The Structure of Neurospora Crassa 3-Carboxy-Cis,Cis-Muconate Lactonizing Enzyme, a ␤ Propeller Cycloisomerase

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Structure, Vol. 10, 483–492, April, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0969-2126(02)00744-X The Structure of Neurospora crassa 3-Carboxy-cis,cis-Muconate Lactonizing Enzyme, a ␤ Propeller Cycloisomerase

Tommi Kajander,1 Michael C. Merckel,1 ing the structural basis for different alternatives for catal- Andrew Thompson,2 Ashley M. Deacon,3 ysis in MLEs could therefore aid in understanding and Paul Mazur,4 John W. Kozarich,4,6 engineering enzymes for degradation of xenobiotic pol- and Adrian Goldman1,5 lutants, such as fluoroaromatics. 1 Institute of Biotechnology MLEs convert cis,cis-muconates to muconolactones Research Program in Structural Biology in soil microbes as part of the ␤-ketoadipate pathway. and Biophysics This aerobic catabolic pathway converts aromatics, such University of Helsinki as the breakdown products of lignin, through catechol FIN-00014 Helsinki and protocatechuate to citric acid cycle intermediates. Finland The two major branches of the ␤-ketoadipate pathway 2 EMBL are the catechol branch, with cis,cis-muconate lactoniz- Grenoble Outstation ing enzymes, and the protocatechuate branch (Figure 38024 Grenoble 1), with carboxy-cis,cis-muconate lactonizing enzymes France (CMLEs). 3 Stanford Synchrotron Radiation Laboratory The evolutionary origins of these enzymes are surpris- Menlo Park, California 94025 ingly divergent, both in terms of sequence similarity and 4 Merck Research Laboratories functional properties (Table 1). Three different classes of Rahway, New Jersey 07065 lactonizing enzymes can be distinguished. The bacterial MLEs contain an unusual TIM barrel [3] and are closely related to mandelate racemase [4]. The bacterial CMLEs belong to the fumarase class II family [5]. Unlike the Summary prokaryotic enzymes, eukaryotic MLEs and CMLEs are related by divergent evolution to each other [1] and differ Muconate lactonizing enzymes (MLEs) convert cis,cis- from the bacterial ones, both in sequence and functional muconates to muconolactones in microbes as part of properties: the eukaryotic MLEs and CMLEs are metal the ␤-ketoadipate pathway; some also dehalogenate cofactor independent, like bacterial CMLEs, but cata- muconate derivatives of xenobiotic haloaromatics. There lyze a syn addition reaction (Table 1) [1, 6, 7], like bacte- are three different MLE classes unrelated by evolution. rial MLEs. We present the X-ray structure of a eukaryotic MLE, Previous work, particularly on Pseudomonas putida Neurospora crassa 3-carboxy-cis,cis-muconate lac- MLE (PpMLE) and on a related enzyme, mandelate ra- ˚ tonizing enzyme (NcCMLE) at 2.5 A resolution, with a cemase [3, 4, 8], has revealed that catalysis by bacterial seven-bladed ␤ propeller fold. It is related neither to MLEs requires a general base and acid, acting to pro- bacterial MLEs nor to other ␤ propeller enzymes, but duce an enol or enolate intermediate in a reverse of a is structurally similar to the G protein ␤ subunit. It ␤ elimination reaction [8–10]. The magnitude of electro- reveals a novel metal-independent cycloisomerase philic or electrostatic catalysis in the 1,2-addition-elimi- motif unlike the bacterial metal cofactor MLEs. To- nation reaction is still unclear [9, 11]; for example, is gether, the bacterial MLEs and NcCMLE structures concerted acid-base catalysis required to stabilize the comprise a striking structural example of functional reaction intermediate or is it achieved by electrostatic convergence in enzymes for 1,2-addition-elimination effects in the active site? Comparison of the Neurospora of carboxylic acids. NcCMLE and bacterial MLEs may crassa CMLE (NcCMLE) metal-independent catalytic enhance the reaction rate differently: the former by motif with that of PpMLE, with a Mn2ϩ cofactor, could electrophilic catalysis and the latter by electrostatic give valuable insight into the possible enzyme catalytic stabilization of the enolate. solutions for such a reaction. The structure of NcCMLE, solved for selenomethionine-substituted (SeMet) protein, reveals a seven-bladed ␤ Introduction propeller, most similar to the WD repeat family member G protein ␤ subunit (G␤) [12, 13] but with a novel re- The muconate lactonizing enzymes (MLEs) are the only peating sequence pattern of conserved hydrophobic known enzymes where it appears that members of sev- positions. We have also identified a family of ORFs dis- eral different superfamilies catalyze the same 1,2-addi- tantly related to the eukaryotic MLEs, which we suggest tion-elimination reactions [1], both with and without a are also ␤ propellers and catalyze ␤ elimination reactions metal cofactor. They thus provide an interesting exam- because the probable catalytic residues are all fully con- ple of convergent functional evolution in enzyme cataly- served among the sequences. Based on substrate dock- sis. The bacterial MLEs also include enzymes capable ing and site-directed mutagenesis, we have been able of dehalogenation of chlorinated substrates [2]. Know- to identify His148 and Glu212 as key catalytic residues,

5 Correspondence: [email protected] 6 Present address: Activx Biosciences, 11025 North Torrey Pines Key words: addition-elimination reaction; ␤ propeller; convergent Road, La Jolla, California 92037. evolution; muconate lactonizing enzyme; X-ray structure Structure 484

tetramer with overall dimensions of approximately 110 A˚ ϫ 110 A˚ ϫ 50 A˚ (Figure 2B). The asymmetric unit actually contains two tetramers related by a noncrystallographic 2-fold. The rmsd of C␣s between monomers in a tetramer is 0.05–0.09 A˚ , and between tetramers is 0.532 A˚ (thus the two tetramers have slightly different conforma- tions). The propeller domain is closed through the con- nection of the N and C termini in the last blade, with a “Velcro” closure [14]. In the case of CMLE, the closure is achieved by a “3 ϩ 1” division of strands from N and C termini (Figure 2A) [15]. The “top side” of the propeller [16], where loops connect the blades, contains two loops which fold out from the propeller. The larger of these loops (residues 241–255) extends out to the solvent, partly folding on itself in a twisted ␤ hairpin-like structure (Figure 2A) and extends at the other end to one of the intratetramer 2-fold subunit interfaces (see below). As in some other seven-bladed ␤ propellers (Tup1, G␤) [12, 13, 17], there is a large solvent channel down the central 7-fold axis of the propeller in CMLE. It is disrupted in CMLE by a single layer of aromatic amino acids (Trp44, Trp197, Phe98) located near the N termini of the A strands of blades II, V, and III (Figure 3A). This layer thus splits the enzyme, on one side into a very large aqueous cavity, and on the other into a smaller pocket of charged residues, which we believe is the active site (see below; Figures 3A and 3B). The seven blades of CMLE superimpose with an rmsd of 0.9–1.8 A˚ for the C␣ atoms of the blades. As a conse- quence, NcCMLE also has a weak identifiable sequence repeat (Figure ): a set of six conserved hydrophobic and aromatic residues, and additional, less well-conserved Figure 1. The ␤-Ketoadipate Pathway in Prokaryotic and Eukaryotic Microorganisms positions in the alignment of its blades. These pack The protocatechuate branches in eukaryotes and bacteria are dis- together (Figure 4), forming a large part of the hydropho- tinct while the catechol pathway is identical [42]. PO, protocatechu- bic contacts between blades such that the aromatic- ate-3,4-dioxygenase; CO, catechol-1,2-dioxygenase; MLE, cis,cis- aliphatic-aromatic (Figure 4, red) sequence in the B muconate lactonizing enzyme; CMLE, 3-carboxy-cis,cis-muconate strands makes contact with three other conserved hy- lactonizing enzyme; CMD, 4-carboxymuconolactone decarboxyl- drophobic positions (Figure 4, yellow) in the C strands ase; MI, muconolactone isomerase; ELH, enol lactone hydrolase; in an adjacent blade (Figure 4B). For instance, Trp55 CMH, 3-carboxymuconolactone hydrolase; CoA, coenzyme A. from the B strand of blade I is in van der Waals contact with Val109 (B strand) and Val126 (C strand) from blade enabling us to propose a model for substrate binding II as well as with Asn53 of the same strand, and Trp62 and catalysis in NcCMLE and compare it to models of of blade I is in contact with Phe124 of blade II C strand catalysis in bacterial MLEs. (Figure 4B). In this way, the hydrophobic core of the CMLE propeller is built up around the repeating, con- Results served amino acids. There are 31 such fully buried residues (accessible surface area Ͻ5%), constituting 27% Description of the Overall Structure of the population of fully buried residues and 8.5% of The NcCMLE monomer forms a compact single domain all the residues in CMLE. structure (41 kDa), folding into a seven-bladed ␤ propeller (Figure 2A). Consistent with earlier results showing Subunit Interfaces that the biologically active form of NcCMLE is a homotet- There are two types of 2-fold subunit interfaces in the ramer [1], the subunits are arranged into a D2-symmetric CMLE tetramer, the A-B and C-D subunit interfaces

Table 1. Classes of MLEs

Stereo- Metal a Enzyme class chemistry requirement Sequence similarity kcat/Km ϫ 106 5.3ف Bacterial MLEs Syn Divalent cation Enolase superfamily ϫ 107 1.5ف Bacterial CMLEs Anti — Class II fumarases Eukaryotic MLEs ϫ 107 2.5ف and CMLEs Syn — none a As exemplified by Pseudomonas putida MLE and CMLE, and Neurospora crassa CMLE [1, 5, 7, 27]. Structure of ␤ Propeller Lactonizing Enzyme 485

Figure 2. The Overall Structure of NcCMLE (A) The fold of an NcCMLE monomer as shown from the top side of the propeller with active site residues (labeled) and loop 241–255 (cyan) shown. The blades are numbered I–VII with the 3 ϩ 1 strands “Velcro” closure shown in the last blade: the C-terminal strand is in blue and N-terminal part of the VIIth blade is in red. Strands in each blade are labeled A–D. (B) The CMLE tetramer viewed along one of its 2-fold axes. The two other 2-folds and the respective subunit interfaces are indicated with arrows, and the monomers A, B, and C are labeled. (C) A stereo image of a portion of the 3FoϪ2Fc electron density map for the refined model showing the fit of the key active site residues His148, Glu212, and Arg196, Arg256, and Arg274. Structure 486

Figure 3. Side View of the NcCMLE ␤ Propeller Structure (A) A side view of the CMLE ␤ propeller showing how Trp44 and Trp196 (yellow) block the CMLE central channel. The catalytic residues (red and blue) have been labeled and the conserved Arg196 and Arg274 are also shown (blue). (B) A slice plane through the CMLE monomer molecular surface along the central channel with electrostatic potential displayed (Ϫ7 kT [red] to ϩ7 kT [blue]) as drawn and calculated with GRASP. The arrow indicates the location of the active site.

(1778 A˚ 2 surface area) and the B-C and D-A interfaces face, as the tetramer has a channel running through it (1116 A˚ 2 surface area). The third 2-fold axis (A-C or D- along this axis (Figure 2B). The A-B interface is formed B) perpendicular to the first two does not form an inter- by hydrophobic interactions, including four prolines in

Figure 4. Structure-Based Sequence Alignment of NcCMLE Blades I–VII (A) Conserved (5/7 similar), hydrophobic (φ), and aromatic (␸) positions (red and yellow), and additionally other positions with four of the same or very similar residues (e.g., E/D) are indicated. Strands in each blade are boxed and strand regions are indicated with an arrow above the alignment. (B) Packing interactions of the conserved repeat residues (red and yellow as in [A]). The box shows residues from blades I and II. Structure of ␤ Propeller Lactonizing Enzyme 487

Figure 5. Alignment of the Related Sequences to NcCMLE and TcMLE The SWISS-PROT or TrEMBL accession numbers for the ORFs: S. pombe O59681, E. coli (YhbE) P52697, B. aphidicola (YhbE) P57380, L. lactis (“orfB”) O86281, B. subtilis (YkgB) O34499, S. aeruginosa Q9HWH7, S. coelicor (B) Q9RD79, S. coelicor (A, secreted) Q9EX09. Fully conserved residues, red; consensus (7/10 conserved), yellow. Active site residues are indicated with upward triangles below each position; the CMLE repeat positions are boxed and arrows above the sequences indicate positions of the CMLE ␤ strands. Numbering is according to NcCMLE. the large top face loop of both subunits and residues fore in structure-based sequence comparisons of evolu- from blade VI (see Supplemental Table S1 at http:// tionarily unrelated ␤ propeller proteins (see below) [15]. images.cellpress.com/supmat/supmatin.htm). The center of this interface is packed with polar residues, of The Location of the Active Site which Ser302s hydrogen bond to each other across The active site of the CMLE ␤ propeller is most likely the interface in the middle (Supplemental Table S1). In located on the top side on the central axis, for the follow- addition, an interesting array of charged residues makes ing reasons. First, this is the location of the active site hydrophobic contacts both adjacent to and in the inter- in all other known ␤ propeller enzymes. Second, the top face. Subunit A Arg346 and Arg349 are stacked between side surface of the propeller in CMLE has a pocket of subunit A Trp333 and subunit B Gln281, and A-Arg346 polar side chains (Tyr115, His148, Arg196, Glu212, Arg256, forms hydrogen bonds with B-Gln281 across the interface Asp258, Arg274, and His310) and positive surface po- (Supplemental Table S1). A PIPES molecule is asymmet- tential capable of attracting and binding the negatively rically bound at this charged interface to residues charged substrate (Figure 3B), whereas the large bottom Arg349 and Glu331 on one side and Arg349 on the other side cleft has negative surface potential and lacks monomer. The B-C/D-A interface is more compact and clearly identifiable carboxylate binding sites. Third, we regular, formed by hydrophobic contacts and hydrogen could dock the substrate into the top side pocket (see bonds involving blade IV (Supplemental Table S1). The below) in a manner consistent with known chemistry large loop of residues 241–255 (Figure 2A) probably has and binding requirements. an important structural role in forming the A-B/C-D sub- The only homologous protein of known function for unit interface. NcCMLE in the sequence databanks is its sister enzyme Trichosporon cutaneum MLE (TcMLE). In addition, we Similarities to Other ␤ Propeller Proteins found eight complete ORFs (two fragments were ex- The structure of NcCMLE aligns best with the WD repeat cluded from analysis) from bacteria and yeast that align proteins G␤ (266 atoms, rmsd 2.09 A˚ ) and Tup1 (267 with NcCMLE with sequence identities ranging from 8% atoms, rmsd 2.05 A˚ ), and with nitrous oxide reductase to 19% (Figure 5). Multiple alignment of these ten se- (265 atoms, 2.02 A˚ ) [18]. CMLE also resembles the WD quences identifies His148, Glu212, Arg196, and Arg274, repeat proteins in having a solvent-filled channel. CMLE all in the smaller top side surface pocket, as 4 of only does not, however, have significant sequence identity 5 fully conserved residues (Figure 5). It therefore seems to these or any other ␤ propeller proteins (Յ10%). The reasonable to assume that these 4 residues are in the canonical “WD” positions in G␤ are often aromatic-polar active site. in CMLE, but such similarities have been observed be- To confirm that this was indeed so, we made the Structure 488

Figure 6. The Fit of the Modeled Substrate into the NcCMLE Active Site and Proposed Reaction Mechanism (A) The 3-carboxy-cis,cis-muconate substrate docked into the CMLE active site. Putative hydrogen bonds are shown with dashed lines, and hydrophobic residues are colored yellow to show the polar charges versus hydrophobic binding pockets of the active site cleft. (B) A surface potential presentation (as in Figure 2) of the active site cleft with the substrate docked.

(C) Numbering of the substrate atoms; the intramolecular nucleophilic attack by 1-CO2 is indicated by the bent arrow. (D) Schematic drawing of the catalytic interactions and the presumed stepwise reverse reaction of an E2 elimination reaction in NcCMLE. The nucleophilic ring closure (step 1, fast) and the completion of the proton addition from His148 and keto-enol tautomerization (step 2, rate limiting) are specified. mutants Glu212Ala and His148Ala. Both variants were had no (Ͻ0.01%) catalytic activity against 3-carboxy- native like. CD spectroscopy (see Supplemental Figure cis,cis-muconate, while Glu212Ala showed very low ac- Ϫ1 Ϫ1 S1) showed that the subunit structure was unaffected; tivity (kcat Ͻ 0.35 s ; Ͻ0.1% of wild-type kcat ϭ 382 s the native and mutant curves superimpose. Native PAGE [1]; data not shown). We therefore conclude that these showed that the quaternary structure, too, was unaf- are indeed key active site residues; there is no change fected (see Supplemental Figure S1). Catalytic activity in structure but dramatic effects on catalytic activity. was, on the other hand, dramatically affected. His148Ala This has allowed us to develop a testable catalytic

Figure 7. Alignment of the MLE and CMLE Active Sites The MLE (cyan) and CMLE (dark gold) active site residues around the docked substrates are shown, with the reactive substrate double bonds aligned, indicating the different binding modes with respect to the positive charge in the two active sites and the conserved hydrophobic pockets between the active sites. Structure of ␤ Propeller Lactonizing Enzyme 489

Table 2. Data Collection and Refinement Statistics CMLE SeMet 1a CMLE SeMet 2

Space group P212121 P212121 Unit cell (A˚ )aϭ 90.4 b ϭ 152.1 c ϭ 247.1 a ϭ 92.3 b ϭ 160.6 c ϭ 237.5 Wavelength 0.9786 A˚ 0.9137 A˚ Resolution 20–3.0 A˚ 20–2.5 A˚ Unique reflections 54480 122302

Rsym (last shell) 6.3% (11.2%) 5.0% (15.6%) I/␴ (last shell) 18.5 (6.6) 13.4 (3.1) Completeness (last shell) 89.8% (60.6%) 92.7% (75.8%)

Rwork/Rfree 32.3%/33.9% 21.4%/25.1% Number of atoms in the model 20392 23781 Number of water molecules — 713 Average main chain B factor — 26.1 A˚ 2 Average side chain B factor — 27.1 A˚ 2 Rmsd in bond lengths 0.0098 A˚ 0.014 A˚ Rmsd in bond angles 1.5Њ 1.8Њ a Preliminary refinement before molecular replacement.

model, consistent with all known experimental data (see Catalytic Mechanism Discussion). The reaction catalyzed by MLEs is the reverse of the elimination of an ␣ proton of a carboxylate group. This reaction is both kinetically and thermodynamically unfa- Discussion vorable [9], as the ␣ proton of the carboxylate has a

high pKa, and the enolate intermediate formed in the The NcCMLE structure is a typical seven-bladed ␤ pro- reaction is very unstable. One possibility is that the eno- peller with almost ideal geometry [19]. The blades of the late is stabilized by formation of an “enolic” intermediate CMLE propeller align well on each other, revealing a through concerted acid-base catalysis and protonation novel but degenerate sequence repeat (Figure 4). The of the enolate [9]. Alternatively, the thermodynamic bar- degeneracy is in contrast to those ␤ propellers where rier could be overcome by electrostatic stabilization and the sequence repeat consists primarily of polar residues, lowered medium polarity [11] without formation of the Gly, Pro, and aromatic residues [15], as in the WD repeat enolic intermediate. If it is the former, a general acid superfamily [20]. In such cases, the sequence motifs are may be required (such as PpMLE Glu372); if it is the unique because they appear to define hydrogen bonds latter, such residues presumably have a different func- and turns. When ␤ propeller structures have conserved tion, such as stabilization of the network of charges in hydrophobic positions, the motifs are more degenerate the active site and hydrogen bonding to the substrate. (above and [21]), possibly because only the type of con- The energy-minimized substrate could be modeled tact between adjacent ␤ sheets is conserved rather than into the active site (Figure 6) with a near “lock-and-key” specific residues. It has been noted earlier that there fit. The substrate fits so that Glu212 is within 2.5–3 A˚ of Ϫ should be no need for special residue patterning for a the 6-CO2 (Figure 6) and could be hydrogen bonded ␤ propeller, but the fold can be determined by general to it. Glu212 could therefore be the general acid as geometric features, for example, periodicity and twist long as it is protonated [9]. This is possible because its [19, 22]. carboxylate is half buried (accessible surface area 22%) The active site of CMLE propeller resides, as is typical, in a hydrophobic environment (next to Leu164). The ob- at the N terminus of the inner strands. Based on the servation that the Glu212Ala variant is almost com- symmetry of the structure and the known properties of pletely inactive along with the fact that Glu212 is one ␤ propellers (see Results), it seemed likely that the active of absolutely conserved residues in related sequences site would be close to the central 7-fold pseudosymme- (Figure 5) supports this idea. try axis. The active site is, however, only the second Chemical considerations, however, suggest that the largest cleft on the surface, the largest cleft by far being general base, not the acid, is essential for catalysis [9]. on the opposite face of the propeller (Figure 3). This is The reaction can, in principle, be accomplished by elec- somewhat unusual; about 80% of the time, enzymes trostatic catalysis without the acid, as has been sug- use the largest cleft as the active site [23]. However, the gested for the bacterial MLE homolog o-succinylben- bottom side large cleft in NcCMLE could not bind a zoate synthase (OSBS) [25]. The His148Ala variant is carboxylate substrate. Furthermore, it should rather be completely inactive; His148 is completely conserved seen as the central channel typical for ␤ propellers, but among related sequences (Figure 5) and so His148 is a one that is blocked by the aromatic Trp197 and Trp44 good candidate for the general base. Structurally, it is at the N termini of two of the A strands (Figure 3). The the only candidate: the N⑀2 of His148 is about 3 A˚ from active site is located on the other side of the channel, the C5 to which the proton is added (Figure 6), and on the top side of the platform formed by these residues proton addition by His148 would occur in syn to produce (Figure 3). This is similar to the ␤ propeller of methyl- the correct enantiomer, 4(S)-3-ML. His148 is the only puta- amine dehydrogenase [24]. tive base positioned to add a proton with the correct regio- Structure 490

and stereospecificity; this requirement, as in MLE (cf., sites that probably bind similar types of substrates and [9, 10]), eliminates other candidates. Finally, Thatcher perform similar reactions. Further mutagenesis studies, and Cain [26] found that the Aspergillus niger CMLE can based on this structural hypothesis, will increase our be inactivated by histidine-blocking reagents, while the understanding of metal-free catalysis in MLEs and the presence of a competitive inhibitor could prevent the role of each conserved active site residue. inactivation, consistent with our structural and biochem- ical results. Biological Implications In our model, the conserved arginines (Arg196 and Arg274) are hydrogen bonded to one of the substrate The muconate lactonizing enzymes (MLEs) form a group Ϫ 1-CO2 oxygens. The presence of the hydrophobic Trp10 of enzymes related by multiply convergent evolution: and Ile97 close to the carboxylate group may raise the three structurally very different protein classes are uti- nucleophilicity of this group (Figure 6). Arg256 (not con- lized to catalyze an analogous reaction. We earlier reserved in TcMLE) positions the substrate by forming an ported the structure of a bacterial MLE [3, 29] which ion pair with the 3-CO2. The presence of Arg256 can contains an ␣/␤ barrel; here, we show that N. crassa explain why the Km of NcCMLE for cis,cis-muconate was CMLE (eukaryotic) is a seven-bladed ␤ propeller, and 2 ϫ 103 higher than that for carboxy-cis,cis-muconate. sequence homology shows that the third MLE class, bacterial CMLEs, is related to all ␣-helical proteins (the class II fumarase superfamily) [5]. This indicates how Active Sites in Bacterial and Fungal flexible and amenable protein structures are for evolu- Lactonizing Enzymes tion and adaptation to new environments. The MLE structures provide a rare example of metal- The MLEs are also the only known enzymes where it dependent catalysis and its metal-independent alterna- appears that members of several different superfamilies tive. Given the large positive point charge on the metal catalyze the same 1,2-addition-elimination reactions, ion, it might follow that the amount of electrostatic stabi- both with and without a metal cofactor [1]. They thus lization available for catalysis is less in the metal-inde- provide an interesting example of convergent functional pendent NcCMLE. As we lack complex structures for evolution in enzyme catalysis. From the structure com- both the MLEs, this remains an open question, but we parison of NcCMLE and PpMLE it is evident that both speculate that this is indeed so based on our mutagene- utilize similar residues to achieve catalysis: Lys/His and sis data and sequence and structure comparisons. Glu as the catalytic base and acid or electrophile. Com- In brief, in PpMLE, the enol intermediate and its transi- parison of the structures supports the idea that it is tion state are positioned toward three positive charges: possible for the same catalytic problem to have two 2ϩ ˚ Ϫ Mn within 4.5 A from the 6-CO2 of the substrate [28], related catalytic answers, depending on the amount of and Lys167 and Lys273 within 3.5 A˚ in our current mod- electrostatic or electrophilic stabilization present. els (see Figure 7) [10]. In NcCMLE, by contrast, the Sequence comparisons also show that NcCMLE is presumed catalytic groups His148 and Glu212 are not the first structure solved from a group of ten bacterial accessible for any intermediate forming near the center and fungal sequences, the others with unknown func- of positive charge density in the active site (Arg196 and tions. Because the key catalytic and substrate binding Arg274). Further, contradicting the requirements of elec- NcCMLE residues (His148, Glu212, Arg196, and Arg256) trostatic catalysis, medium polarity in the NcCMLE ac- are 4 of only 5 fully conserved residues in all sequences, tive site will not be very low, as the active site is solvated we believe that all of the ORFs catalyze similar reactions and polar around the conserved Arg196 and Arg274. In with similar substrates. the bacterial MLEs, the environment probably has a low The structure of NcCMLE reveals a novel catalytic dielectric constant, as the active site is believed to be function for a ␤ propeller domain, and a structure very 2A˚ ) with the unrelated WD repeatف buried and closed upon substrate binding [29]. similar (rmsd for C␣’s Based on our models, PpMLE and CMLE appear to proteins G␤ and Tup1 [12, 13, 17] and nitrous oxide bind their substrates using similar residues (Figure 7). reductase [18]. In both, a hydrophobic pocket is formed predominantly Finally, the bacterial MLEs also include enzymes ca- by aromatic residues, which are similarly disposed around pable of dehalogenation of chlorinated substrates [2]. the substrates. The pocket formed by CMLE Tyr115 and Knowing the structural basis for different alternatives Phe114 finds its counterpart in PpMLE Ile54 and Tyr59 for catalysis in MLEs could therefore aid in understanding (Figure 7). Together with the Schlo¨ mann group, we have and engineering enzymes for degradation of xenobiotic shown [10, 27] that Ile54, Tyr59, and Phe329 (Figure 7) pollutants, such as fluoroaromatics. The binding site hy- are important determinants of substrate specificity in drophobic pocket also seems rather similar in NcCMLE bacterial MLEs [10, 27]. and bacterial MLEs, suggesting that substrate specific- The NcCMLE structure is the first ␤ propeller enzyme ity is governed by a restricted set of residues that might known to catalyze a 1,2-addition-elimination reaction. be transportable between two unrelated systems. This It will be interesting to compare this structure and mech- concept may also be useful for engineering MLEs with anism to that of the third class of MLEs, which have altered substrate specificities. evolved from the class II fumarase family (D. Ringe, personal communication) [5]. The sequence alignment Experimental Procedures (Figure 5) shows that NcCMLE provides the archetypal Production and Purification of SeMet-Labeled NcCMLE structure and catalytic motif for a new sequence family. SeMet-CMLE was expressed in the methionine auxotrophic E. coli All are likely to be ␤ propellers with well-conserved active strain BL21 (DE3) B834 using the New Minimal medium [30] and 50 Structure of ␤ Propeller Lactonizing Enzyme 491

mg/l SeMet and was purified as native CMLE [1], except that 1 Sequence and Structural Alignments mM EDTA and 5 mM ␤-mercaptoethanol were added to buffers to Sequences were identified with iterative PSI-BLAST [39] searches prevent proteolysis and oxidation, and for minor modifications in of the databanks, as implemented at NCBI (http://www.ncbi.nlm.nih. chromatography conditions [31]. gov/BLAST). Sequence alignments were done with ClustalX [40], and hand edited. Structures were aligned using O [41]. Mutagenesis, Characterization of Variants, and Activity Measurements Acknowledgments The CMLE variants His148Ala and Glu212Ala were prepared with the Stratagene QuickChange mutagenesis kit according to the man- We thank Markus Tujula and Petri Va¨ ha¨ koski for computer support, ufacturer’s instructions. Oligonucleotides used for site-directed Maria Rehn for technical assistance, Professor Peter C. Kahn and mutagenesis for His148Ala were 5Ј-CAC-TGG-CAT-CGC-CGG-CAT- Dr. Ilkka Kilpela¨ inen for advice with circular dichroism measure- GGT-GTT-CG-3Ј (forward) and 5Ј-CGA-ACA-CCA-TGC-CGG-CGA- ments, and Sirpa Valjakka for secretarial help. T.K. wishes especially TGC-CAG-TG-3Ј (reverse) and for Glu212Ala, 5Ј-TGC-CCT-GAT- to thank the participants of the 1999 EMBO MAD workshop. The GGC-GGC-CGG-CAA-CCG-3Ј (forward) and 5Ј-CGG-TGG-CCG- Academy of Finland, grants 69520 and 63252, supported this work. GCC-GCC-ATC-AGG-GCA-3Ј (reverse). Mutagenesis results were verified by sequencing. Protein purification was performed as Received: September 25, 2001 above. Enzyme activity and kinetics for the Glu212Ala variant were Revised: January 23, 2002 measured by following the decrease in absorbance at 270 nm with Accepted: February 4, 2002 20 mM PIPES (pH 7.0), 0.1 mM 3-carboxy-cis,cis-muconate using an extinction coefficient of 6000 M-1 cmϪ1 for the substrate, which References takes into account the residual absorbance of the product at 270 nm [1]. CD spectra were measured on a Jasco J-720 spectropolari- 1. Mazur, P., Henzel, W.J., Mattoo, S., and Kozarich, J.W. (1994). meter. The samples were diluted in pure water. Data were collected 3-Carboxy-cis,cis-muconate lactonizing enzyme from Neuros- witha1mmcuvette, with five scans for each spectrum at 298 K. pora crassa: an alternate cycloisomerase motif. J. Bacteriol. 176, 1718–1728. Crystallization and Data Processing 2. Schmidt, E., and Knackmuss, H.-J. (1980). Chemical structure The SeMet protein crystallized under the same conditions as the and biodegradability of halogenated aromatic compounds: con- native [32], except that the ␤-mercaptoethanol concentration was version of chlorinated muconic acids into mateoylacetic acid. increased to 5 mM to protect selenomethionine from being oxidized. Biochem. J. 192, 339–347. The space group for SeMet-CMLE was P2 2 2 with the unit cell 3. Goldman, A., Ollis, D.L., and Steitz, T.A. (1987). Crystal structure 1 1 1 ˚ dimensions as for the native protein, except in the crystal from which of muconate lactonizing enzyme at 3 A resolution. J. Mol. Biol. the initial phases were obtained (Table 2) [31]. Data were indexed 194, 143–153. and integrated using the HKL package (Table 2) [33]. 4. Neidhart, D.J., Kenyon, G.L., Gerlt, J.A., and Petsko, G.A. (1990). Mandelate racemase and muconate lactonizing enzyme are mechanistically distinct and structurally homologous. Nature Structure Determination 347, 692–694. ˚ The selenium sites were located by direct methods at 3.5 A with 5. Williams, S.E., Woolridge, E.M., Ransom, S.C., Landro, J.A., Shake’n’Bake v. 2.0 [34] using the anomalous differences of the Babbitt, P.C., and Kozarich, J.W. (1992). 3-Carboxy-cis,cis-mu- peak wavelength. The resulting phases for the 60 Se positions found conate lactonizing enzyme from Pseudomonas putida is homol- were refined with MLPHARE [35] and the NCS operators were deter- ogous to the class II fumarase family: a new reaction in the mined manually to solve the protein structure with the help of density evolution of a mechanistic motif. Biochemistry 31, 9768–9776. averaging and phase extension [31]. After initial refinement in the 6. Kirby, G.W., O’Laughlin, G.J., and Robins, D.J. (1975). The ste- crystal form for which the phases were solved (Table 2), molecular reochemistry of the enzymatic cyclisation of 3-carboxymuconic replacement was used to place the structure into the dominant acid to 3-carboxymuconolactone. J. Chem. Soc. Chem. Com- crystal form using AMoRE [35, 36]. After rigid body refinement, the mun. 1975, 402–403. R factor was 37.2%, indicating that the solution was likely to be 7. Chari, R.V.J., Whitman, C.P., Kozarich, J.W., Ngai, K.-L., and correct. Refinement was carried out with CNS [37]. After initial cycles Ornston, L.N. (1987). Absolute stereochemical course of the of rigid body refinement, torsion angle dynamics with a starting 3-carboxymuconate cycloisomerases from Pseudomonas put- ϭ temperature of 5000 K resulted in Rwork/Rfree 30.2%/30.9%. ida and Acinetobacter calcoaceticus: analysis and implications. Applying an anisotropic B factor correction and bulk solvent correc- J. Am. Chem. Soc. 109, 5514–5519. tion during refinement lowered the R factors by 3% to 26.9%/27.7%. 8. Landro, J.A., Gerlt, J.A., Kozarich, J.W., Koo, C.W., Shah, V.J., The final model included all residues and 713 water molecules, a Kenyon, G.L., Neidhart, D.J., Fujita, S., and Petsko, G.A. (1994). Ϫ2 PIPES molecule at one 2-fold interface of each tetramer, one SO4 The role of lysine 166 in the mechanism of mandelate racemase ␤ per monomer, and two -mercaptoethanols attached to Cys292 and from Pseudomonas putida: mechanistic and crystallographic Cys317 of each monomer. Most of the waters were built with wARP/ evidence for stereospecific alkylation by (R)-␣-phenylglycidate. ARP [38]. Initially, the individual monomers related by noncrystallo- Biochemistry 33, 635–643. graphic symmetry (NCS) were restrained to be similar with a weight 9. Gerlt, J.A., and Gassman, P.G. (1992). 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