Hexachlorocyclohexane Dehydrochlorinase Lina

PROTEINS:Structure,Function,andGenetics45:471–477(2001)

IdentiﬁcationofProteinFoldandCatalyticResiduesof␥- HexachlorocyclohexaneDehydrochlorinaseLinA

YujiNagata,1* KatsukiMori,2 MasamichiTakagi,2 AlexeyG.Murzin,3 andJirˇı´Damborsky´ 4 1GraduateSchoolofLifeSciences,TohokuUniversity,Sendai,Japan 2DepartmentofBiotechnology,TheUniversityofTokyo,Tokyo,Japan 3 CentreforProteinEngineering,MedicalResearchCouncilCentre,Cambridge,UnitedKingdom 4NationalCentreforBiomolecularResearch,MasarykUniversity,Brno,CzechRepublic

ABSTRACT ␥-Hexachlorocyclohexanedehy- tion.Infact,wehaverevealedthatthreedifferenttypesof drochlorinase(LinA)isauniquedehydrochlorinase dehalogenases,dehydrochlorinaseLinA,4,5 halidohydro- thathasnohomologoussequenceattheaminoacid- laseLinB,6,7 andreductivedehalogenaseLinD,8 arese- sequencelevelandforwhichtheevolutionaryori- quentiallyinvolvedinthedegradationof␥-HCHinUT26.9 ginisunknown.WehereproposethatLinAisa Amongthesethreedehalogenases,LinAisthoughttobea memberofanovelstructuralsuperfamilyofpro- uniquedehydrochlorinase,basedonthefailureofFASTA 5 teinscontainingscytalonedehydratase,3-oxo-⌬ - andBLASTdatabasesearchestofindanysignificantly steroidisomerase,nucleartransportfactor2,and homologoussequencestothelinAgene.4 Thus,theorigin the␤-subunitofnaphthalenedioxygenase—all ofthelinAgeneisofgreatinterest,butisstillunknown. knownstructureswithdifferentfunctions.Thecat- LinAcatalyzestwostepsofdehydrochlorinationfrom alyticandtheactivesiteresiduesofLinAarepre- ␥-HCHto1,3,4,6-tetrachloro-1,4-cyclohexadiene(1,4- dictedonthebasisofitshomologymodel.Ninemu- TCDN)via␥-pentachlorocyclohexene(␥-PCCH;Fig.1).It tantsthatcarrysubstitutionsoftheproposed hasbeenproposedthat1,4-TCDNisconvertedto1,2,4- catalyticresidueswereconstructedbysite-directed trichlorobenzene(1,2,4-TCB)nonenzymatically(Fig.1), mutagenesis.Inadditiontothese,eightmutants because1,4-TCDNhasanunstablediene-typestructure thathaveapotentialtomakecontactwiththesub- andistobechangedintoamoleculewithastablearomatic stratewerepreparedbysite-directedmutagenesis. ring.10 InUT26,partof1,4-TCDNishydrolyticallydechlo- ThesemutantswereexpressedinEscherichiacoli, rinatedbyLinBto2,5-dichloro-2,5-cyclohexadiene-1,4-diol andtheiractivitiesincrudeextractwereevaluated. (2,5-DDOL)via2,4,5-trichloro-2,5-cyclohexadiene-1-ol MostofthefeaturesoftheLinAmutantscouldbe (2,4,5-DNOL).6 explainedonthebasisofthepresentLinAmodel, ThelinAgenewashighlyexpressedinrecombinant indicatingitsvalidity.WeconcludethatLinAcata- Escherichiacolicells,andthegeneproduct(LinA)was lyzestheprotonabstractionviathecatalyticdyad 5 ␥ H73-D25byasimilarmechanismasdescribedfor purifiedtohomogeneity. Inadditionto -HCHand ␥ ␣ ␦ scytalonedehydratase.Theresultssuggestthat -PCCH, -and -isomersofHCHwerealsodehydrochlo- ␤ 5 LinAandscytalonedehydrataseevolvedfroma rinatedbyLinA;however, -HCHwasnot. Theseresults commonancestor.LinAmayhaveevolvedfroman areconsistentwiththoseobtainedusingrestingUT26 10 enzymeshowingadehydrataseactivity.Proteins cells. ItwassuggestedthatdehydrochlorinationbyLinA 2001;45:471–477.©2001Wiley-Liss,Inc. occursstereoselectivelyatatransanddiaxialpairof hydrogenandchlorine.10 Recently,weconfirmedexperi- Keywords:dehydrochlorination;evolution;␥-hexa- mentallythatdehydrochlorinationof␥-HCHproceedsbya chlorocyclohexane;homologymodel- 1,2-antidehydrochlorinationreaction.11 Theotherchlori- ing;LinA;reactionmechanism

INTRODUCTION Abbreviations:2,5-DDOL,2,5-dichloro-2,5-cyclohexadiene-1,4-diol; DDT,1,1,1-trichloro-2,2-bis(p-chloro-phenyl)ethane;2,4,5-DNOL, ␥-Hexachlorocyclohexane(␥-HCH;alsocalledBHCor 2,4,5-trichloro-2,5-cyclohexadiene-1-ol;ECD,electroncapturedetec- lindane)isahalogenatedorganicinsecticidethathasbeen tor;GC,gaschromatography;GSH,glutathione;GST,glutathione S-transferase;HCH,hexachlorocyclohexane;LinA,␥-hexachlorocyclo- usedworldwidesincethe1940s,butthathasbeenprohib- hexanedehydrochlorinase;LinB,1,4-TCDNhalidohydrolase;␥-PCCH, itedinmostcountriesbecauseofitstoxicityandlong ␥-pentachlorocyclohexene;PLP,pyridoxal5Ј-phosphate;1,2,4-TCB, persistenceinsoil.Unfortunately,manycontaminated 1,2,4-trichlorobenzene;1,4-TCDN,1,3,4,6-tetrachloro-1,4-cyclohexadiene. sitesremainthroughouttheworld.Moreover,somecoun- ␥ Grantsponsor:Grant-in-AidforScientiﬁcResearchfromtheMinis- triesarepresentlyusing -HCHforeconomicreasons,and tryofEducation,Science,SportsandCultureofJapan. 1,2 thusnewsitesarecontinuouslybeingcontaminated. *Correspondenceto:YujiNagata,GraduateSchoolofLifeSciences, SphingomonaspaucimobilisUT26degrades␥-HCHun- TohokuUniversity,2-1-1Katahira,Sendai,980-8577,Japan.E-mail: deraerobicconditions.3 Because␥-HCHisahighlychlori- [email protected] natedcompoundhavingsixchlorineatomspermolecule, Received15March2001;Accepted31July2001 dechlorinationisaverysigniﬁcantstepforitsdegrada-

were obtained from Takara Shuzo Co. (Kyoto, Japan). The mutagenesis primers were obtained from Espec-oligo Ser- vice Co. (Tsukuba, Japan). The strain used in this study is E. coli HB101.24

Fig. 1. Proposed degradation pathway of ␥-HCH by LinA. The first and Site-directed Mutagenesis the second reaction steps are catalyzed by LinA. The third reaction is proposed to be nonenzymatic, because 1,4-TCDN has an unstable Site-directed mutagenesis of linA was performed by diene-type structure and is to be changed into a molecule with a stable using the principle of a LA PCR in vitro mutagenesis kit 10 aromatic ring. Compounds: 1, ␥-HCH; 2, ␥-PCCH; 3, 1,4-TCDN; 4, (TaKaRa Shuzo Co. Kyoto, Japan), according to the pro- 1,2,4-TCB. vided protocol except for using KOD (King of DNA) polymer- ase (Toyobo Co., Osaka, Japan) and Pyrobest DNA polymer- nated compounds tested were not dehydrochlorinated by ase (TaKaRa) whose fidelity are very high. All of the the purified enzyme, indicating that the substrate specific- 5 nucleotide sequences of mutants were confirmed by the ity of LinA is narrow. dideoxy-chain termination method with an automated Dehydrochlorinase eliminates HCl from the substrate 12,13 DNA sequencer (ABI PRISM 310 genetic analyzer; Ap- molecule, leading to the formation of a double bond. To plied Biosystems, Foster City, CA). The oligo nucleotides date, the properties of three dehydrochlorinases (including used are as follows: K20Q (5Ј-TAC TCT GAC CAG CTC LinA) have been reported. An eukaryotic dehydrochlorinase ATT GCC-3Ј), K20M (5Ј-TAC TCT GAC ATG CTC ATT isolated from Musca domestica catalyzes the monodehydro- GCC-3Ј), D25N (5-ATT GCC GTA AAC AAG CGC C-3Ј), chlorination of 1,1,1-trichloro-2,2-bis(p-chloro-phenyl)ethane Ј 14,15 D25L (5 -CTC ATT GCC GTA CTC AAG CGC CAA (DDT). 3-Chloro-D-alanine dehydrochlorinase was iso- Ј Ј 16 GAG-3 ), H73Q (5 -GAA TGT ATT CAG TAT GGA ACC- lated from Pseudomonas putida. LinA is, however, very 3Ј), D115N (5Ј-CTC ATA CTC ATT CGT GAA GAC-3Ј), different from these two other dehydrochlorinases. DDT D115L (5Ј-GCG CTC ATA CTC AAG CGT GAA GAC dehydrochlorinase and 3-chloro-D-alanine dehydrochlorinase CGC-3Ј), R129Y (5-CGT GCA TGC GTT GTA CTT AGA Ј require glutathione (GSH) and pyridoxal 5 -phosphate (PLP), GAA CTT-3Ј), R129M (5Ј-CGT GCA TGC GTT CAT CTT respectively, for their activities, whereas LinA does not Ј Ј 5 AGA GAA CTT-3 ) W42L (5 -GCA GAG TTG ACC ATT require any cofactors. Purified LinA did not show gluta- GAG-3Ј), Y50A (5Ј-ATC GGC ACC GCC AAG GGC CCG- thione S-transferase (GST) nor DDT dehydrochlorinase activ- 3Ј), L64A (5Ј-AAT AAC GTA GCC TGG CCA ATG-3Ј), ity in the presence of glutathione. Thus, it seems most likely M67A (5Ј-CTC TGG CCA GCG TTT CAC GAA-3Ј), L96A that LinA is not a GST-type enzyme. LinA is thought to be a (5Ј-ATT TCC AAG GGC AAG GAC GTC-3Ј), F113L (5Ј- unique dehydrochlorinase, and its mechanism of dehydrochlo- ATC CGT GAG GAC CGC AGC-3Ј), F113Y (5Ј-ATC CGT rination is of great interest. GTA GAC CGC AGC-3Ј), and F144L (5Ј-CGG TGC GAG In this study, we propose the protein fold of LinA and ATG AAT GCC-3Ј). identify its catalytic residues. A model of the three- dimensional structure of LinA constructed using homolo- Overexpression of LinA Mutants in E. coli gous proteins as the templates is used for identification of the putative catalytic and the active site residues. The To overproduce LinA mutants in E. coli, plasmids for overexpression were constructed from pAQN, which has mutants designed according to the model are constructed 25 by site-directed mutagenesis. The divergence of the evolu- the same structure as pAQI except for differences in the tion of LinA from the common ancestor with scytalone aqualysin I-coding region. In these plasmids, linA mutants ⌬5 are transcribed by the tac promoter under the control of dehydratase, nuclear transport factor-2,3-oxo- -steroid q isomerase and ␤-subunit of naphthalene 1,2-dioxygenase lacI . E. coli HB101 containing these plasmids for mutants is discussed. were cultured in 10 ml of Luria broth (LB) at 30°C. Cells were harvested after induction with 1 mM IPTG, washed MATERIALS AND METHODS in 50 mM potassium phosphate buffer (pH 7.5), and Identification of the Protein Fold and Computer resuspended in the sample buffer [50 mM potassium Modeling of LinA phosphate buffer (pH 7.5) containing 1 mM 2-mercapto- Sequence comparisons were conducted with a PSI- ethanol and 10% glycerol]. The cells were disrupted by BLAST and BLOSUM62 substitution matrix.17 Threading sonication (Sonifier 250; Branson, Danbury, CT). After searches for remote homologues of LinA were performed centrifugation at 12,000g for 10 min, the supernatant was using Bioinbgu18 and 3D-PSSM19 servers. The homology used as a crude extract. model of LinA dehalogenase reported earlier11 was con- Western Blot Analysis structed according to experimental structures of scytalone dehydratase (1std),20 nuclear transport factor-2 (1oun),21 Antibodies were raised against purified LinA produced 3-oxo-⌬5-steroid isomerase (1opy),22 and naphthalene 1,2- in E. coli.26 Samples were separated by SDS-PAGE and dioxygenase (1ndo).23 transferred to the nitrocellulose membrane Hybond C (Amersham, Arlington Heights, IL). The ECL Western Materials blotting system (Amersham) was used for detection. Rela- All chemicals were purchased from Nakalai tesque tive expression level of mutants to wild-type LinA was (Kyoto, Japan). The enzymes used for DNA manipulations quantified by using a densitometer. STRUCTURE AND CATALYTIC RESIDUES OF LINA 473

Qualitative Analysis for the LinA Activity Gas-chromatography with an electron capture detector was used for detection of LinA activity (i.e., degradation of ␥-HCH, and production of ␥-PCCH and 1,2,4-TCB) as described previously.4

Quantification of the LinA Activity Crude extract was diluted with 50 mM phosphate buffer (pH 7.5) to yield a final volume of 1 ml and incubated with 100 ppm of ␥-HCH at 30°C. The amount of chloride ion released was measured spectrophotometrically at 460 nm with mercuric thiocyanate and ferric ammonium sulfate by the method of Iwasaki et al.27 One unit of LinA activity was defined as the amount of enzyme required for the release of 1 ␮mol of chloride ion per minute. The activity of each mutant relative to wild-type protein was expressed as a percentage. Because the expression level of each mutant was different, the relative activities were revised according to each expression level in crude extract. Experiments were repeated at least three times.

RESULTS Identification of the Protein Fold of LinA and Prediction of Its Structure The striking similarity of the structures of scytalone dehydratase,20 nuclear transport factor 2,21 and 3-oxo-⌬5- steroid isomerase22 (Fig. 2) suggests that these proteins probably descended from a common ancestor, despite the lack of sequence similarity (Fig. 3) and very different function.28 These proteins share many common structural details, suggesting their distant relationships. The domi- nant motif of this superfamily is a central six-stranded ␤-sheet with the strand order 2-1-6-5-4-3. Three of the strands (␤3, ␤4, and ␤5) are unusually long for such small proteins. The ␤-sheet is very curved and is complemented by several ␣-helices resulting in unique mixed barrel-type structure (cone-shaped ␣ϩ␤ barrel). It is suggested that there may be other proteins in this superfamily that were descended from the same ancestor, but that diverged beyond notable sequence similarity and evolved different functions. The sequence libraries GenBank, EMBL, and DDBJ were searched using the sequences of scytalone dehy- Fig. 2. Superposition of the template structures and theoretical model dratase, nuclear transport factor 2, and 3-oxo-⌬5-steroid of LinA. ␥-hexachlorocyclohexane dehydrochlorinase (red; LinA model), isomerase using the PSI-BLAST web server.17 The se- scytalone dehydratase (yellow; 1std), nuclear transport factor-2 (green; 1oun), 3-oxo-⌬5-steroid isomerase (purple; 1opy) and naphthalene 1,2- quences with global hits that were compatible with all of dioxygenase (blue; 1ndo). Only the alpha-trace is displayed for every the conserved structural details of the common fold were structure (A). The alpha-trace of LinA and the side-chains of correspond- filtered out. A small group of sequences—all of which were ing putative catalytic residues are displayed: K20, D25, H73, D115, and R129 of LinA; D31 and H85 of 1std; Y18, D23, and H66 of 1oun; Y16 and hypothetical proteins—were compatible with the two en- D21 of 1opy; D546, D661, and R676 of 1ndo. The structures were 5 zymes, scytalone dehydratase and 3-oxo-⌬ -steroid isomer- presuperimposed manually and the superposition was refined using the ase (data not shown). These were used as queries in the program Swiss-PdbViewer v3.5b31 (B). Detailed view of the putative ␥ next round of searches, which yielded additional se- active site of LinA with docked substrate -hexachlorocyclohexane. Only the side chains of mutated residues: K20, D25, W42, Y50, L64, M67, H73, quences, including two functionally characterized en- L96, F113, D115, R129, and F144 are displayed and labeled. The zymes—naphthalene 1,2-dioxygenase (␤-subunit) and structure is colored by atom type (C). ␥-HCH. The results from the PSI-BLAST searches were further supported by the results from the threading analysis. Top-scoring hits obtained from the threading search top-scoring proteins in the Bioinbgu searches were two for remote homologues of LinA using Bioinbgu18 and different experimental structures of 3-oxo-⌬5-steroid 3D-PSSM19 servers are provided in Table I. First and third isomerase. The first four top-scoring proteins, showing 474 Y. NAGATA ET AL.

Fig. 3. Structurally derived alignment of the amino acid sequences of ␥-hexachlorocyclohexane dehydrochlorinase (LinA), scytalone dehydratase (1std), nuclear transport factor-2 (1oun), 3-oxo-⌬5-steroid isomerase (1opy), and naphthalene 1,2-dioxygenase-beta (1ndo). Corresponding putative catalytic residues are boxed [see caption Fig. 2(B)]. Secondary structure elements are underlined and shaded, ␣-helices are in light, whereas ␤-sheets are in dark. The secondary structure elements of LinA were predicted from the sequence using JPRED.32 The secondary structure elements of 1std, 1oun, 1opy, and 1ndo were assigned from the 3D structures using PROMOTIF.33

TABLE I. Five Top-scoring Proteins from Bioinbgu and 29 3D-PSSM Threading Searches been shown experimentally that enzymatic function can be engineered into the scaffold by positioning appropriate Bioinbgu catalytic residues inside the hydrophobic cavity. E-value PDB-ID_chain Protein PSI-BLAST searches, threading analyses, and predic- 6.9 1isk_a 3-oxo-⌬5-steroid isomerase tion of the secondary elements support our proposal that 5.4 1faf_a N-terminal J domain of murine LinA has ␣ϩ␤ barrel structure. At the same time, the polyomavirus T antigens sequence alignment (Fig. 3) confirms the presence of some ⌬5 4.9 1opy 3-oxo- -steroid isomerase of the catalytic residues of this protein family in LinA 2.5 1ds9_a chlamydomonas outer arm sequence. These evidences together provided the basis for dynein light chain-1 construction of a homology model of LinA based on its 2 2ezh DNA binding domain of MU phage transposase distant relatives. Conformation of the protein backbone and the position of the catalytic residues are expected to be PSSM E-value PDB-ID_chain Protein predicted with significantly higher reliability compared to 0.00785 3std scytalone dehydratase side chains. Side-directed mutagenesis was used to vali- 0.0387 1oun nuclear transport factor-2 date and refine this model as described below. 0.353 1opy 3-oxo-⌬5-steroid isomerase 0.54 1qjg 3-oxo-⌬5-steroid isomerase Construction of LinA Mutants by Site-directed 1.03 2nm_b dnumb Ptb domain Mutagenesis The alignment of the LinA protein sequence with the 95%, 95%, 70%, and 50% certainty based on 3D-PSSM sequences of other proteins from its superfamily (Fig. 3) E-value were scytalone dehydratase, nuclear transport enabled us to propose the catalytic residues of LinA. The factor 2, and two different experimental structures of H73-D25 pair of LinA corresponds to the putative catalytic 3-oxo-⌬5-steroid isomerase (Table I). A theoretical model of dyad of scytalone dehydratase, in which the H85 residue is LinA was constructed by homology modeling using the proposed to act as the general base. The proton abstraction structures of scytalone dehydratase, nuclear transport by the His-Asp pair should be essential for the LinA factor 2, 3-oxo-⌬5-steroid isomerase, and naphthalene activity. H73 is conserved also in nuclear transport factor 1,2-dioxygenase as the templates.11 Although the se- 2. D25 is the single-most conserved residue in the entire quence similarity of these proteins with LinA is well superfamily (Fig. 3). The K20 of LinA is in the same site as beyond the twilight zone, they show highly conserved ␣ϩ␤ the general acid Y16 of 3-oxo-⌬5-steroid isomerase, whereas barrel structure with a distinctive hydrophobic cavity (Fig. the R129-D115 pair aligns with the equivalent pair in 2). This cavity is suited for binding of small ligands.20 ␣ϩ␤ naphthalene dioxygenase (R676-D661). K20 and R129 are barrel structure provides scaffold for catalytic residues, expected to interact with the Cl group and/or to stabilize which must be correctly positioned in the three-dimen- the leaving ClϪ ion. This proposal is consistent with the sional space to fulfill their catalytic function. Not all 1,2-anti dehydrochlorination reaction mechanism.11 D115 proteins in the family catalyze biochemical reactions, nor is hypothesized to assist the proper positioning of R129. do they have fully conserved catalytic residues, but it has The positive charge of R129 and K20 and the negative STRUCTURE AND CATALYTIC RESIDUES OF LINA 475

TABLE II. Relative Expression Levels and Activities [in %] different, the amount of each mutant protein was quanti- of LinA Mutants fied by Western blotting, and the expression level relative to that of the wild-type protein was estimated (Table II). Mutant Relative expressiona Activityb Relative activityc The activities of mutant proteins were revised according to ϩ Wild type 100 100 each expression level, and their activities relative to the K20M 128 ϩ 69 Ϯ 5.6 wild-type enzyme were calculated (Table II). K20Q, W42L, K20Q 122 ϩ 3 Ϯ 2.5 D25N 86 Ϫ and F113Y were almost inactive. F113L showed signifi- D25L 40 Ϫ cant reductions in activity. K20M, D115N, D115L, Y50A, W42L 81 ϩ 5 Ϯ 4.6 M67A, and F144L retained more than half the level of Y50A 46 ϩ 62 Ϯ 3.5 activity shown by the wild-type enzyme. L64A 12 Ϫ M67A 16 ϩ 95 Ϯ 11.2 DISCUSSION H73Q 42 Ϫ L96A 29 ϩ 10 Ϯ 6.3 The predicted three-dimensional structure of LinA be- F113L 58 ϩ 38 Ϯ 3.5 longs to a group of folds called ␣ϩ␤ rolls.30 The polypeptide F113Y 96 ϩ 1 Ϯ 0.6 chain of LinA forms a cone-like ␣ϩ␤ barrel with a distinc- D115L 95 ϩ 69 Ϯ 6.6 tive hydrophobic cavity that in turn forms a binding pocket D115N 90 ϩ 73 Ϯ 2.7 (Fig. 2). The putative catalytic residues H73-D25 and Ϫ R129M 98 putative substrate-binding residues K20 and R129-D115 Ϫ R129Y 54 are positioned inside this cavity. The purpose of the F144L 111 ϩ 102 Ϯ 1.2 mutagenesis analysis of the first nine mutants was to aThe amount of each mutant protein in crude extract was quantified identify the catalytic residues of LinA. D25N, D25L, by Western blotting, and the expression level relative to that of wild H73Q, R129Y, and R129M were inactive as predicted. type protein was estimated. bThe presence of LinA activity was detected by gas chromatography. These residues should be directly involved in the catalysis. cThe relative activities were quantified by measuring the amount of K20Q retained only a very low level of activity. In contrast, chloride ion released spectrophotometrically. K20M, D115L, and D115N retained a high level of activity. K20 is expected to interact with the chlorine atom of the substrate molecule bound in the enzyme-active site. For charges of D25 and D115 are thought to be essential for the role of K20, not only a partial positive charge, but also their roles, respectively. To investigate the roles of above the proper size of the side chain seems to be important. To discussed amino acid residues of LinA, K20M, K20Q, identify the exact role of K20, additional site-directed D25N, D25L, H73Q, D115L, D115N, R129Y, and R129M mutation analyses are necessary. Although D115 was were constructed. expected to assist in proper positioning of R129, the Additional site-directed mutation analyses were con- catalytic activity of D115L and D115N seems not to be ducted to refine the model of LinA and to identify in influenced by the mutations, suggesting that D115 is not greater detail the amino acid residues that are in direct essential for the LinA activity. contact with the substrate. Seven amino acid residues, The refined model could nicely explain the result of the W42, Y50, L64, M67, L96, F113, and F144, were selected following eight mutants. The mutants that retained high for their potential to make contact with the substrate, levels of activity (Y50A, M67A, and F144L) have a muta- according to the initial homology model. Eight mutants, tion in the residues participating in formation of the W42L, Y50A, L64A, M67A, L96A, F113L, F113Y, and entrance to the enzyme active site. It was suggested that F144L, were constructed. neither enzyme-substrate complex formation nor the resulting product release is the rate-limiting step. The largest Relative Activities of Mutant Proteins change in activity was observed in the case of Y50A, which Crude extracts of E. coli expressing LinA and its mu- has the mutation in the most exposed residues of the three tants were prepared. Mutant proteins were assayed for residues within this group. The mutants that showed activity by gas chromatography with an electron capture significantly reduced or lost activities, W42L, L64A, L96A, detector. This assay is relatively sensitive for detecting F113L, and F113Y, have a mutation in the residues in LinA activity, because it can monitor not only depletion of direct van der Waals contact with the substrate. Their ␥-HCH, but also production of ␥-PCCH and 1,2,4-TCB. The mutations possibly influenced the affinity of the enzyme results indicated that D25N, D25L, H73Q, R129Y, R129M, for the substrate. Mutation in L64 either results in nonpro- and L64A were inactive (Table II). The LinA activity of ductive binding of the substrate or in repositioning of H73 10ϫ dilution of crude extract for K20Q could be detected by and prevents efficient extraction of the proton by this this method. All of the mutants listed as inactive (Table II) residue. must have a lower level of activity than K20Q. LinA The overall structure of LinA predicted by the computer activities of other mutants were quantified by measuring modeling was verified by site-directed mutagenesis. The the amount of chloride ion released spectrophotometri- LinA protein apparently has a common ancestry with the cally. The LinA activity of crude extract prepared from E. enzymes scytalone dehydratase and 3-oxo-⌬5-steroid coli expressing wild-type LinA was 1.7 to 5.2 ϫ 10Ϫ2 U/mg isomerase as well as the proteins nuclear transport factor protein. Because the expression level of each mutant was 2 and naphthalene 1,2-dioxygenase (␤-subunit). Catalyti- 476 Y. NAGATA ET AL.

Fig. 4. Comparison of putative reaction mechanism of ␥-hexachlorocyclohexane dehydrochlorinase LinA (A)11 with that of scytalone dehydratase (B).21 The proton abstraction from the substrate molecules is facilitated by the catalytic dyad H73-D25 in LinA and H85-D31 in scytalone dehydratase.

cally inactive proteins nuclear transport factor 2 and 588. ␤-subunit of the naphthalene 1,2-dioxygenasehave retain 2. Iwata H, Tanabe S, Sakai N, Tatsukawa R. Distribution of persistent organochlorines in the Oceanic air and surface seawa- some of the catalytically important residues of LinA, ter and the role of ocean on their global transport and fate. suggesting that these proteins may have originally had a Environ Sci Technol 1993;27:1080–1098. catalytic function that was lost during their evolution. 3. Imai R, Nagata Y, Senoo K, Wada H, Fukuda M, Takagi M, Yano ␥ ␥ Their activity can be reconstituted by mutagenesis experi- K. Dehydrochlorination of -hexachlorocyclohexane ( -BHC) by ␥-BHC-assimilating Pseudomonas paucimobilis. Agric Biol Chem ments, as recently demonstrated in the case of nuclear 1989;53:2015–2017. 29 transport factor 2 by Nixon et al. 4. Imai R, Nagata Y, Fukuda M, Takagi M, Yano K. Molecular cloning of a Pseudomonas paucimobilis gene encoding a 17- CONCLUSION kilodalton polypeptide that eliminates HCl molecules from ␥-hexachlorocyclohexane. J Bacteriol 1991;173:6811–6819. The overall structure of LinA predicted by the computer 5. Nagata Y, Hatta T, Imai R, Kimbara K, Fukuda M, Yano K, modeling was verified by site-directed mutagenesis, and Takagi M. Purification and characterization of ␥-hexachlorocyclo- D25, H73, and R129 were shown to form catalytically hexane (␥-HCH) dehydrochlorinase (LinA) from Pseudomonas important residues of LinA and to be essential for its paucimobilis. Biosci Biotechnol Biochem 1993;57:1582–1583. 6. Nagata Y, Nariya T, Ohtomo R, Fukuda M, Yano K, Takagi M. activity. The requirement of the proton abstraction by the Cloning and sequencing of a dehalogenase gene encoding an Asp-His pair (D25 and H73) is similar to that in the case of enzyme with hydrolase activity involved in the degradation of scytalone dehydratase (Fig. 4).20 These results suggest ␥-hexachlorocyclohexane (␥-HCH) in Pseudomonas paucimobilis. that the enzymatic activity of LinA and scytalone dehy- J Bacteriol 1993;175:6403–6410. 7. Nagata Y, Miyauchi K, Damborsky J, Manova K, Ansorgova A, dratase have the same evolutionary origin. LinA may have Takagi M. Purification and characterization of haloalkane dehalo- evolved from an enzyme showing dehydratase activity. genase of a new substrate class from a ␥-hexachlorocyclohexane- degrading bacterium, Sphingomonas paucimobilis UT26. Appl ACKNOWLEDGMENTS Environ Micobiol 1997;63:3707–3710. 8. Miyauchi K, Suh S-K, Nagata Y, Takagi M. Cloning and sequenc- Part of this work was performed using the facilities of ing of a 2,5-dichlorohydroquinone reductive dehalogenase gene the Biotechnology Research Center, The University of whose product is involved in degradation of ␥-hexachlorocyclohex- Tokyo. AGM was supported by an MRC Senior Fellowship. ane by Sphingomonas paucimobilis. J Bacteriol 1998;180:1354– 1359. REFERENCES 9. Nagata Y, Miyauchi K, Takagi M. Complete analysis of genes and enzymes for ␥-hexachlorocyclohexane degradation in Sphingomo- 1. Blais JM, Schindler DW, Muir DCG, Kimpe LE, Donald DB, nas paucimobilis UT26. J Ind Microbiol Biotechnol 1999;23:380– Rosenberg B. Accumulation of persistent organochlorine com- 390. pounds in mountains of western Canada. Nature 1998;395:585– 10. Nagasawa S, Kikuchi R, Nagata Y, Takagi M, Matsuo M. Stereo- STRUCTURE AND CATALYTIC RESIDUES OF LINA 477

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