Structural Basis of Leukotriene B4 12-Hydroxydehydrogenase/15-Oxo-prostaglandin 13-Reductase Catalytic Mechanism and a Possible Src Homology 3 Domain Binding Loop*

The bifunctional leukotriene B4 12-hydroxydehydrogenase/15-oxo-prostaglandin 13-reductase (LTB4 12-HD/PGR) is an essential enzyme for eicosanoid inactivation. It is involved in the metabolism of the E and F series of 15-oxo-prostaglandins (15-oxo-PGs), leukotriene B4 (LTB4), and 15-oxo-lipoxin A4 (15-oxo-LXA4). Some nonsteroidal anti-inflammatory drugs (NSAIDs), which primarily act as cyclooxygenase inhibitors also inhibit LTB4 12-HD/PGR activity. Here we report the crystal structure of the LTB4 12-HD/PGR, the binary complex structure with NADP+, and the ternary complex structure with NADP+ and 15-oxo-PGE2. In the ternary complex, both in the crystalline form and in solution, the enolate anion intermediate accumulates as a brown chromophore. PGE2 contains two chains, but only the ω-chain of 15-oxo-PGE2 was defined in the electron density map in the ternary complex structure. The ω-chain was identified at the hydrophobic pore on the dimer interface. The structure showed that the 15-oxo group forms hydrogen bonds with the 2′-hydroxyl group of nicotine amide ribose of NADP+ and a bound water molecule to stabilize the enolate intermediate during the reductase reaction. The electron-deficient C13 atom of the conjugated enolate may be directly attacked by a hydride from the NADPH nicotine amide in a stereospecific manner. The moderate recognition of 15-oxo-PGE2 is consistent with a broad substrate specificity of LTB4 12-HD/PGR. The structure also implies that a Src homology domain 3 may interact with the left-handed proline-rich helix at the dimer interface and regulate LTB4 12-HD/PGR activity by disruption of the substrate binding pore to accommodate the ω-chain.

Here we report the crystal structure of the LTB 4 12-HD/ PGR, the binary complex structures with NADP ϩ , and the ternary complex structure of LTB 4 12-HD/PGR with NADP ϩ and 15-oxo-PGE 2 . Based on our findings, we propose a catalytic mechanism of the enzyme. Additionally the structure suggests SH3 domain (Src homology domain 3)-mediated regulation of LTB 4 12-HD/PGR activity. Upper column, biosynthesis of specific prostaglandins is dependent on cell types; most cells (PGE 2 ), brain and mast cells (PGD 2 ), uterus (PGF 2␣ ), endothelial cells (PGI 2 , or prostacyclin), and platelets (TXA 2 ) (12). Leukotrienes are synthesized in inflammatory cells such as polymorphonuclear leukocytes, macrophages, and mast cells, and lipoxins are produced in airway epithelial cells or at inflammation sites (11)(12)(13)(14). Lower column, eicosanoid action is mediated by specific cell surface receptors and is rapidly inactivated by enzymes (2)(3)(4). Two major pathways for LTB 4 inactivation are reported; the oxidative pathway initially catalyzed by LTB 4 20-hydroxylase is present in inflammatory cells (49), whereas in most other cells inactivation is catalyzed by LTB 4 12-HD/PGR (2). LTB 4 12-HD/PGR is emphasized in bold underlined text. The inactivation pathway of PGF 2␣ is identical to that of PGE 2 .

EXPERIMENTAL PROCEDURES
Expression, Purification, and Crystallization-Guinea pig-LTB 4 12-HD/PGR fusion protein was expressed in Escherichia coli strain BL21(DE3) as described (20). A Se-Met derivative was also expressed in E. coli strain BL21-CodonPlus (DE3)-RP-X (Invitrogen) at 20°C. Glutathione S-transferase-LTB 4 12-HD/PGR was purified with a glutathione-Sepharose 4B column (Amersham Biosciences, Uppsala, Sweden), and the N-terminal glutathione S-transferase was cleaved with thrombin (Wako) for 20 h at room temperature, followed by further purification by cation exchange chromatography on a Mono S column (Amersham Biosciences) with 0 -150 mM NaCl gradient. The sample (20 mg/ml) was crystallized by the batch method using the automated crystallization system TERA (24) by mixing equal volumes of protein solution (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 1 mM dithiothreitol) and precipitating solution (100 mM 4-morpholineethanesulfonic acid (pH 5.5-6.8), 17.5-27.5% polyethylene glycol 4000, and 50 mM MgCl 2 ) at 18°C. The binary complex contained 10 mM NADP ϩ in the crystallization solution, whereas the ternary complex included 10 mM NADP ϩ and 3.3 mM 15-oxo-PGE 2 . Crystals appeared within 1 week and belonged to the monoclinic space group P2 1 . The solvent content of the crystals is 48%, calculated from a V m value of 2.37 (25).
Structural Determination and Refinement-x-ray diffraction data were collected at 100 K at SPring-8 with an R-AXIS V detector (Rigaku) for the Se-Met derivative and a Jupiter CCD (Rigaku) for the remaining data sets (Table I). For cryo-cooling, the Se-Met derivative crystal was soaked in the precipitating solution including trehalose, increasing the concentration stepwise from 5% (w/v) up to 30% (w/v), whereas the other crystals were treated with a 1:1 paraffin/Paratone-N mixture (Hampton Research). All data sets were processed using HKL2000 (26). For the Se-Met derivative, 17 of the possible 20 Se sites were identified using Patterson methods in a SOLVE/RESOLVE (27). The phases were improved by non-crystallographic symmetry averaging and solvent flattening with DM (28). The overall figure of merit for the final experimental phases was 0.75. The structural models were built using O (29) and refined by simulated annealing and energy minimization methods with CNS (30). The refinement of other structures was initiated with rigid body refinement in CNS (30) using the atomic coordinates of the Se-Met derivative structure and refining iteratively with O (29) and CNS (30).
Chloride ions were identified by identifying water molecules with very low B-factors compared with surrounding protein residues or those with residual high electron density peaks in ͉F o ͉ Ϫ ͉F c ͉ difference Fourier maps (Table I). If the surrounding coordination sphere was consistent with chloride, the water molecule was changed to a chloride ion. For the apo structure, residues 12-16, 107-108, and 244 -250 of one subunit were not included in the model because of poor or no electron density. For the ternary complex, the N-terminal methionine residue of one subunit is missing. In most models, a part of the N-terminal thrombin digestion site, Ser-Pro-Glu-Phe, was also identified. Several regions exhibited multiple conformations, including residues 124, 213, 280, and 281 in the apo structure and residues 272-274 in the ternary complex. The geometries of the final models were verified using PROCHECK (31), and only one residue, Leu-105 of apo structure, is in generous region of Ramachandran plot. Leu-105 is located in a disordered flexible loop. The average B-factors of the NADP ϩ binding site residues (shown in Fig. 3A) are 24.1, 20.6, and 17.3 Å 2 in the apo, binary, and ternary complexes, respectively, and those of NADP ϩ are 17.7 and 15.0 Å 2 in the binary and ternary complexes, respectively. The average B-factors of the residues lining the -chain binding pore (in Fig. 3D) are 26.4, 24.0, and 20.3 Å 2 in the apo, binary, and ternary complexes, respectively, and that of the -chain of the ternary complex is 38.8 Å 2 . In the ternary complex, only the -chain of 15-oxo-PGE 2 was defined in the electron density map. The -chain was modeled in the keto-form, because resolution was too low to allow distinction between keto-and enolate-forms.
Absorption Spectrum of the Ternary Complex in Solution-Two aliquots of a ternary complex solution (1.6 mg/ml enzyme, 10 mM NADP ϩ , and 0.5 mM 15-oxo-PGE 2 ) were incubated for 3 h in the crystallization solution without the precipitant, polyethylene glycol 4000. An equal volume of solutions with or without 10 mM NADPH was then mixed into the ternary complex solution. A half-volume of 2 N NaOH was further added into both solutions to allow measurement of the residual 15-oxo-PGE 2 (32). The incubated ternary complex solution was ultrafiltered by Millipore Ultrafree®-MC centrifugal filter units (5,000 nominal molecular weight limit) to examine whether the colored material(s) is bound to LTB 4 12-HD/PGR. Absorption spectra were measured for each reaction mixture solution using a Shimadzu MultiSpec-1500. The reaction mixture without the enzyme was used as a reference. For the ternary complex solutions without NADPH addition, a 10ϫ diluted sample was measured after NaOH addition.

RESULTS AND DISCUSSION
Overall Structure of LTB 4 4 12-HD/PGR is a homodimer with 2-fold symmetry (Fig. 2). As a member of the MDR family, it exhibits a typical alcohol dehydrogenase fold (36). Each monomer comprises a catalytic domain (residues 1-122 and 293-329) and a nucleotide binding domain (residues 123-292) arranged around a Rossmann fold. The substrates, NADP ϩ and 15-oxo-PGE 2, are bound at the active site clefts located between the catalytic and nucleotide binding domains (Fig. 2). NADP ϩ Binding to LTB 4 12-HD/PGR in the NADP ϩ Complex Structure-In the binary complex structures, the bound NADP ϩ is in the anti configuration of the ribose-nicotinamide glycosidic bond, as commonly observed in alcohol dehydrogenases (36) (Fig. 3A). NADP ϩ forms a number of hydrogen bonds with LTB 4 12-HD/PGR, some of which are mediated by ordered water molecules. The amino acid residues lining the NADP ϩ binding sites are well conserved in the LTB 4 12-HD/PGRs of all species but not among the MDR family (Fig. 4). This may explain why the substrates directly interact with the bound NADP ϩ (H).

12-HD/PGR-LTB
A well ordered hydrogen bond network around the 2Ј-hydroxyl group of the nicotine amide ribose contains two water molecules (W1 and W2) that are proposed to facilitate catalysis by stabilizing an enolate of the 15-carbonyl group of 15-oxo-PGE 2 through hydrogen bond formation (Fig. 3A). Tyr-245, whose hydroxyl group may be a proton donor to the 2Ј-hydroxyl group, is conserved among known LTB 4 12-HD/PGRs from various species (20,22,37,38). The hydroxyl group of Tyr-245 also makes a hydrogen bond with the main chain nitrogen of Ala-241. Water W1 is a proton acceptor from the main chain nitrogen of Tyr-49 as well as a proton donor to the bound water W2. W2 is a proton donor to the carboxyl group of Asp-47 and the phosphate group of the NADP ϩ .
The LTB 4 12-HD/PGR, NADP ϩ , and 15-Oxo-PGE 2 Ternary Complex-The LTB 4 12-HD/PGR ternary complex crystal has a faint brown color (Fig. 3B), as observed in the enzyme solution with 20 mM NADP ϩ and 0.5 mM 15-oxo-PGE 2 at neutral pH. The ternary complex solution mixture turned a faint brown color over time with a maximum absorption peak of 475 nm, characteristic of an enolate intermediate (Fig. 3C). The solution became colorless upon addition of 10 mM NADPH, whereas the addition of 0.4 M NaOH to the ternary complex mixture resulted in a much denser brown color with an absorption peak at 500 nm (Fig. 3C). These color changes have been utilized as an enzyme assay to quantify the remaining 15-oxo-PGE 2 (32). The addition of NADPH propagates the reduction of the 15-oxo-PGE 2 complex (faint brown) to 13,14-dihydro-15-oxo-PGE 2 (colorless). Ultrafiltration analysis showed that the faint brown chromophore in the ternary complex mixture was retained in the LTB 4   In the crystal structure of the ternary complex, only the -chain (from C12 to C20) of 15-oxo-PGE 2 was defined in the electron density (Fig. 3D). The ␣,␤-unsaturated 15-oxo moiety of the bound 15-oxo-PGE 2 is in direct van der Waals contact with the nicotine amide ring of the bound NADP ϩ (Fig. 3E). The -chain tail is buried in the hydrophobic pore composed of loop ␤E-␣F and helices ␣1 and ␣F at the dimer interface (Figs. 2 and 3D). The 15-carbonyl group of the -chain forms hydrogen bonds with the 2Ј-hydroxyl group of the nicotine amide ribose and the bound water W1 (Fig. 3E). The carbon atoms C12 to C16 including the 15-carbonyl oxygen in the -chain are co-planar with a geometry consistent with the configuration of the conjugated ␣,␤-unsaturated carbonyl. The plane lies parallel to the nicotine amide ring of NADP ϩ with aorbital interaction, which may produce the faint brown chromophore as a charge transfer complex (Fig. 3E). The conjugated 15carbonyl of the -chain is stabilized by a hydrogen bonding network allowing formation of the enolate intermediate (Fig.  3D). An enolate formation should cause electron deficiency of the conjugated C13 atom as the putative hydride ion acceptor FIG. 3. NADP ؉ and 15-oxo-PGE 2 binding to LTB 4 12-HD/PGR. A, a stereo view of the bound NADP ϩ in the binary complex structure. The ͉F o ͉ Ϫ ͉F c ͉ simulated annealing omit electron density map is contoured at 3.0 (orange) and 6.0 (cyan). The carbon atoms of NADP ϩ are yellow, and those of side chains involved in NADP ϩ binding are green. Single-letter codes are used for amino acid residues. The water molecules, oxygen, nitrogen, and phosphorus atoms are colored pink, red, blue, and orange, respectively. The hydrogen bonds are indicated by dashed lines. The main chain nitrogen or carbonyl groups interacting with the bound NADP ϩ are labeled by residue numbers. B, a photo of the ternary complex crystals (0.3 ϫ 0.2 ϫ 0.1 mm). C, absorption spectrum changes of a ternary complex solution mixture. A 1:10 dilution of the ternary mixture after NaOH addition was measured. D, stereo view of the -chain of bound 15-oxo-PGE 2 in the ternary complex structure. The carbon atoms of the -chain of 15-oxo-PGE 2 are colored in cyan, and NADP ϩ in yellow. The residues from the same subunit of the bound NADP ϩ around the -chain hydrophobic pore are colored in green, and those from the other subunit are in magenta. ͉F o ͉ Ϫ ͉F c ͉ simulated annealing omit electron density map is contoured at 2.4 (orange) and 3.5 (blue). The residues Ala-241, Tyr-245, and Met-248 are located on the loop ␤E-␣F. Tyr-273 was refined as two conformers. E, a stereo view of the interaction between the -chain and NADP ϩ . The hydrogen bonds and the orbital interactions between the nicotine amide ring and the conjugated double bonds of the -chain are indicated in dashed lines. The shortest distance of the orbital interactions is 3.2 Å between the carbonyl oxygen atom of 15-oxo-PGE 2 and the nitrogen atom of the nicotine amide ring.
in the reductase reaction. The distance of 3.8 Å between the C13 atom and the C4 position of the nicotine amide ring, the proposed hydride donor, allows for hydride transfer (36) (Figs. 3E and 5).
Hydrophobic residues from both subunits comprise the -chain binding pore at the dimer interface (Fig. 3D). The alkyl chain between C16 and C20 of the -chain is surrounded by Tyr-49 and Ile-52 on helix ␣1, and Ala-241, Tyr-245, and Met-248 on the ␤E-␣F loop and Ile-271 on the edge of strand ␤F of the NADP ϩ -bound subunit, and by Pro-257(B), Glu-258(B), Ile-261(B), and Tyr-262(B) on the helix ␣F of the countersubunit (B). These residues are likely to recognize the -chain and determine LTB 4 12-HD/PGR specificity, as they are highly conserved among LTB 4 12-HD/PGRs but not among MDR families (20,22,37,38) (Fig. 4). 4 12-HD/PGR-Based on the LTB 4 12-HD/PGR crystal structures, a ␤-ketoreductase reaction mechanism is proposed (3, 20, 23) (Fig. 5). 15-Oxo-PGE 2 and 15-oxo-LXA 4 have a conjugated double bond, which is in equilibrium with the ␣,␤-conjugated enolate as the reaction intermediate. The 2Ј-hydroxyl group of the nicotine amide ribose and the water W1 promote enolate formation by forming hydrogen bonds with the O15 carbonyl oxygen of the -chain, which stabilize the intermediate (Figs. 3D and 5). The electrondeficient C13 atom in the conjugated enolate may be directly attacked by the A-side (pro-R) hydride anion (H a ) of the C4 atom of the nicotine amide of NADPH (Fig. 5). The hydride anion should then transfer to the pro-S position of the enolate intermediate (39). Thus, the C14 atom of the enolate anion intermediate would be protonated at the final step of the reaction. The mechanism of the ␤-ketoreductase reaction is identical to that of an enoyl-acyl carrier protein reductase of Mycobacterium tuberculosis, whose enolate anion intermediate is stabilized by hydrogen bonds with the 2Ј-hydroxyl group of the nicotine amide ribose (40). This catalytic mechanism may be a common feature of ␤-ketoreductase reactions.

15-Oxo-PGE 2 and 15-Oxo-LXA 4 Reductase, and LTB 4 Oxidase Reaction Mechanisms of LTB
For the LTB 4 oxidase reaction of LTB 4 12-HD/PGR, some hydrophilic groups would be required as catalytic residues. In the LTB 4 12-HD/PGR structure, Tyr-262 from the other subunit is the only candidate catalytic residue considering both the orientation of the side chain and conservation among species (20,22,37,38) (Fig. 3D).
Domain Movement-Moderate domain movements are observed upon NADP ϩ binding compared with the apo form. The NADP ϩ binding site is wider in both the binary and ternary complexes than in the apo form. Superimposing the nucleotide binding domains of the apo and ternary complex structures of LTB 4 12-HD/PGR revealed that the distances between the center of masses of each domain are 0.41 Å in the nucleotide binding domain and 1.14 Å in the catalytic domain, respectively. This corresponds to a domain rotation of ϳ1.6 o . The most notable structural difference is in the binding site of the adenine moiety of NADP ϩ , residues 316 -323 on the helix ␣3 and the loop ␣3-␤10. Although there are no direct interactions between these residues and NADP ϩ (Fig. 3A), the region is forced to open upon NADP ϩ binding as a result of steric hindrance with the adenine moiety of NADP ϩ . This local conformational change may induce the entire domain movement. Such domain movement is also observed in Thermus thermophilus quinone oxide reductase (QOR) (41) and Candida tropicali enoyl thioester reductase (42), although in these cases domain movement operates to close the NADP ϩ binding site upon NADP ϩ binding. A similar domain movement has also been reported for a zinc-dependent alcohol dehydrogenase (43). It is possible that this phenomenon is common to members of the alcohol dehydrogenase family.
The orientations of each subunit differ between LTB 4 12-HD/ PGR and QOR (Fig. 6). Superimposing the nucleotide binding domains of one subunit of LTB 4 12-HD/PGR and E. coli. QOR (44) revealed that the distance of the center of masses of each nucleotide binding domain is 1.1 Å in the superimposed and 9.3 Å in the countersubunit. Fifty-three residues (16.1%) are identical between guinea pig LTB 4 12-HD/PGR (329 amino acid(s)) and E. coli QOR (328 amino acid(s)) ( Fig. 4). In the dimer of LTB 4 12-HD/PGR, both active sites are closer than those of QOR (Fig. 6). In addition, there is a cavity between subunits (Figs. 2 and 6). Interestingly, the -chain binding pore in LTB 4 12-HD/PGR is in the dimer interface neighboring the cavity (Fig. 2).  (Fig 7A). The sequence of the PPII helix, Leu-251-Pro-Pro-Gly-Pro-Ser-256, is conserved among LTB 4 12-HD/PGRs in all known species (20,23,37,38) (Fig. 7A). Parallels can be drawn to that of the p85 subunit of phosphatidylinositol 3-kinase, which is bound by the SH3 domain of Fyn (45) (Fig. 7B). In the case of LTB 4 12-HD/PGR, Pro-252, Pro-253, and Pro-255 occupy the PϪ1, P0, and P2 positions, respectively, and may be recognized by a proline-rich binding groove of an SH3 domain. SH3 recognition sequences often include a basic residue at either the N-or C-terminal side of the P-X-X-P motif (46). In LTB 4 12-HD/PGR Arg-247 is at the N-terminal side of the PPII helix and forms part of a ␤-turn structure. The guanidino group of Arg-247 occupies a similar position to that of the p85 subunit phosphatidylinositol 3-kinase structure (45) (Fig. 7B). These similar structures suggest that the PPII helix in LTB 4 12-HD/ PGR binds to a specific SH3 domain to regulate enzymatic activity, as described for 5-lipoxygenase. The activity of 5-lipoxygenase is regulated by a tyrosine kinase (47), which modulates binding of the SH3 domain of the growth factor-bound receptor protein 2 (Grb2) to the proline-rich region of 5-lipoxygenase (48).
We propose that binding of an SH3 domain to LTB 4 12-HD/ PGR modulates enzyme activity by causing domain re-orientation. The open conformation of the dimer may allow adequate changes of the dihedral angles of the loop ␤E-␣F residues on both sides of the PPII helix, such that an SH3 domain can be bound to each subunit (Fig. 7C). The SH3 domain binding followed by domain re-orientation should disturb the hydrophobic pore, which accommodates the -chain of 15-oxo-PGE 2 . This would result in an LTB 4 12-HD/PGR⅐SH3 complex, which is no longer able to bind substrate (Fig. 7C). Therefore, we propose that there is a currently unknown protein with an SH3 domain that recognizes the PPII helix and thus regulates LTB 4 12-HD/ PGR activity. The eicosanoid degradation pathway may thus be regulated by a protein with an SH3 domain, similar to that seen for the LTB 4 synthesis pathway.