Sequence Similarity of Mammalian Epoxide Hydrolases to the Bacterial Haloalkane Dehalogenase and Other Related Proteins

Home , Chelatase, Haloalkane dehalogenase

Gii!m

FEBS Letters 338 (1994) 251-256 LETTERS ELSEVIER FEBS 13587

Hypothesis Sequence similarity of mammalian epoxide hydrolases to the bacterial haloalkane dehalogenase and other related proteins

Implication for the potential catalytic mechanism of enzymatic epoxide hydrolysis

Michael Aran@*, David F. Grantb, Jeffrey K. Beethamb, Thomas Friedberg”, Franz Oescha, Bruce D. Hammockb

“Instrtute of Toxrcology, Unrverslty of Mamz, Obere Zahlbacherstr 67, D-55131 Mama, German> bDepartment of Entomology, Unrverslty of Cahfornra, Davis, CA, USA

Received 1.5November 1993

Abstract Direct comparison of the ammo acid sequences of mlcrosomal and soluble epoxlde hydrolase superficially mdlcates that these enzymes are unrelated Both protems, however, share slgmficant sequence slmllanty to a bacterial haloalkane dehalogenase that has earlier been shown to belong to the a@ hydrolase fold family of enzymes The catalytic mechanism for the dehalogenase has been elucidated m detail [Verschueren et al (1993) Nature 363, 693-6981 and proceeds via an ester mtermedlate where the substrate IS covalently bound to the enzyme From these observations we conclude (I) that mlcrosomal and soluble epoxlde hydrolase are distantly related enzymes that have evolved from a common ancestral protein together with the haloalkane dehalogenase and a variety of other proteins specified m the present paper, (u) that these enzymes most likely belong to the a//3 hydrolase fold family of enzymes and (III) that the enzymatic epoxlde hydrolysis proceeds via a hydroxy ester mtermedlate, m contrast to the presently favoured base-catalyzed direct attack of the epoxlde by an activated water

Key words alp Hydrolase fold, Hydrolase, Llpase, Esterase, Luaferase, Chelatase, Peroxldase

1. Introduction cludmg polycychc aromatic hydrocarbons [ 11.This catalytic breakdown protects essential biomolecules like nu- Epoxide hydrolases (EH, EC3.3 2.3) comprise a group cleic acids and proteins from the electrophihc attack by of enzymes that are functionally related m that they cat- these reactive molecules and therefore represents an im- alyze the hydrolytic cleavage of oxu-ane compounds to portant protective metabohc pathway The biochemical yield the corresponding dials. Two major, well charac- properties of mEH, have been studied m detail and the terized EHs are present m the liver of all animal species primary structure and the genomic organization were so far mvestigated elucidated already m the mid eighties [24]. On the other The first EH, called rmcrosomal epoxide hydrolase hand, our understanding of the overall structure of the (mEH,, the broad-specific mEH with the diagnostic sub- mature protem is presently restricted to speculation strate benzo[a]pyrene-4,5-oxide), is located m the endo- about the mode of membrane association/integration of plasmatic reticulum Its major function is thought to be the enzyme [3,.5]. the hydrolysis of epoxides derived from xenobiotics, in- The other major EH is the soluble epoxide hydrolase (sEH), that has often been referred to as cytosohc epoxide hydrolase (cEH) Because accumulatmg evidence has * Correspondmg author Fax (49) 6131 230506 been presented for a simultaneous localization of this enzyme in both the cytosol and the peroxisomal matrix Ahbrevratlons EH, epoxlde hydrolase, sEH, soluble epoxlde hydrolase, of liver cells [&8] the older term ‘soluble epoxide hydro- mEH,, mlcrosomal epoxlde hydrolase, HALO, haloalkane dehalogenase, DMPD, 2-hydroxymucomc semlaldehyde hydrolase lsoenzyme lase’ seems to more precisely characterize the enzyme DMPD_PSEPU, XYLF, 2-hydroxymucomc semlaldehyde hydrolase and is now recommended. While the sEH, like the mEH,, lsoenzyme XYLF_PSEPU accepts some xenobiotic-derived epoxides as substrates

[9,10], it appears that its primary function might be the 3. Results and discussion metabolic transformation of epoxides arismg from en- dogenous substrates, as suggested by the significant en- 3 1 Soluble and mlcrosomal epoxlde hydrolase are both zymatic activity of the enzyme with fatty acid epoxides related to the same bacterral protems [11,12] Recent work of our groups has elucidated the As already pointed out there is no evident similarity primary structure of sEH from rat, mouse and man [13- between sEH and mEH, when compared directly on the 15] Imtrally, direct comparison of these sequences to basis of their ammo acid sequences Instead, screening that of mEH, did not reveal a sigmficant amount of of the SWISS-PROT library with both protein sequences similarity among these enzymes, at first sight. How- from the respective rat enzymes resulted m the identifica- ever, as will be detailed later m this paper, we hypothe- tion of three bacterial proteins as candidates showing a size that there is a distant relationship among these en- significant yet marginal relationship to sEH, namely the zymes that is too obscure to be picked up by standard haloalkane dehalogenase from Xanthobacter autotroph- similarity analysis zcus GJlO (HALO) and two different isoforms of the A third epoxide hydrolase, the mEH,,, that is different 2-hydroxymucomc semraldehyde hydrolases from Pseu- from the mEH, and is also present m the endoplasmic domonas putzda (DMPD and XYLF), while no mEH,- reticulum specifically hydrohzes cholesterol-5,6-epox- related sequences (only the mEH, from rat, rabbit and ides [16] Little mformation is available on this enzyme, human) were picked up. and its relationship to the major EHs is thus largely Interestingly, some degree of similarity between the unclear Two additional epoxide hydrolases are specifi- human mEH, and HALO has earlier been described by cally involved m the metabolism of certain intermediates others [25] Due to minor differences between the rat and of the arachidomc acid cascade Of these, the leukotriene human mEH, sequence this similartty was not noticed by A, hydrolase (LTA, hydrolase) is especially well charac- our first similarity search When the match between the terized [17,18] In contrast to the situation with other dehalogenase and mEH, was taken as the core alignment EHs, hydrolysis by the LTA, hydrolase does not result and the correspondmg fragments of sEH, DMPD and m the formation of vicmal dials The two hydroxyl XYLF were aligned to this, the mterrelationship of all groups introduced during the transformation of LTA, to five proteins became apparent (Fig 1) Over a stretch of LTB, have a spatial distance of eight carbon atoms The about eighty ammo acids, none of the possible compari- available primary sequence of LTA, hydrolase does not sons of any two of the sequences gave a result below 20% show significant similarity to either sEH or mEH, The of similarity With the mtroduction of only two ahgn- fifth enzyme, the hepoxtlme hydrolase, is as yet not suffi- ment gaps, SIX residues were conserved completely ciently characterized to evaluate its relationship to other throughout all of these proteins m the respective region epoxide hydrolases [ 191 and a further fifteen residues were identical m four of the The mechanism of EH-catalyzed epoxide hydrolysis five proteins has not been completely elucidated In the case of mEH, the importance of one specific histidme residue for mam- 3 2 Soluble and mlcrosomal epoxlde hydrolase tenance of catalytic activity could be demonstrated potentially belong to the alp hydrolase-foldfamdy of [20,21] At present, the most accepted hypothesis is that enzymes - lmpllcatlon for the putative reactlon the reaction proceeds via a general base catalysis by the mechanism histidme that is thought to abstract a proton from a The key result of the above alignment is the observa- water molecule The resulting hydroxyl amon subse- tion that the dipeptide DW (see Fig 1) is conserved m quently is thought to attack the oxnane rmg at one of the a similar sequence context among sEH, mEH, and two carbon atoms, the stereochemistry of the resulting HALO, because the specific aspartic acid residue herem product being subJect to the sterical and electronical con- has been identified as the maJor constituent of the cata- straints of the parent compound Several mechanistic lytic center of the haloalkane dehalogenase Two years studies with the sEH are consistent with this mechanism ago, the crystal structure of HALO was determined at a as well [22,23] resolution of 2 4 A [26] This led to the classification of the enzyme as a member of the a/j? hydrolase-fold family This class of enzymes comprises a group of proteins that 2. Materials and methods are structurally, functionally and mechamstlcally related [27] All of them are hydrolytic enzymes that share the Protem sequence retrieval and analysis was carried out employing a same three-dimensional core structure, the a//J hydro- Macintosh IIvx computer Smularlty searches of the SWISS-PROT hbrary, release 25. were performed using the FASTA program, lase-fold While their catalytic specificities are radically V 1 6~22 [24] Dot matnx analysis was conducted using the GeneWorks different - lipids, peptides, haloalkanes, lactones and software package, V 2 1 (IntelhGenetlcs, Mountam View, CA) The acetylcholme are the substrates for the different enzymes same software was used for sequence alignments However, this was mainly done by hand as this yielded better results m the present case, ~ then enzymatic mechanisms appear to be rather siml- due to the comparatively low degree of slmllarlty lar They all possess an active center at correspondmg M Arand et al IFEBS Letters 338 (1994) 251-256 253

A first step of the enzymatic reaction. In the esterase/aml- dase-type enzymes this 1s a serme or cysteme that forms

rnEHb EVICPSIPGY GYSEASSKKG -LNS”ATARI FYKLMTRLGF 217 esters or thloesters with the carboxyhc component of the SEH RVLAIDMKGY GDSSSPPEIE EYAMELLCEE MVTFLNKLGI 324 HALO R"IAPDFFOF OKSDKPVDEE DYTFEFHRNF LLALIERLDL 115 substrate, respectively. In the dehalogenase the nucleo- DMPD RWAPDMLGF GYSERPADAQ -YNRDvwvDH AVGVLDALEI 98 XYLF RVIAPDMLGF GYSERPADGK -YSQARWEH AIGVLDALGI 97 phlle 1s the above mentioned aspartlc acid residue that, Consensus RVIAPD G G S P Y L 40 m analogy, replaces the halide of its substrates thereby forming an ester bond to the remammg alkyl The ester niq QKFYIQGGDW GSLICTNMAQ MVPNHVKGL" LNMAFISRSF Y 258 SE" PQAVFIGHDW AG"L"WNMAL FHPERVRAVA SLNTPLMPPN P 365 intermediates formed by all members of the a/j? hydro- HALO RNITIWQDW GGFLGLTLPM ADPSRFKRLI IMNACLM-TD P 155 DMPD EQADLVGNSF GGGIALALAI RHPERVRRL" LMGSAGV-SF P 138 lase-fold family are subsequently hydrolyzed by a water XYLF QQGDIVGNSF GGGLALALAI RHPERVRRL" LMGSVGV-SF P 137 molecule cooperatively activated by the two other con- COllSenS"S G..GG A PRV L P 81 served residues, a hlstldme and an acldlc ammo acid, either aspartlc or glutamlc acid The single steps m the enzymatic mechanism have recently been confirmed for B the haloalkane dehalogenase m an elegant study employ- mg X-ray analysis of the trapped covalent intermediate [281* The sequence slmllanty among the EHs and HALO, mcludmg the aspartlc acid of the dehalogenase active center, suggests that the EHs potentially belong to the a//? hydrolase-fold family Some additional arguments support this hypothesis First, the other related proteins, the semlaldehyde hydrolases, that do not have the con- C served aspartate residue but a serme instead, catalyze the hydrolytic cleavage of a C-C bond by removmg the aldehyde moiety of the 2-hydroxymucomc semlaldehyde to give 2-oxopent-4-enoate and formic acid The serme 1s a very good candidate for the nucleophlle to form a formylester intermediate All five enzymes of the above alignment fit very well m the proposed consensus for the Rg 1 Analysis of the slmllarlty of expoxlde hydrolases, haloalkane dehalogenase and 2-hydroxymucomc senualdehyde hydrolases m the nucleophlle m the a/,6 hydrolase-fold that 1s sm-x-nu-x- region of the potential catalytic nucleophlle of the enzymes (A) Se- sm-sm, where nu 1s the nucleophlle, sm 1s a small ammo quence alignment The core regons around the potential nucleophlles acid (preferentially glycme), and x 1s any ammo acid (see of the different proteins were aligned by hand Residues conserved m Table 1) Second, the importance of a hlstldme, the sec- all sequences are pnnted m bold letters The potential catalytic nucleo- ond important constituent of the catalytic triad of a/j? phdes are pnnted m big bold letters Residues printed m the consensus are conserved m at least four out of the five sequences Dots m the hydrolase-fold enzymes, for the EH catalysis has long consensus mdlcate posltlons where three out of the five sequences share been accepted For the mEH, the specific hlstldme essen- ldentlcal residues (B) Slmdanty tree The comparison of the different tial for the enzymatic reaction has recently been ldentl- fragments 1s presented structured m subahgnments to exemplify their fied by both blochemlcal analysis and site-directed mut- relatIonship The numbers gve the percentage identity of the respective subahgnment (C) Indlvldual comparison of the different fragments The sequence slmdanty of each mdlvldual comparison of the different fragments ISgiven m percentage IdentIty mEH, = Mlcrosomal epoxide hydrolase from rat, sEH = soluble expoxlde hydrolase from rat, HALO = haloalkane dehalogenase from Xanthobacter autotroptucus mEH, MERGGHFAAFEEP 438 GJ 10, DMPD = 2-hydroxymucomc semlaldehyde hydrolases from sEH IEDCGHWTQIEKP 530 Pseudomonas putrda, lsoform, XYLF = 2-hydroxymucomc semlalde- HALO IADAGHFVQEFGE hyde hydrolases from Pseudonomas purlda, lsoform 296 DMPD FGQCGHWTQIEHA 263 XYLF FGQCGHWTQIEHA 262 Consensus .GH. .Q.E posltlons m the a//? hydrolase-fold motif bearing three largely conserved ammo acid residues that form a cata- Fig 2 Sequence ahgnment ofepoxlde hydrolases, haloalkane dehaloge- lytic triad (these ammo acids are separated from each nase and 2-hydroxymucomc senualdehyde hydrolases m the region of other by a variable number of other residues m the prr- the potential catalytic hlstldme of the enzymes Residues conserved m mary structure of the different enzymes but are, m each all sequences are prmted m bold letters Residues printed m the consensus are conserved m at least four out of the five sequences Dots m the case, m vlcmlty to each other m the correctly folded consensus mdcate positions where three out of the five sequences share proteins) The first of these ammo acids represents a identical residues Note that sEH, DMPD and XYLF are identical m nucleophlle that covalently binds to the substrate m the the eight central residues of the displayed region 254 M Arand et al IFEBS Letters 338 (19941 251-256 agenesis [20,21] Like the catalytic histtdmes of the ol/j3 hydrolase-fold family members its position (Hester) is close to the C-terminus of the enzyme. Stmtlarly, a histidme close to the C-terminus of the sEH (His5’3) is an especially good candidate for the member of a postulated catalytic triad of this enzyme as it is situated wtthm a stretch of eight ammo acid residues that is perfectly conserved among the sEH and the bacterial mucomc semial- dehyde hydrolases (Fig 2) The constraints for the acidic residue (Asp or Glu) that interacts with the His m the water activation are too poorly defined by the present data to identify favourable candidates for this residue m the mEH, or sEH sequence, yet there are several residues m each of the sequences that would fit with the model of a catalytic triad The observed sequence similarity of the epoxide hydrolases to the haloalkane dehalogenase implies that the epoxide hydrolysis catalyzed by these enzymes proceeds via an as yet unexpected two-step mechanism mvolvmg a covalent enzyme-substrate hydroxy ester intermediate (Fig 3) This hydroxy ester mtermediate can be seen as the inverse equivalent to the acyl enzyme of serme hydrolases since the acyl group is contributed by the enzyme rather than by the substrate Fig 3 Potential catalytic mechanism of epoxlde hydrolases The pro- 3 3 Other related enzymes posed reactlon mechanism 1s bdsed on the findings of Verschueren et al [28] for the dehalogenase After entering the activecenter the epoxlde An additional screening of the SWISS PROT data IS polarized by hydrogen bondmg to the hydrogen of a proton donor library with the five protein sequences of the above ahgn- HR yet to be identified and possibly to the tryptophane adjacent to the ment identified further enzymes that display similarity to nucleophde (I) On attack of the nucleophlle the epoxlde rmg opens the alignment m Fig 1 All of these share the above under proton abstractlon leading to the postulated hydroxy ester mter- described consensus around a potential nucleophihc cen- mediate (II) In the following step the water activated by the HIS-ASP/ Clu couple hydrolyzes the ester bond resulting m the release of the ter that lies 40 to 50 ammo acids carboxytermmal to a formed dlol (III) A final necessary regeneration step (not shown) 1s the short sequence loosely fitting with the motif RVIAPD transfer of the proton from the His to the R- to reconstitute the proton that corresponds to p-strand 4 of the haloalkane dehalo- donor The table specifies the respective residues so far ldentdied by genase [26] A third common feature is a short motif either sequence slmllarlty or expenmental work

Table 1 Comparison of the nucleophlle-motif of cr//I hydrolase-fold enzymes to the correspondmg regions of the proteins of the sequence alignment m Fig I

SWISS-PROT entry Protein Source Nucleophlle-motif (sm-x-nu-x-sm-sm)’ a@ hydrolase-fold enzymes ACES_TORCA acetylcholme esterase Torpedo cahjomca GESAGG (structure resolved by X-ray analysis LIPl_GEOCN 11pase Geotruhum candrdum GESAGA

CBP2_WHEAT carboxypeptldase Trztrcum uestrvum GESTAG CLCD_PSEPU dlenelactone hydrolase Preudomonar putldu GTCLGG

HALO_XANAU haloalkane dehdlogenase Xanthobacter autotroph- VQDWGG KU5

Enzymes identified by sequence siml- DMPD_PSEPU hydroxymucomc semi- Pseudomonas putlda GNSFGG larlty to the soluble epoxlde hydrolase aldehyde hydrolase XYLF_PSEPU hydroxymucomc semi- Preudomonas putlda GNSFGG aldehyde hydrolase HYEP_RAT mlcrosomal epoxlde Rattus norvegrcus GGDWGS hydrolase HYES RAT soluble eooxlde hvdrolase Rat&s notvePlcus GHDWAG

“Ammo acid composition of the motif IS gwen m one-letter code except sm = small ammo acid, and nu = nucleophde M Arand et al IFEBS Letters 338 (1994) 251-256 255 between these two with the consensus GxGxS, that is of proteins comprismg a variety of different hydrolytic quite well conserved among the different proteins m enzymes including - according to our deductions - also both, sequence and distance from the other two common epoxide hydrolases. features (see Table 2) The malortty of the proteins iden- In conclusion, sequence compartsons have revealed a tified this way clearly have known hydrolytic function significant relationship of the microsomal and the solu- which fits with the catalytic concept of the a//? hydrolase- ble epoxtde hydrolase to an array of mostly bacterial fold enzymes The possible enzymatic functions of the proteins. The stmilanty among these sequences is mainly magnesium chelatase, the luctferase of Remlla renzformzs restricted to a specific region that corresponds to an and the bromoperoxtdase that relates them to hydro- important part of the active center of the haloalkane lases, however, are as yet obscure dehalogenase, a member of the a//I hydrolase-fold farmly On the basis of these observations we re-examined the of enzymes We conclude from our observations that (1) possible relattonship of LTA, hydrolase with mEH, and there 1s a distant evolutionary relattonshtp between these sEH As shown in Table 2 there is a region m the protem sequences and that (n) probably all these proteins belong sequence of the LTA, hydrolase that loosely fits with the to the a//l hydrolase-fold enzymes, as it is generally ac- above described pattern In contrast to all other enzymes cepted that the three-dimensional structure of proteins addressed above, though, alignments and dot matrix related m function 1s stronger preserved m evolution comparisons of the LTA, hydrolase sequence of the re- than their primary sequence [27]. Thus, the present ob- gion m question to that of the other epoxide hydrolases servations strongly suggest that the enzymatic hydrolysis and the haloalkane dehalogenase did not give conclusive of epoxtdes proceeds via an mtermediate covalently results Thus we cannot rule out a sparse relattonship of bond to the enzyme by an a-hydroxy ester linkage Such LTA, hydrolase to the other epoxide hydrolases but also a mechanism has already been considered as a possible have no convmcmg evidence for it alternative to the base catalysis by Armstrong et al [30] Very recently, Cygler et al. [29] have identified a large but these authors themselves strongly favour the direct number of proteins, mainly esterases and hpases, that are hydrolytic cleavage of epoxides [30,3 11. structurally related to the acetylchohne esterase from Torpedo cahformca and the hpase from Geotrlchum can- Acknowledgements This work was supported by the Deutsche dldum, the two a&l hydrolase-fold enzymes related by Forschungsgememschaft (SFB 302) and the NatIonal Institute of Envl- ronmental Health Saences, NIH (grant numbers ES02710 and sequence stmilartty. The a@ hydrolase-fold family of en- ES05707) J K B was supported m part by the NIH Biotechnology zymes therefore appears to be a rapidly growing group Predoctoral Trammg Grant GM08343

Table 2 Sequences deplcked by slmllanty to the alignment m Fig 1 SWISS-PROT Protein Source RVIAPD”- Distance GxGxS- Distance Nucleophlle- Entry Motif to Next Motif to Next Motif Motif Motif

HYES_RAT soluble epoxlde hydrolase Rattus norvegicus RVLAID 2 GYGDS 33 GHDWAG HYEP_RAT mlcrosomal epoxlde hydrolase Rattus norvegrcus EVICPS 2 GYGYS 32 GGDWGS HALO_XANAU haloalkane dehalogenase Xantobacter autrotrophlcus RVIAPD 2 GFGKS 33 VQDWGG DMPD_PSEPU hydroxymucomc semlaldehyde Pseudomonas putlda RVIAPD 2 GFGYS 32 GNSFGG hydrolase XYLF_PSEPU hydroxymucomc semlaldehyde Pseudomonas putrda RVIAPD 2 GFGYS 32 GNSFGG hydroldse TODF_PSEPU heptadlenoate hydrolase Pseudomonas putlda RVIAPD 2 GFGFT 32 GNSFGG TPES_PSEPU tropmesterase Pseudomonas putrda RYLALD 2 GHGGT 31 GHSMGS BPHD_PSESl blphenyl hydrolase Pseudomonas sp RVLLPD 2 GFNKS 32 GNSMGG PHAB_PSEOL hpase Pseudomonas oleovorans EVIAFD 2 GVGGS 31 GVSWGG ACOC_ALCEU (probable) dlhydrohpoamlde Alcahgenes eutrophus TWALD 2 GHGQS 31 GHSMGG acetyltransferdse LIP3_MORSP hpase Moraxella sp HLIIPD 2 GFGNS 33 GNSMGG BCHO_RHOCA magnesmm chelatase Rhodobacter capsulatw RVIVPD 2 GHGCS 32 GHSAGG LUCI_RENRE luciferase Remlla remformrs RCIIPD 2 GMGKS 33 GHDWGA BPAlSTRAU bromoperoxldase Streptomyces aureofaclens RVITYD 2 GFGQS 31 GFSMGT

LKHA_RATb leukotnene A4 hydrolase Rattus norveglcus RVPIPC 7 GALES 32 AEDLGG d Ammo acid composltlon of the motifs 1s given m one-letter code, %ot plcked up by slmdarlty search (see text) 256 M band et al IFEBS Letters 338 (1994) 2.5~256

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Note added III proof

After submlsslon of the present manuscnpt Lacourclere and Armstrong have pubhshed experimental results which strongly support the enzymatic mechanism proposed here (G M Lacourclere and R N Armstrong (1993) J Am Chem Sot 115. 10466-10467)