Sulfolobus Solfataricus Rhonda L

FEBS 16933 FEBS Letters 384 (1996) 235-239

Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus Rhonda L. Wright, Kathryn Harris, Barbara Solow, Robert H. White, Peter J. Kennelly* Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0308, USA Received 1 March 1996

Inc., Westboro, MA); restriction endonucleases, T4 DNA ligase, pGEM-3Z, Prime-a-Gene DNA labelling kit, and Erase-A-Base kit Abstract A gene, CYPII9, for a potential cytochrome P450 (Promega, Madison, WI); Sequenase version 2.0 DNA sequencing has been isolated and sequenced from the extreme acidothermo- kit, pUC vectors, and shrimp alkaline phosphatase (US Biochemicals, philic archaeon Sulfolobus solfataricus. The gene predicts a Cleveland, OH); GeneAmp PCR core reagent kit (Perkin Elmer, polypeptide of 368 amino acids containing the consensus berne- Branchburg, NJ); E. coli DH5e~ cells (Life Technologies, Inc., binding sequence Phe-Gly-Xaa-Gly-Xaa-His-Xaa-Cys-Xaa-Gly- Gaithersburg, MD); Long Ranger gel solution (J.T. Baker, Phillips- Xaa3-Ala-Arg-Xaa-Glu. It most closely resembles the cyto- burg, NJ); and Deep Vent DNA polymerase (New England Biolabs, Beverly, MA). E. coli 7118 was the generous gift of Dr. Dennis Dean, chrome P450s found in the bacterium Bacillus subtilis, with Virginia Tech. All other reagents were purchased from Sigma Chemi- which it shares 129 identical amino acid residues (35%). This cal Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA). first sequence of a potential archaeal cytochrome P450 represents an important step in tracing the complex evolutionary 2.2. Cloning procedures history of this biologically important enzyme family. S. solfataricus (ATCC 35091) was grown, genomic DNA isolated therefrom, and genomic DNA libraries constructed as described by Key words: Cytochrome P450; Evolution; Archaea; Leng et al. [4]. Restriction enzyme digests, Southern blots, and the Archaebacteria; CYP119 identification and isolation of DNA clones were performed using standard procedures [5]. Oligonucleotide probes were radiolabelled with 32p using the Prime-A-Gene kit following the manufacturer's protocols. DNA sequencing was performed by the dideoxy method of Sanger et al. [6] using a Sequenase version 2.0 kit following the 1. Introduction manufacturer's protocol.

The cytochrome P450s comprise a large family of heme- 2.3. PCR techniques The following oligonucleotide primers were used for PCR: primer containing monooxygenases ubiquitously distributed among A, 5'-d(GGGAATTCCRTDTAYGGWVHHCARTGG)-3'; primer aerobic organisms (reviewed in [1-3]). These enzymes partici- B, 5'-d(GGGATCCCSWHGCDATRTTRAANGG)-Y; primer C, pate in the oxidation of a wide range of endogenous and 5'-d(CCCGGATCCATTTATCTGAATTCCCAAGG)-3'; and pri- exogenous organic compounds. While the oxidation of foreign mer D (SP6 promoter primer), 5'-d(GATTTAGGTACACTATAG)- substances by cytochrome P450 can serve as an important 3'. 'Touchdown' PCR was performed by the procedure of Roux [7] as modified by Leng et al. [4]. The synthesis of the 920 bp primer was step in their neutralization, in man and other organisms the performed according to the standard PCR protocol of Leng et al. [4], oxidation of foreign compounds often results in the creation with the exception that the quantity of DNA used was reduced from of highly toxic and/or carcinogenic metabolites -- hence these 1000 to 500 ng and the quantity of each primer was increased from enzymes have been the subject of intense scrutiny. The genes 100 to 500 pmol. for several hundred cytochrome P450s have now been sequenced. They suggest a complex evolutionary history that 3. Results and discussion crosses phylogenetic divisions, an evolutionary history that is only partially understood [2,3]. One of the barriers to un- The gene for the potential archaeal cytochrome P450 was ravelling the chain of gene duplication, gene transfer, and encountered during an attempt to isolate clones for a thymi- other events is the lack of any data concerning the cyto- dylate synthase gene from the extreme acidophilic archaeon chrome P450s from the third phylogenetic domain, the Ar- S. solfataricus. Briefly, touchdown PCR amplification of geno- chaea. Thus, when we accidentally encountered the partial mic DNA from S. solfataricus was performed using degener- clone of a gene predicting a potential cytochrome P450 ate oligonucleotide primers A and B, which were modelled from the extreme acidothermophilic archaeon Sulfolobus sol- after the sequence of thymidylate synthase from E. coli. The fataricus, we seized upon the opportunity to begin filling a 300 bp product that resulted was used to probe a genomic major gap in the evolutionary history of this important en- DNA library contained in plasmid pGEM-3Z that had been zyme superfamily. constructed using the restriction enzyme HindlII. Several potential clones were isolated and their ends sequenced. A com- 2. Materials and methods puter search of DNA databases using the BLASTX program revealed that the 5'-end of one clone, = 1.5 kb in length, 2.1. Materials predicted a gene product possessing significant sequence Purchased materials included custom oligonucleotides (DNAgency, homology to the members of the cytochrome P450 family. Malvern, PA); radiochemicals (DuPont/New England Nuclear, Bos- ton, MA); nitrocellulose and nylon membranes (Micron Separations, It also was apparent that it encoded only a portion of the presumed structural gene, specifically the first 294 amino acid residues of the protein. Using PCR primers C and D, a *Corresponding author. Fax: (1) (540) 231-9070. 920 bp probe encompassing this partial clone was synthesized E-mail: [email protected] and utilized to probe a second library of S. solfataricus geno-

TAA ~T ATC GTA TTT TTT CTA TAT CTA TAC TTT AAG TTA AGA TAT ATC TAT TTA TTT

ATC TGA AM' CCC AAG GTA TTA CCT ATG TAT GAC TGG TTT AGT GAG ATG AGA AAG AAA GAC CCA GTG ** *** *** ** M Y D W F S E M R KI0 K D P V

TAT TAT GAC GGA AAC ATA TGG CAG GTG TTT TCC TAT AGG TAC ACA AAG GAG GTT TTA AAC AAC TTT Y Y D G N I20 W Q V F S Y R Y T K30 E V L N N F

TCG AAA TTC TCC TCA GAT CTT ACA GGT TAC CAC GAA AGG CTT GAG GAC CTA AGG AAC GGT AAA ATA S K F S $40 D L T G Y E E R !~50 E D L R N G K I

AGG TTC GAC ATC CCC ACT AGG TAT ACC ATG CTG ACC TCA GAT CCC CCT CTC CAT GAT GAG TTA AGA R F60 D I P T R Y T M L T70 S D P P L H D E L Re0

TCA ATG TCA GCA GAT ATA TTC TCG CCT CAA AAG CTA CAG ACA CTT GAG ACA TTT ATT AGG GAG ACC S M S A D I F S P Qg0 K L Q T L E T F I RI00 E T

ACC AGA AGC CTA TTA GAC TCA ATT GAC CCT AGG GAA GAC GAC ATA GTG AAG AAG TTA GCT GTT CCA T R S L L D S Ili 0 D P R E D D I V K K120 L A V P

CTA CCA ATA ATA GTT ATC TCA AAA ATA TTG GGT CTC CCA ATT GAA GAT AAG GAG AAG TTC AAA GAG L P I I V I130 S K I L G L P I E D140 K E K F K E

TGG TCA GAC TTA GTC GCA TTC AGG TTG GGT AAG CCT GGA GAA ATA TTT GAG CTA GGT AAG AAG TAC W S D L150 V A F R L G K P G El60 I F E L G K K Y

CTT GAG TTA ATA GGT TAT GTG AAG GAT CAT CTA AAT TCA GGG ACC GAA GTG GTC AGC AGA GTT GTC L E170 L I G Y V K D E L N180 S G T E V V S R V V190

AAC TCA AAC CTC TCA GAC ATA GAG AAA CTC GGA TAC ATT ATT TTA CTT CTC ATA GCG GGT AAT GAG N S N L S D I E K L200 G Y I I L L L I A G210 N E

ACT ACA ACT AAC TTA ATA TCA AAC TCT GTT ATT GAC TTC ACT AGG TTT AAC CTG TGG CAG AGG ATA T T T N L I S N220 S V I D F T R F N L230 W Q R I

AGG GAA GAG AAC CTC TAC CTT AAG GCT ATC GAA GAG GCT TTA AGG TAT TCT CCT CCT GTG ATG AGG R E E N L Y240 L K A I E E A L R Y250 S P P V M R

ACT GTA AGA AAG ACT AAG GAA AGA GTG AAA TTG GGT GAT CAG ACT ATT GAA GAG GGA GAG TAC GTT T V R K260 T K E R V K L G D Q270 T I E E G E Y V

AGA GTA TGG ATA GCC TCA GCA AAC AGG GAC GAG GAG GTG TTT CAT GAC GGA GAG AAG TTC ATC CCT R V280 W I A S A N R D E E290 V F H D G E K F I P300

GAC AGG AAT CCG AAC CCA CAC TTA AGC TTT GGG TCT GGA ATA CAT CTG TGT TTA ~ GCT CCT TTG D R N P N P E L S F310 G S G I H L C L G A320 P L

GCT AGA TTA GAG GCA AGA ATA GCA ATT GAG GAA TTT TCA AAA AGG TTT AGG CAC ATT GAG ATA TTG A R L E A R I A330 I E E F S K R F R H340 I E I L

GAT ACT GAA AAA GTT CCA AAT GAA GTG CTG AAT GGT TAT AAG AGA CTA GTG GTC AGG TTG AAG AGT D T E K V P350 N E V L N G Y K R L360 V V R L K S

AAT GAA TAA TGC AGA GAG GTT TCT CCA CTT RAT GTT AAA GGA GGG AAG AAA GGT GTT ATT TCT GTT N E end Fig. 1. Complete DNA and inferred amino acid sequence of S. solfataricus cytochrome P450. This sequence, along with portions of the 3' and 5' untranslated regions not shown above for reasons of space, are listed in the GenBank Data Base under accession number U51337. mic DNA. This library was contained in plasmid pUC119 and strong resemblance to the members of the cytochrome P450 had been constructed using the restriction enzyme XbaI. This family. resulted in the isolation of a =2.5 Kb fragment encoding the The open reading frame for the potential archaeal cyto- entire open reading frame for a potential protein possessing a chrome P450, CYPll9, predicts a polypeptide of 368 amino R.L. Wright et aL/FEBS Letters 384 (1996) 235-239 237

O.S. sub~.|olf.- ~i ...... A ...... F- S K ~ ..... ~S~--Y A - - MiKL P MKID EEL P V[ ...... HY~DGN S E K O 2619 ...... ,,T--YA , L • ...... L ZS]~Sa LN ~T,,~A0- SlDIGIVII - - eVG, L~GA, 41 ~s. OX. MOUSe LS ..... LETWVLLAISLVLLYRYGTRKMEL KKQ IPGPK LPF ...... 46 Human ~[~DLA ...... METWLLLAVS LV YLYGTMSHG L~K~L~ I pG pT P F ...... 45 Yeast IEQIIEEVLPYLTKWYTIIFGAA~YFLSIALRNK~Y~YKLKCE YFODAGLFGIPA 61

S. solf. I[~Q~-~qT~qE V[~]N NS F S [~-~"~ ..... T ...... F~I" - E R L E~-~R ...... FNIGF~I 58 B. sub~. - - R LIFIP F P I YL~JDIKIL a E GK ...... IYI D - D V O F VlLIK ...... INIPIKIL 72 S. erygh. Rh. fasc. A F L IVICTC IKR~ERYE D - V R R A N y~KSiNAH ...... FA I P S GG~V ...... - 77 Fus. ox. .... R ...... Mouse Human LGNI L~_~_~KG- - FCMF ECHK KyGKvwGFY GOQ PVLAZ T- - - D FDMI KTVLVK ECY SVF I01 Yeast L I D I I K VIRIK A G Q LAD Y T D TT~D K y p LSS- yDTvAGVLKI VFTVD PENI KAVLATQ_ PNDF 120 S. Moll. B. subt. ....LFGS FS ~F--KP ER __-~P~RV SQR N YE YF- TA - - I~T T ~D D P PKPIL~DT KELRALD~ SA S R AIFITI ~--'~'~ IPIK 0 ~-~IIOAV QK TQIL ~-'E-~ E TIRIII F ~ R~--'~K D V,TIA R ~-~'~'~FIL LIO SE [~A 125 S. eryth. N TSD P P T T %R~S~M L EIF|T V R R~/_E A M R P R V EQIITIA ElL L D~E V 132 Rh. fame. I £D _~V A A M RA T N~L IG~LS D p pIDIMIT RLRK L V S FUS. OX. - - - K Q A A K A I( ~ T P V DSi~ r ~t'~l~IM . V 'P TIPITI~I"AV K NLL~JQ ~ YIIIQ nl~V D~I-L--LIE O s ~27 Mouse L SA~GRRD- FG PVG I MKS K A I S IISIK D DEW K R~RIA L L ~ TIFIT S GIK LIK m M F P VIXIE Q Y G O lILlY X Y L 160 Human T A[~]R AR HP F P V G F M A I S I A E DE E W K "IL R SILL ~ T,FIT S GIK LIK ' M V' IIIIA Q Y G D VIL'V R NL 159 Yeast ALG F[~]P - - L L G D G I FF~LF~lG E G W K H SiRiALMJL ~ p QIFIA R E O I A H V - K A~ffE p H V 0 i LLL.]A K O IL~ 178 S. solf. B. subt. V PRE .... D I I EDFFIAIGPL ~ I A EMIL GIAIF I E DIR H L IIKIT,~JSi "IDIV L v AG A K D S S v K A V A v Isl S. eryth. Rh. fasc. Pus. ox. r LIL sIvlHF NLQJL['~IY L T 0 0 N A - I a T N G S S.I.,A !a~]A S ---~ 187 Mouse K KGK Human Yeast K L N " - KG K TP FI'D'IL0 E L F F R FT VDTAT E F L FIGIE S V - - - H S L Y D E K SG I PN D I P ENVR EA N 234 S. Moll. B. sub~. H Na aoU HA r ...... ~l.~'~'ol~Ir-lq~l~l~Is . Ra ^~ e~ E o ...... LM 2n S. eryth. P.h. fase...... DR RIYIMATLVTRAQ EQ p DD ...... L 212 FI/S. OX. QF pE - - -,.~ ...... [~D[YIL,d~I L V E O R L V E P D D ...... I 210 MOUSe D P LILIF S V V--- L F P F L TPVY EML N I CMF p K D SIIIE, F|KJK F VD RMK E S R L D S KQ K H RVD F II. 272 Human FsLD PF FLS I T- - - VF PFL Z PILEVLNICVFPREVTNFL-~'KSVKRMKESRLEDTQKMRVDFL 271 Yeast T Q HIY LIA T R T Y S Q I F Y W L T N p K E F R DC NA K VH K~A Q[~F V N T A b NAT E K EV E~K S K G G y V F L 295 ~ . solf. ~T R V~'~N ...... ~I~K L~Y~IIA G N E T T T N L I S N S VII D F~EFN -~--~R I~235 subt. L LQ AE - I D G E Y ..... [LIT E EQL I[G[F C-~I L L LI%~A G N E T T T N L I~N~VIR y L D L V~[[~Q VIR~ 268 S] eryth. G~A L I R V Q D D D D G R ..... IL SIA D~IL T S I A V"~-V~k~JLIAL G[~E[~'~'~L I[G'~G~'Y L L L T H S D Q L ][L VIRI268 Rh. fast. TiLARK I G DN ...... IL SIT DIEIL I S I ?~!~G Ht~..~T----~A~'~I ,G L ~L ~ ~ L H H M 265 FUS. ox. L C -_=__T_E Q V K P G N ..... I D K S DAVQ I A F V G" T I AL A AQH PPDEQAAM KI Mouse M M~H N N S K D X V S H K A~'~MF~I T A Q G H LATH I O K K L O 333 Human M I ,DIS[Q N - S K E T E S H K AILS DIL[E[L V A Q $[III F I FIA GIY~T TIS SVL[SIF I MY S LATH PP D V~N K LL~ 331 Yeast Y E L~V~ ...... K Q T R D P K V L~'~_~ ..... Q L L N I M VIA G[R D[T T[A G[L[LISIF A M F £ L A[~N P K I K 345

B. subt. ~ ~ A s P M -ra-~vl~l~ T~-~IREI~m .... 267 N V T Y S Q - A I G V A T~EIDTEILI- 301 Rh.S. eryth.f.... .~DD P S ...... N I I A E T -[]T F A AIEIE~E I - 301 S G L L S V A H S Q P PIRIM A V T EIVI0 I .... 299 Fu ..... AN PS Q L H T A S A L A I KIRIT A~--~I IVID M I - 299 M .... ~i D E-- A L P N K A P P--- T Y D T V M E M E~D M V L NNIE[TILL R~L Y - ~ I A M0L E IRIVC IK[~ IVID E[~- 384 Human DA- -VL PNKAPP- - - TY DTVLQ ME DM T F - z A M LE V ClKIK DME _I - 3B2 Yeast E V N F G L G D E A RV D E I S F E T L K K C EIY LIK A V L MIEITIL RIM Y - S V P I N FIRIT A T R D T T[LIP R G G 405

K G~J-~SIVII-~W I A S A N R D E~--~F[C K P D - C K I - P S Y PP~H L S F G G 347 S i subt.eryth. - .... G V F K Q Y S TIVIL[~-~'N"G"~A N R DP~K OIFIP~P H ...... D - - T R H L S F G G 347 Rh. f .... - - -- -" [ A~S FIVII~- S L LAIA N R DIs , £TD a P D - ] - VAGH L [~]FG G 345 F...... ~l K LV~R AN|EIG I I A S MQIS AN R D E EV,,..,O-~,~;Iml ...... #,,mo L ,o o ,,7 Mouse ..... NGVYIXlPK~I"FT['q]MI t~S~nL--'E"~Dll, 0HWS E r['~ I- 0 FS~ENXG S I DIPIYVYLI.~IPIrGImQI439 H ...... N G M FIII, KIGI, VIVl. I . S Y A L Hi" DIP K Y W T E 'IEI-IK "lSlPIEIRIF S . K N KD "I DIPIY I Yff_PIF G S G, 437 Yeast G K D G N S P I F V P KIGIS SIVIV Y S v Y K T HIRIL K Q F Y G E DAY E~.~ IP[EIRIR W F E P S T R g L G - W A Y[LqP F~-N'~ 465 s. ,oi~. ~--ff]LIC L U A P ', A R L • A~I ^ddE K'~ ~,ammm~i r.[~]E .... v~IN zFg-EIs~qYl~ R L v v ~ LIKIS N ~I B. subt. ,I HIFIC b G2LP L A R L E AINL~ AILS ~ [ ~ ~ 368

FUS. ox. R A S T L Y O P D b K V A V P L K I N Y T~- -~"~R D Vd~_I~'D L P~I F 402 HumanMOUSe P RRNC NIcC['~GG[M L G M R FFIAILA LLMNMK M NN M K LL[AAILLL L Ti KR I M Q NIFIS F Q p C K EITIQ I p L K L S R Q G L|L|Q p E[K]F I~L K V V p R D 498 Yeast p R i ~C-~-GIQ Q FLAIL T~.~S y V-~I~A R V AL Q NIFIS F K P C K~E Q I P L K L S L G G L L~JQ P EIKIP VIVIL K V E~R D 496

Fig. 2. Comparison of the inferred amino acid of the archaeal cytochrome P450 with other members of the cytochrome P450 family. Shown is the derived amino acid sequence of S. solfataricus (S. solf.) cytochrome P450 aligned with the corresponding regions of cytochrome P450s from three bacteria: CYP109 from Bacillus subtilis (B. subt. [10]), CYP107A1 from Saccharopolyspora erythrea (S. eryth. [11]), and ORF1 from the fas locus in Rhodococcusfascians (Rh. fasc. [12]), and four eukaryotes: CYP55 from the fungus Fusarium oxysporum (Fus. ox. [13]), CYP3All from mouse (mouse [14]), CYP3A4 from human (human [15]), and CYP56 from the yeast Candida maltosa (yeast [16]). Sequences were aligned using the BLASTX program from the National Center for Biotechnology Information. Identities with the archaeal cytochrome P450 are en- closed by boxes.

acids (Fig. 1). The first of the two potential N-terminal the predicted initiator methionine. A potential consensus tran- methionines, Met I and Met s, was assigned as the initiator scription termination sequence, TTATTTCT [9], lies in a pyr- methionine based on two factors. First, the codon for Met 1 imidine-rich region 48 bp downstream from the translation is located adjacent to a strong potential ribosome binding termination codon. sequence, AAGGTAATTCC, as is typically the case for The predicted protein product lacks any evidence of an N- many archaeal genes [8]. Second, the sequence between Met 1 terminal membrane targeting sequence. This, along with se- and Met s contains several amino acid residues conserved in quence identity comparisons, indicates that it should be clas- other cytochrome P450s, most notably Phe 5, but also includ- sified as one of the soluble, 'prokaryotic' (sometimes called B ing Asp 3, Trp 4, and Glu 7 (Fig. 2). A sequence, AAACCATT- type [3]) cytochrome P450s found in many bacteria and some TAAA, bearing a strong resemblance to the consensus pro- 'simple' eukaryotes, e.g. CYP55 of the fungus Fusarium oxy- moter sequence for sulfur-dependent, thermophilic archaeons, sporum (Fig. 2). Its closest known homolog is CYP109 from AAANNTTTAAA [8], is located 77 nucleotides upstream of Bacillus subtilis, with which it shares 35% amino acid sequence 238 R.L. Wright et al.IFEBS Letters 384 (1996) 235-239

Table 1 Sequence identity between S. solfataricus CYP119 and selected cytochrome P450s Cytochrome P450 Organism Identities % identity CYP109 B. subtilis 129 35 CYP107A1 S. erythrea 107 29 ORF1 offas operon R. fascians 85 23 CYP55 F. fascians 82 22 CYP3A11 Mouse 70 19 CYP56 C. maltosa 70 19 CYP3A4 Human 68 18 The number of amino acid identities observed between CYPll9 from S. solfataricus and the cytochrome P450s described in Fig. 2 are tabu- lated. Percent identity levels were calculated by dividing the number of identical amino acids by the total number of amino acids in the S. solfatarieus sequence, 368.

identity -- 129 identical residues out of 368 (Table 1). CYP119 have ventured for the first time into the archaeal phylogenetic possesses a complete consensus heme-binding sequence, Phe- kingdom, the putative cytochrome P450 from S. solfataricus Gly-Xaa-Gly-Xaa-His/Arg-Xaa-Cys-Xaa-Gly-Xaaz-Ala-Arg- looks very much like any other prokaryotic cytochrome P450. Glu [1], centered around Cys317 near the C-terminal end. This In fact, construction of an abbreviated dendrogram contain- fact, and the observation that the area of resemblance be- ing a few representative bacterial and eukaryotic cytochrome tween the potential archaeal protein and a set of representa- P450s fails to betray any evidence of its archaeal status (Fig. tive cytochrome P450s (Fig. 2) is spread quite generally 3). Like CYP55 from the fungus F. oxysporum, it groups throughout its sequence, suggests that the gene product very among several bacterial cytochrome P450s as if it were derived likely constitutes a functional enzyme. What might this func- from another bacterium rather than from an archaeon. Since tion be? At this point in time it is difficult to tell, however, it the apparent lineage of this archaeal cytochrome P450 does is noteworthy that in its natural habitat S. solfataricus -- not follow phylogeny, this suggests the occurrence of a hor- which is an obligate aerobe -- obtains energy primarily via izontal gene transfer event. Two things seems apparent con- the oxidation of elemental sulfur to sulfuric acid. Cytochrome cerning the nature of this event. Since the Archaea do not P450s are already known to participate in sulfoxide formation contain subcellular compartments or organelles, acquisition [1] and have been postulated to comprise part of nature's cannot have proceeded via the incorporation of material most ancient respiratory systems [3]. Hence, it is tempting to from the genome of a bacterial endosymbiont into its genome, speculate that the potential product of this open reading as could have been possible for a eukaryote such as F oxy- frame might participate in this metabolically vital sulfur oxi- sporum. Second, the first bifurcation in phylogeny is the se- dation process. paration of the Bacteria from the lineage that eventually gave What can this archaeal gene sequence tell us about the rise to the Archaea and Eukaryotes. Although this means that evolution of the cytochrome P450 family? Even though we S. solfataricus and F. oxysporum are much more closely re- lated to each other than either is to any bacterium, CYPll9 and CYP55 do not cluster together -- once again in defiance S. eryth. of phylogeny. This suggests the occurrence of one or more horizontal gene transfer events after the separation of the Rh. fasc archaeal and eukaryotic lineages one from another. However, the specific partners involved and the direction of the transfer, Fus. ox. bacterium to eukaryote, archaeon to bacterium, etc. remains cryptic. The addition of future cytochrome P450 genes from the Archaea should do much to resolve these issues. S. self. Acknowledgements: This work was supported by Grant 3502 from the B. subt. Council for Tobacco Research, USA. We thank Daniel W. Nebert, University of Cincinnati, and David R. Nelson, University of Tennes- see at Memphis, for their assistance with CYP gene nomenclature. Human References Mouse [1] Nebert, D.W. and Gonzalez, F.J. (1987) Annu. Rev. Biochem. 56, 945-993. Yeast [2] Nebert, D.W. and Nelson, D.R. (1991) Methods Enzymol. 206, I I I I I I I 3-11. 60 50 40 30 20 10 0 [3] Degtyarenko, K.N. and Archakov, A.I. (1993) FEBS Lett. 332, 1-8. Evolutionary Distance [4] Leng, J., Cameron, A.J.M., Buckel, S. and Kennelly, P.J. (1995) J. Bacteriol. 177, 6510q5517. Fig. 3. Phylogenetic relationship of S. solfataricus cytochrome P450 [5] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular to representative bacterial and eukaryotic cytochrome P450s. This Cloning: A Laboratory Manual, 2nd edn., Cold Spring Harbor tree was constructed using the Lasergene computer program from Laboratory, Cold Spring Harbor, NY. DNASTAR, Inc. (Madison, WI) and includes all of the cytochrome [6] Sanger, F., Nicklen, F. and Coulson, A.R. (1977) Proc. Natl. P450s shown in Fig. 2. The abbreviations used are explained in the Acad. Sci. USA 74, 5463 5467. legend to Fig. 2. [7] Roux, K.H. (1994) Biotechniques 16, 812-814. R.L. Wright et al./FEBS Letters 384 (1996) 235-239 239

[8] Brown, J.W., Daniels, C.J. and Reeve, J.N. (1989) CRC Crit. Gotoh, O., Yasui, T. and Shoun, H. (1991) J. Biol. Chem. 266, Rev. Microbiol. 16, 287-338. 10632-10637. [9] Reiter, W.-D., Palm, P. and Zillig, W. (1988) Nucleic Acids Res. [14] Yanagimoto, T., Itoh, S., Muller-Enoch, D. and Kamataki, T. 16, 2445-2458. (1992) Biochim. Biophys. Acta 1130, 329-332. [10] Ahn, K.S. and Wake, R.G. (1991) Gene 98, 107-112. [15] Gonzalez, F.J., Schmid, B.J., Umemo, M., McBride, O.W., [11] Haydock, S.F., Dowson, J.A., Dhillon, N., Cortes, J. and Lea- Hardwick, J.P., Meyer, U.A., Gelboin, H.V. and Idle, J.R. dlay, P.F. (1991) Mol. Gen. Genet. 230, 120-128. (1988) DNA 7, 79-86. [12] Crespi, M., Vereecke, D., Temmerman, W., von Montagu, M. [16] Schunck, W.H., Kaergel, E., Gross, B., Wiedmann, B., Gaestel, and Desomer, J. (1994) J. Bacteriol. 176, 2492 2501. M. and Mueller, H.G. (1989) Biochem. Biophys. Res. Commun. [13] Kizawa, H., Tomura, D., Oda, M., Fukamizu, A., Hoshino, T., 161,843-850.