Crystal structures of native and recombinant yeast fumarase1

Article No. mb981862

J. Mol. Biol. (1998) 280, 431±442

Crystal Structures of Native and Recombinant Yeast Fumarase T. Weaver1, M. Lees1, V. Zaitsev2, I. Zaitseva2, E. Duke2, P. Lindley6 S. McSweeny3, A. Svensson4, J. Keruchenko5, I. Keruchenko5 K. Gladilin5 and L. Banaszak1* 1

Department of Biochemistry University of Minnesota, 4-225 Millard Hall, Minneapolis MN 55455-0347, USA 2

CCLRC Daresbury Laboratory Warrington, WA4 4AD, UK 3

EMBL c/o ILL, F-38043 Grenoble-CEDEX, France 4

Molecular Biophysics Chemical Center, Lund University, P.O.B. 124, Lund Sweden 5

Institute of Biochemistry, RAS 33 Lenisky pr, 117071, Moscow Russia 6

ESRF, BF-220 38043, Grenoble-CEDEX France

Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomyces cerevisiae have been completed to moderate resolution by two separate laboratories. The recombinant form was obtained by the construction of an expression plasmid for Escherichia coli. Despite a high level of amino acid sequence similarity, puri®cation of the eukaryotic enzyme from the wild-type prokaryotic enzyme was feasible. The crystal structure of the native form, NY-fumarase, encompasses residues R22 through M484, while the recombinant form, RYfumarase, consists of residues S27 through L485. Both crystal structures lack the N-terminal translocation segment. Each subunit of the homotetrameric protein has three domains. The active site is formed by segments from each of three polypeptide chains. The results of these studies on the eukaryotic proteins are unique, since the recombinant form was done in the absence of dicarboxylic acid and has an unoccupied active site. As a comparison, native fumarase was crystallized in the presence of the competitive inhibitor, meso-tartrate. Meso-tartrate occupies a position close to that of the bound citrate molecule found in the active site of the E. coli enzyme. This inhibitor participates in hydrogen bonding to an active-site water molecule. The independent determination of the two structures provides further evidence that an activesite water molecule may play an active role in the fumarase-catalyzed reaction. # 1998 Academic Press

*Corresponding author

Keywords: yeast fumarase; Krebs cycle; d-crystallin; E-fumarase multisubunit active site

Introduction Fumarase belongs to a family of homologous enzymes that share amino acid sequence conservation and utilize fumarate as a common substrate/ product during their reaction pathway. Two of the most studied family members are fumarase and aspartase; they are responsible for the addition of water and ammonium, respectively, to the ole®nic bond of fumarate. Typically, fumarase functions as a component of the Krebs cycle responsible for the Abbreviations used: RY-fumarase, recombinant yeast fumarase; NY-fumarase, native yeast fumarase; E-fumarase, Escherichia coli fumarase C; PCR, polymerase chain reaction; PMA, pyromellitic acid; RMS, root-mean-square; PEG, polyethylene glycol. 0022±2836/98/280431±12 $30.00/0

interconversion of fumarate and L-malate as shown below: C1 OOHÿHCˆCHÿC4 OOH ‡ H2 O $ C1 OOH ÿCHOHÿHCHÿC4 OOH The reactions catalyzed by the family members fumarase and aspartase were both shown to proceed through a carbanion transition state, and the conservation of key residues between these two family members suggests a common catalytic mechanism (Porter & Bright, 1980). Most importantly, lysine and asparagine side-chains have been postulated to bind to the C4 carboxylate group of the substrate and stabilize the carbanion species of the transition state (Weaver & Banaszak, 1996). The X-ray data described below show that the # 1998 Academic Press

432 corresponding residues in Y-fumarase are K349 and N351. In eukaryotic cells, the Krebs cycle reactions are operational in the mitochondrial inner membrane matrix, yet fumarase is found also in the cytosol. To provide for this compartmentalization in yeast, a single gene harboring two unique start sites is responsible for coding both the mitochondrial and cytosolic forms of fumarase. The product from the ®rst start site is translocated into mitochondria, while that of the second or shorter form remains in the cytosol (Wu & Tzagoloff, 1987). In prokaryotes, fumarase activity arises from three distinct genes; fumA, fumB and fumC. The gene products of fumarase A and B are examples of class I forms of fumarase. Class I fumarases are homologous, dimeric, heat-labile and irondependent enzymes with a molecular mass of 120 kDa. They are not related to either yeast fumarase or fumarase(c) from Escherichia coli, referred to here as Y-fumarase and E-fumarase(c), respectively. These so-called class II fumarases are heat-stable, iron-independent, tetrameric enzymes with a molecular mass of 200 kDa. The class II fumarases include Y-fumarase and represent the homologous family of enzymes that have been extensively studied in terms of steadystate kinetics and reaction mechanism (Hill & Teipel, 1971). Mutations in fumarase have been implicated in a variety of human diseases, including progressive encephalopathy and fumaric aciduria (Bourgen et al., 1994; De Vivo, 1993). The molecular structure of Y-fumarase is the focus of this work. Other members of class II family catalyze reactions in biochemical processes such as the urea cycle, purine biosynthesis, and certain reactions involved in amino acid metabolism. This broader fumarase family includes aspartase, adenylosuccinate lyase, argininosuccinate lyase, 3carboxy-cis,cis-muconate lactonizing enzyme and d-crystallin. Some of the family members share relatively high levels of sequence identity, even though they catalyze different reactions. For example, Y-fumarase and aspartase from E. coli share 38% identity (Woods et al., 1988). The high degree of conservation of sequence identity is especially evident within three regions of the amino acid sequence. The ®rst region stretches from H154 though T171, the second from I206 through E225, and the third from P337 through E356. The high degree of conservation in the third region has led to a consensus sequence that de®nes this family of proteins. d-Crystallin from avian lens was the ®rst member of the family to have its structure determined by X-ray methods (Simpson et al., 1994). Although it shares approximately 90% sequence identity with argininosuccinate lyase, it lacks any observable enzymatic activity, indicating that it may be an example of a ``hijacked'' enzyme. It is believed that d-crystallin evolved from a common ancestor of modern reptiles and birds by the over-expression

Crystal Structures of Yeast Fumarase

of argininosuccinate lyase in the lens. About the same time, a gene duplication event took place (Piatigorsky & Wistow, 1991). Since that time, the lens gene has accumulated mutations in the coding sequence that have made it enzymatically inactive. The control sequences may have also diverged, allowing independent control of expression. The crystal structure of recombinant human argininosuccinate lyase has recently been reported (Turner et al., 1997). The structure was determined by the molecular replacement method using the coordinates of d-crystallin as a search model. Although the resolution of the data is Ê , the structure has a similar fold to only 4.2 A that of the yeast fumarases, d-crystallin and Efumarase(c) (Weaver et al., 1995). The catalytically active family members have in common the addition of water, NH3, or an organo-nitrogencontaining compound to the ole®nic bond of fumarate. To summarize, the family genotype appears to include a polypeptide chain of 450 to 500 amino acid residues. As noted above, a few family members have evolved with translocation sequences that permit organelle targeting. Within this family of enzymes, all are tetramers with a very unusual core helical domain. Where enzyme activity is present, it appears to be associated with a region at the intersection of three of the four subunits. While one would predict that all family members would be characterized by the same active site, recent crystallographic studies of aspartase leave some uncertainty as to the precise amino acid residues necessary for catalysis (Shi et al., 1997). The common structural motif for the fumarase family can be described as follows. Each subunit of a tetramer is believed to be composed of three domains referred to as D1, D2 and D3. D1 begins at the N terminus, and D3 is located at the C-terminal end of the polypeptide chain. The central domain, D2, is composed of a unique ®vehelix bundle. The association of the D2 domains results in a tetramer with a core of 20 a-helices that are nearly parallel. The other two domains, D1 and D3, cap this helical bundle on opposite ends of the homotetramer. Here, we report the structure determination of both recombinant (RYfumarase) and native forms of yeast fumarase (NY-fumarase) from Saccharomyces cerevisiae by the molecular replacement method. The studies were carried out independently in two laboratories.

Results Monomer structure of NY and RY-fumarase To make a detailed comparison of the two Y-fumarase structures, a few amino acid sequence differences must be taken into account. The two unique start sites within the Y-fumarase gene (Wu

433

Crystal Structures of Yeast Fumarase

& Tzagoloff, 1987) are illustrated below by the letters in bold: M-L-R-F-T-N-C-S-C-K-T-F-V-K-S-S-Y-K-L-N-I-R-R-M-N-S-S-F-R-T-E-T- . . . . 1

10

Residues 1 through 23 are believed to be part of a translocation segment responsible for directing Yfumarase into the mitochondria. The second start site, at M24, is characteristic of the cytosolic form of the enzyme. The mitochondrial enzyme is thought to be proteolytically processed during import into the mitochondria (Wu & Tzagaloff, 1987). The crystal structure of NY-fumarase begins at R22 and is, therefore, likely to be a form of the mitochondrial enzyme. The crystal structure of the recombinant form, RY-fumarase, begins at S26 with the original construct designed to begin with M24. Probably as a result of PCR error, a difference occurs between the two enzymes; RY-fumarase has the following point mutation K289R. A comparison of the crystal structures demonstrates that the mutations do not result in any signi®cant conformational change in or around their respective

20

30

regions. In addition, the recombinant form has a speci®c activity similar to that of the native enzyme. The crystal structures of one subunit of each of the two forms of Y-fumarase are superimposed in Figure 1. Since the origin for the coordinates was selected differently during the X-ray studies and because of differences in the unit cell parameters, the two crystallographic models, as shown, have been superimposed by the method of least-squares. For all of the Ca atoms, the RMS distance separation between corresponding posÊ (Jones et al., 1991). In a similar itions was 1.1 A manner to the enzyme from E. coli, the yeast form is made up of three domains; D1, D2 and D3. The ®rst domain, D1, includes residues 22 through 161, D2 includes residues 162 through 419 and D3 includes residues from 419 through

Figure 1. Crystal structures of NY and RY-fumarase. The stereo-drawing contains a stick model of the Ca atoms from the crystal structures of NY and RY-fumarase. Every 50th residue along with the N and C-terminal residues has been numbered according to the beginning of the amino acid sequence described in the text. The symbols D1, D2 and D3 mark the position of conformational domains described more fully in the text. NY-fumarase has been color-coded in cyan while RY-fumarase is shown in red. Note, there is a short break in the representation of RY-fumarase; residues 144 to 152 are missing. It was not possible to obtain coordinates for this segment, since no electron density was visible. Residues 144 to 152 are absent also in accession number 1yfm in the PDB.

434 485. These three domains are clearly visible in Figure 1, with D3 at the top and D1 at the bottom. The domain labeled D2 forms the core of the tetrameric enzyme and consists of a unique ®ve-helix bundle. A separate independent rigid-body transformation of D2 shows that the agreement between the two crystal structures is better for this domain, Ê . Again giving an RMS distance difference of 0.80 A after separate rigid-body transformations, the RMS distance difference for Ca atoms belonging to NY Ê for D1 and 1.86 A Ê for and RY-fumarase was 1.15 A D3. The only visually obvious difference between the two crystal structures occurs in a loop region between L144 and Q152. The electron density in this region of the map for RY-fumarase was extremely poor; in fact, the atoms in this region were left out of the re®nement procedure and the ®nal coordinates. Somewhat larger differences are observed between the D3 domains of NY and RY-fumarases beginning at P439 and continuing to the C terminus. The most signi®cant differences center around K456 and G457. In this segment, the conformation

Crystal Structures of Yeast Fumarase

is a loop that protrudes into the surrounding solvent and the two residues in question appear at the very tip of that loop. Tetramer structure The enzymatically active form of fumarase has been shown to be a tetramer by a number of studies, which are summarized in a review by Hill & Teipel (1971). The Y-fumarase tetramer has its subunits arranged with 222 point group symmetry, as shown in Figure 2. In the crystal forms of both NY and RY-fumarase, the molecular symmetry is coincident with three crystallographic 2-fold axes. In the cartoon drawing in Figure 2, the subunits themselves have not been color-coded. Instead, three different short segments have been emphasized and, as will be explained below, the positioning of these segments is responsible for formation of the active site. As is partly visible in Figure 2, the ®ve-helix domain of a monomer becomes part of the central 20-helix bundle of the tetramer. The core

Figure 2. The enzymatically active tetramer of RY-fumarase. The cartoon representation illustrates the subunit arrangement in both NY and RY-fumarase. The crystallographic coordinates of RY-fumarase were used to prepare the drawing. In one subunit, regions of high levels of amino acid sequence identity within the fumarase family are colored red and labeled Region 1, 2 or 3. In the upper right-hand corner of the illustration, the same three regions come together to form the active site of the enzyme. Color coding in this region is violet, green and gold for regions 1 through 3, respectively. The tetramer has point symmetry ÿ222. One of the dyad axes passes through the center of the tetramer as shown and would be perpendicular to the plane of the drawing. Intersecting this dyad are the other two, one running horizontally, the other vertically.

Crystal Structures of Yeast Fumarase

helices are nearly parallel, and have an average Ê and contain from 25 to 35 length of about 40 A residues. Most of the a-helices have small

435 amounts of curvature, which appears to accommodate the tight packing at the tetrameric interface.

Figure 3. Active-site NY-fumarase and RY-fumarase crystal structures. The stereo-diagrams depict an identical activesite region in the (a) NY-fumarase and (b) RY-fumarase crystal structures. Hydrogen bonds are represented by broken lines. Backbone atoms are colored: violet, Region 1; green, Region 2; and gold, Region 3. The active-site water molecule is colored green. All other atoms are colored according to the element they represent: carbon, gray; oxygen, red; nitrogen, blue; and sulfur, yellow. The bound inhibitor, meso-tartrate, is visible below T126b. Meso-tartrate is a four-carbon dicarboxylic acid having the covalent structure ÿOOC ±CHOH ± CHOH± COOÿ. The small letters after the residue numbers identify different subunits.

436

Crystal Structures of Yeast Fumarase

Table 1. Conserved hydrogen bonds at the NY, RY and E-fumarase(c) active sites Role Active-site Active-site Active-site Active-site Active-site Inhibitor Inhibitor Inhibitor Inhibitor Inhibitor

water water water water water

Donor

Acceptor

NY-fumarase

Ê) Distance (A RY-fumarase

E-fumarase(c)

W39/W26* W39/W26* W39/W26* W39/W26* W39/W26* N166-OD1/N141-ND2* S165-NH/S140-NH* S165-OG/S140-OG* K349-NZ/K324-NZ* N351-OD1/N326-ND2*

H213-NE2/H188-NE2* N166-OD1/N141-OD1* S124-OG/S98* M-O4/T126-OG1/T100-OG1* M-O1/C-O1* M-O1/C-O3* M-O2/C-O7* M-O2/C-O6* M-O5/C-O4* M-O6/C-O4*

2.4 2.6 2.6 2.9 2.4 3.0 3.4 2.7 3.3 2.9

3.5 2.8 3.4 2.8 ± ± ± ± ± ±

2.6 2.7 2.8 3.0 3.0 3.0 2.9 2.8 3.0 3.1

C, citrate; M, meso-tartrate; *, speci®es E-fumarase(c).

Generation of the multi-subunit active site and conservation of the active-site water molecule For one subunit, the three most highly conserved regions in the fumarase family come together to form a multi-subunit active site, which is located in the crevice between the domains D1 and D3. Based on the presence of meso-tartrate bound to NYfumarase, an independent assessment of the activesite residues is possible. The positions of atoms near the active site are shown in more detail in Figures 3(a) and (b), and 4. Figure 3(a) illustrates the NY-fumarase active site with the bound competitive inhibitor, meso-tartrate. Figure 3(b) illustrates the apo-enzyme as it is found in the structural studies of RY-fumarase. Finally, Figure 4 shows the corresponding segment from a family member that has lost catalytic activity, d-crystallin. The most signi®cant observation is the presence of a water molecule at the active site, which is represented by the central green atom in Figure 3(a) and (b). This active-site water molecule is found in both RY and NY-fumarase structures in about the same position with respect to the other protein atoms. In the apo-form of the enzyme (Figure 3(b), RY-fumarase), the water molecule interacts through hydrogen bonds with atoms in residues H213d, S124b, N166b and T126b. With meso-tartrate present (Figure 3(a), NY-fumarase), T126b is replaced by -OH atoms present in the inhibitor. Nonetheless, the bound water molecule still forms hydrogen bonds with residues H213d, S124b and N166b. The presence of this water molecule at the active site in two different crystalline states of yeast fumarase supports the hypothesis that it may have a role in the catalytic mechanism (Weaver & Banaszak, 1996) and it is not an artifact of the crystallization conditions in the presence of competitive inhibitors. Meso-tartrate is a strong competitive inhibitor of fumarase and was therefore used in the preparation of crystals for the analysis of NY-fumarase (Hill & Teipel, 1971). Its covalent structure is given in the legend to Figure 3(a) (Keruchenko et al., 1992; Wigler & Alberty, 1960). Unlike citrate, it more closely resembles the substrate, L-malate;

only the C3 hydrogen atom of L-malate is replaced by a hydroxyl group. In an jFoj ÿ jFcj electron density map of NY-fumarase, electron density assigned to meso-tartrate was present at the level of 4s. The overall location of bound meso-tartrate is similar to the position of the citrate molecule observed in the crystal structure of E-fumarase(c) (Weaver & Banaszak, 1996). Using the crystal structures of RY and NYfumarase, it is possible to compare important interactions of the inhibitor with the enzyme. The C4 carboxylate group of meso-tartrate is coordinated by N351c-OD1, K349c-NZ and E340c-OE2, while the C1 carboxylate group is hydrogen-bonded to S165b-NH, S165b-OG and N166b-OD1. In addition, meso-tartrate forms two hydrogen bonds to the active-site water molecule. Protein interactions for both the active-site water molecule and the inhibitor are listed in Table 1. The crystal structure of RY-fumarase is the ®rst apo-form of fumarase and helps resolve the question of the tightly bound water molecule. This water molecule is clearly not related to the presence of bound inhibitor. Note from the data in Table 1 that the active-site water molecule retains many of the same hydrogen bonds as found in liganded forms of the enzyme. This apo-form of RY-fumarase contains evidence for four hydrogen bonds to this water molecule: (1) S124b-OG, (2) T126b-OG1, (3) N166b-OD1 and (4) H213d-NE2. S124 and T126 are essentially absent in d-crystallin, and it is the conformational differences in this region that may account for its evolutionary loss of catalytic activity. These differences can be seen by careful comparison of Figures 3(a) and (b), and 4. Using H213 (H160 in d-crystallin) as a marker, the active-site region of Y-fumarase as found in d-crystallin is shown in Figure 4. A large loop region encompassing residues 73 to 89 in d-crystallin is partially deleted, and the polypeptide segment containing S124 and T126 in the fumarases is moved well away from the active-site position in other family members. Hence, while the histidine, lysine and asparagine residues are present, their steric relationships are disposed differently.

Crystal Structures of Yeast Fumarase

437

Figure 4. Non-functional active-site region of d-crystallin. The stereo-diagram contains a representation of atoms in the crystal structure of d-crystallin that are homologous to those in the active site of the Y-fumarases. Atoms are positioned in approximately the same orientation as the NY and RY-fumarase active sites illustrated by Figure 3(a) and (b). Labeling and color coding is the same as in Figure 3. The majority of the active-site residues of d-crystallin are located in approximatley the same orientation as those in NY and RY-fumarase. However, I88 and T90, which represent the active-site residues S124 and T126 in NY and RY-fumarase, are shifted away from the active site and are no longer able to contribute hydrogen bonds to the active-site water molecule.

Discussion Comparison of the active-site components Based on the demonstrated structural homology, N351c, K349c and E356c have been included as active-site residues in Figure 3(a) and (b). Residues K349c and N351c may be critical for the stabilization of the carbanion intermediate that forms during the conversion of malate to fumarate. It is possible to speculate that E356c may form the ®rst part of a charge relay chain between H213d and the active-site water molecule. This histidine sidechain may have two roles within the fumarase active-site: (1) it acts as the catalytic base that activates the active-site water molecule for removal of the C3 proton of L-malate; and (2) it provides part of the binding site for the C1 carboxylate group of incoming substrate. The active-site water molecule has an unusual coordination and position in the Y-fumarase active sites. In the NY-fumarase/meso-tartrate complex, it is within hydrogen bonding distance of ®ve enzyme atoms. Its interactions are similar to those

found in the crystal structure of the E-fumarase(c)/ citrate complex (Weaver & Banaszak, 1996). The reproducible appearance of this water molecule in multiple crystal structures along with its stereochemical location make it a prime candidate to act as one of the bases involved in the catalytic reaction. Based on the assumption that the meso-tartrate location is similar to what would be found for an L-malate:fumarase intermediate, the scissile C3 proton of the substrate would be in a position closest to the active-site water molecule. While water itself is not a strong base, one hypothesis that would circumvent this criticism is that the acid/base properties of the water molecule are dramatically changed by the proximity of H213 and the negatively charged carboxylate group of the substrate. This being the case, one would predict that the mutation of three hydrogen bonding partners to the active-site water molecule, (1) H213, (2) S124 and/or (3) N166, would seriously impair catalytic activity. A careful comparison of the subunit structures of the Y-fumarases with both d-crystallin and aspartase has been done (Simpson et al., 1994; Shi

438 et al., 1997), and in both instances the core domain, D2, has a conformation nearly identical with the yeast and E. coli fumarases. However, for d-crystallin, the other two domains show signi®cant conformational differences. In addition, comparison of NY and RY-fumarase to aspartase shows that all three domains appear to adopt very similar conformations. The packing of the subunits for all of the super-family members includes both a conservation of tertiary and quaternary structure of a tetramer with 222 point symmetry. A detailed comparison of the active-site regions (Figure 4) shows that the positions of the catalytically important residues are roughly similar in the structure of the inactive d-crystallin. The similarities include residues H213, T214, K349, N351 Ê for the and E356, with an RMS deviation of 0.75 A corresponding Ca atoms of d-crystallin. However, the d-crystallin segment I88-Q89-T90, homologous to S124-G125-T126 in RY and NY-fumarase, is in a structurally different location, as illustrated by Figure 4. In addition, S165 of RY and NY-fumarase is replaced by R113 in both d-crystallin and its closely related, enzymatically active partner arginosuccinate lyase. In addition, there is no evidence indicating the presence of an active-site water molecule in the d-crystallin structure (Simpson et al., 1994). A recent report of the crystal structure of aspartase indicated that the active-site water molecule was not present (Shi et al., 1997) and that H213 in

Crystal Structures of Yeast Fumarase

the current yeast structures was replaced by glutamine. Because of the otherwise close similarity in their conformations and catalytic reaction, the discrepancy is dif®cult to rationalize and will have to await further structural studies of both arginosuccinate lyase and aspartase in the presence of inhibitors and/or pseudo-substrates. It is possible that the different family members operate through somewhat different mechanisms, although a major relocation of the active sites seem unlikely. A second dicarboxylic acid binding site In addition to the active site, some of the fumarases and aspartases are activated by substrates or other dicarboxylic acids and anions. Most of the kinetic characterization of this activation phenomenon was done with eukaryotic fumarases. The crystallographic studies of the prokaryotic E-fumarase(c) uncovered a dicarboxylic acid binding site near the active site (Weaver et al., 1997). This secondary site is located on the N-termÊ from the inal end of a short p-helix about 12 A active site and was believed to belong to L-malate used in the puri®cation of the protein. The conformation of NY and RY-fumarase in the region of the secondary site was studied and compared with the coordinates of E-fumarase(c). Since neither form of the crystals was prepared with ``activators'', it is not surprising that no observable electron density belonging to a ligand could be

Figure 5. A comparison of the activator sites from the crystal structures of NY and E-fumarase(c). The stereo-diagram illustrates the results of a superimposition of the crystallographic coordinates of NY-fumarase and E-fumarase(c) in the region of the second dicarboxylic acid binding site. The NY-fumarase positions are colored in violet, while those representing E-fumarase(c) are shaded in gold. The position of the activator, L-malate, as found in the crystal structure of E-fumarase(c) is colored gray and is visible directly above the label H129.

439

Crystal Structures of Yeast Fumarase

found in the region of the secondary site in the crystal structures of the Y-fumarases. Nonetheless, to make a careful comparison of the eukaryotic and prokaryotic enzymes, the method of leastsquares was used to superimpose residues 151 to 176 of NY-fumarase with 125 to 150 of E-fumaraÊ for se(c). The resulting RMS difference was 0.61 A a the C atoms. To avoid dif®culties in viewing, only a subset of this region is shown in stereo in Figure 5. This segment of the crystal structures contains a short segment of a rare p-helix from residues 152 to 159 in NY-fumarase, which should be visible in Figure 5. By homology with the prokaryotic enzyme, the NY-fumarase side-chains for binding at the second site would include K151, H154, N156 and N157; the corresponding side-chains in E-fumarase(c) were R126, H129, N131 and D132 (Weaver & Banaszak, 1996). For N157 (D132), only the peptide nitrogen atom is used in the binding interactions. H154 and N156 would require only torsional movement around w1 to re-locate at the equivalent locations of H129 and D132 in the prokaryotic enzyme. Overall, the positioning of amino acid side-chains at the purported activation site in NY and RY-fumarase suggest that it may have a similar secondary binding site. Although the mechanism is still obscure, the crystallographic studies of the yeast enzymes point to the region shown in Figure 5 as the most probable site of activation. With the legacy of kinetic data on the eukaryotic fumarases and an expression system described below for Y-fumarase, it should be possible to make mutations to test whether or not the proposed second dicarboxylic acid binding site is capable of affecting the turnover number.

Methods and Materials Construction of pASKYMFUM, a plasmid expressing RY-fumarase A 1498 bp fragment containing the coding region for RY-fumarase was generated using the YEp24 plasmid. Two PCR primers were synthesized to obtain RY-fumarase with ¯anking XbaI and PstI restriction sites. The primers used were: 50 -primer, 50 -ATCTATCTAGATACGAGGGCAAAAATGAACTCCTCGTTCAG-30 ; 30 -primer, 50 -CGGAGGGACCACTGCAGAATCACAAA-30 . The 50 -primer contained an XbaI restriction site, underlined above, while the 30 -primer contained a PstI restriction site also underlined. The resulting 1498 bp PCR product and the pASK40 plasmid was subjected to a double restriction digest involving XbaI and PstI (Skerra et al., 1991). A 1% agarose gel separated the coding sequence from the vector and a Bio-Rad Prep-a-Gene Kit was used to purify the products. A ligation reaction consisting of a 3 to 1 molar ratio of PCR product to pASK40 was then used to prepare the expression plasmid. After the ligation reaction at 25 C for 1.5 hour , 10 ml was transformed into competent JM105 cells. To verify the correct construct, ten colonies were subjected to overnight growth in 5.0 ml of LB plus 150 mg/ml ampicillin. Plasmid puri®cation was performed using the Promega Mini-Prep kit, and a double restriction digest was per-

formed using insertion sites, XbaI and PstI. Subsequent nucleic acid sequencing indicated a single point mutation K289R.

Purification and crystallization of RY-fumarase A 5.0 ml LB starter culture containing 150 mg/ml of ampicillin was inoculated with a single colony and allowed to grow overnight. Using this starter culture, a 1:500 dilution was made into one liter of LB in a 2.8 l Fernbach ¯ask containing 150 mg/ml of ampicillin. The cells were induced at an A600 nm of 0.6 to 1.0 with the addition of 2 mM isopropyl thio-b-galactoside. Induction was carried out for four to six hours, at which time the cells were harvested by centrifugation at 4000 g for 30 minutes. Cell pellets were either stored at ÿ80 C or used directly for puri®cation of RY-fumarase. Cell pellets were resuspended (1:2, w/v) in buffer A (50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 3 mM DTT, Protease inhibitor stock). All steps throughout the puri®cation process were maintained at 4 C unless otherwise noted. The cellular resuspension was sonicated for eight minutes at two minute intervals in a solid CO2/isopropanol bath to maintain the temperature at 4 C. The cellular debris was removed by centrifugation at 17,000 g for 40 minutes. For removal of nucleic acids, the supernatant was adjusted to 0.35% polyethylenimine (PEI) and allowed to equilibrate for an additional 20 minutes. The precipitated nucleic acid material was removed by centrifugation at 17,000 g for 20 minutes. The pH of the supernatant was adjusted to 8.5 with the addition of 1 M NaOH and loaded onto a Q-Sepharose column (Pharmacia, Fast-Flow). After washing with buffer A until no further protein emerged, a 500 ml (0 mM to 500 mM NaCl) linear gradient was run through the column. Typically, RY-fumarase eluted between 150 mM and 300 mM NaCl. Fractions that exhibited fumarase activity were pooled, dialyzed against buffer B (20 mM Mes (pH 6.5), 1 mM EDTA, 3 mM DTT) and loaded onto a pyromellitic acid (PMA)-af®nity column. The column was washed to baseline with buffer B, and RY-fumarase typically eluted around 550 mM NaCl during a 0 mM to 600 mM NaCl gradient. Again, the fractions with fumarase activity were pooled and dialyzed against buffer A. E. coli has an endogenous fumarase that appeared to copurify on the PMA-af®nity column. Therefore, the relative pI values of RY-fumarase and E-fumarase(c) were taken into consideration, and a ®nal Mono-Q column was employed to separate these two similar enzymes. After loading the sample onto the Mono-Q (pre-equilibrated with buffer A), a linear NaCl gradient was run from 0 mM to 300 mM over 100 ml. RY-fumarase elutes ®rst between 100 mM and 125 mM NaCl, while Efumarase(c) elutes much later at NaCl concentrations approaching 200 mM. RY-fumarase was judged to be pure by both SDS-PAGE and N-terminal protein sequence analysis. The ®nal speci®c activity of RYfumarase was 320 units/mg. X-ray diffraction-quality crystals of RY-fumarase were grown from solutions of 150 mM Tris-HCl (pH 8.3), 100 mM Li2SO4, and 8 to 10% polyethylene glycol (PEG) 4000. Crystals appeared after two days and reached a maximum size of 0.5 mm  0.3 mm  0.3 mm within one week. The spacegroup was determined to be with the following cell dimensions: P42212 Ê , c ˆ 100.56 A Ê . Estimates showed that a ˆ b ˆ 95.19 A a monomer was present in the asymmetric unit.

440

Crystal Structures of Yeast Fumarase

Data collection and molecular replacement solution of RY-fumarase The RY-fumarase dataset was collected at room temperature using a Siemens-Nicollet area detector and a monochromatic source from a graphite crystal, CuKa Ê ). The X-rays were generated by a rotating (l ˆ 1.542 A anode operating at 45 kV and 200 mA. The data reduction and scaling was carried out with the XENGEN suite of programs (Howard et al., 1987). The data processed as P42212 has an Rsym value equal to 15.5, an average I/s of 6.9, an 8.3-fold redundancy, and is 94.5% Ê . Complete X-ray statistics are provided complete to 2.6 A in Table 2. RY-fumarase crystals were sensitive to X-ray irradiation at room temperture, as evidenced by the decay of the high-resolution data after the ®rst few hours of X-ray exposure. Because of the radiation sensitivity, data from four crystals were merged to obtain a Ê . The relatively high Rmerge complete dataset to 2.6 A value given in Table 2 most likely derives from the combination of sensitivity to the X-ray beam and the number of crystals required to complete the data. The coordinates for E-fumarase(c) were used as a search probe for the molecular replacement solution of the X-ray data from RY-fumarase crystals. Prior to the cross-rotation function calculation, alanine residues were substituted at amino acid positions of non-identity between E-fumarase(c) and RY-fumarase. Cross-rotation functions were calculated in X-PLOR with the search probe placed in an orthorhombic box with the followÊ , b ˆ 250.0 A Ê , c ˆ 100.0 A Ê ing dimensions: a ˆ 180.0 A (BruÈnger, 1990). The angular search was limited to a single asymmetric unit. The highest peak had an Eulerian orientation of y1 ˆ 182.0 , y2 ˆ 87.54 , y3 ˆ 103.3 , and a correlation score equal to 4.37 with a mean of ÿ0.023. Following the cross-rotation function, a threedimensional translation search was performed. A maximum correlation value of 0.508, with a mean of 0.162, was found at the fractional position: x ˆ 0.149, y ˆ 0.064, z ˆ 0.09. Model building and refinement of RY-fumarase The initial Rfactor after the translation search was 45%. The ®rst round of re®nement was carried out prior to replacement of the alanine residues with the corresponding RY-fumarase amino acid residue. The ®rst step of re®nement entailed 50 cycles of Powell minimization for Ê with corresponding jF(hkl)j data between 10 and 4.0 A values greater than 2s. Following the ®rst Powell miniÊ , and an mization, the resolution was increased to 3.5 A Table 2. Data collection statistics for NY and RYfumarase Sample Total number of measurements Number of unique reflections Ê) Resolution (A Rmerge Overall completeness (%) Ê )/ Completeness shell (2.50 ÿ 2.45 A (2.69 ÿ 2.59) (%) hkl with I>3s(I) hkl at I>3s(I) at max resolution

NY-fumarase RY-fumarase 71,089 14,953 2.40 0.078 87.6 84.3 63.9

118,256 14,127 2.59 0.154 94.5 66.7 59.8 N/A

Rmerge ˆ ( [I(h)i ÿ hI(h)ii]/( hI(h)ii, where I(h)i is the observed intensity of a re¯ection (h) and hI(h)ii is the mean intensity of re¯ection h over the i measurements.

additional 150 cycles of positional re®nement were carried out. Immediately after the second round of Powell minimization, a simulated annealing run was performed at 2500 K with cooling increments of 50 K per cycle to a ®nal temperature of 300 K. All data between 10 and Ê were utilized for this simulated annealing run, 2.9 A with the same s cutoff as employed earlier. An additional 150 cycles of Powell minimization followed the simulated annealing procedure, using data again Ê . After this re®nement procedure between 10 and 2.9 A Rfactor was 27.3%, while Rfree was 38.2%. At this stage in the re®nement, alanine side-chains were replaced using the Mutate_Replace command within the program O (Jones et al., 1991). Further adjustments were made using both the RSR_Rotamer databank and Tor_Residue. Once all of the alanine residues had been replaced with the appropriate side-chains corresponding to the RY-fumarase sequence, 150 cycles of Powell minimization were performed. At this stage in the re®nement the jFoj ÿ jFcj maps, contoured at 3s, clearly showed a number of potential water molecules. Water molecules were added with the assistance of the Wat_PekPik command within O13 if they displayed proper hydrogen bonding geometry, 1s 2jFoj ÿ jFcj, and 3s jFoj ÿ jFcj difference electron density. 100 cycles of Powell minimization followed each round of water addition. Immediately following the Powell minimization, a grouped B-factor re®nement step was added for Ê , excluding the water molall data between 10 and 2.9 A ecules. A water molecule was removed if either of the following occurred: (1) the distance to its hydrogenÊ ; and/or (2) there was a bonding partner exceeded 3.5 A lack of 2jFoj ÿ jFcj difference density contoured at 1s. Following these steps, iterations of manual model rebuilding with the O program (Jones et al., 1991) were followed with 100 cycles of Powell minimization and grouped B-factor re®nement (BruÈnger, 1990). The ®nal re®nement scheme utilized the bulk solvent correction within X-PLOR and included data between 30 Ê . The ®nal Rfactor was 18.8%, with a correspondand 2.6 A ing Rfree of 29.8% (using 10% of the data). Table 3 provides relevant re®nement statistics. The Ramanchandran plot indicates 327 out of a possible 373 non-glycine residues within the most-favored regions, while 41 out of the 373 non-glycine residues lie within the additionally allowed areas of the plot. Only F381 falls within the disallowed region of the plot, and its conformation has been shown to be involved in the positioning of H213 within the active site (Weaver et al., 1997). The atomic coordinates for the recombinant form of yeast fumarase

Table 3. Final re®nement statistics for NY and RYfumarase Sample Ê) Resolution (A Number of reflections used Number of restraints Number of protein atoms Number of water molecules Number of positional parameters Rfactor Rfree Ê) Bond lengths (A Angles ( ) Ê 2) Overall B-factor (A

NY-fumarase

RY-fumarase

12±2.45 14,791 9287 3486 57 10,458

10 ±2.6 11,555 NA 3441 27 NA

19.3 26.4 0.026 2.5 28.27

18.8 29.8 0.014 2.4 24.3

441

Crystal Structures of Yeast Fumarase have been deposited in the Protein Data Bank under the identi®cation code 1yfm (Berstein et al., 1971).

the Y-fumarase structure through the molecular replacement method.

Crystallization of NY-fumarase

Structure determination of NY-fumarase and refinement

NY-fumarase (fumarate hydratase, EC 4.2.1.2.) has been puri®ed from a wild-type strain of S. cerevisiae by a method including DEAE ion-exchange and dye-ligand af®nity chromatography (Keruchenko et al., 1992). Crystals suitable for X-ray structure analysis were grown by the vapor-diffusion method (sitting drops) from a solution of 1.0% (w/v) protein, 9% (w/v) PEG-6000 in the presence of 25% ethylene glycol and 4 mM meso-tartaric acid, in 20 mM Mops-buffer (pH 7.5) at room temperature. A streak-seeding technique was applied to initiate crystal growth. The crystals grew to typical dimensions of 0.5 mm  0.3 mm  0.2 mm in approximately one week. The crystals belong to the tetragonal space group Ê , c ˆ 105.9 A Ê and one P42212 with a ˆ b ˆ 93.6 A monomer in the asymmetric unit. The cell dimensions were comparable, but not identical with those for RYfumarase. Data collection and initial phasing of NY-fumarase Over 50 heavy-atom compounds were tested in the search for isomorphous heavy-atom derivatives. In most cases the soaking resulted in a severe deterioration of the crystal, but in a few favorable cases the diffraction Ê ). pattern was measurable at low resolution (3.5 to 4.0 A Several data sets from native and potential derivative crystals were collected on a Siemens Area Detector (Molecular Biophysics, Lund University). For two derivatives, KAu(CN)2 and Hg(CH3)2, self-consistent heavy-atom site solutions were found using Patterson and difference Ê mulFourier techniques. From these initial sites, a 3.5 A tiple isomorphous replacement map was calculated. The Ê resolution of the derivative data was extended to 2.8 A using a synchrotron radiation source at CCLRC Daresbury Laboratory. Although the NY-fumarase crystals diffracted to Ê , they were extremely radiation-sensitive and 2.4 A Ê within a few frames. diffraction fell to less than 3.0 A Crystals of sizes greater than 0.25 mm disintegrated when ¯ash-freezing was attempted. However, crystals of dimensions 0.15 mm  0.07 mm  0.07 mm were successfully frozen at 100 K in the native mother liquor containing 25% ethylene glycol. X-ray data from one native crystal was collected on Station 9.5, SRS Daresbury, at Ê wavelength using a 18 cm Mar-Research image 0.98 A plate detector. Data were processed using the MOSFLM program suite (Leslie, 1992) and the ®nal scaling and reduction was achieved with the CCP4 program package (CCP4, 1994). The freezing of the crystal led to a shrinkage of the Ê (room temperacell along the a and b-axes from 93.6 A Ê (frozen). The data were determined to be ture) to 92.2 A Ê and the principal data collection statuseful to 2.45 A istics are provided in Table 2. This extended data set was used in conjunction with the DM program, incorporating solvent ¯attening, histogram matching and phase extension to produce a new map (Cowtan, 1994). This map clearly showed the molecular boundary and various secondary structure components, but the overall connectivity was poor and the structure was only partly interpretable. At this stage, the X-ray coordinates of the E-fumarase(c) became available, and were used to solve

Solutions for the rotation and translation functions were found with the AMORE program package (Navaza, 1994). The search model consisted of one subunit of the tetrameric structure of the E. coli enzyme (57% sequence identity; Weaver et al., 1995). The crossrotation function was successful only when calculated using the Fobs expanded to the space group P1. This enabled the radius of integration to be increased from 32 Ê , thus Ê (for space group P42212) to 47 to 50 A to 35 A including as many self-vectors as possible from an Ê  56 A Ê  102 A Ê ) search model. The elongated (36 A translation function gave a unique solution for the position of the monomer in the asymmetric unit with the correlation coef®cient of 0.49 and Rfactor of 48%. Cycles of rigid-body re®nement and graphics rebuilding resulted in the localization of about 150 side-chains in the NYfumarase structure that had been mutated to alanine residues in the search model. Re®nement was carried out using a combination of XPLOR (BruÈnger, 1990) and RESTRAIN (Driessen et al., 1989). The ®nal value of the Rfactor was 19% with a correlation coef®cient of 0.95 for Ê . Rfree was all data in the resolution range 12.0 to 2.45 A 26% using 3% of the data. The re®nement statistics are given in Table 3.

Acknowledgments The studies of RY-fumarase were aided signi®cantly by Ed Hoeffner and his vigilant maintenance of the X-ray and computer facilities. The RY-fumarase studies were supported by grants from the NSF (MCB-9603656) and the Minnesota Supercomputer Institute. The NY-fumarase studies were supported by the grants from ISF, N14000, 14300, and RFFI, 93-046871 (Russia). The NY-fumarase structure analysis was undertaken as part of a joint biology program at CCLRC Daresbury Laboratory supported by the EPSRC, BBSRC and MRC.

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442

Crystal Structures of Yeast Fumarase

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Edited by D. Rees (Received 7 January 1998; received in revised form 30 March 1998; accepted 7 April 1998)