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Hexachlorocyclohexane Dehydrochlorinase Lina

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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-cyclohexa- diene. 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- ©2001WILEY-LISS,INC. 472 Y. NAGATA ET AL. 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
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