Leukotriene A4 Hydrolase: Selective Abrogation of Leukotriene B4 Formation by Mutation of Aspartic Acid 375

Leukotriene A4 hydrolase: Selective abrogation of leukotriene B4 formation by mutation of aspartic acid 375

Peter C. Rudberg*, Fredrik Tholander*, Marjolein M. G. M. Thunnissen†, Bengt Samuelsson*, and Jesper Z. Haeggstro¨ m*‡

*Department of Medical Biochemistry and Biophysics, Division of Chemistry II, Karolinska Institutet, S-171 77 Stockholm, Sweden; and †Department of Biochemistry and Biophysics, University of Stockholm, Arrhenius Laboratories A4, S-106 91 Stockholm, Sweden

Contributed by Bengt Samuelsson, February 14, 2002

Leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14-cis-eico- Glu-296 and Tyr-383 acting as the base and proton donor, satetraenoic acid) hydrolase (LTA4H)͞aminopeptidase is a bifunc- respectively. Moreover, in a recent study, Glu-271 was identified tional zinc metalloenzyme that catalyzes the ﬁnal and rate-limiting as the recognition site for the N-terminal amino group of the step in the biosynthesis of leukotriene B4 (LTB4, 5S,12R-dihydroxy- peptidase substrate (8). 6,14-cis-8,10-trans-eicosatetraenoic acid), a classical chemoattrac- Unlike the aminopeptidase activity, the epoxide hydrolase tant and immune modulating lipid mediator. Two chemical fea- activity is restrained by suicide inactivation that involves binding tures are key to the bioactivity of LTB4, namely, the chirality of the of LTA4 to Tyr-378, which is located within a 21-residue active 12R-hydroxyl group and the cis-trans-trans geometry of the con- site-peptide denoted K21 (9, 10). The epoxide hydrolase reaction jugated triene structure. From the crystal structure of LTA4H, a of LTA4H (i.e., the conversion of LTA4 to LTB4) is also unique hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 was in the sense that the stereoselective introduction of water to the identiﬁed in a narrow and otherwise hydrophobic pocket, believed carbon backbone of LTA4 occurs several methylene units away to bind LTA4. In addition, Asp-375 belongs to peptide K21, a from the epoxide moiety, presumably proceeding by means of a previously characterized 21-residue active site-peptide to which carbocation intermediate (11). The structural requirements for LTA4 binds during suicide inactivation. In the present report we controlling such a reactive species in the active site and for used site-directed mutagenesis and x-ray crystallography to show obtaining sufficient stereochemical specificity are of fundamen- that Asp-375, but none of the other candidate residues, is specif- tal biochemical as well as enzymological interest. However, only ically required for the epoxide hydrolase activity of LTA4H. Thus, very few residues have been shown to be involved in the epoxide mutation of Asp-375 leads to a selective loss of the enzyme’s ability hydrolase activity of LTA4H. In fact, no single amino acid to generate LTB4 whereas the aminopeptidase activity is pre- residue specifically required for the epoxide hydrolase activity served. We propose that Asp-375, possibly assisted by Gln-134, acts has yet been identified. as a critical determinant for the stereoselective introduction of the We recently determined the x-ray crystal structure of LTA4H 12R-hydroxyl group and thus the biological activity of LTB4. at 1.95-Å resolution in complex with bestatin ([(2S,3R)-3-amino- 2-hydroxy-4-phenylbutanoyl]-L-leucine) (12). At the active cen- he development and maintenance of inflammation are gov- ter, an extended hydrophobic pocket was found that potentially Terned by a complex network of cellular and humoral factors could bind LTA4. In this cavity, a hydrophilic patch comprising to which belong the leukotrienes, a class of structurally related the residues Gln-134, the phenolic hydroxyl group of Tyr-267, paracrine hormones derived from the oxidative metabolism of and Asp-375 was identified, potentially contributing polar resi- arachidonic acid (1, 2). In the biosynthesis of leukotrienes, dues involved in catalysis in an otherwise lipophilic environment 5-lipoxygenase converts arachidonic acid into the unstable ep- (Fig. 1). Interestingly, Asp-375 also belongs to peptide K21, in BIOCHEMISTRY ͞ oxide leukotriene A4 (LTA4, 5S-trans-5,6-oxido-7,9-trans-11,14- which it is located within a stretch of charged Asp Glu residues, cis-eicosatetraenoic). This intermediate may in turn be conju- any one of which could act as a nucleophile in enzyme catalysis. gated with glutathione by a specific leukotriene C4, LTC4 In the present article we used site-directed mutagenesis, (5S-hydroxy-6R-S-glutathionyl-7,9-trans-11,14-cis-eicosatetra- complemented with x-ray crystallography, to analyze the func- enoic acid) synthase to form the spasmogenic LTC4 or hydro- tion of all residues within the hydrophobic pocket or peptide K21 lyzed into leukotriene B4 (LTB4, 5S,12R-dihydroxy-6,14-cis- that could potentially participate in the epoxide hydrolase 8,10-trans-eicosatetraenoic acid), in a reaction catalyzed by and͞or aminopeptidase reaction. We show that mutation of LTA4 hydrolase (LTA4H). LTB4 is a classical chemoattractant Asp-375 selectively eliminates the epoxide hydrolase activity, of human neutrophils and triggers adherence and aggregation of suggesting that this residue plays a key role in this enzyme leukocytes to the endothelium at only nM concentrations (3). mechanism. On the other hand, exchange of Gln-134 for a These effects are signaled by means of a specific, high-affinity, nonpolar residue selectively enhances the epoxide hydrolase G protein-coupled receptor for LTB4 (BLT1) (4). In addition, an activity, suggesting a regulatory function for this residue. Based ORF encoding a second receptor for LTB4 (BLT2) was recently on the present data and previous conclusions regarding the discovered in the promoter of the gene for BLT1 (5). The active site structure and catalytic mechanisms, we propose that functional role of BLT2 is presently not known. LTA4H (EC 3.3.2.6) is a bifunctional zinc metalloenzyme, exhibiting an aminopeptidase activity in addition to its epoxide Abbreviations: LTA4, leukotriene A4 (5S-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid); LTB4, leukotriene B4 (5S,12R-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic hydrolase activity, i.e., the hydrolysis of the unstable allylic acid); LTA4H, LTA4 hydrolase; PGB, prostaglandin B. epoxide LTA4 into LTB4 (for a review see ref. 6). A physiological Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, substrate has not yet been discovered for the aminopeptidase www.rcsb.org (PDB ID code 1gw6). activity but certain arginyl dipeptides and tripeptides as well as ‡To whom reprint requests should be addressed. E-mail: [email protected]. p-nitroanilide derivatives of Ala and Arg are hydrolyzed with The publication costs of this article were defrayed in part by page charge payment. This high efficiency (7). Several lines of evidence indicate that the article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. aminopeptidase activity follows a general base mechanism with §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.072090099 PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 ͉ 4215–4220 Downloaded by guest on September 30, 2021 Asp-375, possibly assisted by Gln-134, is responsible for the The final wash consisted of 0.8 bed volumes of 50 mM Tris⅐HCl, stereospecific introduction of the 12R-hydroxyl group in LTB4. pH 8.0, containing 100 mM imidazole. The (His)6-tagged protein In this role, Asp-375 will also be an important functional element was eluted with 0.6 bed volumes of 50 mM Tris⅐HCl, pH 8.0, that governs the generation of bioactivity in the product LTB4. containing 100 mM imidazole. If required, a final step of anion-exchange chromatography on a Mono Q HR5͞5 column Experimental Procedures was used. The resin was equilibrated with 10 mM Tris⅐HCl, pH Materials. Oligonucleotides were synthesized by Cybergene, 8.0 and eluted with a linear gradient of KCl (0–0.5 M) in 10 mM ⅐ Stockholm. LTA4 methyl ester was synthesized as described (13) Tris HCl, pH 8.0. The purity of the final preparation was assessed ͞ or purchased from Biomol, Plymouth Meeting, PA. LTA4 methyl by SDS PAGE on an Amersham Pharmacia Phast system by ester was saponified in tetrahydrofurane with 1 M LiOH (6% using 10–15% gradient gels, subsequently stained with Coomas- vol͞vol) for 48 h at 4°C. Alanine-p-nitroanilide, isopropyl-␤-D- sie brilliant blue. Protein concentrations were determined by the thiogalactopyranoside, PMSF, soybean trypsin inhibitor, and Bradford method (BioRad Coomassie brilliant blue, G-250) with streptomycin sulfate were purchased from Sigma. Nickel-nitrilo a MCC͞340 96-well multiscan spectrophotometer and BSA as triacetic acid resin was from Qiagen, Chatsworth, CA. standard (15).

Mutagenesis of Human LTA4H cDNA. Site-directed mutagenesis was Aminopeptidase Activity Assays. The aminopeptidase activity was carried out by PCR on the recombinant plasmid pT3MB4 determined in a spectrophotometric assay at 405 nm, using a ͞ designed for expression of (His)6-tagged LTA4H in Escherichia MCC 340 multiscan spectrophotometer, essentially as described coli (11). Briefly, it involves two consecutive steps of PCR (16). Briefly, the enzyme (1–20 ␮g) was incubated at room comprising four different primers, according to the ‘‘mega- temperature in 96-well microtiter plates with alanine-p- primer’’ method (14). PCRs were carried out in a total volume nitroanilide as substrate in 50 mM Tris⅐HCl, pH 8.0, containing of 50 ␮l, using 1ϫ Pfu polymerase buffer, 125–150 ng each of 100 mM KCl. The absorbance at 405 nm was measured at 10-min primers A and B, 1 unit Pfu polymerase, 10 nmol each dNTPs, intervals. Different concentrations of substrate (0.125, 0.25, 0.5, and 100 ng template. In the second reaction, 15–20 ␮l of the first 1, 2, 4, and 8 mM) were used for kinetic determinations. PCR mix was used to supply the reaction with the megaprimer, Spontaneous hydrolysis of the substrate was corrected for by which was used together with a primer C (125–150 ng). The subtracting the absorbance of blank incubations without enzyme. amplification program included an initial round of denaturation at 94°C (60 s), annealing at 55–66°C (60 s), and elongation at Epoxide Hydrolase Activity Assays. The epoxide hydrolase activity 72°C (90 s), followed by 30 cycles of denaturation (45 s), was determined from incubations of enzyme (1–20 ␮g) in 100 ␮l ⅐ ␮ annealing (30 s), and elongation (60 s) on a Perkin–Elmer of 10 mM Tris HCl, pH 8.0, with LTA4 (2.5–125 M) for 30 s on GeneAmp PCR System 2400. ice. Reactions were quenched with 200 ␮l of MeOH, followed by ͞ ͞ For generation of [Q134A L N]LTA4H, and [H139Q]- the addition of 0.4 nmol of prostaglandin (PG) B 1 or PGB2 as LTA4H, a SalI and BglII site were used whereas a BglII and a internal standard. Samples were acidified with 5 ␮l of acetic acid BfrI site were used for [Y267F]LTA4H, [D368N]LTA4H, (10%), and metabolites were extracted on solid-phase Chroma- ͞ ͞ [D371N]LTA4H, [D373N]LTA4H, [D375A E N]LTA4H, and bond C18 columns. Enzymatic metabolites of LTA4 were iden- [E384Q]LTA4H. DNA fragments were cleaved by SalI͞BglII or tified and quantified by reverse-phase HPLC. BglII͞BfrI and purified by agarose gel electrophoresis (1.5%) followed by extraction (QIAEX II Gel Extraction Kit). Mutated Reverse-Phase HPLC. Metabolites of LTA4 were separated by fragments were ligated into pT3MB4 (T4 DNA Ligase Protocol), isocratic reverse-phase HPLC on a Waters Nova-Pak C18 col- opened with the respective pair of restriction enzymes. Compe- umn eluted with a mixture of methanol͞acetonitrile͞water͞ tent E. coli cells (JM101) were transformed with mutated acetic acid (30:30:40:0.01 by vol) at a flow rate of 1.2 ml͞min. The recombinant plasmid and grown in LB medium containing UV detector was set at 270 nm, and metabolites were quantified ampicillin (100 ␮g͞ml). Stock cultures were kept at Ϫ70°Cina by using CHROMATOGRAPHY STATION for Windows version 1.7 ͞ ͞ 1:1 mixture of culture medium and 40% (vol vol) glycerol computer software. Known amounts of PGB1 or PGB2 were used 0.75% (wt͞vol) NaCl, respectively. Recombinant plasmids were as internal standard. Calculations were based on peak area purified by using Wizard Minipreps Plus, and all of the mutated measurements by using an extinction coefficients of 30,000 MϪ1 ϫ Ϫ1 inserts were sequenced by using a Dyenamic ET terminator cycle cm for the internal standards PGB1 and PGB2 and 50,000 Ϫ1 ϫ Ϫ1 sequencing kit (Amersham Pharmacia) to confirm that no M cm for LTB4. nucleotide alterations had occurred in addition to the desired mutation. Crystallization. Plate-like crystals of [D375N]LTA4H were ob- tained by liquid-liquid diffusion in capillaries, as described (12). Protein Expression and Purification. Mutated enzymes were ex- Briefly, 5 ␮l of precipitation solution [28% (vol͞vol) PEG8000, pressed as N-terminal (His)6-tag fusion proteins in E. coli JM101 0.1 mM Na acetate, 0.1 mM imidazole buffer, pH 6.8, and 5 mM cells grown at 37°C in M9 medium (50 mM Na2HPO4,22mM YbCl3] was injected into the bottom of a melting point capillary ͞ ⅐ KH2PO4,20mMNH4Cl, 8.5 mM NaCl), pH 7.4, containing 0.4% and an equal volume of LTA4H (5 mg ml) in 10 mM Tris HCl, ͞ ͞ (wt vol) glucose, 0.2% (wt vol) casamino acids, 2 mM MgSO4, pH 8, was layered on top. The crystals belonged to space-group Ϸ ␤ ϭ ϭ ϭ and 0.1 mM CaCl2.AtOD620 0.2, isopropyl- -D-thiogalacto- P212121 with cell dimensions a 78.430, b 87.060, c 99.070, pyranoside was added to a final concentration of 500 ␮M. Cells ␣ ϭ ␤ ϭ ␥ ϭ 90° at 100 K. Ϸ ϫ were harvested at OD620 1.8, pelleted at 1,000 g, and resuspended in 30 ml homogenization buffer (50 mM Tris⅐HCl, Data Collection and Structure Determination. For data collection, pH 8.0, containing soybean trypsin inhibitor) supplemented with crystals were soaked in 15% PEG8000, 50 mM imidazole buffer 1 mM PMSF. Nucleic acids were removed by streptomycin (pH 6.8), 50 mM sodium-acetate, 2.5 mM YbCl3, and 25% sulfate precipitation. After centrifugation (10,000 ϫ g for 15 glycerol. Data were collected at the beamline I711 of the min), the supernatant was filtered (0.22 ␮m) and applied to a Max-Lab, Lund, Sweden. A complete set of data were collected nickel-nitrilo triacetic acid resin. The column was washed with 1 from a single crystal. Statistics on data collection and quality are bed volume of 50 mM Tris⅐HCl (pH 8.0), 50 mM sodium given in Table 1. Processing, scaling, and merging of data were phosphate buffer (pH 6.8), 0.5 M NaCl, and 50 mM Tris⅐HCl (pH carried out by using the program MOSFLM (34) and programs 8.0) with each solution supplemented with 10 mM imidazole. from CCP4 (17). As a starting structure for refinement, the

4216 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072090099 Rudberg et al. Downloaded by guest on September 30, 2021 and the first four N-terminal residues. The model exhibits a highly restrained but acceptable stereochemistry with 97.7% of the residues lying in the most favored or additionally allowed regions of the Ramachandran plot. Root mean square deviations for bond lengths and angles are 0.010 Å and 1.51°, respectively. Results Mutagenetic Replacements, Expression, and Purification of Recombi- nant Proteins. To detail their function we exchanged Asp-375 for an Ala, Asn, or Glu residue, Gln-134 for an Asn, Leu, and Ala, and Tyr-267 for a Phe by site-directed mutagenesis (Table 2). This procedure generated the mutants [D375A͞E͞N]LTA4H, [Q134A͞L͞N]LTA4H, and [Y267F]LTA4H. As a control for mutants in position 134, His-139 was exchanged for Gln to produce [H139Q]LTA4H. In the wild-type structure, His-139 is also hydrogen-bonded to Asp-375. In addition, the remaining acidic residues within peptide K21, Asp-368, Asp-371, Asp-373, and Glu-384 were exchanged for an Asn, Asn, Asn, and Gln, respectively, generating mutants [D368N]LTA4H, [D371N]LTA4H, [D373N]LTA4H, and [E384Q]LTA4H. The resulting 12 mutants were all expressed as (His)6-tagged fusion proteins in E. coli, to allow rapid purification on nickel-nitrilo triacetic acid resins. The level of expression was similar for wild-type enzyme and all mutants, with a final yield of about 1–5 mg purified protein per liter cell culture. It may be noted that expression of [Y267F]LTA4H resulted in generation of varying Fig. 1. Structure of wild-type LTA4H, spatial relationships at the catalytic amounts of a protein fragment of about 30 kDa, in addition to center. At the active center, a narrow (6–7 Å wide) L-shaped, hydrophobic the regular 69-kDa LTA4H. Therefore, this mutant was more pocket extends about 15 Å into the protein. At the angle of the cavity, a extensively purified than others, carefully selecting the active hydrophilic patch composed of Gln-134, Tyr-267, and Asp-375 is located. In 69-kDa subfractions. The phenomenon might indicate the in- addition, the positions of the other acidic residues along peptide K21, Asp- troduction of a structural weakness upon mutation at resi- 368, Asp-371, Asp-373, and Glu-384 are indicated. Amino acids are depicted in due 267. stick-and-ball representation. The substrate LTA4 (yellow) does not belong to the structure and has been modeled into its tentative binding mode. The Effects of Mutation of Asp-375. catalytic zinc is depicted in gold, adjacent to the epoxide moiety of LTA4. The Exchange of Asp-375 for an Ala, figure was generated with SWISS-PDBVIEWER (33) and POV-RAY (http:͞͞ Asn, or Glu resulted in a complete to near-complete loss of www.povray.org). epoxide hydrolase activity (Table 2). In contrast, the aminopeptidase reaction was diminished only in [D375E]LTA4H. For the epoxide hydrolase activity, exchange of Asp-375 for an Ala [E271Q]LTA4H mutant was used (8). All refinement was done resulted in an enzyme with a low, but detectable, catalytic by using the CNS package (18). Manual model building and activity with Vmax (kcat) and Km of 4% and 40% of wild-type interpretation of electron density maps were performed by using control, respectively. Exchange of Asp-375 for a Glu or Asn the program XTALVIEW (19). During the refinement 440 water resulted in inactive enzymes for which kinetics could not be 2ϩ determined. For the aminopeptidase reaction, exchange of Asp- molecules, one Zn ion, one imidazole molecule, one acetate BIOCHEMISTRY molecule, and one Yb3ϩ ion were identified. The final R factor 375 for a Glu resulted in an enzyme with Vmax (kcat) and Km of 2% and 152% of wild-type enzyme, respectively. Exchange of was 17.4% and the R factor was 22.04%. Most of the model free Asp-375 for either an Ala or an Asn gave rise to active enzymes of [D375N]LTA4H is in good density except for the (His) tag 6 that, however, did not exhibit saturation kinetics within the substrate intervals used (0.125–8 mM). Mutation of His-139, Tyr-267, or any of the acidic residues, Asp-368, Asp-371, Asp- Table 1. Data collection and refinement statistics 373, or Glu-384, along peptide K21, were all compatible with a Data collection statistics significant, albeit variable, epoxide hydrolase activity (Table 2). Diffraction limit, Å 2.2 For these mutants, values of Vmax (kcat) and Km ranged between Wavelength, Å 0.991 25% and 165% and 3% and 167%, respectively. Likewise, Completeness, % 97.2 [H139Q]LTA4H, [Y267F]LTA4H, [D368N]LTA4H, Mean I͞␴ (I) 7.3 [D371N]LTA4H, [D373N]LTA4H, and [E384Q]LTA4H main- Multiplicity of observation 3.4 tained a variable aminopeptidase activity with values of Vmax Rmerge*, % 8.3 (kcat) and Km between 7% and 119% and 64% and 359%, Refinement statistics respectively. R factor†,% 17.4 ‡ Rfree ,% 22.04 Effects of Mutation of Gln-134. Exchange of Gln-134 for an unpolar rms deviation in bond distance, Å 0.010 Leu residue resulted in significantly increased epoxide hydrolase rms deviation in bond angle, ° 1.51 activity with Vmax (kcat) and Km of 721% and 364% of wild-type enzyme, respectively. Exchange of Gln-134 for an Ala also led to *Rmerge ϭ⌺h⌺i͉Ii(h) Ϫ I(h)͉͞⌺h⌺iIi(h), where Ii(h) is the ith measurement of reflection h and I(h) is the weighted mean of all measurements of h. a hyperactive enzyme although it did not display saturation † R factor ϭ⌺h͉Fobs(h) Ϫ Fcalc(h)͉͞⌺hFobs(h), where Fobs and Fcalc are the observed kinetics within the concentrations of substrate used (6.3–101 and calculated factor amplitudes, respectively. ␮M). On the other hand, a conservative replacement of Gln-134 ‡ Rfree is the R factor calculated for the test set of reflections that are omitted for Asn resulted in a Vmax (kcat) and Km of 89% and 148% of during the refinement process. wild-type enzyme, respectively. With respect to the aminopep-

Rudberg et al. PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 ͉ 4217 Downloaded by guest on September 30, 2021 Table 2. Mutagenetic replacements in LTA4H, effects on enzyme activities, and kinetic constants Epoxide hydrolase activity Aminopeptidase activity

Mutant kcat Km kcat͞Km kcat Km kcat͞Km

Q134A Ͼ1,000* Ͼ1,000* — 18 Ϯ 1 157 Ϯ 17 12 Q134L 721 Ϯ 52 364 Ϯ 70 198 13 Ϯ 0.4 87 Ϯ 615 Q134N 89 Ϯ 6 148 Ϯ 27 60 206 Ϯ 4 108 Ϯ 5 190 H139Q 68 Ϯ 6 167 Ϯ 51 40 7 Ϯ 0.6 359 Ϯ 42 2 Y267F 165 Ϯ 858Ϯ 17 285 61 Ϯ 364Ϯ 796 D368N 83 Ϯ 672Ϯ 16 116 99 Ϯ 4 107 Ϯ 10 93 D371N 57 Ϯ 551Ϯ 14 112 66 Ϯ 477Ϯ 11 86 D373N 45 Ϯ 443Ϯ 13 104 119 Ϯ 6 101 Ϯ 12 118 D375A 4 Ϯ 140Ϯ 25 9 421 Ϯ 145 1,436 Ϯ 574 29 D375E ND ND — 2 Ϯ 0.1 152 Ϯ 22 1 D375N ND ND — Ͼ1,000* Ͼ1,000* — E384Q† ϳ25 Ͻ3 Ͼ80 11 Ϯ 0.7 71 Ϯ 12 16

Mutated recombinant enzymes were expressed in E. coli, puriﬁed by afﬁnity chromatography, and assayed for epoxide hydrolase and aminopeptidase activity. Apparent kinetic constants are expressed in % of those of wild-type enzyme. Each data point was calculated as mean Ϯ SD, triplicate determinations. ND, not detectable. For the epoxide hydrolase activity, the values of kcat and Km for the wild-type enzyme varied between 0.21 and 0.47 sϪ1 and 6 and 28 ␮M, respectively. For the aminopeptidase activity, the corresponding values of Ϫ1 kcat and Km for the wild-type enzyme were 1.16–1.81 s and 2.3–3.8 mM, respectively. *The enzyme activity of this mutant exhibited unsaturable enzyme kinetics within the range observed. These values are estimations. †Mutant E384Q exhibited substrate inhibition of the epoxide hydrolase activity at higher substrate concentrations (Ͼ21 ␮M). The value of Vmax is estimated to be equal to the calculated plateau of V between 5 and 21 ␮M LTA4.

tidase activity, all of the mutants [Q134L͞A͞N]LTA4H dis- conformation and the interactions this inhibitor makes with the played significant aminopeptidase activities with values of Vmax mutated protein are nearly identical to the complex with wild- (kcat) and Km ranging between 13% and 206% and 87% and type LTA4H. 157% of wild-type enzyme, respectively (Table 2). Only very small changes in conformation are observed near the active site (Fig. 2). Near the mutated D375N residue, the Structure of [D375N]LTA4H. The structure of the [D375N]LTA4H hydrogen-bonding network has been slightly altered. This alter- mutant is very similar to that of the native LTA4H (12). Overall ation is caused mainly by a rotation around the C␣-C␤ bond of rms difference of all C␣ atoms between the two structures is 0.45 the Asn-375 side chain, which causes the formation of a direct Å. The only loop that has changed its conformation is loop weak hydrogen bond between the N␦2 of Asn-375 and the OH 175–185, which is located near the surface and far away from the of Tyr-267. In the native protein there was no direct contact active site. This is probably caused by a difference in crystal between these two residues, instead a bridging water molecule contacts because the native protein crystallizes in a different was observed. This water molecule is still present in the structure space group. Like in the native structure, the aminopeptidase of the mutant but has a slightly altered position. The distance inhibitor bestatin is bound to the Zn2ϩ ion in the active site. The between the O␦1 of Asn-375 and the putative nucleophilic water

Fig. 2. Structure of the mutant [D375N]LTA4H. (A) Superpositioning of wild-type LTA4H and [D375N]LTA4H. Stereo image of selected residues of the active site of wild-type LTA4H complexed with bestatin superimposed onto [D375N]LTA4H. The wild-type and [D375N]LTA4H structures are shown in pale blue and red, respectively. The Zn2ϩ ions, the bestatin, and water molecules of wild type (pale blue) and [D375N]LTA4H (red) are also shown. The image shows very subtle conformational changes and thus a high degree of structural preservation after mutation of Asp-375 to Asn-375. See text for further discussion. The ﬁgure was generated with SWISS-PDBVIEWER (33) and POV-RAY (http:͞͞www.povray.org). (B) Comparison of the hydrogen-bonding networks around Asp-375 and Asn-375. The side chain of Asn-375 is indicated in red. Likewise the interactions with His-139, Tyr-267, and two water molecules of Asp-375 and Asn-375 are depicted in dotted black and red lines, respectively. Distances are given in Å.

4218 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.072090099 Rudberg et al. Downloaded by guest on September 30, 2021 Fig. 3. Sequence alignment of members of the M1 family of the MA clan of metallopeptidases. Shown are residues subjected to mutational analysis and vicinal stretches of amino acids. Asp-375 (boxed) is the only residue speciﬁcally involved in the epoxide hydrolase reaction.

molecule has been somewhat increased in comparison to the the presence of the catalytic metal, as expected from the native structure (from 2.46 Å in native to 3.31 Å in the mutant preserved aminopeptidase activity, and an almost completely structure) and the angle is now unfavorable for the formation of conserved overall architecture, particularly at the active site (Fig. a proper hydrogen bond. 2). Apparently, the loss of epoxide hydrolase activity is site- specific and can be attributed to the removal of the carboxylate Discussion from the side chain of Asp-375. We conclude that Asp-375 is LTB4 is a potent chemoattractant and immune modulating lipid involved in a key step of the epoxide hydrolase reaction. Hence, mediator. To elicit these biological effects, LTB4 signals by by a single point mutation at position 375 we can dissociate the means of a high-affinity, G protein-coupled, seven-transmem- two catalytic activities of LTA4H with preservation of the brane receptor present on the surface of a variety of immuno- aminopeptidase activity. competent cells, e.g., peripheral leukocytes, including neutro- LTA4H is a member of the M1 family of the MA clan of phils and eosinophils, as well as peritoneal macrophages (20, 21). metallopeptidases, which includes enzymes such as aminopep- Studies of structure activity relationships for LTB4 and related tidase A, aminopeptidase B, aminopeptidase N, and angiotensin compounds have revealed that two chemical properties are key converting enzyme. Members of this family share a common zinc to the bioactivity of this molecule, namely the cis-trans-trans binding signature and a proteolytic activity, which seems to geometry of the conjugated triene system and the R stereochem- proceed according to a zinc-assisted general base mechanism istry of the hydroxyl group at C12 (22–24). It is therefore of (25, 26). In LTA4H, Glu-296 and Tyr-383 are catalytic in the particular interest to learn the mechanisms by which LTA4H aminopeptidase reaction in which they act as a general base manages to incorporate these structural features into LTB4 catalyst and proton donor, respectively (27, 28). In addition, during enzymatic hydrolysis of LTA4. Glu-271 is involved in the reaction and appears to bind the Recently, we determined the x-ray crystal structure of LTA4H N-terminal amino group of the peptidase substrate (8). This in complex with bestatin at 1.95-Å resolution (12). Behind the residue is also catalytic for the epoxide hydrolase reaction in inhibitor a narrow extended hydrophobic cavity was found that which it seems to assist in the initial activation and opening of potentially could bind LTA4. In addition, a hydrophilic patch, the epoxide ring. However, besides the catalytic zinc and Glu- composed of Gln-134, the hydroxyl group of Tyr-267, and 271, very little is known about the functional elements that are Asp-375, is present in that cavity, each of which could potentially required for the conversion of LTA4 into LTB4. In fact, Asp-375 act as a general base catalyst in the epoxide hydrolase reaction. is the first example of an amino acid that is specifically involved Moreover, Asp-375 is located within an acidic stretch of five in the epoxide hydrolase reaction. Yet, Asp-375 is not unique for Asp͞Glu residues (Asp-368, Asp-371, Asp-373, Asp-375, and LTA4H and is present in related aminopeptidases (Fig. 3). One Glu-384) in peptide K21, a previously identified active site such example is aminopeptidase B, which seems to lack an BIOCHEMISTRY peptide. In fact, peptide K21 carries Tyr-378, the binding site for epoxide hydrolase activity, although conflicting data are present ͞ LTA4 during suicide inactivation covalent modification of in the literature (29, 30). Apparently, from a noncatalytic LTA4H (9, 10). To possibly identify a nucleophilic amino acid function in certain early aminopeptidases, evolution has gradu- involved in the epoxide hydrolase reaction we mutated all of the ally brought Asp-375 into a key role for the epoxide hydrolase candidate residues of the hydrophilic patch of the putative LTA4 reaction in LTA4H. In this context, it is interesting to note that binding pocket as well as those of peptide K21. Asp-375 is indeed present in Saccharomyces cerevisiae LTA4H, a product of an ancestral gene for mammalian LTA4H that Asp-375 Is a Catalytic Residue Specifically Required for the Epoxide exhibits both a leucyl aminopeptidase and a primitive epoxide Hydrolase Reaction. Exchange of Asp-375 for an Asn, Ala, or Glu hydrolase activity against LTA4 (31, 32). residue essentially eliminated the ability of LTA4H to convert Exchange of Gln-134 for a nonpolar residue such as Leu or Ala LTA4 into LTB4 (Table 2). The only mutant with detectable led to an unexpected increase in epoxide hydrolase efficiency, epoxide hydrolase activity was [D375A]LTA4H, which may whereas a conservative replacement, Gln 3 Asn, had minimal reflect that an unpolarized water molecule may slip into the extra effects. The proper interpretation of these results is not obvious space near the substrate, gained by the reduced side-chain length but the data may be taken as evidence that the polar side chain caused by the mutation. In contrast, a significant aminopeptidase of Gln-134 plays a regulatory role in the epoxide hydrolase activity was observed in [D375A]LTA4H and [D375N]LTA4H mechanism. The location of Gln-134 in the hydrophilic patch is whereas the activity of [D375E]LTA4H was greatly reduced, in line with this hypothesis. although clearly detectable. Mutations of Tyr-267, Gln-134, and the remaining acidic residues in peptide K21 were all compatible Potential Role of Asp-375 in the Epoxide Hydrolase Reaction. The with significant, albeit variable, epoxide hydrolase and amino- bioactivity of LTB4 depends on the presence of a 12R-hydroxyl peptidase activities (Table 2). To explore possible structural group and a cis-trans-trans-conjugated triene system. After the alterations induced by the mutations of Asp-375, we determined determination of the crystal structure of LTA4H we are now the crystal structure of the mutant with the most conservative beginning to understand the mechanisms by which the enzyme replacement, namely [D375N]LTA4H. This structure revealed controls the generation of this chemistry during conversion of

Rudberg et al. PNAS ͉ April 2, 2002 ͉ vol. 99 ͉ no. 7 ͉ 4219 Downloaded by guest on September 30, 2021 of a hydrophilic patch positioned close to the point where the L-shaped cavity bends. If LTA4 is modeled into this hydrophobic pocket with a head-to-tail orientation that positions the allylic epoxide close to the zinc, the C7-C20 olefinic tail fits snugly and occupies almost the entire cavity. With this orientation of the substrate, C12 gets close to the hydrophilic patch and Asp-375, suggesting an involvement of this residue in the introduction of the 12R-hydroxyl group (Fig. 4). In line with this hypothesis, the structure of LTA4H in complex with bestatin revealed the presence of a water molecule hydrogen-bonded to Asp-375 that could be the origin of the hydroxyl moiety. Hence, assuming that LTA4H operates according to an SN1 mechanism, we propose that Asp-375 is a major determinant for the stereospecific nucleophilic attack at C12 of a carbocation intermediate to generate the 12R-hydroxyl group of LTB4. In this capacity, Asp-375 is also of fundamental importance for the ability of LTA4H to catalyze the formation of a biologically active Fig. 4. Role of Asp-375 in the epoxide hydrolase reaction. LTA4 is modeled into the L-shaped hydrophobic pocket with its ␻-end pointing to the bottom product. of the cavity. After activation and opening of the epoxide by the Zn2ϩ and Because exchange of the polar side chain of Gln-134 for an Glu-271 according to an SN1 mechanism, a carbocation is formed whose alkyl moiety resulted in a greatly enhanced epoxide hydrolase charge will be delocalized over the conjugated triene system. At C12, a activity, it is also possible that this residue has a regulatory, albeit nucleophilic attack can be mediated by means of Asp-375 to produce the not critical, role in this reaction. Further studies are needed to 12R-hydroxyl group of LTB4 (for further details see text). For an alternative elucidate the detailed molecular mechanisms by which Asp-375 role of Glu-271 involving an ester intermediate, see ref. 8. governs the stereoselective chemistry catalyzed by LTA4H.

We thank personnel at beamline I711 of MAX-Lab for help during data LTA4 into LTB4. A narrow, L-shaped hydrophobic pocket has collection and Eva Ohlson for technical assistance. The work was funded been found at the active center, which may be the binding site for by the Swedish Medical Research Council (O3X-10350), The European LTA4, and into which LTA4 has been modeled (Fig. 1). Indeed, Union (QLG1-CT-2001-01521), The Swedish Foundation for Strategic several residues within the active site-peptide K21 are located Research, Konung Gustav V:s 80-Årsfond, and the Swedish Natural along this cavity, including Asp-375, which is also a component Sciences Research Council.

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