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Molecular evolution of yeast thiolase isozymes
JOURNAL OF FERMENTATION AND BIOENGINEERING
Vol. 78, No. 4, 279-282. 1994
Molecular Evolution of Yeast Thiolase Isozymes NAOKI KANAYAMA, MITSUYOSHI UEDA, H A R U Y U K I ATOMI, TATSUO KURIHARA, AND ATSUO TANAKA*
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01, Japan Received 9 May 1994/Accepted 12 July 1994 The coexistence of two thiolase isozymes (acetoacetyl-CoA thiolase and 3-ketoaeyl-CoA thiolase), essential for the complete degradation of fatty acids, only in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (Kurihara et al., J. Bioehem., 106, 474-478, 1989) is unique in eukaryotic cells. As one of the methods of analysis of molecular information from these isozymes, the calculation of the evolutional distance among thiolases from various organisms suggested that yeast peroxisomai thlolase isozymes are important enzymes in examining the molecular evolution of the fatty acid metabolic pathway and the biogenesis of peroxisomes.
Thiolases catalyze the final step of fatty acid ~-oxidation in prokaryotic (1) and eukaryotic cells (2-6) and are classified into two types, acetoacetyl-CoA thiolase (ACT), which is specific to acetoacetyl-CoA, and 3-ketoacyl-CoA thiolase (KCT), which has broad substrate specificity. For the complete and efficient degradation of fatty acids having various chain lengths, the coexistence of these two types of thiolases is important. In eukaryotes, thiolases exhibit diverse subcellular localization. In rat liver cells, there exist two distinct KCTs, one localized in peroxisomes responsible for long-chain acylCoAs (4) and another in mitochondria for middle-chain acyl-CoAs (2), whereas ACTs are localized in cytosol (2) and in mitochondria (2). We were the first to find the presence of both ACT and KCT in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (5-7) and their absence in its mitochondria. The peroxisomal localization of the two thiolases is unique and provides a basis for examining the evolution of the fatty acid p-oxidation system. Since the fatty acid p-oxidation system was also exclusively detected in peroxisomes in the yeast cells (8), fatty acids could be completely degraded with cooperation of these peroxisomal ACT and KCT (6, 7). These facts are quite different from the case of mammalian cells, which possess two E-oxidation pathways: a peroxisomai one involved in long-chain fatty acid degradation and a mitochondrial one responsible for medium- or short-chain fatty acid degradation (9-11). We have cloned and sequenced the genes of peroxisomal ACT and KCT of C. tropicalis (12, 13), and determined their primary protein structures. By calculating the evolutional distance among thiolases from various sources (14, 15), we have constructed a phylogenetic tree of thiolases and have discussed the molecular evolution of thiolase isozymes. The symmetric divergent branch points of this tree strongly suggested that the ancestors of peroxisomal ACT and KCT had occurred at the same time in the yeast C. tropicalis, as predicted by the endosymbiotic theory of peroxisomes (16-18).
MATERIALS AND METHODS Alignment of amino acid sequences The multiple alignment of protein sequences and the number of similarity between sequences were obtained with the program A L I G N contained within the ODEN program provided by DNA Databank Japan (DDBJ). Nucleotide sequences of thiolases have been deposited in DDBJ with accession numbers. Construction of phylogenetic tree The number of amino acid substitutions was estimated with the DISTA program (19). The distance matrix was made with the DMATA program (19). The phylogenetic tree was constructed by the neighbor-joining method with the TREENJ program (20). Bootstrap resampling was performed with the BSTRAP program (21). These programs were contained within the ODEN program provided by DDBJ.
RESULTS AND DISCUSSION We compared all of the primary sequences of thiolases, encoded by the open reading frames of respective DNAs, from various organisms reported so far (Fig. 1) and constructed a phylogenetic tree of thiolases based on the number of amino acid substitutions (19) and the neighbor-joining method (20) (Fig. 2). The phylogenetic tree clearly indicates that three types of thiolases, acetoacetyl-CoA thiolase (ACT), 3-ketoacyl-CoA thiolase (KCT), and mitochondrial 3-ketoacyl-CoA thiolase (mtKCT), diverged simultaneously from one original enzyme. We can find that the divergence of one original thiolase to thiolases with different substrate specificities--that is, to KCT and ACT--occurred before the divergence of prokaryotic and eukaryotic thiolases. mtKCT may have followed a completely different evolutionary pathway after its divergence. This indicates that mitochondria have their own fatty acid p-oxidation system. Surprisingly, the divergent branch point and branch length of prokaryotic ACT and eukaryotic CTPACT (peroxisomal ACT of C. tropicalis) closely resemble those of prokaryotic KCT and eukaryotic CTPKCT (peroxisomal KCT of C. tropicalis). A slightly shorter branch length of ACT than that of KCT is probably due
* Corresponding author. 279
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KANAYAMA ET AL.
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
MO-RLNQLSGQLK---PNA . . . . . . . . KQSIL-QKNPD . . . . . . . . . DVVIV-AAYRTAI MGQRLQSIKDHLV---ESAMGKGESKRKNSLL-EKRPE . . . . . . . . . DVVIV-AANRSAi MH-RLQVVLGHLAGRSESSSALQAAPCSAGFP-QASAS. . . . . . . . . DVVVV-HGQRTPI M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. . . . . . . . . QVVIV-DAIRTPM M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5LNPR. . . . . DVVIV-DFGRTP~ M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . . . LLRGVFIV-AAKRTPF M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . Lp---PVYIV-STARTPI M.................................... S. . . . . Q---NVYIV-$TARTPI MA-ALAVLHGVVRRPLLRGLLQEVRCLGRSYASKPTLN. . . . . . . . . DVVIV-SATRTPI M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . . DVVIV-SAARTAV M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . TPSIVIASARTAV
37 46 45 14 15 17 16 15 49 14 15
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RflACT AEACT ZRACT
KAAKAVAAGAFKSEILPIRSI IRNSDGTEKEI-I-VDTDEGPREGV-TAESLGKLRPAFKAYKAKNEGLFEDE ILP IKL . . . . PDGS. . . . . I -CO SDEGPRPNV- TAESLSSI RPAF I KAAS AQSKGCFRAEIVPVTTTVLD DKGDRKT I -T-VS QOEGVRPST-TMEGLAKLKPAFRAWAATQSAAFKNE I I P TGGHOAD. . . . . GVL-K QFNYDEVl RPET- TVEALATLRP AFD LAHKATVEGKFKDE I IPMQGYDEN. . . . . GFL-K IFD YDETIRPDT- TLE SLAALKPAFN RWKAANEAGYFNEEMAP I EVKTK) . . . . . GKQ-T-MQ VDEHARPQT- TLEQLQNLPPVFK KAGKALSEGKFKSE I APVTI KGFR. . . . . GKP-D -TV I ENOEEI GKFNEERLK$ARTVFQ KSQO5QKEGKFDNEIVP VT IKGFR. . . . . GKP-D -TO VTNDEEPARLHVEKLKSARTVFQ RSKEAWDAGKFANE I TP I T I SVKG. . . . . KPD-V-VVKEDEEYKRV-DFSKVPKLKTVFQ KAEAAOKAGKFDEEIVPVLIPQRK. . . . . GDPVA-FK TDEFVRQGA-TLDSMSGLKPAFD KAEAAORDGRFKDEIVPFIVKGRK. . . . . GDi -T-VDADEYI RHGA-TLDSMAKLRPAFD * * = *
255
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
G-KGFKGSFRSVRSEFI LTEFLKEFI K) . . . . T- NID P-SLIEDVA[ GNVL-N(]AA- GAT G-KGFKGAFKDVNTDYLLYNFLNEFIGRFPEPL- RADL-NLI EEVACGNVL-NVGA- GAT G-RAGRGGFKDTTPOELLSAVLTAVLQO. . . . V- KPKP-ECLGD I SVGNVL-QPGA- GAA GRSKG-GAFRNVRAEDLSAHLMRSLLAR. . . . NPALEA-AALDD I YWGCVQ-QTLEQGFN GRSXG-GMHRNTRAEDMSAHL ISKVLER. . . . NSKVDP-G EVEDVIWGCVN-QTLEQGWN G-AY G-GLLKD FTATDLTEFAARAALSA. . . . GKV-P P-ETI DSVIVGNVMQSSSD- AAY G-SFQ-GSLSSLTYSDLGAHAVKAALAK--- -VPQ I K P-QDVDE IVFGGVL-QANV- GIlA G-SFQ-GSLSSKTAVELGAAALKGALAK . . . . VPELDASKDFDE I IFGNVL-SANL- GQA G-SFL-GSLASOPATKLGT IA IQGAI EK. . . . A- GI P K-EEVKEVYMGNV I -QGGE- GQA G-KFG-GSLAK I PAPELGAW IKAALER. . . . AGV-K P-EQVSEV IMGQVL-TAGS- GQN G-SFN-GAFANTPAHELGATV I SAVLER. . . . AGV-A A- GEVNEVI LGQVL-PAGE- GON
85 101 99 67 71 65 67 67 99 64 65
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
--DGTTTAGNASQVSDGAAAVLLMKRSLAEAKGYPIIGKYVLCSTAGVPPEIMGVGPAYA KDRGTTTAGNASQVSDGVAGVLLARRSVANQLNLPVLGRYIDFQTVGVPPEIMGVGPAYA KDGGSTTAGNSSQVSDGAAAVLLARRSKAEELGLPILGVLRSYAVVGVPPDIMGIGPAYA PVNGMVTAGTSSALSOGAAAMLVMSESRAHELGLKPRARVRSMAVVGCOPSIMGYGPVPA PKGGTVTAGTSSQITDGASCMIVMSAQRAKDLGLEPLAVIRSMAVAGVDPAIMGYGPVPA KE-GTVTAGNASGMSDGAGVVIIASEDAVKKHNFTPLARVVGYFVSGCDPAIMGIGPVPA KENGTVTAPNASKLNDGGAALVLVSEAKLKQLGLKPLAKISGWGEAARTPFDFTiAPALA RENGTVTAANASPINDGAAAilLVSERVLKEKNLKPLAIVKGWGEAAHLPADFTWAPSLA KENGTVTAANASTLNDGAAAVVLIdTAEAAQRLKVKPLARIAAFADAAVDPIDFPLAPAYA KA-GTVTAANASGLNDGAAAVWMSAAKAKELGLTPLATIKSYANAGVOPKVMGMGPVPA KE-GTVTAGNASGLNDGAAAALLMSEAEASRRGIQPLGRIVSWATVGVDPKVMGTGPIPA
313 322 324 257 291 299 300 500 329 296 294
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
EHRGACLAAG I PYTAAF I AVNRFCSSGLMAI SD I ANK I KTGEI ECGLAGGAESMS-- -TN EHRAACLASG I PYSTPFVALNRQCSSGLTAVNDI ANK I KVGQI D I GLALGVESMT-- -NN MARl AQFLSGI PETVPLSAVNRQCSSGLQAVANI AGG I RNGSYDI GMACGVES~T-- -LS [ARNAALLAEVPHSVPAVTVNRLCGSSMQALHDAARM II~(TGDAOACLVGGVEHMGHVPMS I ARMASLMTQI PHTSAAQTVSRLCGSSMSALHTAAQA IMTGNGDVFVVGGVEHMGHVSMM LARHVGLRVGVPTETGALTLNRLCGSGFQS I VSGCQEICSKDAEVVLCGGTESMSQSPYS PARQVALKAGLPDS [VAST IN KVCASGMKAVI I GAQNI I CGTSD I VVVGGAESMSNTPYY PARQVALTAGLGNH WATTVNKVCASAMKAI I LGAQ$ I KCGNADVVVAGGCESMTNAPYY P TRQATLGAGLP I ATPCTTVNKVCASGMKAi MMASQSLMCGHQDV~IVAGGMESMSNVPYV PARQAAI KAGLPAMVPAMTtNKVCGSGLKAVWLAANA IMAGDAE I VVAGGQENMSAAPHV PARQAAMKAGVPQEATAWGMNQLCGSGLRAVALGMQ(]IATGDASI IVAGGMESMSMAPHC
145 158 156 1 27 131 128 127 127 159 124 125
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA $UCACT RMACT AEACT ZRACT
IPEVLKRTGLTV-DOIDVFEINEAFAAQCLYSAE---QVNVPEEKLNINGGAIALGHPLG IPKVLEATGLQV-QDIDIFEINEAFAAQALYCIH---KLGIDLNKVNPRGGAIALGHPLG IPAALQKAGLTV-NDIDIFEINEAFASQALYCVE---KLGIPAEKVNPLGGAIALGHPLG SKLALKKAGLSA-SDIGVFEMNEAFAAQILPCIKDLGLIEQIDEKINLNGGAIALGHPLG TQKALKRAGLNM-ADIDFIELNEAFAAQALPVLKDLKVLDKMNEKVNLHGGAI ALGHPFG ITGALKKAGLSL-KDMDLIDVNEAFAPQFLAVQK---SLDLDPSKTNVSGGAIALGHPLG VPKAVKHAGLTV-ORVDFFELNEAFSVVGLANAE---LVNIPLEKLNVYGGAVAMGHPLG VPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTK---ILKLDPSKVNVYGGAVALGHPLG VPKVLKYAGLKK-EOIAMWEVNEAFSVWLANIK---MLEiDPQKVNVHGGAVSLGHPIG SKRALSRAEWTP-QDLDLMEINEAFAAQALAVHQ---QMGWOTSKVNVNGGAIAIGHPIG SRKALERAGWKI-GDLDLVEANEAFAAQACAVNK---DLGWOPSIVNVNGGAIAIGHPIG
380 346 350 355 356 357 385 352 350
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
Y--RD--PRVAPRIOP-HLADDAQMEKC-LIPMGITNENVANQFNISRERQOEFAAKSYN Y--KN--VNPLGMISSEELQKNREAKKC-LIPMGITNENVAANFKISRKDODEFAANSYO E--RGNPGNISSRLLENEKARDCL . . . . . . IPMGITSENVAERFGISRQKQOAFALASQQ H . . . . . GVDFHPGLSRNVAKAAGM. . . . . . . . MGLTAEMLARMHGISREMQDAFAARSHA H . . . . . GVDPNPHMSLYAAKASGM. . . . . . . . MGLTAEMLGKMHGISREQQDAFAVRSHQ VRNVRFGTKFGLDLKLEDTLWAGLTDQHVKLPMGMTAENLAAKYN[SREDCDRYALQSQQ LPSARSGARYGDAIMVDGVQKDGLLDVYEEKLMGVAAEKCAKDHGFSREDQDNFAINSYE MPAARGGAKFGQTVLIDGVERDGLNDAYOGLAMGVHAEKCARDWD[TRDQQDSFAIESYQ M--SRGATPYGGVKLEDLIVKDGLTDVYNKIHMGNCAENTAKKLSISREEQDKYAIGSYT LPGSRDGFRMGDAKLVDTMIVDGLWDVYNQYHMGITAENVAKEYGITREAQDEFAVGSQN --AHLAGVKMGDFKMJDTMIKDGLTDAFYGYHMGTTAENVAKQWQLSRDEQDAFAVASON
199 213 206 174 178 188 187 187 217 184 183
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
ETGARQYATIIPLLKP--GO-IGLTSMC-IGSGMGSASILV-R . . . . . . . . E CTGARQVATILRELKK--DO-IGVVSMC-IGTGMGAAAIFI-K . . . . . . . . E CTGARQVVTLLNELKRRGRRAYGVVSMC-IGTGMGAAAVFE-Y. . . . . . PGN CSGARISTTLLNLMERKDVQ-FGLADGCVSGLGQGIATVFE-R . . . . . . V - CSGARISGTLLNVMKQNGGT-FGLSTMC-IGLGQGIATVFE-R . . . . . . V - GSGSRITAHLVHELRRRGGK-YAVGSAC-tGGGGGISLIIQNT. . . . . . A-CSGARIIVTLLSVLTQEGGR-FGVAGVC-NGGGGASAVVIE-KIDADAKL-CSGARVVVTLLSILQQEGGK-IGVAAIC-NGGGGASSVVIE-K . . . . . . L - MSGARWVHLAHALKQ--GE-FGLASIC-NGGGGASAVLIE-K . . . . . . L - ASGCRILVTLLHEMKRRDAK-KGLASLC-IGGGMGVALAVERK . . . . . . . . . ASGARILNTLLFEMKRRGAR-KGLATLC-IGGGMGVAMCIESL . . . . . . . . .
262 264 227
231 240 240 240 269 237 255
369
378
408 417 424 388 391 397 403 398 424 393 391
FIG. 1. Alignment of amino acid sequences of thiolases from various sources. All sequences are shown by the one-letter amino acid notation. The stars mark the active site cysteines. Identical residues among these thiolases are indicated with (*). Abbreviations: CTPKCTA, C. tropicalis peroxisomal 3-ketoacyl-CoA thiolase A; SCPKCT, Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase; RPKCTB, rat peroxisomal 3-ketoacyl-CoA thiolase B; ECKCT, Escherichia coli 3-ketoacyl-CoA thiolase; PFKCT, Pseudomonas fragi 3-ketoacyl-CoA thiolase; RMKCT, rat mitochondrial 3-ketoacyl-CoA thiolase; CTPACTA, C. tropicalis peroxisomal acetoacetyl-CoA thiolase A; SUCACT, S. uvarum cytosolic acetoacetyl-CoA thiolase; RMACT, rat mitochondrial acetoacetyl-CoA thiolase; AEACT, Alcaligenes eutrophus acetoacetylCoA thiolase; ZRACT, Zoogloea ramigera acetoacetyl-CoA thiolase. Accession numbers in DDBJ: D17320 for CTPKCTA, X53946 for SCPKCT, J02749 for RPKCTB, J05498 for ECKCT, D90447 for PFKCT, X05341 for RMKCT, D 13470 for CTPACTA, X07976 for SUCACT, D00511 for RMACT, J04987 for AEACT, and J02631 for ZRACT. to the very strict substrate specificity o f ACT, which slowed its evolution. The symmetrical divergence of C T P A C T and CTPKCT isozymes suggests that original thiolases corresponding to CTPKCT and C T P A C T appeared at the same time and independently in an ancestral microorganism o f C. tropicalis, which had a function o f fatty acid assimilation. This simultaneous appearance o f two different enzymes (isozymes) in the evolution, having similar functions in the fatty acid metabolism, in a single cell may be explained by endosymbiosis. Furthermore, by introducing gene duplication (22, 23), we could help explain the molecular evolution of thiolase isozymes. The high similarity o f eukaryotic ACTs beyond species and subcellular localization [seen in C T P A C T (111, calculated from the origin of the divergence on one branch in Fig. 2), S U C A C T (118) and R M A C T (113)] indicates that gene duplication has recently given rise to these genes (15). Although rat mitochondrial KCT (mtKCT) revealed its own divergence,
it has also been suggested that rat mitochondrial ACT (RMACT) (at present, ACT is not found in mammalian peroxisomes) has been derived from the gene duplication o f peroxisomal ACT (CTACT). As shown in Fig. 1, the highest similarity is observed between C T P A C T and Saccharomyces uvarum cytosolic A C T (SUCACT). Cytosolic A C T has a similar alignment as peroxisomal C T A C T but lacks carboxy-terminal residues containing the three-residue -Ala-Lys-LeuCOOH (12), which is similar to the -Ser-Lys-Leu-COOH motif known as a peroxisomal targeting signal (24). These facts suggest that a gene duplication event o f cytosolic A C T from the peroxisomal one occurred more recently. Rat peroxisomal KCT (RPKCT-A and -B), CTPACT (A and B) and CTPKCT (A and B) have two very similar genes, respectively. Each pair of genes may be the products of a recent gene duplication, and they may be in the transition period o f diversification.
VOL. 78, 1994
MOLECULAR EVOLUTION OF THIOLASE ISOZYMES
44°/~ SCTPACTA U C A C T I Eukaryote 3.....--I RMACT 33/ 43../ AEACT 9/_23~ 50 ZRACT Prokaryote
~
mtKCT E RMKCT
I00
94 0~f ~ - - ~
'k ~
49
281
ACT
ECKCT ~] p - PFKCT rokaryote
zT~,, ~- RPKCTB I 5tk ~ SCPKCT Eukaryote • CTPKCTA
KCT
FIG. 2. Phylogenetic relationship of thiolases from various sources. The phylogenetic tree of thiolases with all aligned sequences (Fig. 1) containing amino-terminal and carboxy-terminal extensions, which are considered to be important in examining the evolution, was constructed. Therefore, the main replicates in 100 bootstrap replicates are low (40%). The number shows the relative branch length to RMKCT. The mark (*) demonstrates the origin of the divergence. Abbreviations used are as in Fig. 1. There are some differences in the localization o f the fatty acid E-oxidation system a m o n g eukaryotes. F o r example, C. tropicalis has only the peroxisomal/~-oxidation system, while rat has the peroxisomal and mitochond r i a l / % o x i d a t i o n systems. Coexistence o f A C T and K C T is essential for the complete d e g r a d a t i o n o f fatty acids. A c c o r d i n g to our analysis o f the molecular evolution o f thiolase isozymes, the final step enzymes o f the ~-oxidation system, the p-oxidation systems had existed b o t h in peroxisomes and in m i t o c h o n d r i a immediately after the endosymbiosis o f a specific m i c r o o r g a n i s m which evolved to peroxisomes in eukaryotes. However, the m i t o c h o n d r i a l system was degenerated in C. tropicalis because the peroxisomal system had a b r o a d e r substrate specificity than the m i t o c h o n d r i a l one (25, 26). On the other hand, in rat liver cells, the m i t o c h o n d r i a l /~-oxidation system remained to participate in the d e g r a d a t i o n o f short- and middle-chain substrates (by R M K C T as shown in Fig. 2), p r o b a b l y due to the occurrence o f R M A C T and the deletion o f p e r o x i s o m a l A C T . D N A has not been detected in peroxisomes and these organelles have only a single-membrane structure. These characteristics differ from those o f m i t o c h o n d r i a and chloroplasts, a b o u t 90% o f whose D N A s have been transferred to the nucleus b y so-called horizontal gene transfer (27, 28). E n d o s y m b i o s i s o f m i t o c h o n d r i a and chloroplasts has been suggested judging f r o m the fact that they contain characteristic D N A s (17). Peroxisomes, however, differ in these respects, de Duve and Borst et al. have discussed the possibility o f endosymbiosis o f peroxisomes based on the mechanism o f posttranslational protein t r a n s p o r t and the primitive or bacterial character o f the metabolic functions (15-18). The phylogenetic tree o f thiolases and peroxisomal localization o f these C T P A C T and C T P K C T isozymes in C. tropicalis s u p p o r t the concept presented in refs. 15-18 regarding o f molecular evolution. The construction o f the following m o d e l o f the evolution o f peroxisomes in C. tropicalis is also possible f r o m the phylogenetic tree. First, a euk a r y o t e and a specific microorganism, having a peroxisomal function, diverged from one original organism (an ancestor). Second, endosymbiosis o f the specific microorganism into eukaryotes and development to p r o k a r y otes independently occurred. The specific m i c r o o r g a n i s m evolved to peroxisomes in eukaryotes as a result o f endosymbiosis and, on the other hand, the m i c r o o r g a n i s m de-
veloped to p r o k a r y o t e s This hypothesis could m a n y as yet u n k n o w n peroxisomal enzymes phylogenetic trees.
which can assimilate fatty acids. be tested t h r o u g h analysis o f a m i n o acid sequences o f other and the c o m p a r i s o n o f their
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KANAYAMA ET AL. J., Teranishi, Y., and Tanaka, A.: Comparison of molecular structures and regulation of biosynthesis of unique thiolase isozymes localized only in peroxisomes of n-alkane-utilizing yeast, Candida tropicalis. J. Ferment. Bioeng., 78, 273-278 (1994). Yong, S.Y., Yang, X.Y.H., Healy-Louie, G., Schulz, H., and Elzinga, M.: Nucleotide sequence of the fadA gene-primary structure of 3-ketoacyl-coenzyme A thiolase from Escherichia coli and the structural organization of the fadAB operon. J. Biol. Chem., 265, 10424-10429 (1990). Iguai, J.C., Gonzales-Bosch, C., Dopazo, J., and PerezOritin, J . E . : Phylogenetic analysis of the thiolase family. Implications for the evolutionary origin of peroxisomes. J. Mol. Evol., 35, 147-155 (1992). Borst, P.: Peroxisome biogenesis revisited. Biochim. Biophys. Acta, 100g, 1-13 (1989). Cavalier-Smith, T.: The simultaneous symbiotic origin of mitochondria, chloroplasts, and microbodies. Ann. N.Y. Acad. Sci., 503, 55-71 (1987). de Duve, C.: Microbodies in the living cell. Sci. Am., 248, 7484 (1983). Kimura, M.: The neutral theory of molecular evolution. Cambridge University Press, Cambridge (1983). Saltou, N.M. and Nei, M.: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol.
J. FERMENT. BIOENG., Evol., 4, 406-425 (1987). 21. Felsenstein, J.: Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783-791 (1985). 22. Ohno, S.: Evolution by gene duplication. Springer-Verlag, Berlin, New York (1970). 23. Kimura, M. and Ohta, T.: On some principles governing molecular evolution. Proc. Natl. Acad. Sci. USA, 71, 2848-2852 (1974). 24. Gould, S.J., Keller, G.A., Hosken, M., Wilkinson, J., and Subramani, S.: A conserved tripeptide sorts proteins to peroxisomes. J. Cell Biol., 108, 1657-1664 (1989). 25. Osumi, T., Hashimoto, T., and Ui, N.: Purification and properties of acyl-CoA oxidase from rat liver. J. Biochem., 87, 1735-1746 (1980). 26. Hryb, D.J. and Hogg, J. F.: Chain length specificities of peroxisomal and mitochondrial E-oxidation in rat liver. Biochem. Biophys. Res. Commun., 87, 1200-1206 (1979). 27. Nugent, J . M . and Palmer, J . D . : RNA-mediated transfer of the gene coxII from the mitochondrion to the nucleus during flowering plant evolution. Cell, 66, 473-481 (1991). 28. Brinkmann, H., Martinez, P., Qulgley, F., Martin, W., and Cerff, R.: Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize. J. Mol. Evol., 26, 320-328 (1987).
Vol. 78, No. 4, 279-282. 1994
Molecular Evolution of Yeast Thiolase Isozymes NAOKI KANAYAMA, MITSUYOSHI UEDA, H A R U Y U K I ATOMI, TATSUO KURIHARA, AND ATSUO TANAKA*
Laboratory of Applied Biological Chemistry, Department of Synthetic Chemistry and Biological Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-01, Japan Received 9 May 1994/Accepted 12 July 1994 The coexistence of two thiolase isozymes (acetoacetyl-CoA thiolase and 3-ketoaeyl-CoA thiolase), essential for the complete degradation of fatty acids, only in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (Kurihara et al., J. Bioehem., 106, 474-478, 1989) is unique in eukaryotic cells. As one of the methods of analysis of molecular information from these isozymes, the calculation of the evolutional distance among thiolases from various organisms suggested that yeast peroxisomai thlolase isozymes are important enzymes in examining the molecular evolution of the fatty acid metabolic pathway and the biogenesis of peroxisomes.
Thiolases catalyze the final step of fatty acid ~-oxidation in prokaryotic (1) and eukaryotic cells (2-6) and are classified into two types, acetoacetyl-CoA thiolase (ACT), which is specific to acetoacetyl-CoA, and 3-ketoacyl-CoA thiolase (KCT), which has broad substrate specificity. For the complete and efficient degradation of fatty acids having various chain lengths, the coexistence of these two types of thiolases is important. In eukaryotes, thiolases exhibit diverse subcellular localization. In rat liver cells, there exist two distinct KCTs, one localized in peroxisomes responsible for long-chain acylCoAs (4) and another in mitochondria for middle-chain acyl-CoAs (2), whereas ACTs are localized in cytosol (2) and in mitochondria (2). We were the first to find the presence of both ACT and KCT in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (5-7) and their absence in its mitochondria. The peroxisomal localization of the two thiolases is unique and provides a basis for examining the evolution of the fatty acid p-oxidation system. Since the fatty acid p-oxidation system was also exclusively detected in peroxisomes in the yeast cells (8), fatty acids could be completely degraded with cooperation of these peroxisomal ACT and KCT (6, 7). These facts are quite different from the case of mammalian cells, which possess two E-oxidation pathways: a peroxisomai one involved in long-chain fatty acid degradation and a mitochondrial one responsible for medium- or short-chain fatty acid degradation (9-11). We have cloned and sequenced the genes of peroxisomal ACT and KCT of C. tropicalis (12, 13), and determined their primary protein structures. By calculating the evolutional distance among thiolases from various sources (14, 15), we have constructed a phylogenetic tree of thiolases and have discussed the molecular evolution of thiolase isozymes. The symmetric divergent branch points of this tree strongly suggested that the ancestors of peroxisomal ACT and KCT had occurred at the same time in the yeast C. tropicalis, as predicted by the endosymbiotic theory of peroxisomes (16-18).
MATERIALS AND METHODS Alignment of amino acid sequences The multiple alignment of protein sequences and the number of similarity between sequences were obtained with the program A L I G N contained within the ODEN program provided by DNA Databank Japan (DDBJ). Nucleotide sequences of thiolases have been deposited in DDBJ with accession numbers. Construction of phylogenetic tree The number of amino acid substitutions was estimated with the DISTA program (19). The distance matrix was made with the DMATA program (19). The phylogenetic tree was constructed by the neighbor-joining method with the TREENJ program (20). Bootstrap resampling was performed with the BSTRAP program (21). These programs were contained within the ODEN program provided by DDBJ.
RESULTS AND DISCUSSION We compared all of the primary sequences of thiolases, encoded by the open reading frames of respective DNAs, from various organisms reported so far (Fig. 1) and constructed a phylogenetic tree of thiolases based on the number of amino acid substitutions (19) and the neighbor-joining method (20) (Fig. 2). The phylogenetic tree clearly indicates that three types of thiolases, acetoacetyl-CoA thiolase (ACT), 3-ketoacyl-CoA thiolase (KCT), and mitochondrial 3-ketoacyl-CoA thiolase (mtKCT), diverged simultaneously from one original enzyme. We can find that the divergence of one original thiolase to thiolases with different substrate specificities--that is, to KCT and ACT--occurred before the divergence of prokaryotic and eukaryotic thiolases. mtKCT may have followed a completely different evolutionary pathway after its divergence. This indicates that mitochondria have their own fatty acid p-oxidation system. Surprisingly, the divergent branch point and branch length of prokaryotic ACT and eukaryotic CTPACT (peroxisomal ACT of C. tropicalis) closely resemble those of prokaryotic KCT and eukaryotic CTPKCT (peroxisomal KCT of C. tropicalis). A slightly shorter branch length of ACT than that of KCT is probably due
* Corresponding author. 279
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KANAYAMA ET AL.
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
MO-RLNQLSGQLK---PNA . . . . . . . . KQSIL-QKNPD . . . . . . . . . DVVIV-AAYRTAI MGQRLQSIKDHLV---ESAMGKGESKRKNSLL-EKRPE . . . . . . . . . DVVIV-AANRSAi MH-RLQVVLGHLAGRSESSSALQAAPCSAGFP-QASAS. . . . . . . . . DVVVV-HGQRTPI M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. . . . . . . . . QVVIV-DAIRTPM M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5LNPR. . . . . DVVIV-DFGRTP~ M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . . . LLRGVFIV-AAKRTPF M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. . . . Lp---PVYIV-STARTPI M.................................... S. . . . . Q---NVYIV-$TARTPI MA-ALAVLHGVVRRPLLRGLLQEVRCLGRSYASKPTLN. . . . . . . . . DVVIV-SATRTPI M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. . . . . . . . . DVVIV-SAARTAV M. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. . . . . . . . . TPSIVIASARTAV
37 46 45 14 15 17 16 15 49 14 15
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RflACT AEACT ZRACT
KAAKAVAAGAFKSEILPIRSI IRNSDGTEKEI-I-VDTDEGPREGV-TAESLGKLRPAFKAYKAKNEGLFEDE ILP IKL . . . . PDGS. . . . . I -CO SDEGPRPNV- TAESLSSI RPAF I KAAS AQSKGCFRAEIVPVTTTVLD DKGDRKT I -T-VS QOEGVRPST-TMEGLAKLKPAFRAWAATQSAAFKNE I I P TGGHOAD. . . . . GVL-K QFNYDEVl RPET- TVEALATLRP AFD LAHKATVEGKFKDE I IPMQGYDEN. . . . . GFL-K IFD YDETIRPDT- TLE SLAALKPAFN RWKAANEAGYFNEEMAP I EVKTK) . . . . . GKQ-T-MQ VDEHARPQT- TLEQLQNLPPVFK KAGKALSEGKFKSE I APVTI KGFR. . . . . GKP-D -TV I ENOEEI GKFNEERLK$ARTVFQ KSQO5QKEGKFDNEIVP VT IKGFR. . . . . GKP-D -TO VTNDEEPARLHVEKLKSARTVFQ RSKEAWDAGKFANE I TP I T I SVKG. . . . . KPD-V-VVKEDEEYKRV-DFSKVPKLKTVFQ KAEAAOKAGKFDEEIVPVLIPQRK. . . . . GDPVA-FK TDEFVRQGA-TLDSMSGLKPAFD KAEAAORDGRFKDEIVPFIVKGRK. . . . . GDi -T-VDADEYI RHGA-TLDSMAKLRPAFD * * = *
255
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
G-KGFKGSFRSVRSEFI LTEFLKEFI K) . . . . T- NID P-SLIEDVA[ GNVL-N(]AA- GAT G-KGFKGAFKDVNTDYLLYNFLNEFIGRFPEPL- RADL-NLI EEVACGNVL-NVGA- GAT G-RAGRGGFKDTTPOELLSAVLTAVLQO. . . . V- KPKP-ECLGD I SVGNVL-QPGA- GAA GRSKG-GAFRNVRAEDLSAHLMRSLLAR. . . . NPALEA-AALDD I YWGCVQ-QTLEQGFN GRSXG-GMHRNTRAEDMSAHL ISKVLER. . . . NSKVDP-G EVEDVIWGCVN-QTLEQGWN G-AY G-GLLKD FTATDLTEFAARAALSA. . . . GKV-P P-ETI DSVIVGNVMQSSSD- AAY G-SFQ-GSLSSLTYSDLGAHAVKAALAK--- -VPQ I K P-QDVDE IVFGGVL-QANV- GIlA G-SFQ-GSLSSKTAVELGAAALKGALAK . . . . VPELDASKDFDE I IFGNVL-SANL- GQA G-SFL-GSLASOPATKLGT IA IQGAI EK. . . . A- GI P K-EEVKEVYMGNV I -QGGE- GQA G-KFG-GSLAK I PAPELGAW IKAALER. . . . AGV-K P-EQVSEV IMGQVL-TAGS- GQN G-SFN-GAFANTPAHELGATV I SAVLER. . . . AGV-A A- GEVNEVI LGQVL-PAGE- GON
85 101 99 67 71 65 67 67 99 64 65
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
--DGTTTAGNASQVSDGAAAVLLMKRSLAEAKGYPIIGKYVLCSTAGVPPEIMGVGPAYA KDRGTTTAGNASQVSDGVAGVLLARRSVANQLNLPVLGRYIDFQTVGVPPEIMGVGPAYA KDGGSTTAGNSSQVSDGAAAVLLARRSKAEELGLPILGVLRSYAVVGVPPDIMGIGPAYA PVNGMVTAGTSSALSOGAAAMLVMSESRAHELGLKPRARVRSMAVVGCOPSIMGYGPVPA PKGGTVTAGTSSQITDGASCMIVMSAQRAKDLGLEPLAVIRSMAVAGVDPAIMGYGPVPA KE-GTVTAGNASGMSDGAGVVIIASEDAVKKHNFTPLARVVGYFVSGCDPAIMGIGPVPA KENGTVTAPNASKLNDGGAALVLVSEAKLKQLGLKPLAKISGWGEAARTPFDFTiAPALA RENGTVTAANASPINDGAAAilLVSERVLKEKNLKPLAIVKGWGEAAHLPADFTWAPSLA KENGTVTAANASTLNDGAAAVVLIdTAEAAQRLKVKPLARIAAFADAAVDPIDFPLAPAYA KA-GTVTAANASGLNDGAAAVWMSAAKAKELGLTPLATIKSYANAGVOPKVMGMGPVPA KE-GTVTAGNASGLNDGAAAALLMSEAEASRRGIQPLGRIVSWATVGVDPKVMGTGPIPA
313 322 324 257 291 299 300 500 329 296 294
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
EHRGACLAAG I PYTAAF I AVNRFCSSGLMAI SD I ANK I KTGEI ECGLAGGAESMS-- -TN EHRAACLASG I PYSTPFVALNRQCSSGLTAVNDI ANK I KVGQI D I GLALGVESMT-- -NN MARl AQFLSGI PETVPLSAVNRQCSSGLQAVANI AGG I RNGSYDI GMACGVES~T-- -LS [ARNAALLAEVPHSVPAVTVNRLCGSSMQALHDAARM II~(TGDAOACLVGGVEHMGHVPMS I ARMASLMTQI PHTSAAQTVSRLCGSSMSALHTAAQA IMTGNGDVFVVGGVEHMGHVSMM LARHVGLRVGVPTETGALTLNRLCGSGFQS I VSGCQEICSKDAEVVLCGGTESMSQSPYS PARQVALKAGLPDS [VAST IN KVCASGMKAVI I GAQNI I CGTSD I VVVGGAESMSNTPYY PARQVALTAGLGNH WATTVNKVCASAMKAI I LGAQ$ I KCGNADVVVAGGCESMTNAPYY P TRQATLGAGLP I ATPCTTVNKVCASGMKAi MMASQSLMCGHQDV~IVAGGMESMSNVPYV PARQAAI KAGLPAMVPAMTtNKVCGSGLKAVWLAANA IMAGDAE I VVAGGQENMSAAPHV PARQAAMKAGVPQEATAWGMNQLCGSGLRAVALGMQ(]IATGDASI IVAGGMESMSMAPHC
145 158 156 1 27 131 128 127 127 159 124 125
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA $UCACT RMACT AEACT ZRACT
IPEVLKRTGLTV-DOIDVFEINEAFAAQCLYSAE---QVNVPEEKLNINGGAIALGHPLG IPKVLEATGLQV-QDIDIFEINEAFAAQALYCIH---KLGIDLNKVNPRGGAIALGHPLG IPAALQKAGLTV-NDIDIFEINEAFASQALYCVE---KLGIPAEKVNPLGGAIALGHPLG SKLALKKAGLSA-SDIGVFEMNEAFAAQILPCIKDLGLIEQIDEKINLNGGAIALGHPLG TQKALKRAGLNM-ADIDFIELNEAFAAQALPVLKDLKVLDKMNEKVNLHGGAI ALGHPFG ITGALKKAGLSL-KDMDLIDVNEAFAPQFLAVQK---SLDLDPSKTNVSGGAIALGHPLG VPKAVKHAGLTV-ORVDFFELNEAFSVVGLANAE---LVNIPLEKLNVYGGAVAMGHPLG VPKALKHAGIEDINSVDYFEFNEAFSVVGLVNTK---ILKLDPSKVNVYGGAVALGHPLG VPKVLKYAGLKK-EOIAMWEVNEAFSVWLANIK---MLEiDPQKVNVHGGAVSLGHPIG SKRALSRAEWTP-QDLDLMEINEAFAAQALAVHQ---QMGWOTSKVNVNGGAIAIGHPIG SRKALERAGWKI-GDLDLVEANEAFAAQACAVNK---DLGWOPSIVNVNGGAIAIGHPIG
380 346 350 355 356 357 385 352 350
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
Y--RD--PRVAPRIOP-HLADDAQMEKC-LIPMGITNENVANQFNISRERQOEFAAKSYN Y--KN--VNPLGMISSEELQKNREAKKC-LIPMGITNENVAANFKISRKDODEFAANSYO E--RGNPGNISSRLLENEKARDCL . . . . . . IPMGITSENVAERFGISRQKQOAFALASQQ H . . . . . GVDFHPGLSRNVAKAAGM. . . . . . . . MGLTAEMLARMHGISREMQDAFAARSHA H . . . . . GVDPNPHMSLYAAKASGM. . . . . . . . MGLTAEMLGKMHGISREQQDAFAVRSHQ VRNVRFGTKFGLDLKLEDTLWAGLTDQHVKLPMGMTAENLAAKYN[SREDCDRYALQSQQ LPSARSGARYGDAIMVDGVQKDGLLDVYEEKLMGVAAEKCAKDHGFSREDQDNFAINSYE MPAARGGAKFGQTVLIDGVERDGLNDAYOGLAMGVHAEKCARDWD[TRDQQDSFAIESYQ M--SRGATPYGGVKLEDLIVKDGLTDVYNKIHMGNCAENTAKKLSISREEQDKYAIGSYT LPGSRDGFRMGDAKLVDTMIVDGLWDVYNQYHMGITAENVAKEYGITREAQDEFAVGSQN --AHLAGVKMGDFKMJDTMIKDGLTDAFYGYHMGTTAENVAKQWQLSRDEQDAFAVASON
199 213 206 174 178 188 187 187 217 184 183
CTPKCTA SCPKCT RPKCTB ECKCT PFKCT RMKCT CTPACTA SUCACT RMACT AEACT ZRACT
ETGARQYATIIPLLKP--GO-IGLTSMC-IGSGMGSASILV-R . . . . . . . . E CTGARQVATILRELKK--DO-IGVVSMC-IGTGMGAAAIFI-K . . . . . . . . E CTGARQVVTLLNELKRRGRRAYGVVSMC-IGTGMGAAAVFE-Y. . . . . . PGN CSGARISTTLLNLMERKDVQ-FGLADGCVSGLGQGIATVFE-R . . . . . . V - CSGARISGTLLNVMKQNGGT-FGLSTMC-IGLGQGIATVFE-R . . . . . . V - GSGSRITAHLVHELRRRGGK-YAVGSAC-tGGGGGISLIIQNT. . . . . . A-CSGARIIVTLLSVLTQEGGR-FGVAGVC-NGGGGASAVVIE-KIDADAKL-CSGARVVVTLLSILQQEGGK-IGVAAIC-NGGGGASSVVIE-K . . . . . . L - MSGARWVHLAHALKQ--GE-FGLASIC-NGGGGASAVLIE-K . . . . . . L - ASGCRILVTLLHEMKRRDAK-KGLASLC-IGGGMGVALAVERK . . . . . . . . . ASGARILNTLLFEMKRRGAR-KGLATLC-IGGGMGVAMCIESL . . . . . . . . .
262 264 227
231 240 240 240 269 237 255
369
378
408 417 424 388 391 397 403 398 424 393 391
FIG. 1. Alignment of amino acid sequences of thiolases from various sources. All sequences are shown by the one-letter amino acid notation. The stars mark the active site cysteines. Identical residues among these thiolases are indicated with (*). Abbreviations: CTPKCTA, C. tropicalis peroxisomal 3-ketoacyl-CoA thiolase A; SCPKCT, Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase; RPKCTB, rat peroxisomal 3-ketoacyl-CoA thiolase B; ECKCT, Escherichia coli 3-ketoacyl-CoA thiolase; PFKCT, Pseudomonas fragi 3-ketoacyl-CoA thiolase; RMKCT, rat mitochondrial 3-ketoacyl-CoA thiolase; CTPACTA, C. tropicalis peroxisomal acetoacetyl-CoA thiolase A; SUCACT, S. uvarum cytosolic acetoacetyl-CoA thiolase; RMACT, rat mitochondrial acetoacetyl-CoA thiolase; AEACT, Alcaligenes eutrophus acetoacetylCoA thiolase; ZRACT, Zoogloea ramigera acetoacetyl-CoA thiolase. Accession numbers in DDBJ: D17320 for CTPKCTA, X53946 for SCPKCT, J02749 for RPKCTB, J05498 for ECKCT, D90447 for PFKCT, X05341 for RMKCT, D 13470 for CTPACTA, X07976 for SUCACT, D00511 for RMACT, J04987 for AEACT, and J02631 for ZRACT. to the very strict substrate specificity o f ACT, which slowed its evolution. The symmetrical divergence of C T P A C T and CTPKCT isozymes suggests that original thiolases corresponding to CTPKCT and C T P A C T appeared at the same time and independently in an ancestral microorganism o f C. tropicalis, which had a function o f fatty acid assimilation. This simultaneous appearance o f two different enzymes (isozymes) in the evolution, having similar functions in the fatty acid metabolism, in a single cell may be explained by endosymbiosis. Furthermore, by introducing gene duplication (22, 23), we could help explain the molecular evolution of thiolase isozymes. The high similarity o f eukaryotic ACTs beyond species and subcellular localization [seen in C T P A C T (111, calculated from the origin of the divergence on one branch in Fig. 2), S U C A C T (118) and R M A C T (113)] indicates that gene duplication has recently given rise to these genes (15). Although rat mitochondrial KCT (mtKCT) revealed its own divergence,
it has also been suggested that rat mitochondrial ACT (RMACT) (at present, ACT is not found in mammalian peroxisomes) has been derived from the gene duplication o f peroxisomal ACT (CTACT). As shown in Fig. 1, the highest similarity is observed between C T P A C T and Saccharomyces uvarum cytosolic A C T (SUCACT). Cytosolic A C T has a similar alignment as peroxisomal C T A C T but lacks carboxy-terminal residues containing the three-residue -Ala-Lys-LeuCOOH (12), which is similar to the -Ser-Lys-Leu-COOH motif known as a peroxisomal targeting signal (24). These facts suggest that a gene duplication event o f cytosolic A C T from the peroxisomal one occurred more recently. Rat peroxisomal KCT (RPKCT-A and -B), CTPACT (A and B) and CTPKCT (A and B) have two very similar genes, respectively. Each pair of genes may be the products of a recent gene duplication, and they may be in the transition period o f diversification.
VOL. 78, 1994
MOLECULAR EVOLUTION OF THIOLASE ISOZYMES
44°/~ SCTPACTA U C A C T I Eukaryote 3.....--I RMACT 33/ 43../ AEACT 9/_23~ 50 ZRACT Prokaryote
~
mtKCT E RMKCT
I00
94 0~f ~ - - ~
'k ~
49
281
ACT
ECKCT ~] p - PFKCT rokaryote
zT~,, ~- RPKCTB I 5tk ~ SCPKCT Eukaryote • CTPKCTA
KCT
FIG. 2. Phylogenetic relationship of thiolases from various sources. The phylogenetic tree of thiolases with all aligned sequences (Fig. 1) containing amino-terminal and carboxy-terminal extensions, which are considered to be important in examining the evolution, was constructed. Therefore, the main replicates in 100 bootstrap replicates are low (40%). The number shows the relative branch length to RMKCT. The mark (*) demonstrates the origin of the divergence. Abbreviations used are as in Fig. 1. There are some differences in the localization o f the fatty acid E-oxidation system a m o n g eukaryotes. F o r example, C. tropicalis has only the peroxisomal/~-oxidation system, while rat has the peroxisomal and mitochond r i a l / % o x i d a t i o n systems. Coexistence o f A C T and K C T is essential for the complete d e g r a d a t i o n o f fatty acids. A c c o r d i n g to our analysis o f the molecular evolution o f thiolase isozymes, the final step enzymes o f the ~-oxidation system, the p-oxidation systems had existed b o t h in peroxisomes and in m i t o c h o n d r i a immediately after the endosymbiosis o f a specific m i c r o o r g a n i s m which evolved to peroxisomes in eukaryotes. However, the m i t o c h o n d r i a l system was degenerated in C. tropicalis because the peroxisomal system had a b r o a d e r substrate specificity than the m i t o c h o n d r i a l one (25, 26). On the other hand, in rat liver cells, the m i t o c h o n d r i a l /~-oxidation system remained to participate in the d e g r a d a t i o n o f short- and middle-chain substrates (by R M K C T as shown in Fig. 2), p r o b a b l y due to the occurrence o f R M A C T and the deletion o f p e r o x i s o m a l A C T . D N A has not been detected in peroxisomes and these organelles have only a single-membrane structure. These characteristics differ from those o f m i t o c h o n d r i a and chloroplasts, a b o u t 90% o f whose D N A s have been transferred to the nucleus b y so-called horizontal gene transfer (27, 28). E n d o s y m b i o s i s o f m i t o c h o n d r i a and chloroplasts has been suggested judging f r o m the fact that they contain characteristic D N A s (17). Peroxisomes, however, differ in these respects, de Duve and Borst et al. have discussed the possibility o f endosymbiosis o f peroxisomes based on the mechanism o f posttranslational protein t r a n s p o r t and the primitive or bacterial character o f the metabolic functions (15-18). The phylogenetic tree o f thiolases and peroxisomal localization o f these C T P A C T and C T P K C T isozymes in C. tropicalis s u p p o r t the concept presented in refs. 15-18 regarding o f molecular evolution. The construction o f the following m o d e l o f the evolution o f peroxisomes in C. tropicalis is also possible f r o m the phylogenetic tree. First, a euk a r y o t e and a specific microorganism, having a peroxisomal function, diverged from one original organism (an ancestor). Second, endosymbiosis o f the specific microorganism into eukaryotes and development to p r o k a r y otes independently occurred. The specific m i c r o o r g a n i s m evolved to peroxisomes in eukaryotes as a result o f endosymbiosis and, on the other hand, the m i c r o o r g a n i s m de-
veloped to p r o k a r y o t e s This hypothesis could m a n y as yet u n k n o w n peroxisomal enzymes phylogenetic trees.
which can assimilate fatty acids. be tested t h r o u g h analysis o f a m i n o acid sequences o f other and the c o m p a r i s o n o f their
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