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Sequence of a Giardia lamblia gene coding for the glycolytic enzyme, pyruvate,phosphate dikinase
MOLECULfS
iii%mMIcAL PARAsIToLoGy Molecular
and Biochemical
Parasitology
77 (1996) 217--223
Sequence of a Giardia Zamblia gene coding for the glycolytic enzyme, pyruvate,phosphate dikinase’ Leena Nevalainen, The Rockrjtller Received
Ivan Hrdjr’, Miklbs
Uniuersiry,
15 December
1230 York Awnre,
1995; revised 4 March
Nets
Miiller*
York .YY.
1996: accepted
ISA
7 March
1996
Abstract The sequence of the gene coding for pyruvate,phosphate dikinase (EC 2.7.9.1) in Giurdiu lurnhliu (syn. G. duodmulis) has been established. The deduced amino acid sequence is very similar to all of its known homologs from the protist, Entamoeha histolytica, the eubacterium, Clostridiwn synhiosurr~ and plant chloroplasts. Phylogenetic reconstruction with neighbor-joining and maximum parsimony methods reveals that the sequences form two clades, one comprising the anaerobic microorganisms, and the other the chloroplast enzymes. Kqworcls: Giardiu lanzhliu; gDNA
library;
Pyruvate,phosphate
Biochemical studies disclosed an unexpected diversity in the enzymes and subcellular organization of the energy metabolism of various parasitic ,4hhreriariorzs: Pyruvate dikinase. pyruvate.phosphate dikinase: PEP, phosphoenolpyruvate: PEP synthase. phosphoenolpyruvate synthase: PCR, polymerase chain reaction. *Tel: + 1 212 3278153; fax: + 1 212 3277974: e-mail: mmulleri$rockvax.rockefeller.edu ’ fort: The nucleotide sequence obtained, including stretches of the 5’- and 3’-untranslated regions of the gene (126 and 22 bp, respectively) not discussed in this paper, has been submitted to the GenBankTM database with the accession No. u43531. ’ Present address: Department of Physiology and Developmental Biology, Charles University, Vinicna 7, 128 00 Prague 2. Czech Republic.
$15.00
c) 1996 Elsevier
PII SO166-6851(96)02604-7
Science
B.V. All rights
Diplomonad;
Amitochondriate _____
protist
Certain species lack mitochondria and hydrogenosomes thus their metabolism, which is fermentative, is not compartmentalized [l]. The detailed exploration of one of these organisms, Entamoeba Izistolyticcl, revealed that several enzymes of its core metabolism differ from those of typical eukaryotic glycolysis [2]. Restricted to a single, however well studied, species these results were interpreted as reflecting an exceptional, ancestral metabolic machinery [2]. Subsequently Giardia lamblia, another amitochondriate parasite belonging to a different protist lineage [3-51, was found to share a number of metabolic characteristics with E. histolytica [6-81. This finding raised the possibility of a broader distribution of this unusual metabolic type [l] and the question of its evolutionary origin in representatives of two unrelated lineages [9]. protists.
1. Introduction
0166.6851/96#
dikinase:
reserved
218
L. Neualainen
et al. / Molecular
and Biochemical
One of the striking deviations in the glycolysis of
Purusitologv 77 11996) _‘I 7-22
2. Materials
and methods
E. histolytica from the typical eukaryotic pattern is
the reaction leading from phosphoenolpyruvate (PEP) to pyruvate. In most organisms this is catalyzed by pyruvate kinase (EC 2.7.1.40): PEP + ADP --) Pyruvate
+ ATP,
while in E. histolytica it is effected by the action of pyruvate,phosphate dikinase (pyruvate dikinaseEC 2.7.9.1) [lo]: PEP + PPi + AMP ++ Pyruvate
+ Pi + ATP.
In addition to E. histolytica, this enzyme is known from various bacteria and chloroplasts [l l-131. In certain bacteria and in chloroplasts its function is anabolic, providing PEP as a substrate for CO2 fixation [ 11,131. When acting as a catabolic enzyme, pyruvate dikinase utilizes inorganic pyrophosphate resulting in increased glycolytic ATP yield, a circumstance regarded as especially significant for organisms that rely exclusively on fermentation [ 13- 151. Pyruvate dikinase is not a homolog of pyruvate kinase [l I] but is closely related to phosphoenolpyruvate synthase (PEP synthaseEC 2.7.9.2) studied in detail from Escherichia coli [ 161 and Pyrococcus furiosus [ 171, which catalyzes an analogous reaction: PEP + Pi + AMP ++ Pyruvate
+ ATP.
These two enzymes in turn are closely related to Enzyme I of the bacterial PEP:sugar phosphotransferase system [l 1,121.These proteins together comprise the Enzyme I protein family [12], members of which participate in an unusually wide array of cellular functions [ 181. The presence of pyruvate dikinase in G. Iamblia has been demonstrated with biochemical methods [ 191, underscoring the metabolic similarities between this organism and E. histolytica. No pyruvate kinase was found yet in this species [7,19]. In this communication we report the deduced primary structure of G. lamblia pyruvate dikinase, its similarity to the E. histolytica enzyme [20,21] and its close relationship to all related enzymes for which sequence information is available. While this study was underway, the sequence of the pyruvate dikinase gene has been established from a Giardia isolate from sheep with results highly similar to those reported here [22].
Giardia lamblia (syn. (;, duodenalis), strain WB, clone C6 (ATCC 30957) was used in this study. gDNA and a AZAPII gDNA library have been kindly provided by Drs. F.D. Gillin and S.B. Aley (University of California, San Diego, CA). A partial nucleotide sequence of the pyruvate dikinase, corresponding to the region from Gly-28 to Phe-87 in the final sequence was obtained by Dr. J.E. Gray and kindly communicated to us by Dr. F.R. Doolittle (University of California, San Diego, CA). Based on this information, two nondegenerate polymerase chain reaction (PCR) primers were designed (sense: S-GGCAAGGGGAATTTCTCTTG-3’; antisense: 5’-GAAAGTCTTCCCCATCTT-3’) and with the use of gDNA as template, a I80-bp PCR product was obtained. This product, labeled with [32P]dATP by random priming, was used as a probe to screen the gDNA library. The pBluescript insert from one of the positive clones was excised with helper phage following the manufacturer’s instructions (Stratagene) and amplified in Escherichia coli XL-l Blue. Both strands of the purified phagemid were sequenced by the dideoxy chain termination method. Standard molecular methods have been used throughout this study. Since the first insert isolated contained an incomplete open reading frame, with approximately 100 carboxyl-terminal amino acid residues missing, we rescreened the library with a 300-bp PCR product corresponding to a carboxyl-terminal area of the molecule. The primers used were a nondegenerate one corresponding to a part close to the carboxylterminus of the previously obtained incomplete sequence and a degenerate one designed from a conserved stretch in the missing area of the enzyme (sense: 5’-GAGGTGGGGACCATGAT-3’; antisense: 5’- CCRTGYTCNCCRCADAT-3’). The sequences obtained were assembled and edited with Eyeball Sequence Editor [23]. Numbering of the amino-acid residues is based on the derived G. Iamblia sequence. Alignment with related sequences and formatting of the data for phylogenetic reconstruction were done with the MUST suite of programs (version 1.O) [24]. Phylo-
219
L. Nevalainen et al. 1 Molecular and Biochemical Parasitology 77 (1996) 217-2X
[l 1,121, are well conserved in the G. famblia enzyme, too (Fig. 1). Domain A contains the GGXTSH motif strictly conserved in all these proteins [12] which includes His-464, the residue known to be phosphorylated in all these enzymes and also the conserved Thr-462. The latter residue is known to play a role in the regulation of this enzyme, observed so far only in plant dikinases [25]. It remains to be established, whether in G. lamblia it has a regulatory function. The functional role of conserved domains B-D has not been firmly established yet. They are implicated in PEP binding and transfer of phosphoryl groups [ 11,2629]. The flexible Q-linker region, assumed to connect the two functional halves of these proteins [12] and a putative P-loop are also clearly recognizable [29] (Fig. 1). Two sequence signatures have been proposed for this protein family [12]. The first located in domain A (residues 456-480) is fully conserved, while the second one in domain C (residues 769780) shows some deviation, since the two first residues are Gln-769 and Tyr-770 instead of (DE)(FG). Note that E. histolytica pyruvate dikinase also deviates slightly from the proposed signature, having Ser-Phe in this position [20,21]. The G. lamblia sequence was about equally divergent from all other pyruvate dikinases, with 46-52x amino acid differences (Table 1). Its distance from the E. histo(vtica enzyme (52%) was
genetic reconstruction was done with the neighbor-joining (NJ) method of the MEGA package (version 1.0) and by a parsimony (PROTPARS) method in the PHYLIP package (version 3.55~).
3. Results and discussion The assembled sequence contained an open reading frame coding for a putative protein of 884 amino acid residues (Fig. 1). This sequence differed from that from the sheep isolate [22] in 13 nucleotide positions. Most of the differences were silent. The putative translations differed only in amino-acid three residues (E76K, R219K. G228A). The sheep isolate is referred to as G. duodenalis [22]. This name is synonymous with G. lamblia, thus the two studies explored the same species from different vertebrate hosts. The putative G. lumblia protein showed great similarity to all available dikinase sequences (Table 1). An almost complete colinearity was noted, with only a minimal number of short gaps needed to obtain a convincing alignment. This colinearity indicated that this G. lamblia gene contains no introns, thus it does not break the current view on the absence of introns in genes of this organism. Four highly conserved domains (A-D), previously noted in other Enzyme I family members
Table I Positional
amino
acid identity
for Giardia lamhlia pyruvate
Species
Accession
number
Giardia Iamhliab
u 43531
Giardia duodenalis’ Entamoeba histolytica Clo.rlridiunt symbioszmz’ Ftaveria trinerciu (C,-plant) F. pringlei (C,-plant) Mesentbr).ant/lemunz cr~stallinum Zea mays
2 54168 u 02529. x 14596 J 02595 x 57141 X 75516 x 78347 J 03401
dikinase Reference
This study VI t20,211 [I I.281 1331 ]341 [351 [361
“For 859 residues remaining after the elimination of gaps ‘The two names are synonymous and refer to the same species, ‘Formerly Bacreroides symbiosus. The sequence was corrected in reference
[28].
Percent identity” G.1. G.d. E.h.
C.S.
F.t.
F.p.
M.c.
99.5 49.5 53.7 54.1 54.3 54.6 52.7
46.2 54.6 57.0 55.0
95.9 83.0 78.6
84.3 80.3
79.2
49.1 53.6 54.0 54.1 54.5 52.6
51.6 48.1 48.9 48.3 48.9
220
L. Nevalainen
Cisrdls
lsmblis
et al. 1 Molecular
and Biochemical
ParasitologJa
77 (1996)
217-223
MSTIZRVYFFC ETPENQPANSELCRKVLGIKL CMPVPUFTI TCQTCVEYQ*KTASWPEGlXEEVASNLKLL 79 MQ-.-A-* ******EDCDCTNK-L---- -AC-CT-T-I-L---Q--V--TEM-KQFIANCN)!J4----H ---KKEYQ-V 71 ,‘,K"--K-e***ti*ERCNAS)(-NL---.-CN-.E-TI----I-Q---V-TEA-T--Y-N SCKQITQEIQLXJIFEAIW- 72 (+ 77 as) TAKK--IT--+**KCRSECNPiDliKSL----AN--E-%1 -LS--P-L--STEA-E---Q NCK.L-P-SWD-ISEC-DYV154
Enrsmoebrhistolytics Closcrldiumsymblosum Flsveris C. E. C. F.
trinervis
lsmblis hlstolytlcs symbiosum trlncrvls
C. lsmblis E. histolytlcs C. rymbiosum
EEWGD NTNPLLVSVRSGAAVSMPGHHDTILNLGLNDESVKClAAVTGNARFAYDSYRRFMQHFCDVCLGIDHDKFEHALDAVKTR149 -K-S.-V--CRE----.--- --..,,.------.---.---KT-VA--KL-N-R..-----.-.VSL-.KIA-),ACDm ,,m-m.VE 161 --w..,(.-. QKS-SAS,__-
TED.-.-.-PSK.--L-..
-A-m.-.-..--I-..--
---------. ---V.---.-
-VA-&F-u ..V-A.--CK
---p-...-S-*-----.-
---.I--yS-
-wPu”-
----U---N
-VII--P-SLDEK-EQN-AE243
.KII.-“.EE
162
F.
trlnervis
YGRXTDPELTADELEEVCW YRKICVAA*TCKTFFQCPHEQLEUINAVF RSWTNPRAQCYRTLNKLD**HN?ICTAV?JVQ SMTFCNTCDD256 K-V-L-T--D -NDNK-IAQVFI-KTEEPc-KQP--VD-YA ---F--C------ICK--VD--REF-ITPEQAD----S-V ---Y--H-N-250 --.D--.-IV -.m.DIPt* CD,,------T-----K-m 250 K-VHF-TD-- --D.K-LA-KFKAVYKE-NN-EE---E-KD--NC-VK--K.I"L-TD-. .AD.KOLV-K.KJ,VY-E-*K -EK--TD.KK--.--V----D--DS-.-M( --SI.QIW CLI[-----I------H-NT330
G. E. C. F.
lsmblis hlstolytics symbiosum trIner!fls
YCEFIQNAQC EDWACIRTP VNUEMPTIN ASWKACYDELSLIYAKLEGYYNDHVDLEm VENCKLUNUJSCTCVCFTRCPKTCEKFSYL346 -A-.-C---D-C---NMI*FF--Y.K-.------------QIISK-Arm mm..eQu)-m.--.. FHEVQ-P... 1.,I,&-Y--337 __-.-A---N.S---.G**I-.-Y.I---.----.-V---QPImLH*E mPD.-KQF ,$DIDw(---,C,, Fit--Q-H--I-E---YF-335 .-_--L---N.S_--.K**-.--..I--.. --.-------ED-m--E TQIPEA-K-."&JCRI.-R,,-K-.,,-I---QENR-..--415
C. E. C. F.
1smblIs histolytics symblosum trinervls
ARACKRTCFAMVRIAIDMCKEGNLTEEWL IRIDANKINEFLFKRFDPS VKPnWLCK CIPASPCMV CVICFCPMRTCEUEQCKKV 433 T-N--MNAT-I--TGV--VE --LI-K-Q-IIi--APQSVDQ L-H-NW-A NYA*EAP-V- .L-..--.-T-AW.DADDA V-~AK-..- 423 T-N-.--Ap-AL+-C-LVD -.-I...-V V--E-KSUIQL-HPT-*N-AAUACE-I-S AL-------A .KVY.TADEA KAAH-K-ER-424 C-T--.--KCA..--V--VN --LIDTRT-IK-VETQHLDqL-HP+E--- AYK*SH-VAT -L---------QV--SARDARTUHA---SA504
C. E. C. F.
lsmblis histolytlcs symblosum
<_----0-.O-.--.---.-.--.-> A ILTRIETSPEDILCMDRAVGILTARCCQTSHAAWARCHG KCCVACADCCQINYATKTLV ICDRKFKECDFISINCTTCEIYNCAVQTIE523 L-L-E.-K-. --,,-Fm-E.---C.--K--.--.-.-.---p-.S--RCIKVDV-K.IAK -.S---. ILT.D-S--CV-K-R-PLE-,513 __V_L____-..E_.~-E- ---V_.."---..----...T---S.CCEIK--Kp&-FE L-CHT-A_.-Y..m.S.-K --K-DIR-Q-513 _..----M-.-_--__.-,J. --.-S-MI RV_DD,,-In ----VI-.-."L-L.---..VIL.KQ,Up 594 --V-T_-..._VC__,,A_A_
trlnenrie
C. lsmblis E. histolytics C. symbiosum F. CrlnervIs
<-------.---------..> Q-linker <_.._.-_-.O->p-loop PGITDD*l&TIMDWSDKYRVLKIRTNADTPIIDAAVARKFG AECICLCRTEHtiFFM*DRIXAMRENILSDDECARRTALNKLLPFqREDF611 ----..----QVCSCYFG--LX-ANEIKKICVFMC-L- SA-KK-LE-R--N-V*L-LPIVVK----NTLRE-KKY--E-H-L-KQ--602 RSVSCS*FER .-V-A.-F-T-.V--.----R_TLN_V_L_ ___-__-.---__.E-*----KI_K_-_--SVR--R&-- E-I.--KC-.602 -AMSN-*-EI,?-S-A-QA.K -.I,,@,-_--N-.LT--NN_ -Q------------SDE--K-V-K--HAVTPEQ-KV--DL.--Y--S--683
C. E. C. F.
lsmblis hlstolytics symblosum trinervfe
ICIFKAHDGKCVNIRLLDPPLHEFLPHTRDUJK*******KIAEDHNKICH RHIHRRVEDLHE'JNPI'ILCFR CVRUXVYPE ISEHQVIUIL694 --LL.T.N.Lp-TV----.--.----TLEE-HREIFEMKLSCKTECIAEKEVVIJW-KEfl-----I-H-I---l-TN--Y---I--F-692 --F---“-H-C--AVT---AK--T--VH695 KAMY--LE-R PMTV-Y----...-V-.-EEE-A****+ttE--KN-CLTLA--DEE__-R----Lp-T-...-.--.----EGDLE,,IV*r***N E--V-TCI(SA DE-YSKI.N-S-----.----C--.-S---LT.------F 768
C. E. C. F.
lsmblia histolycics symblosum trlnervia
<.-------.---.-----------------.-..--EAACIVSRE*CVTVKPEIMIPVLFSENEW EIMHALVNRVAASVFKWCT TVDYEv_GMIELPRACVMADKIAQTAQYFSFGTNDLTQTT762 --TRE-IK-*-INDHR-----NVTEV--LrNlRKNVLEP-HEE-E-KY-IK-PFSY---V -CV--ALT---.-T&SF-- -------.G-761 774 ---IE-KE-T-IDIV------LVGEKK-*LKFVKDV-VE- -EQ-K--K-SDNQ-HI---- -I--.ALT--A..EE-EF-- .-------,,Q-.VSMTNQ* --.-I--_.V -LVCTPQ-*L K~IQISVIRC--N--A-M-V -LE-K------I---ALI-EE-CKE-DF-- --------M-856
C. lsmblls E. hisrolytics C. symbiosum F. trlnervla C. lsmblls E. histolytlcs C.
F.
symbiosum
trlncrvle
____> c C--> D FCYSPDDAG*KFIPKYIDRCIFKVDPFVTLDQQCVCAUK MAIECCRSTRTDMKIGICCE'QTDPASILPw(IClNYVS CSPYRVPt'AR 860 -S_--E-SEN..-_--VEU(-LPAN_-EI.-Rp-.-m-R I-V,'K--Q-PE,J,V-.--. ,,@X-S--E,, C-b,------. .-S--I---861 pc~-c----m--S-V,jc--V-.------F---I-863 _-F_..__.,, __rJ,s-yKAK -YES-_-~. -.T---Q-VE --W--Q-_--_--.I,-* _-WI-UQ. .,&H---E,,1 --K---Q-I--T-K--m PSL-V----“C$iE-S-VAFXV--D--- ---F---I--945
VAAAIAAIKARTNQ I---Q-Q.RHPREN L---Q--UN K L---QVIV
884 885 874 953
Fig, I, Comparison of the deduced amino-acid srquence of Giardia larnblia pyruvate dikinase gene with related sequences (Table 1). Dashes (-) represent residues identical to those in the G. lumblia sequence. Asterisks (*) represent gaps. Stretches used to design oligonucleotides for PCR are underlined. Parts (A with the conserved Thr and His (0. B. C and D) highly conserved in all Enzyme I molecules [12] are marked, as are the putative P-loop with the conserved Arg (0) [29] and the putative fexilble Q-linker [IZ].
much greater than distances between the chloroplast enzymes ( < 23%). Alignment with the more distant PEP synthases of E. coli [ 161 and P. fuvio sus [17] was convincing in a number of functional
areas, but not elsewhere. Phylogenetic reconstructions with a neighbor-joining method (Fig. 2) based on an amino acid distance matrix and with a maximum parsimony method (not shown) gave
L. Nwulainen et al.
i Molecular and Biochemical Parasitolog.t 77 (1996) 217-223
Giardia lamblia Entamoeba histolytica
CF 60
100
97
Clostridium symbiosum Flaveria trinervia Flaveria pringlei
Mesembryanthemum Zea mays
100
crystallinum
o-1
Fig. 2. Unrooted phylogenetic tree of pyruvate dikinases from anaerobic microorganisms and plants reconstructed with the neighbor-joining (NJ) method implemented in the MEGA package. The tree is based on an amino-acid distance matrix calculated for 830 common positions (Table 1). Bootstrap proportions for the nodes are in percentages based on 500 replicates. Bar denotes ten amino acid differences. A tree with identical topology was obtained with the amino acid parsimony method (PROTPARS) of the PHYLIP package. One most parsimonious tree was found, which required a total of 1599 steps.
trees with identical topologies. They revealed the relationship of the protist and the bacterial sequences, and showed a clear monophyly for all plant enzymes. When E. coli PEP synthase was also included in the analysis, considering only stretches that could be convincingly aligned [12], the root of the pyruvate dikinase tree was between the plant lineage and the clade comprising the two protist and the C. .y’rnbiosus enzymes (not shown). Southern analysis of G. ~amb~ia DNA digested with a number of restriction enzymes (Bum HI, Eco RI. Hind III and Sal I) and probed with the PCR product used in the initial screening, showed single hybridizing bands in each lane (not shown), indicating that the gene coding for pyruvate dikinase is a single copy gene in G. hnbh. The data obtained here and in the companion paper [22] thus demonstrate that pyruvate dikinase of G. lamblia (syn. G. duodenulis) is closely related to dikinases known from other organisms. Through this relationship their membership in the Enzyme I family is also documented [ 121. The results support the conclusions reached from biochemical data that G. lurnblitr contains a pyruvate dikinase [19]. In this species and in E. histolytica [lo], the enzyme has a catabolic function in the direction of pyruvate
231
formation. In the hydrogenosome containing amitochondriate parabasalid flagellate, Trichornonus vaginalis, pyruvate kinase is responsible for pyruvate formation [30], although this organism shares with G. larnblia and E. histo&tica the presence of another PPi-dependent enzyme, PPi-phosphofl-uctokinase [15] and a number of other metabolic features [I]. Thus two amitochondriate protists exhibit a unique mecha-nism of pyruvate formation, which is not found in hydrogenosomeor mitochondrion-containing eukaryotes. These two species are, in fact, the only eukaryotes without significant metabolic compartmentation that have been studied in any detail with biochemical methods [l]. Both species are parasitic thus it is also unknown whether their metabolic characters were acquired as adaptations to a parasitic mode of life or inherited from their presumably free-living ancestors. Further studies on other amitochondriate organisms, including free-living species [5,31,32], are necessary to establish the taxonomic distribution of this type of metabolic organization and obtain clues to its evolutionary origin. The paths of this enzyme leading to its catabolic role in certain organisms and to an anabolic one in others present a fascinating question prompting further comparative studies.
Acknowledgements We thank Dr. Peter Kiihler (Ziirich, Switzerland) for his friendly cooperation in coordinating the publication of his work with ours and sharing his data during the development of these studies. His critical comments on this manuscript are also appreciated. We thank Jeffrey E. Gray and Russell F. Doolittle (San Diego, CA) for generously giving us the partial dikinase sequence which enabled us to perform this study and Frances D. Gillin and Stephen B. Aley (San Diego, CA) for providing Ginrciia larnblia DNA and gDNA library. This study was supported by USPHS National Institutes of Health grant AI 11942.
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[21 Reeves, R.E. (1984) Metabolism
homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli. Gene 160, IOl- 103. [I81 Saier, M.H., Jr. and Reizer. J. (1994) The bacterial phosphotransferase system: new frontiers 30 years later. Mol. Microbial. 13, 7555764. E. (1993) Giardia v91 Hrdy. I., Mertens, E. and Nohynkova, ititestinalis: Detection and characterization of a pyruvate phosphate dikinase. Exp. Parasitol. 76, 4388441. I. and Tannich, E. (1993) Primary structure [201 Bruchhaus, of the pyruvate phosphate dikinase in Enntamoeba histo/W.a. Mol. Biochem. Parasitol. 62. 153- 156. E. and Perez-Montfort, R. (1994) [211 Saavedra-Lira. Cloning and seqeunce determination of the gene coding for the pyruvate phosphate dikinase of Erztamoeba histollka. Gene 142, 2499251. I221 Bruderer, T., Wehrli, C. and Kohler. P. (1996) Cloning and characterization of the gene encoding pyruvate phosphate dikinase from Giardia duodenalis. Mol. Biochem. Parasitol. 77, 109- 117. [231 Cabot, E.L. and Beckenbach, A.T. (1989) Simultaneous editing of multiple nucleic acid and protein sequences with ESEE. Comput. Appl. Biosci. 5, 233-234. [241 Philippe, H. (1993) MUST. a computer package of Management Utilities for Sequences and Trees. Nucl. Acids Res. 21. 5264-5272. ~251Roeske, C.A., Kutny, R.M., Budde. R.J.A. and Chollet, R. (1988) Sequence of the phosphothreonyl regulatory site peptide from inactive maize leaf pyruvate,orthophosphate dikinase. J. Biol. Chem. 263, 66833 6687. [261 Carroll, L.J., Xu. Y., Thrall, S.H., Martin, B.M. and Dunawdy-Mariano, D. (1994) Substrate binding domains in pyruvate phosphate dikinase. Biochemistry 33. 11341142. D. and Mar[271 Xu, Y., McGuire, M., Dunaway-Mariano, tin. B.M. (1995) Separate site catalysis by pyruvate phosphate dikinase as revealed by deletion mutants. Biochemistry 34, 219552202. [281 Xu. Y., Yankie, L.. Shen, L., Jung, Y.-S., Mariano, P.S., Dunaway-Mariano, D. and Martin, B.M. (1995) Location of the catalytic site for phosphoenolpyruvate formation within the primary structure of Closrridium symbiosum pyruvate phosphate dikinase. I. Identification of an essential cysteine by chemical modification with [ 1-“‘C]bromopyruvate and site-directed mutagenesis. Biochemistry 34, 2181-2187. D. (1995) Location ~291Yankie. L. and Dunaway-Mariano, of the catalytic site for phosphoenolpyruvate formation within the primary structure of Clostridium symbiosum pyruvate phosphate dikinase. 2. Site-directed mutagenesis of an essential arginine contained within as apparent P-loop. Biochemistry 34. 2188-2194. E. and Miiller, M. (1992) [301 Mertens. E., Van Schaftingen, Pyruvate kinase from Trichomoruxs vagina/is, an allosteric enzyme stimulated by ribose 5-phosphate and glycerate 3-phosphate. Mol. Biochem. Parasitol. 54, 13-20. T. (I 991) Archamoebae: the ancestral [311 Cavalier-Smith, eukaryotes?. BioSystems 25, 25-38.
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[32] Fenchel, T. and Finlay, B.J. (1995) Ecology and Evolution in Anoxic Worlds. Oxford University Press, Oxford, New York. Tokyo. [33] Rosche, E. and Westhoff. P. (1990) Primary structure of pyruvate. orthophosphate dikinase in the dicotyledonous C, plant Flareria rrinewia. FEBS Lett. 273. 116121. [34] Rosche. E.. Streubel. M. and Westhoff, P. (1994) Primary structure dikinase analysis
of the photosynthetic pyruvate orthophosphate of the C, plant Flawvia pringlei and expression of pyruvate orthophosphate dikinase sequences
in C,, C,-C,
223
and C, Fluueria species. Plant Mol. Biol. 26,
163-769. [35] Fisslthaler, B., Meyer, C., Bohnert. H.J. and Schmitt, J.M. (1995) Age dependent induction of pyruvate, orthophosphate dikinase in Mesenlhr?~ant/rrmun~ cry.stullinum. Planta 196. 492-500. [36] Matsuoka, M.. Ozeki. Y., Yamamoto. N., Hirano. H.. Kano-Murakami, Y. and Tanaka, Y. (1988) Primary structure of maize pyruvate,orthophosphate dikinase as deduced from cDNA sequence. J. Biol. Chem. 263. 11080~11083.
iii%mMIcAL PARAsIToLoGy Molecular
and Biochemical
Parasitology
77 (1996) 217--223
Sequence of a Giardia Zamblia gene coding for the glycolytic enzyme, pyruvate,phosphate dikinase’ Leena Nevalainen, The Rockrjtller Received
Ivan Hrdjr’, Miklbs
Uniuersiry,
15 December
1230 York Awnre,
1995; revised 4 March
Nets
Miiller*
York .YY.
1996: accepted
ISA
7 March
1996
Abstract The sequence of the gene coding for pyruvate,phosphate dikinase (EC 2.7.9.1) in Giurdiu lurnhliu (syn. G. duodmulis) has been established. The deduced amino acid sequence is very similar to all of its known homologs from the protist, Entamoeha histolytica, the eubacterium, Clostridiwn synhiosurr~ and plant chloroplasts. Phylogenetic reconstruction with neighbor-joining and maximum parsimony methods reveals that the sequences form two clades, one comprising the anaerobic microorganisms, and the other the chloroplast enzymes. Kqworcls: Giardiu lanzhliu; gDNA
library;
Pyruvate,phosphate
Biochemical studies disclosed an unexpected diversity in the enzymes and subcellular organization of the energy metabolism of various parasitic ,4hhreriariorzs: Pyruvate dikinase. pyruvate.phosphate dikinase: PEP, phosphoenolpyruvate: PEP synthase. phosphoenolpyruvate synthase: PCR, polymerase chain reaction. *Tel: + 1 212 3278153; fax: + 1 212 3277974: e-mail: mmulleri$rockvax.rockefeller.edu ’ fort: The nucleotide sequence obtained, including stretches of the 5’- and 3’-untranslated regions of the gene (126 and 22 bp, respectively) not discussed in this paper, has been submitted to the GenBankTM database with the accession No. u43531. ’ Present address: Department of Physiology and Developmental Biology, Charles University, Vinicna 7, 128 00 Prague 2. Czech Republic.
$15.00
c) 1996 Elsevier
PII SO166-6851(96)02604-7
Science
B.V. All rights
Diplomonad;
Amitochondriate _____
protist
Certain species lack mitochondria and hydrogenosomes thus their metabolism, which is fermentative, is not compartmentalized [l]. The detailed exploration of one of these organisms, Entamoeba Izistolyticcl, revealed that several enzymes of its core metabolism differ from those of typical eukaryotic glycolysis [2]. Restricted to a single, however well studied, species these results were interpreted as reflecting an exceptional, ancestral metabolic machinery [2]. Subsequently Giardia lamblia, another amitochondriate parasite belonging to a different protist lineage [3-51, was found to share a number of metabolic characteristics with E. histolytica [6-81. This finding raised the possibility of a broader distribution of this unusual metabolic type [l] and the question of its evolutionary origin in representatives of two unrelated lineages [9]. protists.
1. Introduction
0166.6851/96#
dikinase:
reserved
218
L. Neualainen
et al. / Molecular
and Biochemical
One of the striking deviations in the glycolysis of
Purusitologv 77 11996) _‘I 7-22
2. Materials
and methods
E. histolytica from the typical eukaryotic pattern is
the reaction leading from phosphoenolpyruvate (PEP) to pyruvate. In most organisms this is catalyzed by pyruvate kinase (EC 2.7.1.40): PEP + ADP --) Pyruvate
+ ATP,
while in E. histolytica it is effected by the action of pyruvate,phosphate dikinase (pyruvate dikinaseEC 2.7.9.1) [lo]: PEP + PPi + AMP ++ Pyruvate
+ Pi + ATP.
In addition to E. histolytica, this enzyme is known from various bacteria and chloroplasts [l l-131. In certain bacteria and in chloroplasts its function is anabolic, providing PEP as a substrate for CO2 fixation [ 11,131. When acting as a catabolic enzyme, pyruvate dikinase utilizes inorganic pyrophosphate resulting in increased glycolytic ATP yield, a circumstance regarded as especially significant for organisms that rely exclusively on fermentation [ 13- 151. Pyruvate dikinase is not a homolog of pyruvate kinase [l I] but is closely related to phosphoenolpyruvate synthase (PEP synthaseEC 2.7.9.2) studied in detail from Escherichia coli [ 161 and Pyrococcus furiosus [ 171, which catalyzes an analogous reaction: PEP + Pi + AMP ++ Pyruvate
+ ATP.
These two enzymes in turn are closely related to Enzyme I of the bacterial PEP:sugar phosphotransferase system [l 1,121.These proteins together comprise the Enzyme I protein family [12], members of which participate in an unusually wide array of cellular functions [ 181. The presence of pyruvate dikinase in G. Iamblia has been demonstrated with biochemical methods [ 191, underscoring the metabolic similarities between this organism and E. histolytica. No pyruvate kinase was found yet in this species [7,19]. In this communication we report the deduced primary structure of G. lamblia pyruvate dikinase, its similarity to the E. histolytica enzyme [20,21] and its close relationship to all related enzymes for which sequence information is available. While this study was underway, the sequence of the pyruvate dikinase gene has been established from a Giardia isolate from sheep with results highly similar to those reported here [22].
Giardia lamblia (syn. (;, duodenalis), strain WB, clone C6 (ATCC 30957) was used in this study. gDNA and a AZAPII gDNA library have been kindly provided by Drs. F.D. Gillin and S.B. Aley (University of California, San Diego, CA). A partial nucleotide sequence of the pyruvate dikinase, corresponding to the region from Gly-28 to Phe-87 in the final sequence was obtained by Dr. J.E. Gray and kindly communicated to us by Dr. F.R. Doolittle (University of California, San Diego, CA). Based on this information, two nondegenerate polymerase chain reaction (PCR) primers were designed (sense: S-GGCAAGGGGAATTTCTCTTG-3’; antisense: 5’-GAAAGTCTTCCCCATCTT-3’) and with the use of gDNA as template, a I80-bp PCR product was obtained. This product, labeled with [32P]dATP by random priming, was used as a probe to screen the gDNA library. The pBluescript insert from one of the positive clones was excised with helper phage following the manufacturer’s instructions (Stratagene) and amplified in Escherichia coli XL-l Blue. Both strands of the purified phagemid were sequenced by the dideoxy chain termination method. Standard molecular methods have been used throughout this study. Since the first insert isolated contained an incomplete open reading frame, with approximately 100 carboxyl-terminal amino acid residues missing, we rescreened the library with a 300-bp PCR product corresponding to a carboxyl-terminal area of the molecule. The primers used were a nondegenerate one corresponding to a part close to the carboxylterminus of the previously obtained incomplete sequence and a degenerate one designed from a conserved stretch in the missing area of the enzyme (sense: 5’-GAGGTGGGGACCATGAT-3’; antisense: 5’- CCRTGYTCNCCRCADAT-3’). The sequences obtained were assembled and edited with Eyeball Sequence Editor [23]. Numbering of the amino-acid residues is based on the derived G. Iamblia sequence. Alignment with related sequences and formatting of the data for phylogenetic reconstruction were done with the MUST suite of programs (version 1.O) [24]. Phylo-
219
L. Nevalainen et al. 1 Molecular and Biochemical Parasitology 77 (1996) 217-2X
[l 1,121, are well conserved in the G. famblia enzyme, too (Fig. 1). Domain A contains the GGXTSH motif strictly conserved in all these proteins [12] which includes His-464, the residue known to be phosphorylated in all these enzymes and also the conserved Thr-462. The latter residue is known to play a role in the regulation of this enzyme, observed so far only in plant dikinases [25]. It remains to be established, whether in G. lamblia it has a regulatory function. The functional role of conserved domains B-D has not been firmly established yet. They are implicated in PEP binding and transfer of phosphoryl groups [ 11,2629]. The flexible Q-linker region, assumed to connect the two functional halves of these proteins [12] and a putative P-loop are also clearly recognizable [29] (Fig. 1). Two sequence signatures have been proposed for this protein family [12]. The first located in domain A (residues 456-480) is fully conserved, while the second one in domain C (residues 769780) shows some deviation, since the two first residues are Gln-769 and Tyr-770 instead of (DE)(FG). Note that E. histolytica pyruvate dikinase also deviates slightly from the proposed signature, having Ser-Phe in this position [20,21]. The G. lamblia sequence was about equally divergent from all other pyruvate dikinases, with 46-52x amino acid differences (Table 1). Its distance from the E. histo(vtica enzyme (52%) was
genetic reconstruction was done with the neighbor-joining (NJ) method of the MEGA package (version 1.0) and by a parsimony (PROTPARS) method in the PHYLIP package (version 3.55~).
3. Results and discussion The assembled sequence contained an open reading frame coding for a putative protein of 884 amino acid residues (Fig. 1). This sequence differed from that from the sheep isolate [22] in 13 nucleotide positions. Most of the differences were silent. The putative translations differed only in amino-acid three residues (E76K, R219K. G228A). The sheep isolate is referred to as G. duodenalis [22]. This name is synonymous with G. lamblia, thus the two studies explored the same species from different vertebrate hosts. The putative G. lumblia protein showed great similarity to all available dikinase sequences (Table 1). An almost complete colinearity was noted, with only a minimal number of short gaps needed to obtain a convincing alignment. This colinearity indicated that this G. lamblia gene contains no introns, thus it does not break the current view on the absence of introns in genes of this organism. Four highly conserved domains (A-D), previously noted in other Enzyme I family members
Table I Positional
amino
acid identity
for Giardia lamhlia pyruvate
Species
Accession
number
Giardia Iamhliab
u 43531
Giardia duodenalis’ Entamoeba histolytica Clo.rlridiunt symbioszmz’ Ftaveria trinerciu (C,-plant) F. pringlei (C,-plant) Mesentbr).ant/lemunz cr~stallinum Zea mays
2 54168 u 02529. x 14596 J 02595 x 57141 X 75516 x 78347 J 03401
dikinase Reference
This study VI t20,211 [I I.281 1331 ]341 [351 [361
“For 859 residues remaining after the elimination of gaps ‘The two names are synonymous and refer to the same species, ‘Formerly Bacreroides symbiosus. The sequence was corrected in reference
[28].
Percent identity” G.1. G.d. E.h.
C.S.
F.t.
F.p.
M.c.
99.5 49.5 53.7 54.1 54.3 54.6 52.7
46.2 54.6 57.0 55.0
95.9 83.0 78.6
84.3 80.3
79.2
49.1 53.6 54.0 54.1 54.5 52.6
51.6 48.1 48.9 48.3 48.9
220
L. Nevalainen
Cisrdls
lsmblis
et al. 1 Molecular
and Biochemical
ParasitologJa
77 (1996)
217-223
MSTIZRVYFFC ETPENQPANSELCRKVLGIKL CMPVPUFTI TCQTCVEYQ*KTASWPEGlXEEVASNLKLL 79 MQ-.-A-* ******EDCDCTNK-L---- -AC-CT-T-I-L---Q--V--TEM-KQFIANCN)!J4----H ---KKEYQ-V 71 ,‘,K"--K-e***ti*ERCNAS)(-NL---.-CN-.E-TI----I-Q---V-TEA-T--Y-N SCKQITQEIQLXJIFEAIW- 72 (+ 77 as) TAKK--IT--+**KCRSECNPiDliKSL----AN--E-%1 -LS--P-L--STEA-E---Q NCK.L-P-SWD-ISEC-DYV154
Enrsmoebrhistolytics Closcrldiumsymblosum Flsveris C. E. C. F.
trinervis
lsmblis hlstolytlcs symbiosum trlncrvls
C. lsmblis E. histolytlcs C. rymbiosum
EEWGD NTNPLLVSVRSGAAVSMPGHHDTILNLGLNDESVKClAAVTGNARFAYDSYRRFMQHFCDVCLGIDHDKFEHALDAVKTR149 -K-S.-V--CRE----.--- --..,,.------.---.---KT-VA--KL-N-R..-----.-.VSL-.KIA-),ACDm ,,m-m.VE 161 --w..,(.-. QKS-SAS,__-
TED.-.-.-PSK.--L-..
-A-m.-.-..--I-..--
---------. ---V.---.-
-VA-&F-u ..V-A.--CK
---p-...-S-*-----.-
---.I--yS-
-wPu”-
----U---N
-VII--P-SLDEK-EQN-AE243
.KII.-“.EE
162
F.
trlnervis
YGRXTDPELTADELEEVCW YRKICVAA*TCKTFFQCPHEQLEUINAVF RSWTNPRAQCYRTLNKLD**HN?ICTAV?JVQ SMTFCNTCDD256 K-V-L-T--D -NDNK-IAQVFI-KTEEPc-KQP--VD-YA ---F--C------ICK--VD--REF-ITPEQAD----S-V ---Y--H-N-250 --.D--.-IV -.m.DIPt* CD,,------T-----K-m 250 K-VHF-TD-- --D.K-LA-KFKAVYKE-NN-EE---E-KD--NC-VK--K.I"L-TD-. .AD.KOLV-K.KJ,VY-E-*K -EK--TD.KK--.--V----D--DS-.-M( --SI.QIW CLI[-----I------H-NT330
G. E. C. F.
lsmblis hlstolytics symbiosum trIner!fls
YCEFIQNAQC EDWACIRTP VNUEMPTIN ASWKACYDELSLIYAKLEGYYNDHVDLEm VENCKLUNUJSCTCVCFTRCPKTCEKFSYL346 -A-.-C---D-C---NMI*FF--Y.K-.------------QIISK-Arm mm..eQu)-m.--.. FHEVQ-P... 1.,I,&-Y--337 __-.-A---N.S---.G**I-.-Y.I---.----.-V---QPImLH*E mPD.-KQF ,$DIDw(---,C,, Fit--Q-H--I-E---YF-335 .-_--L---N.S_--.K**-.--..I--.. --.-------ED-m--E TQIPEA-K-."&JCRI.-R,,-K-.,,-I---QENR-..--415
C. E. C. F.
1smblIs histolytics symblosum trinervls
ARACKRTCFAMVRIAIDMCKEGNLTEEWL IRIDANKINEFLFKRFDPS VKPnWLCK CIPASPCMV CVICFCPMRTCEUEQCKKV 433 T-N--MNAT-I--TGV--VE --LI-K-Q-IIi--APQSVDQ L-H-NW-A NYA*EAP-V- .L-..--.-T-AW.DADDA V-~AK-..- 423 T-N-.--Ap-AL+-C-LVD -.-I...-V V--E-KSUIQL-HPT-*N-AAUACE-I-S AL-------A .KVY.TADEA KAAH-K-ER-424 C-T--.--KCA..--V--VN --LIDTRT-IK-VETQHLDqL-HP+E--- AYK*SH-VAT -L---------QV--SARDARTUHA---SA504
C. E. C. F.
lsmblis histolytlcs symblosum
<_----0-.O-.--.---.-.--.-> A ILTRIETSPEDILCMDRAVGILTARCCQTSHAAWARCHG KCCVACADCCQINYATKTLV ICDRKFKECDFISINCTTCEIYNCAVQTIE523 L-L-E.-K-. --,,-Fm-E.---C.--K--.--.-.-.---p-.S--RCIKVDV-K.IAK -.S---. ILT.D-S--CV-K-R-PLE-,513 __V_L____-..E_.~-E- ---V_.."---..----...T---S.CCEIK--Kp&-FE L-CHT-A_.-Y..m.S.-K --K-DIR-Q-513 _..----M-.-_--__.-,J. --.-S-MI RV_DD,,-In ----VI-.-."L-L.---..VIL.KQ,Up 594 --V-T_-..._VC__,,A_A_
trlnenrie
C. lsmblis E. histolytics C. symbiosum F. CrlnervIs
<-------.---------..> Q-linker <_.._.-_-.O->p-loop PGITDD*l&TIMDWSDKYRVLKIRTNADTPIIDAAVARKFG AECICLCRTEHtiFFM*DRIXAMRENILSDDECARRTALNKLLPFqREDF611 ----..----QVCSCYFG--LX-ANEIKKICVFMC-L- SA-KK-LE-R--N-V*L-LPIVVK----NTLRE-KKY--E-H-L-KQ--602 RSVSCS*FER .-V-A.-F-T-.V--.----R_TLN_V_L_ ___-__-.---__.E-*----KI_K_-_--SVR--R&-- E-I.--KC-.602 -AMSN-*-EI,?-S-A-QA.K -.I,,@,-_--N-.LT--NN_ -Q------------SDE--K-V-K--HAVTPEQ-KV--DL.--Y--S--683
C. E. C. F.
lsmblis hlstolytics symblosum trinervfe
ICIFKAHDGKCVNIRLLDPPLHEFLPHTRDUJK*******KIAEDHNKICH RHIHRRVEDLHE'JNPI'ILCFR CVRUXVYPE ISEHQVIUIL694 --LL.T.N.Lp-TV----.--.----TLEE-HREIFEMKLSCKTECIAEKEVVIJW-KEfl-----I-H-I---l-TN--Y---I--F-692 --F---“-H-C--AVT---AK--T--VH695 KAMY--LE-R PMTV-Y----...-V-.-EEE-A****+ttE--KN-CLTLA--DEE__-R----Lp-T-...-.--.----EGDLE,,IV*r***N E--V-TCI(SA DE-YSKI.N-S-----.----C--.-S---LT.------F 768
C. E. C. F.
lsmblia histolycics symblosum trlnervia
<.-------.---.-----------------.-..--EAACIVSRE*CVTVKPEIMIPVLFSENEW EIMHALVNRVAASVFKWCT TVDYEv_GMIELPRACVMADKIAQTAQYFSFGTNDLTQTT762 --TRE-IK-*-INDHR-----NVTEV--LrNlRKNVLEP-HEE-E-KY-IK-PFSY---V -CV--ALT---.-T&SF-- -------.G-761 774 ---IE-KE-T-IDIV------LVGEKK-*LKFVKDV-VE- -EQ-K--K-SDNQ-HI---- -I--.ALT--A..EE-EF-- .-------,,Q-.VSMTNQ* --.-I--_.V -LVCTPQ-*L K~IQISVIRC--N--A-M-V -LE-K------I---ALI-EE-CKE-DF-- --------M-856
C. lsmblls E. hisrolytics C. symbiosum F. trlnervla C. lsmblls E. histolytlcs C.
F.
symbiosum
trlncrvle
____> c C--> D FCYSPDDAG*KFIPKYIDRCIFKVDPFVTLDQQCVCAUK MAIECCRSTRTDMKIGICCE'QTDPASILPw(IClNYVS CSPYRVPt'AR 860 -S_--E-SEN..-_--VEU(-LPAN_-EI.-Rp-.-m-R I-V,'K--Q-PE,J,V-.--. ,,@X-S--E,, C-b,------. .-S--I---861 pc~-c----m--S-V,jc--V-.------F---I-863 _-F_..__.,, __rJ,s-yKAK -YES-_-~. -.T---Q-VE --W--Q-_--_--.I,-* _-WI-UQ. .,&H---E,,1 --K---Q-I--T-K--m PSL-V----“C$iE-S-VAFXV--D--- ---F---I--945
VAAAIAAIKARTNQ I---Q-Q.RHPREN L---Q--UN K L---QVIV
884 885 874 953
Fig, I, Comparison of the deduced amino-acid srquence of Giardia larnblia pyruvate dikinase gene with related sequences (Table 1). Dashes (-) represent residues identical to those in the G. lumblia sequence. Asterisks (*) represent gaps. Stretches used to design oligonucleotides for PCR are underlined. Parts (A with the conserved Thr and His (0. B. C and D) highly conserved in all Enzyme I molecules [12] are marked, as are the putative P-loop with the conserved Arg (0) [29] and the putative fexilble Q-linker [IZ].
much greater than distances between the chloroplast enzymes ( < 23%). Alignment with the more distant PEP synthases of E. coli [ 161 and P. fuvio sus [17] was convincing in a number of functional
areas, but not elsewhere. Phylogenetic reconstructions with a neighbor-joining method (Fig. 2) based on an amino acid distance matrix and with a maximum parsimony method (not shown) gave
L. Nwulainen et al.
i Molecular and Biochemical Parasitolog.t 77 (1996) 217-223
Giardia lamblia Entamoeba histolytica
CF 60
100
97
Clostridium symbiosum Flaveria trinervia Flaveria pringlei
Mesembryanthemum Zea mays
100
crystallinum
o-1
Fig. 2. Unrooted phylogenetic tree of pyruvate dikinases from anaerobic microorganisms and plants reconstructed with the neighbor-joining (NJ) method implemented in the MEGA package. The tree is based on an amino-acid distance matrix calculated for 830 common positions (Table 1). Bootstrap proportions for the nodes are in percentages based on 500 replicates. Bar denotes ten amino acid differences. A tree with identical topology was obtained with the amino acid parsimony method (PROTPARS) of the PHYLIP package. One most parsimonious tree was found, which required a total of 1599 steps.
trees with identical topologies. They revealed the relationship of the protist and the bacterial sequences, and showed a clear monophyly for all plant enzymes. When E. coli PEP synthase was also included in the analysis, considering only stretches that could be convincingly aligned [12], the root of the pyruvate dikinase tree was between the plant lineage and the clade comprising the two protist and the C. .y’rnbiosus enzymes (not shown). Southern analysis of G. ~amb~ia DNA digested with a number of restriction enzymes (Bum HI, Eco RI. Hind III and Sal I) and probed with the PCR product used in the initial screening, showed single hybridizing bands in each lane (not shown), indicating that the gene coding for pyruvate dikinase is a single copy gene in G. hnbh. The data obtained here and in the companion paper [22] thus demonstrate that pyruvate dikinase of G. lamblia (syn. G. duodenulis) is closely related to dikinases known from other organisms. Through this relationship their membership in the Enzyme I family is also documented [ 121. The results support the conclusions reached from biochemical data that G. lurnblitr contains a pyruvate dikinase [19]. In this species and in E. histolytica [lo], the enzyme has a catabolic function in the direction of pyruvate
231
formation. In the hydrogenosome containing amitochondriate parabasalid flagellate, Trichornonus vaginalis, pyruvate kinase is responsible for pyruvate formation [30], although this organism shares with G. larnblia and E. histo&tica the presence of another PPi-dependent enzyme, PPi-phosphofl-uctokinase [15] and a number of other metabolic features [I]. Thus two amitochondriate protists exhibit a unique mecha-nism of pyruvate formation, which is not found in hydrogenosomeor mitochondrion-containing eukaryotes. These two species are, in fact, the only eukaryotes without significant metabolic compartmentation that have been studied in any detail with biochemical methods [l]. Both species are parasitic thus it is also unknown whether their metabolic characters were acquired as adaptations to a parasitic mode of life or inherited from their presumably free-living ancestors. Further studies on other amitochondriate organisms, including free-living species [5,31,32], are necessary to establish the taxonomic distribution of this type of metabolic organization and obtain clues to its evolutionary origin. The paths of this enzyme leading to its catabolic role in certain organisms and to an anabolic one in others present a fascinating question prompting further comparative studies.
Acknowledgements We thank Dr. Peter Kiihler (Ziirich, Switzerland) for his friendly cooperation in coordinating the publication of his work with ours and sharing his data during the development of these studies. His critical comments on this manuscript are also appreciated. We thank Jeffrey E. Gray and Russell F. Doolittle (San Diego, CA) for generously giving us the partial dikinase sequence which enabled us to perform this study and Frances D. Gillin and Stephen B. Aley (San Diego, CA) for providing Ginrciia larnblia DNA and gDNA library. This study was supported by USPHS National Institutes of Health grant AI 11942.
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