Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity

Transcription

1 Pro. NtI. Ad. Si. USA Vol. 85, pp , Otoer 1988 Biohemistry Proteins from eight eukryoti ytohrome P-450 fmilies shre segmented region of sequene similrity (ytohrome P450/onsensus sequene/evolution/p450m/superfmily) VERNON F. KALB AND J. C. LOPER Deprtment of Miroiology nd Moleulr Genetis, University of Cininnti College of Mediine, 231 Bethesd Avenue, Cininnti, OH Communited y M. J. Coon, July 5, 1988 (reeivedfor review Mrh 30, 1988) ABSTRACT Proteins from eight eukryoti fmilies in the ytohrome P-450 superfmily shre one region of sequene similrity. This region egins mino ids from the mino terminus of eh P-450, ontinues for =170 residues, nd ends mino ids efore the roxyl terminus. The region n e divided into four domins of sequene similrity, eh possessing its own pttern of invrint, onserved, nd vrile mino ids. The four domins re 56, 20, 59, nd 28 residues long nd re onneted y three shorter segments of limited sequene similrity. The numer of residues in these short segments vries with the P-450 protein ut rnges from 0 to 20 residues. Consensus sequenes sed on these similrities n e used to determine whether the sequene of n unidentified peptide resemles tht expeted for P-450. Sequene similrities etween proteins sometimes reflet onstrints imposed y the requirements of ommon funtion. The fourth domin of the P-450s, for exmple, ontins n invrint ysteine tht provides the xil thiolte lignd to the heme iron. Other reltionships etween the four domins nd P-450 funtion n e exmined y in vitro mutgeni proedures tht lter the onserved mino ids or modify the distne etween domins. The P-450 ytohromes re omponents in monooxygense systems tht tlyze wide vriety of oxidtive retions in prokryotes nd eukryotes. These retions inlude steps in the synthesis nd degrdtion of suh ompounds s holesterol, steroid hormones, nd prostglndins. P-450 proteins re lso involved in the metolism of drugs nd in the tivtion nd intivtion of rinogens. A reent nlysis of >60 P-450s from eukryotes nd one prokryote led to the orgniztion of the known P-450s into 10 different fmilies with totl of 15 sufmilies. Amino id sequenes within P-450 fmily re >36% similr, while sequenes within sufmily re :70% similr (1). Although sequene reltedness etween P-450 fmilies is low, these fmilies re still grouped into one P-450 superfmily ording to stndrd riteri (2). The eukryoti rnh of the ytohrome P-450 superfmily rose >0 million yers go (1, 3) nd sequene similrities mong these nient fmilies might identify onserved domins of struture or funtion. Sine omplete mino id sequenes hd een pulished for proteins from eight eukryoti P-450 fmilies, we used representtive sequene from eh fmily in series of sequene omprisons. A multiple lignment of the eight sequenes reveled one region of similrity shred y ll. Sequene similrities in this region reside in four domins onneted y short segments of vrile length. The region will provide fous for experiments tht proe the reltionship of mino id sequene to struture nd funtion in the P-450 superfmily. The pulition osts of this rtile were defryed in prt y pge hrge pyment. This rtile must therefore e herey mrked "dvertisement" in ordne with 18 U.S.C solely to indite this ft METHODS Amino Aid Sequenes. The soure for eh eukryoti P-450 sequene used in these omprisons ws s follows:, rt liver (4);, rt liver (5); 17, ovine drenl ortex (6); s, ovine drenl ortex (7); pnl, rt liver (8);, ovine drenl ortex (9);, rt liver (10);, Shromyes erevisie (11). The teril P-450 sequene ws P-450m from Pseudomons putid (12). Eh mino id sequene ws derived from the orresponding nulei id sequene with the N-terminl methionine ounted s residue 1. Some nlyses used mino id sequenes of the Protein Identifition Resoure Protein Sequene Dtse.* Constrution of the Multiple Alignment. The multiple lignment ws onstruted fter exmintion nd mnipultion of numer of different pirwise lignments for the eight P-450 proteins. We generted these lignments y using the Wilur nd Lipmn lgorithm (13) s implemented t Bionet in the IFIND progrm. The prmeters WORD-LENGTH, GAP- PENALTY, WINDOW, DENSITY, nd FAST were set t 1, 2, 30, LESS, nd NO. The sore for two ligned sequenes equls the numer of mthing residues minus the numer of gps multiplied y the GAP-PENALTY. Similrities shred y ll eight sequenes were pprent only in the roxylterminl portions of these proteins nd only these portions re displyed in the multiple lignment. The onstrution of the multiple lignment ws not strightforwrd sine different pirwise lignments sometimes yielded inonsistent results. One suh se involved the three pirwise lignments of P-450, P-450, nd P-450pnl. Although the lignment of P-450pnl with P- 450 required one gp nd the lignment of P-450 with P-450 required two gps, the lignment of P-450pnl with P-450 did not require gps. Suh inonsistenies were eliminted y mnully sliding or removing gps in eh pirwise lignment nd then heking the multiple sequene lignment to find onfigurtion tht yielded onsistent gp plement nd mximized the numer of mthing residues. This proess removed losely positioned gps, yielding multiple lignment of eight sequenes tht ontined only three gps. The suess of this pproh suggests tht pirwise lignment lgorithms might e improved y inorporting n djustle prmeter tht ould e used to penlize losely positioned gps. Consensus Sequenes. Sequene similrities re summrized y four tenttive onsensus sequenes. To onstrut these onsensus sequenes, we ssumed tht eh mino id would pper n verge of 1 time in 20 t ny position in ny of the eight ligned mino id sequenes. We then lulted (14) the expeted frequeny for every omintion of eight mino ids tht ould our t single position in the multiple lignment. The 22 possile omintions rnge from the se where ll eight mino ids re identil to the se *Protein Identifition Resoure (1987) Protein Sequene Dtse (Ntl. Biomed. Res. Found., Wshington, DC), Relese 12.

2 7222 Biohemistry: Kl nd Loper where ll eight re different. The 6 most ommon ptterns nd their expeted frequenies re s follows: (i) no pirs of identil mino ids, 0.198; (ii) one pir of identil mino ids, 0.427; (iii) two pirs of identil mino ids, 0.229; (iv) three pirs of identil mino ids, 0.031; (v) three identil mino ids, 0.061; nd (vi) three identil mino ids plus one pir of identil mino ids, When 1 of these ommon ptterns ourred, no onsensus mino id ws seleted. Eh of the remining 16 ptterns is expeted to our t frequeny <1%. When 1 of these less ommon ptterns ourred, we seleted the identil mino ids in tht pttern s the onsensus mino ids t tht position in the multiple lignment. We expeted tht typil eukryoti P-450 protein would mth these onsensus sequenes t mny ut not neessrily ll of the onsensus positions. Comprisons of Consensus Sequenes to Proteins. Using sliding window pproh, eh onsensus sequene ws ompred to trget protein or to rndomly permuted version of the trget protein. To omplish this omprison, the first position in the onsensus sequene ws pled ove the first mino id in the sequene of the protein nd the numer of positions t whih oth sequenes shred n identil mino id ws determined. The first position in the onsensus sequene ws then pled ove the seond mino id in the protein nd the numer of mthing residues ws gin determined. The stepwise movement of the onsensus sequene ws ontinued to the end of the protein. The highest numer of mthing residues found during this proess eme the sore for tht protein. When the trget protein ws P-450, the onsensus sequene ws lso ompred to rndomly permuted vrints of this P450 protein. A men nd stndrd devition were then lulted for the numer of mthing residues found during the steps in the omprisons to the rndomized P-450 sequenes. Using this men nd stndrd devition, z vlue ould e lulted tht quntitted the differene etween the sore for n unrndomized P-450 nd the men numer of mthing residues ourring in rndom vrints of the P-450: z = (sore - men)/stndrd devition. An lgorithm (15) ws used to generte the rndom numers tht were required to permute (16) the mino id sequenes. Consensus sequenes were lso ompred to the proteins in the Protein Identifition Resoure dtse. The distriution of sores ws plotted nd the men nd stndrd devition of these sores were determined. RESULTS AND DISCUSSION We performed pirwise lignments of eight eukryoti ytohrome P-450 sequenes to exmine sequene similrities t the level of individul mino id residues. Eh of the eight hosen sequenes ws from different P-450 fmily sine we wished to find similrities ommon to ll eukryoti P-450s. These pirwise lignments were mnully djusted s desried in Methods to onstrut multiple sequene lignment. The multiple lignment (Fig. 1) revels the detils of region of sequene similrity found in ll eight sequenes. This shred region egins mino ids from the mino terminus of eh P-450 nd ontinues to within mino ids of the roxyl terminus. Sequene similrities in this region reside in four domins tht ontin 56, 20, 59, nd 28 mino id residues. These domins re joined y short segments of dissimilr mino id sequene tht vry in size mong the individul P-450 sequenes. The four domins (A, B, C, nd D in Fig. 1) define the region of sequene similrity found in ll eight eukryoti P-450 fmilies. The oundries of this region re mrked y deresed sequene Pro. Ntl. Ad. Si. USA 85 (1988) similrity. No onsensus mino id ours in the 30 positions immeditely efore the strt of the region. The yest nd mmmlin sequenes diverge t the end of the region. Three of the four domins inlude elements desried y previous workers. In the A domin, residues A-11 through A-27 orrespond to segment in the prokryoti P-450m tht ws demonstrted y x-ry rystllogrphy to spn the heme distl surfe nd to ontin residues tht ontt the sustrte mphor (17). In ddition, ntiodies direted ginst the dodepeptide, whih orresponds to residues A-8 through A-19 of P-450, were shown to ross-ret with P-450 (21). In the B domin, residues B-8 through B-20 orrespond to onserved tridepeptide (18). The sequene D-7 through D-27 is the onserved peptide (19) tht ontins the thiolte lignd to the heme (22, 23). These previously identified peptides ontin numer of invrint or nerly invrint residues; the multiple lignment hs identified dditionl highly onserved residues, whih inlude those t A-36, A-44, A-45, C-24, C-29, C-43, C-46, C-49, C-51, C-53, nd C-54. Four onsensus sequenes were derived from the ligned mino id sequenes nd eh ontins its own pttern of onserved nd invrint mino ids. These hrteristi ptterns do not rise merely euse of some is in the mino id omposition of P-450s s is shown y the dt in Tle 1. This tle lists the sores otined when the four onsensus sequenes were ompred to eight eukryoti P-450s nd one teril P-450. For the eukryoti P-450s, eh sore ws lwys more thn 8 stndrd devitions ove the men numer of mthing residues oserved in rndomized P450 sequenes. On the other hnd, sores for the teril P-450m were lower, espeilly the sore of 5 otined with the B onsensus sequene. Results of the rndomiztion experiments indited tht n verge rndomized vrint of P-450m ontins out three segments with five mthes to the B onsensus sequene. Furthermore, the segment in P-450m tht est mthed the B onsensus sequene did not lie etween the A nd D domins. The highest soring segment etween these two domins hd only four mthes with the B onsensus. This numer of mthes is expeted to our =13 times in n verge "shuffled" P-450m sequene. Thus, no segment in P-450m mthes the B onsensus sequene to signifint extent. The four onsensus sequenes were lso tested for speifiity y omprison ginst the proteins in the Protein Identifition Resoure dtse. A imodl distriution of sores ws oserved (Fig. 2). Sores for ll full-length eukryoti P-450s fell into the high group while non-p-450 sores did not. These results indite tht the onsensus sequenes ould e used to determine whether the sequene of n unidentified peptide resemled tht expeted for eukryoti P-450. Sores for the teril P-450m were not s esy to tegorize s those for the eukryoti P-450s. Using the D onsensus sequene, the sore for P-450m ws higher thn tht for ny non-p-450 protein. When either the A or C onsensus sequene ws ompred to P-450m, the sores were higher thn the sores for ll ut few of the non-p-450 proteins, nd the sum of the A nd C sores for P-450m exeeded the sum for ny non-p-450 sequene. However, for the B onsensus sequene, lmost 60% of the non-p-450 proteins hd sores tht equled or exeeded tht of P- 450m. Consequently, s ws lso indited y the results of the shuffling experiments desried ove, P-450m does not ontin peptide tht resemles the B onsensus sequene to signifint degree. All P450 proteins shre some properties, while other properties re speifi to susets of these proteins. For exmple, they ll ind heme in mnner tht yields

3 Biohemistry: Kl nd Loper 17 pnl sc m A E I I A F K I LL L L F A D SSS I L LA QKIR V Al MSD V A V L6IG1ETTVLSW V ML1HP QRRLQEELD VLG P LLEGHVHMSVVDLFIGGTETTASTLSWAVAFLLHHPEIQRRLQEELDRELGPGASC LSDKDLRAEVDTFMFEGHD1TASGVSWIFYALATHPKHQQRCREEVQSVLGDGSSI LSNRHMLATIGDIFGAGVEMSVIKWIVAYLLHHPSLKKRIQDDIDQIIGFNRTP LSDI4EITAQSIIFIFAGYEPTSSTLSFVLHSLATHPDTQKKLQEEIDRALPNKAPP LSDDKVITIVFDLFGAGFDTITTAISWSLxYLVTNPRIQRKIQEELDTVIGRDRQP FHHENLMISLLSLFFAGTETSSTTLRYGFLLMLKYPHVAEKVQKEIIDQVIGSHRLP MLLEDVKANITEMLAGGvNTTSMTLQWHLYEMARSLNVQEMLREEVLNARRQAEGD MTDQEIANLLIGVLmGGQHTSAATSAWILLHLAERPDVQQELYEEQMRVLDGGKKE ITSDEAKRMCGLLLVGGLDTVVNFLSFSMEFLAKSPEHRQELI ERPERIPAACEEL Pro. Ntl. Ad. Si. USA 85 (1988) 7223 [SRVi I C L I S H 0 A PIP L R T D I A P L E F N F G *PVVS*-VPH * DV * GY*LPKG- V*V. HRDP W P -FRPERWL... K B D K L L LL A T T-SDR- MPYT-M-I*EVLR TYKDRARLPLLNATIAEVLR TwDHLDQIPYTTMCIKEALR TISDRNRLvLLEATIREVLR TYDTVEMNEYLDMVLNETLR I RLSDRPQLPYLEAFILETFR [ TLDDRSKMPYTDAvIHEIQR I ISKMLQIEvPLLKAsIKETLR L TYDLLQEMPLLNQTIKETLR [ I 17 pnl s m 1 LI RPVVPLALPHRTTRPSSIFGYDIPEGMVVIPNLQGAHLDETVWEQPYEFRPDRFLEPGA [ I LI YPPVPGIVRELSTSVTFPDGRSLPKGIQVTLSIYGLHHNPKVWPNPEVFDPSRFAPDSP [RH 1 I] RPVAPTLIPHKAVIDSSIGDLTIDKGTDVVVNLWALHHSEKEWQHPDLFMPERFLDPTG [TQLISP I LYPIGNRLERVCKKDVEINGVFMPKGSVVMIPSYALHRDPQHWPEPEEFRPERFSKENK [GSID I HI SSFVPFTIPHSTIRDTSLNGFYIPKGHCVFVNQWQVNHDQELWGDPNEFRPERFLTSSG [TLDKHL F] SDLVPIGVPHRVTKDTMFRGYLLPKNTEVYPILSSALHDPQYFDHPDSFNPEHFLDANG [ALKK [ LHPISVTLQRYPESDLVLQDYLIPAKTLVQVAIYAMGRDPAFFSSPDKFDPTRWLSKDK [DLI ] [MH ] HPLHSLFRKVMKDMHVPNTSYVIPAGYHVLVSPGYTHLRDEYFPNAHQFNIHRWNKDSA [SSYSVGEEVDYGFGAISKGVI [ LI RRFSLVADGRILTSDYEFHGVQLKKGDQILLPQMLSGLDERENACPmHVDFSRQ D I F G L I LFL L S * LPFS-G6R-CVGE-LAR-EMKVFM 17 pnl s m NPSALAFGCGARVCLGESLARLELFVVLLRLLQAFTLLPPPVGALPSLQPDPYCGVNLKVQPFQVRLQPRGVEAGAWESASAQ-COOH SHSFLPFSGGARNCIGKQFAKSEMKVIVALTLLRFELLPDPTKVPIPLPRLVLKSKNGIYLYLKKLH-COOH SLSYLPFGAGPRSCVGEMLARQELFLFMSRLLQRFNLEIPDDGKLPSLEGHASLVLQIKPFKVKIEVRQAWKEAQAEGSTP-COOH PYvYLPFGNGPRNCIGMRFALMNMKLALTKVLQNFSFQPCKETQI PLKLSRQGLLQPTKPI ILKWPRDEI ITGS-COOH SEKVILFGLGKRKCIGETIGRLEVFLFLAILLQQMEFNVSPGEKVDMTPAYGLTLKHARCEHFQVQmRSSGPQHLQA-COOH SEAFMPFSTGKRICLGEGIARNELFLFFTT ILQNFSVSSHLAPKDIDLTPKESGIGKIPPTYQ ICFSAR-COOH HFRNLGFGWGVRQCVGRRIAELEMTLFLIHI LENFKVEMQHIGDVDTIFNLILTPDKPIFLVFRPFNQDPPQA-COOH SSPYLPFGGGRHRCIGEHFAYCQLGVLMSIFIRTLKWHYPEGKTVPPPDFTS4VTLPTGPAKI IWEKRNPEQKI -COOH KVSHTTFGHGSHLCLGQHLARREI IVTLKEWLTRIPDFSIAPGAQIQHKSGIVSGVQALPLVWDPATTKAV-COOH FIG. 1. Sequene lignment of the roxyl-terminl portion of nine P-450s. These prtil P-450 sequenes egin mino ids from the mino terminus of the prent P-450 ut re otherwise omplete from their eginning in the A domin on through the roxyl terminus of the prent protein. Squre rkets enlose residues tht lie etween domins. A horizontl line, positioned eneth the onsensus sequene, spns eh domin. A period in these onsensus sequenes identifies position tht lks onsensus mino ids. Boldfe type indites mino ids tht mth the onsensus sequene. Vertil lines on the horizontl lines mrk every 10th mino id within eh domin. The horizontl line tht spns eh domin thikens to mrk previously reported regions of sequene similrity. These regions inlude segment in P-450m tht trverses the heme distl surfe (17) in the A domin; onserved tridepeptide (18) in the B domin; nd onserved ysteinyl peptide (19) in the D domin. The lignment for P-450m ws guided y the eukryoti multiple lignment. The mino id residue in eh protein tht egins the A domin is s follows:, Leu-275;, Leu-305; 17, Leu-287; pnl, Leu-291;, Leu-306;, Phe-283; SCC, Met-310;, Met-299; nd m, Ile-234. The N-terminl methionine in the dedued mino id sequene of eh protein is residue 1 in this numering system. The stndrd single letter nottion is used (20). The I helix in P-450m orresponds to residues A-2 through A-35, the J helix orresponds to residues A-36 through A-44, while the L helix strts t D-16 nd ontinues for 20 residues (17). The highest perentge of invrint positions mong the P-450s ours in the first hlf of the onserved ysteinyl peptide in the D domin. This high density of invrint residues llows the peptide to e deteted with reltive ese in vrious P-450 sequenes. Ptterns for the A nd C domins re less pprent sine the density of invrint nd highly onserved residues is reltively low in these domins. hrteristi differene spetrum in the presene of ron monoxide nd reduing gents (24). On the other hnd, mitohondril P-450s reeive eletrons from n iron-sulfur redoxin, while the mirosoml P-450s reeive eletrons from P-450 redutse. The mirosoml P-450 redutse from one speies n donte eletrons to P-450 from nother speies oth in vitro (25) nd in vivo (26). These ommon properties might e refleted in the sequene similrities identified in the multiple lignment displyed in Fig. 1. These sequene similrities n e ltered in vitro to exmine the effets on struture nd funtion. Highly onserved residues, suh s the phenyllnines t C-63 nd D-7, re ttrtive trgets for site-direted mutgenesis. This pproh llowed Ling et l. (27) to show tht the phylogenetilly onserved Phe-87 in the yest iso-1-ytohrome is involved in the trnsfer of eletrons etween hemoproteins. Eh of the four domins identified in the lignment might ply speifi role in P-450 struture or funtion. A series of himeri P-450 proteins ould e onstruted y swpping DNA sequenes tht enode these domins etween different P-450 proteins. These onstruts, when expressed in vivo, might llow the mpping of speifi funtions to speifi domins nd might lso nswer questions onerning the funtionl independene of the domins. The joints tht onnet the heterologous mino id sequenes in these himers would lie etween the domins. Sine segments etween the domins vry in oth size nd sequene mong the P-450s, ny perturtion in tertiry struture used y these joints is likely to e tolerted. Furthermore, sine the sping etween domins vries from one P-450 to nother, it seems likely tht hnges to this sping in single P-450 would not severely disrupt t lest some of the hrteristi P-450 properties. It would e

4 7224 Biohemistry: Kl nd Loper Tle 1. Sores from omprisons of the four onsensus sequenes to nine P-450s Gene Consensus sequene fmily P450 A B C D XXI 32 (14.1) 14 (10.6) 28 (14.6) 17 (11.1) IV 29 (13.3) 11 ( 8.7) 22 (11.4) 16 (10.9) XVII (12.3) 13 (10.1) 26 (13.9) 20 (13.7) III pnl 31 (14.5) 10 ( 7.6) 24 (12.5) 15 (10.1) I 31 (14.2) 12 ( 9.5) 26 (13.9) 16 (10.9) II 26 (11.4) 12 ( 9.2) 27 (14.3) 18 (11.8) XI SCC 25 (11.3) 11 ( 8.6) 25 (13.1) 15 (10.1) LI 23 (10.3) 12 ( 9.6) 19 ( 9.7) 15 (10.4) CI m 13 ( 4.7) 5 ( 3.3) 12 ( 5.3) 12 ( 8.0) The sore nd z vlue (in prentheses) were lulted s desried. Gene fmily designtions re those of Neert nd Gonzlez (3). informtive to lter the distne etween the domins nd determine the effets on heme inding, the differene spetrum, nd the intertion with ytohrome P-450 redutse. Even though lrge vritions in the distne etween the four onserved domins might e tolerted for some P-450 properties, the three vrile segments might hve some funtionl signifine with respet to nother property suh s sustrte speifiity nd this ould e tested. The lignment my lso id in the design of syntheti polypeptides for ntiody prodution. Polylonl ntiodies to syntheti dodepeptide in P-450 (orresponding to residues A-8 through A-19) ross-reted with oth P-450 nd P-450 (21), lthough these two proteins shre only five identil mino ids in this segment. A smll pnel of polylonl ntiodies tht ross-rets with mny different P-450s might e used, for exmple, to isolte dditionl P-450 genes from expression lirries. As dditionl P-450 sequenes eome ville, it will e interesting to see how they further define the four linked domins of sequene similrity tht we hve found in ll eight eukryoti P-450 sequenes. The prokryoti P-450m shres three of these domins. Additionl prokryoti sequenes should help deide whether or not the differenes ~A- Pro. Ntl. Ad. Si. USA 85 (1988) seen in P-450m re speifi to tht P-450 or result from the divergene etween prokryoti nd eukryoti P-450s. We expet tht sequene dt from new eukryoti P-450 fmilies my result in hnges to the pttern. Suh hnges might inlude (i) minor modifitions in onsensus mino id residues nd (ii) resolution of the onserved domins into sudomins. Indeed, prtil dedued mino id sequene for romtse, the first known memer of newly disovered P-450 fmily (28), suggests tht the C domin n e divided into two sudomins with the oundry ourring immeditely fter residue C-40. Detiled informtion out the tertiry struture of the P-450 proteins will id in understnding struture nd funtion for this diverse protein superfmily. Poulos nd his ollegues (17) hve determined the rystl struture of P-450m nd hve shown tht the heme in this teril protein is sndwihed etween two helies. Eh helix ontins segment exhiiting sequene similrity to similrly positioned segments in mmmlin P-450s. Poulos hs suggested tht the rystl struture of P-450m might e useful in modeling the tertiry struture of eukryoti P-450s. Suh modeling requires the orret lignment of the two sequenes (29) sine inorret lignments n led to flwed three-dimensionl strutures (30). The multiple lignment presented in this work my e useful in this ontext even though P-450m lks segment tht resemles the B onsensus sequene. In their work on P-450 evolution nd memrne topology, Nelson nd Stroel (31, 32) reently presented n lignment of P-450 sequenes tht inluded the teril P-450m nd memers from seven of the eight eukryoti P-450 fmilies tht we hve nlyzed. They inluded lrge numer of sequenes from the P-4501 nd P-450II fmilies in their lignment, nd the resultnt intrfmily similrities tend to osure similrities ommon to ll P-450 fmilies. Beuse their lignment ws not hrterized y using rndomiztions or onsensus sequenes, speifi differenes with our lignment re hrd to evlute. In ny event, their lignment ontins gps nd insertions in the roxyl-terminl portion of the P-450 sequenes tht re not neessry y our nlysis B 0~.0 16 z p 10I 5i =- =.. 0 *1 * Im. 0 m m _ I * I I. ȧd FIG. 2. Distriution of sores for pro- * teins in the Protein Identifition Resoure dtse. Eh onsensus sequene ws ompred to eh of the proteins in the dtse. Results of these omprisons us- ing the A, B, C, nd D onsensus sequenes._,.._,,. * l. - re plotted in their respetive pnels. (Bottom) Numer of proteins versus the sum of the A nd C sores for these proteins. In A+C - eh pnel, vertil r mrks the point orresponding to the highest sore for - non-p-450 protein nd n rrow mrks the point orresponding to P-450m. The ver-. ~~~~~~~~~~~~~~til xis swithes from rithmeti to logrithmi for vlues lrger thn 10. Men _ sores (with stndrd devitions in pren- *./_1 _ * "_"19,- _"" _t - theses) using the A, B,C, D, nd A + C onsensus sequenes were 8.9 (2.9), 4.7 (1.2), 7.2 (2.5), 5.4 (1.6), nd 16.1 (5.1), Sore respetively.

5 Biohemistry: Kl nd Loper Furthermore, we detet sequene similrity ommon to ll eight eukryoti P-450 fmilies only in the roxyl-terminl portion. One proposed mehnism for gene evolution suggests tht exons enode funtionl domins tht ssort to form new genes vi reomintion within flnking introns (33). These domins re short mino id segments tht rry out limited funtion, suh s the inding of heme y the peptide enoded in the entrl exon of the 0-gloin gene (34). Although the pttern of exon orgniztion vries from one P-450 gene fmily to nother (23), there re exmples of introns interrupting eh of the four domins. At lest two of these introns interrupt eukryoti P-450 genes in regions tht orrespond to known funtionl domins in the teril P-45Qm. One intron splie position interrupts the odon for residue A-17 in memers of the P-450I fmily (35). In P-450m, this residue is thought to e one of the two residues in the oxygen inding site tht ontts moleulr oxygen (36). Another intron splie position interrupts the odon for residue D-10 in memers of the P-45011B sufmily (37). In P-450m, this odon lies etween residues D-7 nd D-15, whih provide hydrophoi ontts to the heme proximl surfe (17). A segmented pttern of sequene similrity lso ours in the gloin superfmily. Bshford et l. (38) ligned nd nlyzed ll known gloin sequenes. The 226 sequenes inluded not only the hemogloins nd myogloins of higher nimls ut lso gloins from insets, invertertes, nd plnts. Sequene similrities in these gloins n e found in six domins tht re residues long. Thus, in oth the P-450 nd gloin superfmilies, evolution hs onserved short domins tht ontin speifi ptterns of invrint, onserved, nd vrile mino id residues. The sene of smller domins in these superfmilies my imply tht there is lower size limit for heritle elements of protein struture or funtion. We thnk Dvid J. Lipmn nd F. Peter Guengerih for helpful disussions while the mnusript ws in preprtion. Prts of this work were supported y grnts from the U.S. Environmentl Protetion Ageny nd the University of Cininnti, Groundwter Reserh Institute. Computer resoures were provided y BIONET Ntionl Computer Resoure for Moleulr Biology. 1. Neert, D. W., Adesnik, M., Coon, M. J., Estrook, R. W., Gonzlez, F. J., Guengerih, F. P., Gunslus, I. C., Johnson, E. F., Kemper, B., Levin, W., Phillips, I. R., Sto, R. & Wtermn, M. R. (1987) DNA 6, Dyhoff, M. O., Brker, W. C. & Hunt, L. T. (1983) Methods Enzymol. 91, Neert, D. W. & Gonzlez, F. J. (1987) Annu. Rev. Biohem. 56, Hrdwik, J. P., Song, B.-J., Huermn, E. & Gonzlez, F. J. (1987) J. Biol. Chem. 262, Sogw, K., Gotoh, O., Kwjiri, K. & Fujii-Kuriym, Y. (1984) Pro. Nti. Ad. Si. USA 81, Zuer, M. X., John, M. E., Okmur, T., Simpson, E. R. & Wtermn, M. R. (1986) J. Biol. Chem. 261, Morohshi, K., Fujii-Kuriym, Y., Okd, Y., Sogw, K., Pro. Ntl. Ad. Si. USA 85 (1988) 7225 Hirose, T., Inym, S. & Omur, T. (1984) Pro. Ntl. Ad. Si. USA 81, Gonzlez, F. J., Neert, D. W., Hrdwik, J. P. & Ksper, C. B. (1985) J. Biol. Chem. 260, Yoshiok, H., Morohshi, K., Sogw, K., Ymne, M., Kominmi, S., Tkemori, S., Okd, Y., Omur, T. & Fujii- Kuriym, Y. (1986) J. Biol. Chem. 261, Fujii-Kuriym, Y., Mizukmi, Y., Kwjiri, K., Sogw, K. & Murmtsu, M. (1982) Pro. Ntl. Ad. Si. USA 79, Kl, V. F., Woods, C. W., Turi, T. G., Dey, C. R., Sutter, T. R. & Loper, J. C. (1987) DNA 6, Unger, B. P., Gunslus, l. C. & Sligr, S. G. (1986) J. Biol. Chem. 261, Wilur, W. J. & Lipmn, D. J. (1983) Pro. Ntl. Ad. Si. USA 80, Snedeor, W. W. & Cohrn, W. G. (1980) Sttistil Methods (Iow Stte Univ. Press, Ames), pp Wihmnn, B. & Hill, D. (1987) Byte 12, MLhln, A. D. & Boswell, D. R. (1985) J. Mol. Biol. 185, Poulos, T. L., Finzel, B. C., Gunslus, I. C., Wgner, G. C. & Krut, J. (1985) J. Biol. Chem. 260, Ozols, J., Heinemnn, F. S. & Johnson, E. F. (1981) J. Biol. Chem. 256, Gotoh, O., Tgshir, Y., lizuk, T. & Fujii-Kuriym, Y. (1983) J. Biohem. 93, IUPAC-IUB Commission on Biohemil Nomenlture (1968) Eur. J. Biohem. 5, Frey, A. B., Wxmn, D. J. & Kreiih, G. (1985) J. Biol. Chem. 260, Blk, S. D. & Coon, M. J. (1986) in Cytohrome P450, ed. Oritz de Montellno, P. R. (Plenum, New York), pp Morohshi, K., Sogw, K., Omur, T. & Fujii-Kuriym, Y. (1987) J. Biohem. 101, Omur, T. & Sto, R. (1964) J. Biol. Chem. 239, Aoym, Y., Yoshid, Y., Kuot, S., Kumok, H. & Furumihi, A. (1978) Arh. Biohem. Biophys. 185, Oed, K., Skki, T. & Ohkw, H. (1985) DNA 4, Ling, N., Pielk, G. J., Muk, A. G., Smith, M. & Hoffmn, B. M. (1987) Pro. Ntl. Ad. Si. USA 84, Simpson, E. R., Evns, C. T., Corin, C. J., Powell, F. E., Ledesm, D. B. & Mendelson, C. R. (1987) Mol. Cell. Endorinol. 52, Brlow, D. J., Blundell, T. L., Edwrds, M. S., Sind, B. L., Sternerg, M. J. E., Tylor, W. R. & Thorton, J. M. (1986) in Protein Engineering, eds. Inouye, M. & Srm, R. (Ademi, New York), pp Blundell, T. L., Sind, B. L., Sternerg, M. J. E. & Thornton, J. M. (1987) Nture (London) 326, Nelson, D. R. & Stroel, H. W. (1987) Mol. Biol. Evol. 4, Nelson, D. R. & Stroel, H. W. (1988) J. Biol. Chem. 263, Gilert, W. (1978) Nture (London) 271, Crik, C. S., Buhmn, S. R. & Beyhok, S. (1981) Nture (London) 291, Gonzlez, F. J., Kimur, S. & Neert, D. W. (1985) J. Biol. Chem. 260, Poulos, T. L., Finzel, B. C. & Howrd, A. J. (1987) J. Mol. Biol. 195, Suw, Y., Mizukmi, Y., Sogw, K. & Fujii-Kuriym, Y. (1985) J. Biol. Chem. 260, Bshford, D., Chothi, C. & Lesk, A. M. (1987) J. Mol. Biol. 196,