phosphate site is almost identical for inorganic phosphate,9,10 and the phosphate moities of the three ligands described below. The peptide NH groups of G212 and G233 make hydrogen bonds, and the 1-ammonium group of K53 a salt-bridge to the phosphate moiety. These interactions will not be emphasized further on. As shown below, the catalytic side-chains of K53, E51 and K110 form a salt-bridge triad in the ribulose/triose binding site. On the opposite side of the active-site cavity, moreover, residues E159 and N180 form a hydrogen-bonded triad with the invariant residue S211. N180 orients the carboxyl group of E159, assisted by S211. Two distinct but adjacent hydrophobic pockets are involved in binding the substrate or the product, where certain hydrophobic side-chains contact the benzene rings of rCdRP and CdRP, others the benzene ring of IGP, and some contact both. The structural arrangements of these catalytically important side-chains are presented for several sIGPS:ligand complexes in the following.

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Catalytic Mechanism of IGPS

Table 2. Residues at the active site of IGPS Residuea Positionb

Conservationc

Phosphate-binding site K53 K55 G212 G216 G233 G236 S234 S237

b1a1 b7a7 a80 a80

inv inv inv cns (S . E)

Ribulose/triose-binding site E51 E53d K53 K55d K110 K114d E159 E163d N180 N184d

b1 b1a1 b3 b5 b6

inv inv inv inv inv

a0 b1a1 b2a2 b6a6 b6a6

var inv inv inv inv

b2a2 b3 b3a3 b4 b6a6

inv inv inv cns (I , M) inv

S. solfataricus

E. coli

Anthranilate-binding pocket W8 V4 P57 P59 F89 F93 R182 R186d L184 L188 Indole-binding pocket F89 F93 K110 K114d F112 F116 I133 M137 L184 L188

a Residues correlated by structure-based sequence alignment.9 b Location of the residue according to secondary structural elements, biai, loop between structural elements b-strand i and the subsequent a-helix i. c Conservation assessed from alignment of 25 orthologous sequences. inv, invariant residue; cns, conserved residue (the relative frequency of the two major residues is indicated); var, variable residue. d Catalytically important residues.12

Structure of the enzyme: rCdRP complex ˚ resolution crystal structure of the In the 2.05 A sIGPS:rCdRP complex (Figures 3(a) and 4(a)), bound rCdRP assumes a bent conformation. PRAI from Thermotoga maritima (tPRAI)19,20 also binds rCdRP, albeit as a product analogue. Its conformation in the tPRAI:rCdRP complex is rather similar to that in Figure 3(a) (M.H., unpublished results), and might represent a low-energy conformation. In this complex, binding of the stereoisomer with the configuration S is preferred. Out of the six hydrophobic side-chains shown in Figure 3(a), the benzene ring of rCdRP contacts only the residues W8, P57, F89, and L184 (Table 2). The negative charge of the carboxylate group is compensated, however, by a salt-bridge to the 1-ammonium group of K53, which simultaneously hydrogen bonds to the C30 hydroxyl group of rCdRP. E51 also hydrogen bonds to the C30 hydroxyl group, and forms a salt-bridge with K110 (for N – O distances see Table 3). Importantly, the 1-amino group of K110 hydrogen bonds to the C20 hydroxyl group of rCdRP, which mimicks the crucial C20 carbonyl group of CdRP (Figure 1(a)). Moreover, the well-defined electron density map presented in Figure 4(a) reveals the absolute configuration at rCdRP’s extra chiral center at C20

to be R, most likely because only this diastereomer hydrogen bonds to K110. We conclude that one diastereomer binds preferentially. The NH group of rCdRP, on the other hand, is too far from the carboxylate group of E159 and in a geometrically unfavourable orientation for hydrogen-bonding to it. The positively charged side-chain of R182, moreover, is close to, but does not form a salt-bridge with, the carboxylate group of the anthranilate moiety. Structure of the enzyme: IGP complex Stable complexes of sIGPS with IGP can be formed, because the conversion of CdRP to IGP is ˚ resolution crystal strucirreversible. In the 2.0 A ture of the sIGPS:IGP complex, (Figure 3(b)), the electron density of the ligand is well defined (Figure 4(b)). Note that the carbon atoms of the triosephosphate moiety of IGP have similar conformations and locations as the carbon atoms C30 , C40 and C50 of the ribityl moiety of rCdRP (cf. Figures 3(a) and 4(a)). The benzene ring of the indole moiety, however, fills an adjacent but different binding pocket, compared to that of the anthranilate moiety of rCdRP (Figure 3(a) and (b)). As listed in Table 2, the indole moiety contacts mainly the side-chains of residues F89, K110, F112, I133 and L184. Note that the triple salt-bridge cluster involving K53, E51 and K110 occupies about the same position as in the enzyme:rCdRP complex, with the N –O distance of the hydrogen bond between K53 and the C30 -hydroxyl group similar to that in the sIGPS:rCdRP complex (Table 3). Although indole provides fewer hydrogen-bonding possibilities than anthranilate, due to the missing carboxylate and C20 hydroxyl groups, the carboxylate group of E159 now hydrogen bonds to the favourably oriented NH of the indole moiety. E159 is itself stabilised by hydrogen bonding to N180, which, in turn, hydrogen bonds to the sidechain of S211. The side-chain of R182, moreover, occupies practically the same position as in the sIGP:rCdRP complex. Structure of the enzyme:CdRP complex PRAI, the enzyme preceding IGPS in the biosynthesis pathway of tryptophan, first generates a transient fluorescent product (CdRPp),21 which spontaneously isomerizes to a stable, non-fluorescent species (CdRP8). The latter is the exclusively competent substrate of eIGPS, and has been identified by one and two-dimensional NMR as the ketoamine tautomer of CdRP (Figure 1(a); U. Hommel, K. Rumpel & K.K., unpublished results). By comparison to eIGPS18 at 37 8C, sIGPS has a higher affinity for its substrate CdRP CdRP ¼ 0:045 mMÞ,18 but a 100-fold smaller turnðKM over number ðkcat ¼ 0:033 s21 Þ: The values of sIGPS favour the formation of a metastable

Catalytic Mechanism of IGPS

761

Figure 3. Stereo views of specific ligands bound to the active site of sIGPS: Ball-and-stick representation with white, grey and black spheres for carbon, nitrogen and oxygen, respectively. The sticks for the ligands are drawn in green. Hydrogen bonds that are important for catalysis and discussed in the text are drawn as broken lines. N – O distances are given in Table 3. (a) Complex with substrate analogue rCdRP. The residues W8, P57, F89, R182 and L184 line the anthranilate pocket (Table 2). (b) Complex with the product IGP. The residues F89, K110, F112, I133 and L184 line the indole pocket adjacent to the anthranilate pocket (Table 2). (c) Complex with the substrate CdRP. The anthranilate pocket is shown as in (a). The Figure was produced with MOLSCRIPT.30

enzyme-substrate complex in the crystal at low temperature. Figure 4(c) displays the Fo 2 Fc difference electron ˚ resolution of the crystal structure density at 2.4 A of the complex of sIGPS with the substrate CdRP, calculated with the ligand omitted. Comparison with Figure 4(a) (complex with rCdRP) shows that the phosphate and carboxyphenylamino moieties of the substrate are well defined, while the electron density of the ribulose moiety is very weak even at the 2.5s level. This must be attributed to the high mobility of the ribulose moiety. The atomic model of CdRP that was built into the density is, therefore, not very accurate in this region. However, the requirement of connectivity with the visible portions restricts the freedom of conformation,

and the result strongly resembles the structure of rCdRP (Figures 3(a) and 4(a)). Some additional density at the right top corner shows indole-like shape and may appear due to weak occupancy of the product IGP. Therefore, the model of IGP was superimposed as well (Figure 4(b)). We cannot exclude, however, that the extra density is due to solvent molecules. It is likely that the same activesite groups interact with the various substrate atoms that were identified as such in the complex with rCdRP. Figure 3(c) displays these interactions in one of a small number of very similar models that are compatible with the electron density of Figure 4(c). The corresponding N – O distances are certainly less accurate than for the complexes with rCdRP and IGP (Table 3).

762

Catalytic Mechanism of IGPS

Figure 4. Stereoview of the difference electron density of the ligands bound to sIGPS, as it appears in electron density omit maps. These were computed with phases resulting from refinement, in which the ligand was omitted. The ligands are shown in a ball-and-stick representation with carbon, oxygen, nitrogen and phosphorus coloured white, ˚ map, contoured at 4s. (b) IGP on a 2.0 A ˚ map, contoured red, blue and magenta, respectively, (a) rCdRP on a 2.05 A ˚ at 4s. (c) CdRP and IGP on a 2.4 A map, contoured at 2.5s. The Figure was produced with MOLOC.29

Model building of the putative catalytic intermediates I1 and I2 ˚ ) between the catalytic Table 3. The N – O distances (A side chains of sIGPS and its ligands Ligand Side chain K53– NH K53– NH K110–NH K110–NH E159– CO2 2

a

Ligand group 2 2

C1 –CO C30 –OH C20 –O C20 –OH vN–H

rCdRP

CdRP

I1b

I2b

IGP

3.0 2.7 – 2.5 7.0c

3.0 2.7 2.6 – 6.7c

2.9 3.0 – 2.6 3.6

– 2.7 – 2.6 3.0

– 2.9 – – 2.8

Hydrogen bonds or salt links identified by dashed lines between side chains and ligand groups in Figures 3 and 5. a Atom numbering and ligand groups as in Figure 1. b Note that NI1 and I2 are models (see Figure 5) and not experimental structures. c ˚. No interaction because O–N distance .4 A

Because CdRP is bound in an unproductive mode, and a non-reactive analogue of I2 (Figure 1(c)) was not available for cocrystallization, we decided to model both I1 and I2 into the active site of sIGPS. The models shown in Figure 5 are based on all the available chemical and structural information. The conformations of the contact and catalytic residues at the active site (Table 2), as well as of those of the phosphate and triose moieties of the respective ligand, were fixed to positions identical with those observed in the crystal structures of bound CdRP for I1 and of bound IGP for I2. As carbon atom C1 of CdRP approaches carbon atom C20 to initiate the formation of the new covalent bond, the benzene ring is simultaneously distorted to that of cyclohexadiene, moving the carboxylate group out of the plane of the

Catalytic Mechanism of IGPS

763

Figure 5. Molecular models of the bound catalytic intermediates I1 and I2, generated as described in Materials and Methods, viewed in stereo within the active site of sIGPS. The view is identical with that of Figure 3. See the text for details. (a) I1: one of four possible diastereomers at the position of the original carbon atoms C1 and C20 of CdRP. (b) I2: one of two possible enantiomers involving the original carbon atom C20 of CdRP. Note that the positions of the six-membered ring moieties of I1 and I2 are intermediate between those of CdRP (Figure 3(c)) and IGP (Figure 3(b)).

ring (Figure 5(a)). Maintenance of the critical N – O distances between the catalytic groups and the substrate (Table 3) selects one of the two possible chiral configurations. After energy minimization, the cyclohexadiene moiety of I1 is at a position intermediate between that of anthranilate in CdRP (Figure 3(c)) and indole in IGP (Figure 3(b)). Since

both K53 and K110 maintain their original hydrogen bonds to the C1 carboxylate and the C20 hydroxyl groups, respectively, their orientations and N – O distances define the intermediate I1 as that diastereomer, in which C1 has the absolute configuration S, and C20 that of R. Importantly, the positively charged NH group of I1 now forms

Figure 6. The proposed roles of K53, K110 and E159 of sIGPS in catalyzing the conversion of CdRP to IGP. Hydrogen bond distances are given in Table 3. ( p ) Asymmetric carbon atoms generated transiently. The arrows show how the reactions are assisted by the indicated catalytic residues. See the text for details. (a) The substrate bound unproductively as observed in the sIGPS:CdRP complex (Figure ˚. 3(c)), with C1 – C20 distance ¼ 4.8 A (b) The intermediate I1 as in Figure 5(a). (c) The intermediate I2 as in Figure 5(b). (d) The sIGPS:IGP complex as in Figure 3(b).

764

a hydrogen-bonded salt-bridge to the carboxylate group of E159. The benzene ring of the modelled intermediate I2 (Figure 5(b)) lies close to the position it has in IGP (Figure 3(b)). I2 is in a less strained conformation compared to I1, but the pyrrolidine ring of I2 is tilted out of the plane of the benzene ring. As observed with I1 (Figure 5(a)), the original C20 hydroxyl group still hydrogen bonds to K110. Yanofsky3,22 showed that not only 4-methylanthranilate, but also the 5-methyl and 5-fluoroderivatives are readily converted enzymatically to the corresponding IGP products. The modelled structures of I1 and I2, as well as the experimental structures of CdRP (Figure 3(c)) and IGP (Figure 3(b)), are consistent with these observations, showing that there is enough room to accommodate the extra substituent groups in the active site.

Catalytic Mechanism of IGPS

restores the p-electron system of the benzene ring and neutralizes the positive charge of the NH group. Both K110 and E159 maintain in I2 their original hydrogen bonds to I1, and K53 maintains its hydrogen bond to the C30 hydroxyl group. As illustrated in Figure 6(c), the dehydration of I2 could be catalyzed once more by the ammonium group of K110, donating a proton to the C20 hydroxyl group, which is eliminated as a water molecule. In contrast, the carboxylate group of E159 could now exercise an active catalytic function by accepting the C10 proton, thus generating the p-electron system of the annellated pyrrole ring. In order to regenerate E159 as a general base after the third step, N180 could participate in its deprotonation, possibly assisted by S211, the third member in this hydrogen-bonded triad of invariant residues.

The enzymatic catalysis of indole synthesis The structures of the three experimental and two modelled sIGPS –ligand complexes (Figures 3 and 5) assign unique roles to three out of the six invariant residues previously identified12,17 as catalytically important. Figure 6(a) represents the unproductive sIGPS:CdRP complex (Figure 3(c)), where the separation between the carbon atoms ˚ ) is too large to initiate C – C C1 and C20 (4.8 A bond formation. The protonated 1-amino group of K110, active as a general acid, polarizes the C20 carbonyl carbon atom of CdRP by hydrogen-bonding to the carbonyl oxygen atom. The arrows show how the electrophilic C20 atom could attack the p electrons of the benzene ring. The weak electron density of the ribulose moiety in the sIGPS:CdRP complex (Figure 4(c)) indicates that this region of the substrate is mobile. It is likely, then, that the transition state of the condensation reaction step is attained by thermal motion. The salt-bridge between K53 and the anthranilate carboxyl group seems to guide the C1 atom towards the C20 atom ˚ ). As the to within bond-forming distance (, 2 A condensation of CdRP proceeds, the positive charge developing on the NH group is increasingly compensated by the incipient salt-bridge to the carboxylate group of E159 (Figure 6(b)). Importantly, it appears that their long side-chains allow both K53 and K110 to maintain their original hydrogen bonds to CdRP during the reaction. It is conceivable that the triple salt-bridge cluster among K53, E51 and K110 mediates the reprotonation of the 1-amino group of K110, which was deprotonated in the CdRP to I1 conversion, thus restoring it to a general acid. The observed interaction of K53 with the carboxyl group of CdRP and the intermediate I1 explains, moreover, why the carboxyl group of CdRP is needed for the biosynthesis of IGP,4,5 as well as why K53 is catalytically essential.17 The arrows in Figure 6(b) show how the decarboxylation of the intermediate I1 could be catalyzed. The movement of electron pairs eliminates the carboxyl group in the form of CO2,

Conclusion The biochemical synthesis of indole from an a-arylamino ketone may proceed, as originally postulated by Parry,1 by means of the same intermediates proposed to occur in the chemical synthesis of indole.2 The strongly acidic conditions required for the chemical process seem to be replaced in IGPS by a general acid (K110) and a general base (E159).These seem to act in a spatially adapted environment, and stabilize the three sequential transition states optimally by interacting with assistant catalytic residues and a set of invariant hydrophobic contact residues. K110 is hydrogen-bonded to the C20 carbonyl or hydroxyl groups and E159 is hydrogen-bonded to the NH group in all three reaction steps. The overall movement of the benzene ring from the anthranilate to the indole pocket seems to be guided by the long side-chain of K53 that maintains a salt-bridge to the carboxylate groups of both CdRP and I1 in the first two steps of the reaction, as well as by a hydrogen bond to the hydroxyl group at C30 .

Materials and Methods Production of sIGPS sIGPS from S. solfataricus was overexpressed in E. coli and purified as described.9

Crystallization and X-ray data collection Crystallization was performed using the hanging drop vapour diffusion method. Crystals were mounted in glass capillaries and X-ray data sets for all complexes were measured on a modified Marconi-Elliott GX-20 rotating anode generator (E. Bratschi, unpublished results) with a copper anode operated at 40 KV and 50 mA. The data were evaluated with the MOSFLM program package.23 Details of the data collection are summarized in Table 1.

765

Catalytic Mechanism of IGPS

sIGPS:rCdRP complex

Model building

rCdRP was synthesised by selective reduction of the keto group of CdRP with NaBH415 and was cocrystallized with the enzyme. In a vapour diffusion experiment, drops containing 5 ml of protein solution (20 mg/ml of IGPS, 50 mM phosphate buffer (pH 5.0), 20 mM rCdRP) and 5 ml of reservoir solution (50 mM phosphate buffer (pH 5.0), 1.3 M ammonium sulphate) were mixed at 4 8C and equilibrated against the reservoir solution. Trial runs with the same buffer, but varying pH had previously established pH 5.0 as optimal. Crystals were first observed about three days after setting up the drops, and were allowed to grow for at least two weeks. Their maximum dimensions are 0.3 mm £ 0.3 mm £ 1.0 mm ˚ resolution. and they diffract to about 2.0 A

The sIGPS:I1 complex was obtained by model building studies using the molecular graphic package MOLOC,29 which was also used for the energy minimization calculations. Starting from the experimental structure of the substrate complex, the ligand molecule was modified to the I1 intermediate. By keeping the conformations of the contact and catalytic residues, as well as those of the phosphate and the carbon atoms C20 to C50 of the ribulose moiety of CdRP strictly rigid, its conformation was manually changed to that of I1, at the same time satisfying approximately the distance requirements for the catalysis. Subsequently, the conformation of the ligand was optimized by geometry and energy minimization. The sIGPS:I2 complex was modelled in a similar way, but with the crystal structure of the sIGPS:IGP complex as the starting structure. Again the conformation of the enzyme was fixed as well as the position of the phosphate moiety of the ligand. The remaining atoms were built and finally the conformation of the ligand was refined by geometry optimization and energy minimization.

sIGPS:IGP complex IGP was produced enzymatically using ePRAI/ IGPS.21 The enzymes present in the preparation were removed by ultrafiltration. A crystal of sIGPS with dimensions of 0.3 mm £ 0.3 mm £ 1.5 mm was soaked for two hours in a stabilizing solution (1.3 M ammonium sulphate, 50 mM phosphate buffer (pH 6.0)) containing 2 mM IGP.

Protein Data Bank accession numbers The coordinates were deposited in the Protein Data Bank with the accession numbers 1IGS, 1LBF and 1LBL.

sIGPS:CdRP complex CdRP was produced enzymatically and purified by reversed phase HPLC.24 Crystals of sIGPS were grown as described.9 A crystal of sIGPS was used for a soaking experiment, which was performed at 4 8C. Several test experiments were carried out first in order to find the best conditions for the soaking procedure. The best result was obtained with the following procedure. First, the crystal was mounted in a glass capillary. Second, the crystal was soaked at 25 8C for five minutes in a solution containing 3 mM CdRP, 50 mM phosphate buffer (pH 6.5) and 1.3 M ammonium sulphate. Third, the soaking solution was removed and the capillary closed. Fourth, the crystal was transferred immediately into a cooled air stream of about 0 8C. Data from a crystal with dimensions 0.25 mm £ 0.25 mm £ 1.0 mm were collected in 12 hours with an exposure time of 600 seconds and oscillation range of 18. Freezing of the crystals at lower temperature (liquid N2) has not been possible because a freezing procedure for these crystals was not available.

Structure determination and refinement Phases were obtained using the refined structure of sIGPS,9 without solvent molecules (water and phosphate ion), as starting model for difference Fourier methods. All reflections, without application of a s cut-off, were used in the refinement of the structure. After several cycles of restrained positional and individual temperature factor refinement, using XPLOR,25,26 the molecular structure was inspected and rebuilt with the molecular graphics program O,27 using SIGMAA-weighted Fo 2 Fc and 2Fo 2 Fc electron density maps.28 Details of the refinement parameters are summarized in Table 1. In all three structures, S211 is in the energetically disfavoured region of the Ramachandran plot as it was observed also in the uncomplexed structure of sIGPS.

Acknowledgments We thank Professor A. Vasella for stimulating discussions, and both R. Mu¨ller and E. Bratschi for expert technical assistance. This work was supported by the Swiss National Science Foundation grants 31-36432.92 to J.N.J. and 31-32369.91 to K.K.

References 1. Parry, R. J. (1972). Biosynthesis of compounds containing an indole nucleus. In The Chemistry of Heterocyclic Compounds in Indoles (Houlihan, W. J., ed.), part II, vol. 25, pp. 1 – 64, Wiley-Interscience, New York. 2. Brown, R. T., Joule, J. A. & Sammes, P. G. (1979). Indoles and related systems. In Comprehensive Organic Chemistry, vol. 4, pp. 411– 492, Pergamon Press, Oxford, UK. 3. Yanofsky, C. (1955). On the conversion of anthranilic acid to indole. Science, 121, 138– 139. 4. Smith, O. H. & Yanofsky, C. (1960). 1-(o-Carboxylphenyl-amino) 1-deoxyribulose 5-phosphate, a new intermediate in the biosynthesis of tryptophan. J. Biol. Chem. 235, 2051– 2057. 5. Smith, O. H. & Yanofsky, C. (1962). Enzymes involved in the biosynthesis of tryptophan. Methods Enzymol. 5, 794– 806. 6. Nichols, B. P. (1996). Evolution of genes and enzymes of tryptophan biosynthesis. In Escherichia coli and Salmonella (Neidhardt, F. C., ed.), vol. 2, pp. 2638 –2648, ASM Press, Washington, DC. 7. Priestle, J. P., Gru¨tter, M. G., White, J. L., Vincent, M. G., Kania, M., Wilson, E. et al. (1987). Threedimensional structure of the bifunctional enzyme N-(50 -phosphoribosyl)anthranilate isomerase-indole-

766

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Catalytic Mechanism of IGPS

3-glycerol phosphate synthase from Escherichia coli. Proc. Natl Acad. Sci. USA, 84, 5690 –5694. Wilmanns, M., Priestle, J. P., Niermann, T. & Jansonius, J. N. (1992). Three-dimensional structure of the bifunctional enzyme phosphoribosyl anthranilate isomerase: indoleglycerol phosphate synthase ˚ resolution. from Escherichia coli refined at 2.0 A J. Mol. Biol. 223, 477– 507. Hennig, M., Darimont, B., Kirschner, K. & Jansonius, ˚ structure of indole-3-glycerol phosJ. N. (1995). 2.0 A phate synthase from a hyperthermophile: possible determinants for protein stability. Structure, 3, 1295– 1306. Kno¨chel, T. R., Hennig, M., Merz, A., Darimont, B., Kirschner, K. & Jansonius, J. N. (1996). The crystal structure of indole-3-glycerol phosphate synthase from the hyperthermophilic archaeon Sulfolobus solfataricus in three different crystal forms: effects of ionic strength. J. Mol. Biol. 262, 502– 515. Eberhard, M., Tsai-Pflugfelder, M., Bolewska, K., Hommel, U. & Kirschner, K. (1995). Indoleglycerol phosphate synthase – phosphoribosyl anthranilate isomerase: comparison of the bifunctional enzyme from Escherichia coli with engineered monofunctional domains. Biochemistry, 34, 5419– 5428. Darimont, B., Stehlin, C., Szadkowski, H. & Kirschner, K. (1998). Mutational analysis of the active site of indole glycerol phosphate synthase from Escherichia coli. Protein Sci. 7, 1221– 1232. Andreotti, G., Tutino, M. L., Sannia, G., Marino, G. & Cubellis, M. V. (1994). Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes. Biochim. Biophys. Acta, 1208, 310– 315. Merz, A., Yee, M-C., Szadkowski, H., Pappenberger, G., Crameri, A., Stemmer, P. W. C. et al. (2000). Improving the catalytic activity of a thermophilic enzyme at low temperatures. Biochemistry, 39, 880– 889. Bisswanger, H., Kirschner, K., Cohn, W., Hager, V. & Hansson, E. (1979). N-(5-Phosphoribosyl)anthranilate isomerase –indoleglycerol phosphate synthase. 1. A substrate analogue binds to two different binding sites on the bifunctional enzyme from Escherichia coli. Biochemistry, 18, 5946– 5953. Cohn, W., Kirschner, K. & Paul, C. (1979). N-(5-Phosphoribosyl)anthranilate isomerase– indoleglycerol phosphate synthase. 2. Fast-reaction studies show that a fluorescent substrate analogue binds independently to two different sites. Biochemistry, 18, 5953– 5959. Eberhard, M. & Kirschner, K. (1989). Modification of a catalytically important residue of indoleglycerol-

18.

19.

20.

21.

22. 23. 24.

25. 26. 27.

28. 29.

30.

phosphate synthase from Escherichia coli. FEBS Letters, 245, 219– 222. Stehlin, C., Dahm, A. & Kirschner, K. (1997). Deletion mutagenesis as a test of evolutionary relatedness of indoleglycerol phosphate synthase with other TIM barrel enzymes. FEBS Letters, 403, 268–272. Sterner, R., Kleemann, G. R., Szadkowski, H., Lustig, A., Hennig, M. & Kirschner, K. (1996). Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer. Protein Sci. 5, 2000– 2008. Hennig, M., Sterner, R., Kirschner, K. & Jansonius, ˚ resolution of J. N. (1997). Crystal structure at 2.0 A phosphoribosyl anthranilate isomerase from the hyperthermophile Thermotoga maritima: possible determinants of protein stability. Biochemistry, 36, 6009– 6016. Hommel, U., Eberhard, M. & Kirschner, K. (1995). Phosphoribosyl anthranilate isomerase catalyzes a reversible Amadori reaction. Biochemistry, 34, 5429– 5439. Yanofsky, C. (1956). The enzymatic conversion of anthranilic acid to indole. J. Biol. Chem. 223, 171– 184. Collaborative Computing Project Number 4 (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallog. sect. D, 50, 760– 763. Kirschner, K., Szadkowski, H., Jardetsky, T. S. & Hager, V. (1987). Phosphoribosylanthranilate isomerase-indoleglycerol phosphate synthase from Escherichia coli. Methods Enzymol. 142, 386– 397. Bru¨nger, A. T. (1992). X-PLOR Version 3.1. A System for X-ray Crystallography and NMR, Yale University Press, New Haven and London. Engh, R. & Huber, R. (1991). Accurate bond and angle parameters for X-ray protein structure refinement. Acta Crystallog. sect. A, 47, 392– 400. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallog. sect. A, 47, 110 – 119. Read, R. J. (1986). Improved Fourier coefficients for maps using phases from partial structures with errors. Acta Crystallog. Sect. A, 42, 140– 149. Gerber, P. R. (1992). Peptide mechanics: a force field for peptides and proteins working with entire residues as smallest units. Biopolymers, 32, 1003– 1017. Kraulis, P. J. (1991). MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallog. 24, 946– 950.

Edited by R. Huber (Received 14 January 2002; received in revised form 5 April 2002; accepted 10 April 2002)