Structure of Dihydroorotate Dehydrogenase B

Structure, Vol. 8, 1227–1238, December, 2000, 2000 Elsevier Science Ltd. All rights reserved.

PII S0969-2126(00)00530-X

Structure of Dihydroorotate Dehydrogenase B: Electron Transfer between Two Flavin Groups Bridged by an Iron-Sulphur Cluster Paul Rowland,*§ Sofie Nørager,* Kaj Frank Jensen,† and Sine Larsen*‡ * Centre for Crystallographic Studies Department of Chemistry University of Copenhagen DK-2100 Copenhagen Denmark † Center for Enzyme Research Institute of Molecular Biology University of Copenhagen DK-1307 Copenhagen Denmark

Summary Background: The fourth step and only redox reaction in pyrimidine de novo biosynthesis is catalyzed by the flavoprotein dihydroorotate dehydrogenase (DHOD). Based on their sequences, DHODs are grouped into two major families. Lactococcus lactis is one of the few organisms with two DHODs, A and B, belonging to each of the two subgroups of family 1. The B enzyme (DHODB) is a prototype for DHODs in Grampositive bacteria that use NAD⫹ as the second substrate. DHODB is a heterotetramer composed of two different proteins (PyrDB and PyrK) and three different cofactors: FMN, FAD, and a [2Fe-2S] cluster. Results: Crystal structures have been determined for DHODB and its product complex. The DHODB heterotetramer is composed of two closely interacting PyrDB-PyrK dimers with the [2Fe-2S] cluster in their interface centered between the FMN and FAD groups. Conformational changes are observed between the complexed and uncomplexed state of the enzyme for the loop carrying the catalytic cysteine residue and one of the lysines interacting with FMN, which is important for substrate binding. Conclusions: A dimer of two PyrDB subunits resembling the family 1A enzymes forms the central core of DHODB. PyrK belongs to the NADPH ferredoxin reductase superfamily. The binding site for NAD⫹ has been deduced from the similarity to these proteins. The orotate binding in DHODB is similar to that in the family 1A enzymes. The close proximity of the three redox centers makes it possible to propose a possible electron transfer pathway involving residues conserved among the family 1B DHODs. Introduction Dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate. This oxidation is the fourth reaction in the universal pathway for de novo pyrimidine nucleotide biosynthesis. Although all DHODs are flavoproteins, the ‡ To whom correspondence should be addressed (e-mail: [email protected].

ku.dk). § Present address: Department of Structural Biology, SmithKline Beecham Pharmaceuticals, New Frontiers Science Park (North), Harlow, Essex CM19 SAW, UK.

properties of enzymes from different organisms vary considerably [1, 2]. The enzymes from Gram-positive bacteria, archaea, and some lower eukaryotes, e.g., Saccharomyces cerevisiae, are soluble proteins, which use soluble substances as electron acceptors. They are members of family 1. The DHODs belonging to family 2 are mainly from eukaryotic organisms and Gramnegative bacteria related to Escherichia coli. These enzymes are attached to membranes and donate the electrons from oxidation of the substrate to respiratory quinones [3]. At the sequence level, the soluble and membrane bound DHODs have less than 20% amino acid sequence identity, and the protein chains of the membrane-associated enzymes are extended by more than 40 residues in their N termini, which have no resemblance to the soluble family 1 proteins. These extensions target the enzymes for transport to mitochondria in eukaryotic cells and, in addition, they appear responsible for attachment to membranes and communication with the respiratory quinones [1, 3, 4]. Furthermore, as the sequence similarity between the N-terminal extensions of the family 2 DHODs of eukaryotic cells and the Gram-negative bacteria is quite limited, and the biological diversity between the different DHODs is substantial [1, 3]. This great diversity between DHODs of different organisms makes these enzymes promising targets for chemotherapy and allows the development of compounds that selectively inhibit pyrimidine biosynthesis in some organisms and leave others unaffected. For instance, the immunosuppressive drug leflunomide, which has been approved for treatment of rheumatoid arthritis [5–8], inhibits the human DHOD with little if any affect on the enzymes from enteric and other bacteria in the human body. The soluble DHODs also diverge considerably from each other and have been divided into two subgroups, family 1A and family 1B, which have about 30% sequence identity [1, 2, 9, 10]. The family 1A enzymes are found in anaerobic yeasts [17], perhaps also in some protozoa [18], and in milk-fermenting bacteria such as Lactococcus lactis [9, 11, 12] and Enterococcus faecalis [13, 14]. It is noteworthy that the latter organisms also contain a family 1B DHOD [15, 16]. The DHODs belonging to family 1A are dimeric proteins with one FMN molecule bound tightly to each subunit [10, 12, 14] and are able to use fumarate as an electron acceptor [2, 11, 14, 17]. In contrast to other types of DHODs, the DHODs belonging to family 1B are prevalent in Gram-positive bacteria and are able to use NAD⫹ as an electron acceptor [1, 16]. A representative of family 1B is the DHODB enzyme from Lactococcus lactis. This enzyme was found to be a heterotetramer composed of two PyrDB and two PyrK subunits. The PyrDB subunits are encoded by the pyrDb gene and are homologous to the family 1A DHODs [9], while the PyrK subunits that are encoded by the cotranscribed pyrK gene lack similarity to the DHODs in general [11, 16]. The heterotetrameric enzyme contains 2 FMN, 2 FAD, and 2 [2Fe2S] iron-sulfur clusters as tightly bound prosthetic groups, and it is able to use NAD⫹ as an electron acceptor [16]. The PyrDB subunit contains FMN and can be considered to be the dihydroorotate dehydrogenase proper. The PyrK protein in the tetramer binds the FAD and the iron sulfur cluster and is responKey words: ferredoxin reductase superfamily; flavoproteins; iron sulphur cluster; protein-protein interaction; pyrimidine biosynthesis

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dihydroorotate dehydrogenases of Gram-positive bacteria and have enabled us to suggest a pathway for the transfer of electrons between the three different redox centers associated with the two subunits in the family 1B DHODs. Results and Discussion Structure Determination The crystal structure of the DHODB was determined by the multiple isomorphous replacement (MIR) method, for which two derivatives were used. The structure was first solved with synchrotron radiation data to 2.2 A˚ resolution collected on a crystal cooled to 277 K; however, this model left some small parts of the structure undefined. We were finally able to trace all but one residue of the two protein chains using data to 2.1 A˚ resolution collected with synchrotron radiation on a cryocooled crystal (120 K). The structural description will be based on the latter structure. Information about the substrate binding was obtained from the structure of DHODB complexed with the product orotate. This structure was determined with an inhouse data set to 2.4 A˚ resolution collected on a crystal cooled to 288 K.

Figure 1. Possible Pathway for the Transfer of Electrons from the Substrate Dihydroorotate (DHO) to the Electron Acceptor NAD⫹ The bold lines represent the flow of electrons in a reaction favored at higher pH values. However, the reaction is reversible, and at lower pH values the enzyme readily reduces orotate to dihydroorotate at the expense of [NADH ⫹ H⫹]. The transfer of electrons from DHO to FMN takes place in the PyrDB subunit; the PyrK subunit binds the [2Fe-2S] cluster and FAD. The latter cofactor is necessary for the ability to use NAD⫹ as an electron acceptor.

sible for channeling the electrons from the oxidation of dihydroorotate from the FMN redox center in the PyrDB subunit to NAD⫹ [16, 19]. This suggests that electron transfer between the two flavin cofactors in DHODB may involve the [2Fe-2S] cluster as illustrated in Figure 1. The DNA sequences from several Gram-positive bacteria indicate that the architecture of DHODB is representative for many Gram-positive bacteria as these often have an open reading frame with high similarity to pyrK located upstream of their pyrD gene [11]. Furthermore, purification and analyses of the DHODs from Bacillus subtilis [20] and E. faecalis [15] have revealed that these enzymes are composed like DHODB from L. lactis and that the subunits have similar functions. Also, the dihydroorotate dehydrogenase in Zymobacterium oroticum (now renamed Clostridium oroticum), which was discovered by A. Kornberg and collaborators in 1953 [21] during their pioneering work of clarifying the pathway for pyrimidine nucleotide biosynthesis, has recently been shown to be a family 1B enzyme [22]. Although L. lactis and E. faecalis contain both an A and a B type DHOD, it is clear that the B type enzyme alone is sufficient for optimal growth in pyrimidine-free media [11] and that Bacillus subtilis only contains the B type DHOD [20, 23]. We have previously described the crystal structure of dihydroorotate dehydrogenase A from L. lactis (DHODA) both of the free enzyme [10] and of the enzyme in complex with the product, orotate [24]. In this paper we report the similar structures for the heterotetrameric dihydroorotate dehydrogenase B from L. lactis (DHODB). These first structures of a family 1B enzyme provide general insight into the reaction catalyzed by

The Architecture of the Heterotetrameric DHODB The heterotetrameric DHODB is shown in Figure 2a. It is composed of two PyrDB and two PyrK subunits. A crystallographic 2-fold axis relates the two halves of the heterotetramer. Its central core consists of two PyrDB subunits interacting across the 2-fold axis in an arrangement that resembles the other dihydroorotate dehydrogenase from L. lactis (DHODA) [10]. Each of the PyrDB subunits is in close contact with a PyrK protein. The two PyrK subunits protrude from the central core of the PyrDB proteins (Figure 2a). This arrangement offers an explanation for the larger B values of the PyrK subunit (Table 4). The DHODB PyrDB Subunits The PyrDB subunit has an ␣/␤ barrel fold (Figure 2b) and resembles the PyrDA subunit in the L. lactis DHODA [10]. The two proteins are both composed of 311 amino acid residues but show only 30% sequence identity; nevertheless, they display a very similar fold including both the ␣/␤ barrel and the secondary structural inserts as shown on their sequence alignment in Figure 3. The ␣1 helix in PyrDA is replaced by two 310 helices in PyrDB. As in PyrDA, the region between ␤2 and ␤C folds into a ␤ turn with a conserved cis peptide bond between Thr60 and Pro-61. A second cis peptide bond between Met-195 and Ile-196 at the end of ␤6 matches a cis peptide found in an equivalent position in PyrDA. Significant differences between PyrDB and PyrDA are found in the highly conserved region between strand ␤4 and helix ␣4, which contains an additional 310 helix (␣E) in pyrDB. The long barrel insert between ␤6 and ␣6, which forms the link to the other PyrDB subunit, displays less secondary structure in PyrDB than in PyrDA containing only strands ␤E and ␤H. Helix ␣D comprising the last part of the polypeptide chain is considerably longer in PyrDB than in PyrDA. In addition to the secondary-structural elements, DHODB contains salt bridges that connect oppositely charged residues from different secondary elements. There are five salt bridges with interatomic distances less than 3 A˚, which may play a role in stabilizing the PyrDB subunit. These salt bridges are as follows: Arg-6–Asp-292 (2.8 A˚, N⫺term /␣8); Lys-160–Asp-191 (2.6 A˚, ␣4/coil-␤6); Lys-226–Asp-255 (2.8 A˚, ␣6b/␣7); Lys-226–

Dihydroorotate Dehydrogenase B 1229

Figure 2. Structure of Dihydroorotate Dehydrogenase B (a) The overall structure of DHODB viewed along the crystallographic 2-fold axis. The secondary-structural elements are colored according to B factors. Residues with B factors below 20 A˚2 are dark blue, and residues with B factors above 40 A˚2 are bright red. The intersubunit salt bridges are shown as a ball-and-stick representation. The FMN and FAD groups are represented as stick models in chartreuse and pale turquoise, respectively. The [2Fe-2S] cluster is shown as brown (Fe) and yellow (S) spheres. (b) The PyrDB subunit color ramped, with the N terminus in blue and the C terminus in red. The labels of the secondary-structural elements correspond to those in Figure 3. (c) The PyrK subunit colored according to increasing numbers in the sequence, as in Figure 2b. The labels of the secondary-structural elements correspond to those in Figure 3.

Glu-258 (2.9 A˚, ␣6b/␣7), and Asp-276–Lys-282 (2.7 A˚, ␣C/␣8). The fact that Lys-226, Asp-255, and Glu-258 are completely conserved among the family 1 DHODs indicates the significance of these interactions.

Alignment of the 270 C␣ atoms from the uncomplexed PyrDA and PyrDB subunits has a rmsd of 1.9 A˚ (ALIGN [25]). The result from this superposition is shown in Figure 4a. The region between ␤4 and ␣4 that covers the active site shows the largest Figure 3. Sequences of the Two Subunits in DHODB The top shows the alignment of the sequences of PyrDA and PyrDB. The residues shown in magenta are conserved in both family 1 and family 2 of the dihydroorotate dehydrogenases; those marked in spring green are only conserved in family 1. The secondary-structural elements in PyrDA are shown in green, and those in PyrDB are in blue. The symbol ␣ is used for both ␣ helices and 310 helices. The bottom of the figure contains the sequence of the PyrK protein with the secondary-structural elements marked. The residues marked in red are also conserved in the NADPH ferredoxin reductase superfamily. The cysteine ligands of the iron-sulfur cluster are shown in yellow. This figure was prepared with ALSCRIPT [51].

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Figure 4. Comparison of the Subunits, PyrDB and PyrK, to Related Proteins (a) A superposition of the C␣ traces of the PyrDB (blue) with the PyrDA subunit (red) from DHODA. The FMN groups are represented in green. (b) C␣ trace of the PyrK subunit (blue) with the structure of PDR (magenta, apart from the [2Fe-2S] domain shown in yellow) superimposed. The [2Fe-2S] cluster and the FAD of PyrK are represented as green ball-andstick models; the [2Fe-2S] cluster and the FMN group of PDR are colored black. (c) The C␣ trace of the two subunits in the closely linked PyrDB-PyrK dimer is displayed in stereo. The C␣ traces for both subunits are color ramped. The PyrDB subunit is at the top with colors going from red to blue and the numbers of the residues given in black, and the PyrK subunit is at the bottom with colors from blue to violet and the residue numbers given in red.

deviation in the protein backbones. In PyrDA this loop closes in on the FMN group, while in the uncomplexed PyrDB it is in an open position, which could allow easy diffusion of substrate into the active site.

The DHODB PyrK Subunits Though PyrK has a 49 amino acid shorter peptide chain, it occupies almost as much space as the PyrDB subunit. This observation reflects its more open structure composed of three

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domains (Figure 2c). Each domain associated with a different region of the primary sequence appears to have a distinct function. The structural alignment of PyrK that was performed with DALI [26] revealed its similarity to the proteins belonging to the NADPH ferredoxin reductase superfamily, which was characterized thoroughly by Ingelman and Eklund [27]. The nomenclature used for the assignment of the secondary-structural elements in this family has been employed for the PyrK subunit (Figure 2c). The N-terminal domain contains the first 100 amino acids and constitutes the most rigid part of the PyrK subunit. It forms a flattened ␤ cylinder composed of six antiparallel ␤ strands (F␤1–F␤6) capped by two short helices (F␣0 and F␣1). The opening in the cylinder between strands F␤4 and F␤5 serves as the binding site for FAD, as shown in Figure 2c. Apart from the additional 310 helix (F␣0) located at the base of the N-terminal end of the cylinder, this domain resembles the flavin binding domains in other members of the ferredoxin reductase superfamily. The peptide bond that links Gly-96 and Pro-97, which adopts a cis conformation, is in a region with well-defined electron density. The second domain in the PyrK subunit comprises an open ␣/␤ structure. It resembles the NAD binding domains of the ferredoxin reductase family and shows some significant differences from the typical NAD binding fold of many other dehydrogenases [28]. Knowing that DHODB is able to utilize NAD⫹ as an oxidant in the enzymatic reaction, we suggest that NAD⫹ binding occurs at this domain. Its core consists of a 5 strand parallel ␤ sheet (N␤1–N␤5) that is sandwiched between two layers of ␣ helices, N␣1–N␣2 and N␣3–N␣4. The [2Fe-2S] iron-sulfur cluster binds to the last domain in the PyrK subunit. This domain at the interface between the flavin and NAD binding domains contains three ␤ strands. The long loop between N␤5 and I␤1 connects the two domains and contains two of the cysteine residues that bind the iron-sulfur cluster (Cys-226 and Cys-231). This loop leads into the 2 strand antiparallel ␤ sheet, which is made up of strands I␤1 and I␤2. The two other cysteine residues that bind to the iron-sulfur cluster are neighbors to this sheet. These residues are Cys234 just before the start of I␤1 and Cys-249 immediately following I␤2. The iron-sulfur cluster is localized in a well-ordered part of this domain close to the FAD binding site. At the start of I␤3, Gly-252 and Pro-253 are linked by a cis peptide bond. Strand I␤3 runs antiparallel to the 5 strand parallel ␤ sheet of the NAD binding domain, and this creates a linkage between the NAD and the iron-sulfur binding domains. Four well-defined salt bridges connect secondary-structural elements and have distances between the oppositely charged side chains that are less than 3.0 A˚. These salt bridges are Arg52–Asp-93 (2.7 A˚, F␤4a/F␤6), Lys-151–Glu-154 (2.6 A˚, N␣2a/ N␣2b), Lys-247–Glu-250 (2.9 A˚, I␤2/coil), and Asp-238–His-244 (3.0 A˚, I␤1/I␤2). The sequence alignment of PyrK proteins from other species, Bacillus subtilis, Bacillus caldolyticus, and E. faecalis, showed that the majority of these residues are conserved among the PyrK proteins, which suggests that they are likely to contribute to the stabilization of the structure of the PyrK protein [15]. PyrK Belongs to the NADPH Ferredoxin Reductase Family The structural alignment showed that the PyrK subunit belongs to the NADPH ferredoxin reductase superfamily. Structures are known for several members of this family [27, 29–33]. These proteins all assist in electron transfer reactions involving other proteins or another protein domain. The members of this family

also have in common the presence of a flavin group as a cofactor, which is FMN in phthalate dioxygenase reductase (PDR) [29] and FAD in the other members of the family, and the ability to bind the pyridine nucleotides, NADH or NADPH, as reductants of the flavin group. Despite the low sequence similarity in the ferredoxin reductase family, detailed structural comparisons of the members have revealed common features of their flavin binding sites [27, 32, 33]. The main structural differences between these proteins are in their relative orientation in the flavin and NAD binding domains, extra external structural elements, and variation in the length of the loops. From a structural alignment of six known structures, ten completely conserved residues were identified [27]. If PyrK is included in the alignment, the number of conserved residues reduces to seven (Figure 3). It is noteworthy that PyrK lacks the tyrosine residue found two positions upstream from the completely conserved Arg-53, which was shown to be important for flavin binding in the ferredoxin reductase structures. Of the currently known structures in this superfamily, PDR is the one most similar to the PyrK subunit because it is the only protein that contains a [2Fe-2S] binding domain in addition to the flavin and NAD binding domains. PDR catalyzes the transfer of electrons from an NADH molecule via an FMN molecule and a [2Fe-2S] cluster to a Rieske [2Fe-2S] cluster in phthalate dioxygenase. The first two domains of the two structures are very similar, and the common part of the two flavins (FAD in PyrK and FMN in PDR) superimposes well; the differences seem to be related to the larger cofactor in the DHODB. Though the iron-sulfur cluster binding domain in PDR is also present as a third domain at the C-terminal end of the protein sequence, it is distinctly different from the [2Fe-2S] binding domain in PyrK. An alignment of their first 220 residues gives 183 C␣ target pairs, which align with an rmsd of 1.83 A˚ (ALIGN [25]). Figure 4b shows the structural alignment of the PyrK subunit and PDR with the program O [34]. The alignment between the first two domains of PyrK and PDR shows that the [2Fe-2S] clusters are in the same overall position but the loops surrounding the iron-sulfur clusters are rotated almost 180⬚ relative to each other (Figure 4b). Intersubunit Interactions The heterotetrameric DHODB in Figure 2a can be considered to be composed of two heterodimers formed by the PyrDB and PyrK subunits with a small hydrophobic cavity located in the center. A complete list of residues from one subunit that are within 3.6 A˚ from another subunit is given in Table 1. The majority of the interactions are between the two PyrDB subunits (PyrDB and PyrDB⬘), which leads to an arrangement that resembles the homodimeric DHODA. The ␤ sheet region (␤E– ␤H) from one subunit that extends from the top of the ␣/␤ barrel interacts with the C terminus of the other subunit. In DHODB two pairs of salt bridges, Arg-203–Glu-258⬘ (3.2 A˚) and Arg-209–Glu-305⬘ (2.7 A˚), serve as the links between the two PyrDB subunits. Only Glu-258 belongs to the completely conserved residues in the family 1 DHODs. A single pair of salt bridges that serves the same function is found in DHODA, but the positions of these residues in the sequence do not match any of the salt bridges in DHODB. When one considers the great similarity between DHODA and PyrDB, it is also noteworthy that only 5 of the 30 residues involved in the PyrDB–PyrDB⬘ interface belong to the residues conserved in family 1 (Figure 3). The natural redox partners for the ferredoxin reductases are other proteins, but structures have not yet been reported for any of these protein-protein complexes. This makes it of spe-

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Table 1. Residues Found Closer Than 3.6 A˚ to Each Other at the Three Different Interfaces of the Heterotetramer in the Orotate-Free Structure PyrDB–PyrDB⬘ Thr-A66 Ala-A67 Ser-A68 Thr-A147 Val-A175 Thr-A176 Met-A200 Gly-A201 Val-A202 Arg-A203 Phe-A204 Leu-A206 Leu-A207 Arg-A209 Gln-A210 Pro-A211 Pro-A223 Lys-A226 Pro-A227 Val-A228 Leu-A230 Lys-A231 Glu-A258 Met-A261 Ala-A262 Tyr-A294 Arg-A295 Leu-A302 Glu-A305 Lys-A311

PyrDB–PyrK Pro-C227 Ser-C68, Pro-C223 Ala-C67, Ser-C68 Thr-C176 Thr-C176 Thr-C147, Val-C175, Thr-C176 Val-C228 Pro-C227 Pro-C227, Leu-C230, Lys-C231 Lys-C226, Glu-C258 Glu-C258, Ala-C262, Met-C261 Glu-C258 Arg-C295 Met-C261, Tyr-C294, Arg-C295, Leu-302, Glu-C305 Lys-C311 Lys-C311 Ala-C67 Arg-C203 Thr-C66, Gly-C201, Val-C202 Met-C200 Val-C202 Val-C202 Arg-C203, Phe-C204, Leu-C206 Phe-C204, Arg-C209 Phe-C204 Arg-C209 Lys-C207 Arg-C209 Arg-C209 Gln-C210, Pro-C211

Gly-A28 Glu-A32 Tyr-A33 (Lys-A35) Tyr-A36 (Lys-A48) Phe-A56 Thr-A60 Arg-A62 Val-A63 Glu-A65 Thr-A66 Ser-A68 Ile-A74 Gln-A77 Pro-A223 Ala-A275 (Pro-A277) (Phe-A278)

PyrDB⬘–PyrK Ala-B230 Ile-B228, Gly-B229, Ala-B230 (Gly-B226), Ile-B228, Ala-B230 (Glu-B250) Leu-B4, Met-B95, Ile-B228 (Tyr-B232) His-B237, (Glu-B242), (Ser-B243), Ala-B245 Val-B235 Tyr-B232, Ala-B233, (Val-B235) Ala-B233 Met-B49, Leu-B50, Leu-B51, Arg-B53, FAD-B263 Ala-B48 Ala-B48, (Leu-B50) Cys-B231 Tyr-B232, Val-B235, Ala-B245 Leu-B50 (Leu-B50), Arg-B52 (Met-B95) (Ser-B2)

Ser-A68 Asn-A252 Gln-A254

Ala-D48 Thr-D77 Asp-D75, Glu-D76, Thr-D77

A and C refer to residues from PyrDB subunit; B and D refer to residues from PyrK. Hydrogen bonds are indicated in bold. Some of the PyrDB–PyrK contacts change when orotate is bound. These are marked as follows: italics, only in the orotate complex; parentheses, only in the free enzyme.

cial interest to examine the interactions between the PyrDB and PyrK proteins. Each PyrK subunit is closely associated with one of the PyrDB subunits in the heterotetramer, but in addition Asp-75 and Glu-76 from PyrK make hydrogen bonds to Gln-254 from the other PyrDB molecule in the heterotetramer (PyrDB⬘) (Table 3), with Gln-254 being one of the residues that are highly conserved among the PyrD proteins (Figure 3). The binary complex between the PyrDB and PyrK subunits is illustrated in Figure 4c. The interface between the subunits includes residues from the top of the ␣/␤ barrel of the PyrDB subunit and from the flavin and [2Fe-2S] cluster binding domains in the PyrK subunit. The intersubunit interactions displayed in Table 1 comprise hydrophobic interactions between the ␣1a helix from PyrDB and the [2Fe-2S] binding domain. Similar interactions are observed between the PyrDB ␣C helix and three different sections of the FAD domain in PyrK. The ␤C and the loop leading into ␣C are in contact with both the FAD and the [2Fe-2S] binding domains of PyrK, and this interface contains a number of hydrogen bonds. The most significant appears to be the intersubunit salt bridge (2.8 A˚) between Glu65 from the PyrDB subunit and Arg-53 from the PyrK subunit. Both residues are completely conserved among the family 1B DHODs. The interactions between the PyrDB subunit and the FAD and [2Fe-2S] domains of PyrK seem to be important for the binding of FAD and the [2Fe-2S] cluster to the PyrK subunit. These cofactors bind very poorly, if at all, to the isolated PyrK subunit, but they bind strongly to the tetramer, and the maintenance of a stable tetrameric structure of DHODB depends on the content of FAD [19].

The Three Cofactors [2Fe-2S] Cluster The [2Fe-2S] cluster is found close to the interface between the PyrDB and PyrK subunits, as shown in Figure 5a. Four cysteine ligands from the PyrK subunit attach the [2Fe-2S] cluster to the protein; Cys-226 and Cys-231 are coordinated to Fe1, and Cys-234 and Cys-249 are coordinated to Fe2. The overall negative charge of the [2Fe-2S] cluster is partly compensated by the two side chains from His-41 and Lys-247 that are placed at opposite sides of the [2Fe-2S] cluster. The isoalloxazine rings from FAD in the PyrK subunit and FMN in the PyrDB subunit are arranged almost symmetrically around the [2Fe-2S] cluster (Figure 5a). The distances from the center of the [2Fe-2S] cluster to N5 of FMN and FAD are 12.7 and 14.6 A˚, respectively. From FMN the shortest distance is between a methyl group and Fe1 in the [2Fe-2S] cluster (Fe1– C7M, 7.8 A˚); a methyl group of FAD has similar contacts to and Fe2 and S1 (8.6 and 8.3 A˚, respectively). FMN The environment of the FMN group in the PyrDB subunit is illustrated in Figure 6a. The cofactor adopts a conformation that is very similar to the one seen in the structure of the other family 1 enzyme, DHODA. The ribityl and phosphate moieties (Figure 6a) are interacting with Ile-196, and the residues Gly222, Gly-249, Gly-270, Thr-271, Ser-24, and Lys-170 are completely conserved among the family 1 DHODs. These residues create an environment for the ribityl and phosphate groups that is almost identical to the one seen in the structure of the DHODA enzyme. However, due to a slightly different orientation of the

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Figure 5. The PyrDB-PyrK Interface (a) The part of the interface between the PyrDB and the PyrK subunits that contain the three redox centers in the uncomplexed DHODB structure. The residues from the PyrDB subunit are labeled with a red “A,” and those from the PyrK subunit are labeled with a green “B.” The violet broken line indicates the interface between the two subunits. (b) A superposition of the environment of the FMN and [2Fe-2S] clusters in the uncomplexed (yellow) and complexed (violet) structures. Residues from the two subunits are distinguished as in Figure 5a.

Lys-170 side chain, its ammonium group is not able to interact with O2 and O2* as in DHODA but only with O3*. In this orientation it is also farther away from N1 of the isoalloxazine ring. It therefore cannot make a direct hydrogen bond like in DHODA, but it is close enough to stabilize the anionic hydroquinone form of FMN. The significance of a lysine residue in this position was demonstrated for DHODA; a substitution of the lysine with an alanine gave a mutant protein that was devoid of any activities and unable to bind FMN [35]. As in the DHODA structure, there are no close protein contacts to the hydrophobic dimethylbenzene part of the isoalloxazine ring, but there are differences between PyrDA and PyrDB in their hydrogen bonds to the hydrophilic part of FMN. O2 and N3 are hydrogen bonded to Asn-104; in DHODA O2 interacts with the equivalent of Lys-

170, and N3 interacts with the side chain of Ser-44. The most prominent difference between the FMN environment in DHODA and PyrDB is seen in the Lys-48 side chain. In DHODA the equivalent residue, Lys-43, was in a conformation enabling it to make hydrogen bonds to N5 and O4 in the native enzyme and to make an additional hydrogen bond to the carboxylate group of orotate in the complex structure [24]. In the uncomplexed PyrDB Lys-48 has swung away from the isoalloxazine ring so that it points into the PyrK-PyrDB interface and makes hydrogen bonds to three water molecules. In DHODB O4 is hydrogen bonded to the backbone NH of Ala-49 and a water molecule that interacts with N5. The catalytic Cys-135 residue is farther away from the FMN group than the equivalent residue in DHODA due to the different conformation of the section between ␤4 and ␣4. This conformation leaves the active site open with free access to the FMN group. FAD The FAD bound to the PyrK subunit is in an unusual conformation because it folds into a U shape with the adenine and ribityl moieties almost parallel (Figure 6d). This is very different from the extended conformation normally observed in other FAD proteins. It differs also from the bent conformation of FAD in the E. coli flavodoxin reductase structure [27]. Contributing to the stabilization of this unusual conformation is the intramolecular hydrogen bond between two hydroxy groups from the two different ribose parts (Figure 6d and Table 2) and the intersubunit salt bridge [Glu-65(PyrDB)–Arg-53(PyrK)], which is conserved in all family 1B DHODs and is found at the bottom of the FAD binding pocket (Figures 5a and 6d). This interaction would obviously prevent FAD from taking its usual extended conformation. FMN can functionally replace FAD in the PyrK subunit of DHODB and the equivalent PyrDII subunit of tetrameric B. subtilis DHOD, but the binding is very weak [19, 20]. The hydrogen bonding interactions of the FAD group are listed in Table 2; they comprise both direct contacts to PyrK residues and to water molecules that serve as bridges to the PyrK subunit. Some of the water molecules surrounding the FAD group connect the adenine and ribose moieties of FAD to the backbone of Met-224 and Arg-52. Three arginine residues (52, 53, and 72) that are completely conserved among the PyrK proteins are found in the vicinity of the pyrophosphate group [14]. Only four direct hydrogen bonds are formed between the FAD isoalloxazine ring and the surrounding protein. In addition to the side chain of Ser-56, they involve the backbone atoms of Ser-56, Leu-70, and Arg-72. The most favorable electron transfer pathway shown in Figure 1 involves a transfer of electrons from the reduced FMN group via FAD to NAD⫹. This is opposite to the electron transfer in most NADPH ferredoxin reductases, which implies a difference in their FAD redox potentials. Among the factors that could influence the redox potential of FAD are its unusual conformation, the larger number of water molecules surrounding FAD in the PyrK subunit, and the difference in hydrogen bonds to N5 and O4 of the isoalloxazine ring system. In the ferredoxin reductase structures a tyrosine residue is part of the hydrogen bonding network involving N5 and O4; in PyrK these atoms are in contact with water molecules and backbone atoms. Comparison between the Native and Orotate Bound Structures Considering the difference in resolution and temperature between the native structure and the orotate complex structure,

Structure 1234

Figure 6. The Environment of the Two Flavin Groups and the Orotate Binding Site in DHODB (a) The environment of the FMN group in the uncomplexed PyrDB subunit with an open catalytic loop. Water molecules are shown as cyan spheres. (b) The DHODB-orotate complex structure showing the same view of the FMN group as in Figure 6a and containing a closed but slightly disordered catalytic loop. (c) A closeup view of the environment of the bound orotate in the complexed structure. (d) The environment of the FAD group in PyrK subunit in the uncomplexed structure. No significant changes were observed in the structure of the orotate complex.

we will focus only on the most prominent differences between the two structures. They are seen in the flexible loop carrying the catalytic cysteine residue and in the orotate binding site. The catalytic cysteine residue (Cys-135) is localized in the segment (133–142) between ␤4 and ␣4, which was well defined in the native structure and contained two short helical segments, ␣A and ␣E. Compared to the DHODA structure, it was in an open conformation where the catalytic, active Cys-135 is turned away from the orotate binding site. In contrast, the

Table 2. Hydrogen Bonds Connecting FAD to the PyrK Subunit and the Surrounding Water Molecules FAD Atom

Hydrogen Bond Contact

O2 N3 O4 N5 O2* O3* O4* O1P O2P AO1 AO2 AO2* AO3* AO4* AN1 AN3 AN6

Arg-B72 N (2.8), Wat-51 (2.9) Leu-B70 O (2.8) Wat-66 (3.4), Wat-126 (2.7) Ser-B56 N (2.8), Ser-B56 O␥ (2.9), Wat-66 (3.5) Pro-B54 O (2.7) Wat-51 (2.8), Wat-199 (2.7) FAD AO2* (2.8) Arg-B53 N⑀ (3.0), Gly-B79 N (2.7), Thr-B80 N (2.8) Thr-B80 O␥1 (3.0), Wat-142 (2.6) Arg-B53 N␩2 (3.0), Wat-114 (2.7) Wat-310 (3.0) FAD O4* (2.8), Wat-28 (2.9), Wat-34 (3.2) Wat-32 (2.7), Wat-34 (2.7) Wat-16 (3.3), Wat-310 (3.4) Wat-226 (3.4) Wat-28 (3.3) Wat-199 (3.34)

The values given in parentheses are the corresponding distances in A˚.

orotate-DHODB complex has a poorly defined electron density in this region; it is without the two helices and lacks residues 138–141. However, the traceable part of the map clearly shows that Cys-135 has shifted position; it is now oriented as in DHODA and is able to catalyze the reaction. Orotate stacks with the FMN group in the same way as in the equivalent complex of DHODA [10, 24], but some noteworthy differences between the complexed and uncomplexed structure are seen in the FMN environment (Figures 6a and 6b). In the orotate complex the side chain of Lys-170 has changed its orientation. It makes hydrogen bond interactions with the FMN group that are similar to those seen for the equivalent lysine residue in both the native and complexed structure of DHODA. The side chain of Met-247 has been reoriented so that its thioether group is unable to interact with FMN O3* and Lys-170. The most dramatic change is observed for Lys-48. This residue has been shown to be important for orienting the substrate in the active site [24]. In the native structure its side chain points away from the FMN group toward the PyrK subunit (Figures 5a and 6a), with the N⑀ atom about 3.5 A˚ from the C␦1 and C⑀1 atoms of Tyr-232 from the PyrK subunit. Lys-247 from the PyrK subunit and Lys-170 from the PyrDB subunit are almost symmetrically positioned around the aromatic plane of Tyr-232. In the orotate complex the side chain of Lys-48 has rotated so that it points toward the FMN group and makes hydrogen bonds with N5 and O4 of the isoalloxazine ring, as in DHODA (Figures 5b and 6b). It also forms a hydrogen bond to the carboxy group of orotate (Figure 6c). A superposition of the environment of Lys-48 in the two structures in Figure 5b shows that orotate binding also affects Tyr-232 from the PyrK

Dihydroorotate Dehydrogenase B 1235

Table 3. Data Collection and Phasing Statistics for the Various Data Sets Used to Solve the DHODB Structure

Data collection temperature (K) Soaking conditions Resolution (A˚) Observations Unique reflections Completeness (%) (I ⬎ 3␴I) Rmerge (%) Riso (%) Heavy-atom sites Phasing power (acentric/centric) Rcullis (acentric/centric)

Uranium

Platinum

288 2 mM, 24 hr 2.8 33,073 15,405 97.7 (58.4) 12.0 15.8 2 0.83/0.67 (1.50/1.13)a 0.86/0.80 (0.71/0.65)a

288 0.5 mM, 6 hr 3.0 16,854 11,554 89.8 (52.9) 12.0 15.1 2 0.54/0.57 (0.88/0.65)a 0.94/0.89 (0.88/0.84)a

In-House Native

Synchrotron Native

In-House Complex

Synchrotron Native

288

277

288

120

2.6 68,884 19,174 98.0 (79.3) 9.6

2.2 75,830 29,987 92.8/56.2 9.8

2.4 89,776 21,335 86.0 9.9

2.1 270,295 35,821 98.7 7.3

a Results for data up to 4 A˚. Rmerge is defined as ⌺|Ij ⫺ ⬍Ij⬎|/⌺Ij, where Ij is the intensity of an observation of reflection j and ⬍Ij⬎ is the average intensity for reflection j. Riso is defined as ⌺||FPH| ⫺ |FP||/⌺|FP|, where FPH is the structure factor amplitude of the derivative crystal and FP is that of the native crystal. The phasing power is the root-mean square (|FH|/E), where FH is the calculated structure factor amplitude due to scattering by the heavy atoms, and E is the residual lack of closure error. Rcullis ⫽ lack of closure/isomorphous difference.

subunit. This residue and both Phe-29 and Glu-31 from the PyrDB subunit have moved toward each other to fill the space occupied by Lys-48 in the native structure (Figure 5b). The hydrogen bonding patterns of orotate in DHODA and DHODB (Figure 6c) are almost identical; however, it should be noted that Asn-137 is not well defined in the DHODB-orotate complex. The conformational changes in the PyrDB subunit that are associated with the binding of orotate also cause some small changes in the hydrophobic contacts between the subunits, but they do not affect the hydrogen bond interactions (Table 1). Insight into Enzyme Catalysis and Electron Transfer Pathways The structures of the two Lactococcus lactis dihydroorotate dehydrogenases (DHODA and DHODB) enable us to explain many of the unique features that are characteristic for the family 1 DHODs. The FMN group in the PyrDB subunit is in an environment similar to the one seen in family 1A, and the two enzymes bind orotate in an almost identical manner. The difference in conformation of the loop carrying the catalytic cysteine residue in the free enzymes seems to be linked to a change in the conformation of the catalytically important lysine (Lys48) residue interacting with N5 from the isoallozaxine ring in DHODB. The reorientation seems to be linked to the presence of the PyrK subunit as it points right into the interface between the PyrDB and PyrK subunits in the free enzyme. This could explain why a similar reorientation is not seen in DHODA. The structural similarity of the PyrK subunit to enzymes belonging to the ferredoxin reductase superfamily lends additional support to the identification of the outermost domain in the heterotetramer as the binding site for NAD⫹. The present structure explains also why the ability to use NAD⫹ as electron acceptor is connected to the PyrK subunit. The available space to accommodate NAD⫹ and the close proximity of the NAD binding domain to the quite exposed isoalloxazine ring of FAD would facilitate electron transfer from FAD to NAD⫹. A detailed model for this process awaits knowledge of the structure with bound NAD⫹ or NADH. The electron transfer pathway outlined in Figure 1 is in accordance with the structure of DHODB. The separation of the

cofactors in DHODB shows distinct similarity to the arrangement of redox centers in the fumarate reductase respiratory complex [36]. The cofactors in this complex, which include FAD and [2Fe-2S] are separated by 11–13 A˚. The fact that the [2Fe-2S] cluster is found at approximately the same distance from N5 of the isoalloxazine rings of FMN and FAD makes it likely that it plays a role in electron transfer. The relative position of the three redox centers (Figure 5a) suggests a possible electron transfer pathway. Both flavin groups are oriented so that their dimethylbenzene moieties are pointing toward each other and the [2Fe-2S] cluster. Each of the isoalloxazine ring systems has a phenylalanine and methionine side chain in its vicinity in an arrangement that almost displays 2-fold symmetry, which is a characteristic feature of the interface between the two subunits. The phenylalanine side chains are the closest neighbors to the methyl groups of the FMN and FAD. The shortest distance from Phe-274 of PyrDB to FMN C7M is approximately 4.0 A˚, whereas C8M is 4.4 A˚ from Met-70. An equivalent arrangement is seen at the PyrK part of the interface. Phe-39 from the PyrK subunit has short contacts to the [2Fe2S] cluster (6–7 A˚), C7M (4–5 A˚) in the FAD isoalloxazine ring, and the methyl group of Met-224. The latter group is very close to the methyl groups of the dimethylbenzene moiety (4.7 A˚). Since both the phenylalanine and methionine residues are completely conserved among the family 1B enzymes, they may be involved in the electron transfer pathway between the FMN and FAD redox centers. Biological Implications In de novo biosynthesis of pyrimidine nucleotides, the fourth and only redox step in the pathway is catalyzed by dihydroorotate dehydrogenase (DHOD). All DHODs are flavoproteins, and according to their sequences, they can be classified into two major families, which also reflect their location in the cell. Family 1 enzymes are cytosolic, and those belonging to family 2 are membrane associated. The nature of the second substrate, the natural electron acceptor, is remarkably different between the DHODs from different organisms. This property makes DHODs a promising target for drug developments, as it may be possible to develop compounds that inhibit pyrimidine bio-

Structure 1236

Table 4. Refinement Summary

Resolution (A˚) R factor (%) Rfree (%) Nonhydrogen protein atoms Flavin atoms (FAD/FMN) Iron-sulphur cluster atoms Orotate atoms Water molecules

Synchrotron 277 K

Synchrotron 120 K

Complex 288 K

40–2.2 20.2 25.6 4,285 31/53 4

20–2.1 19.5 23.7 4,300 31/53 4

195

466

38.5–2.4 19.0 24.1 4,256 31/53 4 11 144

0.011 1.76

0.009 1.43

0.009 1.64

21.6/53.2 25.5/55.5 12.2/42.2 32.0

21.2/42.3 22.8/43.2 13.7/25.2 24.1

38.9

39.6

25.1/55.1 28.6/57.8 16.0/37.7 32.1 20.6 38.0

Rmsd from Ideality Bond lengths (A˚) Angles (⬚) Average B Factors (A˚2) Main-chain PyrD/PyrK Side-chain PyrD/PyrK Flavin atoms (FMN/FAD) Iron-sulphur cluster Orotate Water molecules

synthesis in some organisms and leave others unaffected. The drug leflunomide, which has been approved as a prescription drug for treatment of rheumatoid arthritis and may have applications for treatment of other inflammatory conditions and as an immunosuppressive drug [5, 6, 8], has been shown to inhibit human DHOD [7]. An analog of leflunonomide binds to the N-terminal region of the enzyme [4], and though it has been suggested that leflunomide inhibits the DHOD through competition with the respiratory quinones [7], other measurements do not support this interpretation [37]. Lactococcus lactis and the closely related bacterium Enterococcus faecalis are so far the only two organisms known to contain two DHODs, proven to function in pyrimidine biosynthesis. The enzymes are representatives of the two family 1 subgroups 1A and 1B. Both of these bacteria grow well in milk and have applications in the dairy industry. It is not known if their content of two types of DHODs is due to their ability to grow in milk, which contains very little iron and high amounts of orotic acid and riboflavins. In this study we have completed the structural picture of the family 1 DHODs by presenting the structure of DHODB, the first known structure of a family 1B enzyme. DHODB is a heterotetramer composed of two PyrDB proteins, which are the dehydroorotate dehydrogenase proper, and two iron-, sulfur-containing PyrK proteins. The dimer of PyrDB that forms the core of the heterotetramer is very similar to the family 1A enzyme from Lactococcus lactis (DHODA). Each PyrK subunit is closely associated with a PyrDB. PyrK serves as the binding site for the cofactor FAD, and a possible binding site close to FAD for the electron acceptor NAD⫹ can be deduced from the structure. This explains why FAD must be present for NAD⫹ to function as an electron acceptor [19]. Not only the overall dimeric arrangement of the PyrDB subunits but also the environment of the FMN group are very similar to the structure of DHODA. The catalytic cysteine residue is located in a loop on top of the active site in both enzymes. In the uncomplexed DHODB this loop has moved away and left the active site exposed to the substrate, whereas it is closed, like in DHODA, when DHODB is complexed with the product orotate. This change in the conformation of the loop is accompanied by significant shifts of side chains. These shifts are most pronounced for Lys-48,

which is important for substrate binding and is able to interact with the FMN N5, which gets protonated in the reaction. The amino acid sequence of the PyrDB subunit is very closely related to a segment of the mammalian dihydropyrimidine dehydrogenases, which initiate the degradation of the pyrimidine ring structure by reducing the 5,6-double bond at the expense of NADPH [38]. In fact, the sequence of the PyrDB subunit of DHODB is more closely related to the (degradative) dihydropyrimidine dehydrogenases (sequence identity about 32%) than it is related to the membrane bound (biosynthetic) dihydroorotate dehydrogenases belonging to family 2 (sequence identity less than 20%) [1]. In contrast to this, the PyrK subunit of DHODB shows no sequence similarity to the dihydropyrimidine dehydrogenases, which also are iron-sulfur cluster–containing proteins [39, 40]. Instead, a structural alignment revealed that the PyrK protein belongs to the NADPH ferredoxin reductase family, whose members have other proteins as natural redox partners and often are involved in shunting electrons between NAD(P)H and respiratory or photosynthetic electron transfer chains. The intersubunit interactions between PyrK and PyrDB may also contribute to the understanding of the function of these proteins since no structural information is yet available about their protein-protein complexes. The interface between the PyrDB and PyrK subunits contains a [2Fe-2S] cluster, which is placed close to and almost symmetrically between the two flavin cofactors, FMN from PyrDB and FAD from PyrK. The biological function of DHODB and its ability to use NAD⫹ as an electron acceptor depends on the three cofactors being in close proximity, and we have been able to propose possible electron pathways that involve residues conserved among the family 1B DHODs. Experimental Procedures Enzyme Preparation and Crystallization The heterotetrameric Lactococcus lactis DHODB was expressed and purified as described previously [16, 19]. The brown, rod-shaped crystals of L. lactis DHODB were grown by the hanging-drop vapor diffusion technique from solutions containing 2.4 M ammonium sulfate and 0.1 M sodium acetate (pH 4.6) [41]. The crystals belong to the rhombohedral space group R32. The asymmetric unit contains one PyrDB subunit and one PyrK subunit, and this gives an estimated crystal solvent content around 50%. We pre-

Dihydroorotate Dehydrogenase B 1237

pared heavy-atom derivatives by adding a solution of the heavy-atom compound in water directly to drops containing crystals in their original mother liquor. To be able to collect data under cryogenic conditions, we soaked the crystals for a few seconds in a cryoprotecting solution containing 2.4 M ammonium sulfate, 0.1 M sodium acetate (pH 4.6), and 30% glycerol. Data Collection and Processing The 2.6 A˚ native data set, the 2.4 A˚ orotate complex data set, and the two heavy-atom derivative data sets were collected with an R-axis II imaging plate system. As an X-ray source, a Rigaku RU200 rotating anode generator with a Cu-target was used (␭ ⫽ 1.5418 A˚). The generator was operated at 50 kV and 180 mA, and a graphite monochromator was used to select CuK␣ radiation. A 0.5 mm collimator was used for the data collections. Two further native data sets were collected later at DESY in Hamburg. Both data collections were performed with a MAR Research imaging plate detector. The first data set extended to 2.2 A˚ resolution and was collected at beamline X11 (␭ ⫽ 0.912 A˚) on a crystal cooled to 277 K. The second data set was collected under cryogenic conditions (120 K) at the BW7B beamline (␭ ⫽ 0.8469 A˚) at a resolution of 2.1 A˚. The three data sets conform to the previously established rhombohedral space group R32 and unit cell dimensions for the native crystal [40]. The hexagonal unit cell dimensions for the cryocooled crystals are a ⫽ b ⫽ 119.9 A˚ and c ⫽ 80.3 A˚; the equivalent values for the complex crystal are 202.4 A˚ and 80.9 A˚. In all cases the diffraction images were processed with DENZO and SCALEPACK [42]. Structure factors were derived from the reflection intensities with the program TRUNCATE [43]. Details for the data collection and processing are given in Table 3. Structure Determination and Refinement The structure of native DHODB was solved using the multiple isomorphous replacement method based on two heavy-atom derivatives. We obtained the first derivative by soaking a crystal with uranyl nitrate, UO2(NO3)2, and two heavy-atom sites could be identified from the 2.8 A˚ difference Patterson map. The second derivative was obtained with red platinum, 2-hydroxyethanethiolato(2,2’,2“-terpyridine)platinum(II) nitrate, and two Pt sites were revealed in the cross difference Fourier electron density between the native data set phased from the first derivative. The final MIR phasing process included these two derivatives scaled to the in-house native data set. Scaling was done with the program SCALEIT [43]. The heavy-atom positions, occupancies and temperature factors were refined simultaneously with the program MLPHARE [43]. All data from 40 A˚ up to the maximum resolution of the two derivative data sets were employed without any inclusion of the anomalous differences. The phasing powers and Rcullis for the two derivatives are listed in Table 3. For data in the range from 40 to 2.8 A˚, the overall mean figure of merit was 0.24 (0.42 for data up to 4 A˚). The program DM [43] was used to improve the MIR phases for the data extending to 2.8 A˚ in 50 cycles of solvent flattening (55% solvent content) and histogram matching. The resulting 3 A˚ resolution electron density map was not immediately interpretable. However, after several months of unsuccessful experiments to prepare other heavy-atom derivatives, we returned to the 3 A˚ solvent-flattened map. We eventually located the eight helices and the eight strands of the barrel and identified the well-defined electron density at the top of the barrel that corresponds to FMN. This allowed superposition of a monomer of DHODA into the electron density for DHODB and substitution of the side chains to match those of DHODB. It was much more difficult to build the PyrK subunit into the electron density as it is less well ordered than the PyrDB subunit. An initial model for the PyrK subunit was built following a procedure consisting of model building followed by phase combination with SIGMAA [44] and simulated annealing refinement with XPLOR [45]. The residues were included a few at a time, and phase-combined maps were calculated to 3 A˚ with the inhouse native data set, while all refinement and subsequent generation of electron density maps was carried out to 2.2 A˚ with the synchrotron native data set. After approximately half of the PyrK model had been built, the search for structurally similar proteins [26] revealed that the PyrK subunit was similar to the structure of phthalate dioxygenase reductase [29]. This structure was used for guidance in assigning the chain direction of the ␤ strands in the PyrK subunit and the connections between secondary-structural elements. The subsequent structure refinement involved careful model building, phase combination, positional refinement, simulated annealing, and B factor refinement, with the maximum resolution (2.2 A˚) of the synchrotron native data set used for the later refinement stages. Three positions in the electron

density map were inconsistent with the published sequence of the PyrDB subunit [9]. Resequencing of the pyrDb gene confirmed these discrepancies; the amino acids were Ala-123 (not arginine), Asp-255 (not valine), and Ala266 (not arginine). The final model from this data set has been deposited as 1EP1. The data collected under cryogenic conditions and extending to 2.1 A˚ were used in a refinement with CNS [46] starting with the model derived from the 2.2 A˚ data set. The final model from the 2.1 A˚ data included 466 water molecules and was refined to R ⫽ 19.5% and Rfree ⫽ 23.7%. It has been deposited as 1EP3. Table 4 gives a summary of the final refinement statistics. Of the residues, 89.1% are in the most favored regions of the Ramachandran plot (PROCHECK, [47]). Ile-216 of the PyrDB subunit is the only residue in a disallowed region, although it has very good electron density to support its φ/␺ values. The DHODB-orotate complex structure was solved from the 2.2 A˚ native model (1EP1). The structure was refined with X-PLOR [45] in the resolution range 38–2.4 A˚ to a final R factor of 19.0% and Rfree ⫽ 24.1%. The refinement statistics are shown in Table 4. The electron density corresponding to residues 138–141 in the PyrDB subunit was so poorly defined that it did not allow any modeling of these residues. The model of the orotate complex has been deposited with the code 1EP2. The illustrations of the molecular structure of DHODB were prepared with MOLSCRIPT [48], BOBSCRIPT [49], and RASTER3D [50]. Acknowledgments The authors are very grateful for the funding from the Danish National Research Foundation, which enabled this research. The authors thank Dr. Finn S. Nielsen for providing the first samples of DHODB for structure determination. We thank the EMBL outstation in Hamburg for beam time and Dr. Ryepnewski, Dr. Paul Tucker, and Dr. Victor Lamzin for their help during the experiments. Received: July 28, 2000 Revised: September 29, 2000 Accepted: October 3, 2000 References 1. Jensen, K.F., and Bjo¨rnberg, O. (1998). Evolutionary and functional families of dihydroorotate dehydrogenases. Paths to Pyrimidines 6, 20–28. 2. Bjo¨rnberg, O., Gru¨ner, A.C., Roepstorff, P., and Jensen, K.F. (1999). The activity of Escherichia coli dihydroorotate dehydrogenase is dependent on a conserved loop identified by sequence homology, mutagenesis, and limited proteolysis. Biochemistry 38, 2899–2908. 3. Rawls, J., Knecht, W., Diekert, K., Lill, R., and Lo¨ffler, M. (2000). Requirements for the mitochondrial import and localization of dihydroorotate dehydrogenase. Eur. J. Biochem. 267, 2079–2087. 4. Liu, S., Neidhardt, E.A., Grossman, T.H., Ocain, T., and Clardy, J. (2000). Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents. Structure Fold. Des. 8, 25–33. 5. Infante, R., and Lahita, R.G. (2000). Rheumatoid arthritis. New diseasemodifying and anti-inflammatory drugs. Geriatrics 55, 39–40. 6. Prakash, A., and Jarvis, B. (2000). Leflunomide: a review of its use in active rheumatoid arthrithis. Drugs 58, 1137–1164. 7. Davis, J.P., Cain, G.A., Pitts, W.J., Magolda, R.L., and Copeland, R.A. (1996). The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35, 1270–1273. 8. Tugwell, P., et al., and Thompson, A. (2000). Clinical improvement as reflected in measures of function and health-related quality of life following treatment with leflunomide compared with methotrexate in patients with rheumatoid arthrithis: sensitivity and relative efficiency to detect a treatment effect in a twelve-month, placebo-controlled trial. Arthrithis Rheum. 43, 506–514. 9. Andersen, P.S., Jansen, P.J., and Hammer, K. (1994). Two different dihydroorotate dehydrogenases in Lactococcus lactis. J. Bacteriol. 176, 3975–3982. 10. Rowland, P., Nielsen, F.S., Jensen, K.F., and Larsen, S. (1997). The crystal structure of the flavin containing enzyme dihydroorotate dehydrogenase A from Lactococcus lactis. Structure 5, 239–252. 11. Andersen, P.S., Martinussen, J., and Hammer, K. (1996). Sequence analysis and identification of the pyrKDbF operon from Lactococcus lactis

Structure 1238

12.

13. 14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26. 27.

28.

29.

30.

31.

32.

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