APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 93-98
Vol. 58, No. 1
0099-2240/92/010093-06$02.00/0 Copyright © 1992, American Society for Microbiology
Molecular Cloning of the Isocitrate Dehydrogenase Gene of an Extreme Thermophile, Thermus thermophilus HB8 KENTARO MIYAZAKI,* HIDETAKA EGUCHI, AKIHIKO YAMAGISHI, TAKAYOSHI WAKAGI, AND TAIRO OSHIMA Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 227, Japan
Received 21 June 1991/Accepted 22 October 1991
The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G+C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.
The NADP-dependent threo-Ds-isocitrate dehydrogenase (threo-DS-isocitrate:NADP oxidoreductase [EC 1.1.1.42])
means to elucidate the molecular basis of the evolution of substrate specificity. We previously cloned and sequenced the isopropylmalate dehydrogenase gene of T. thermophilus (14, 28), characterized the thermophile isopropylmalate dehydrogenase (30), and also purified isocitrate dehydrogenase from the same organism (6). Only one enzyme activity was detected in the thermophile extract, suggesting the absence of an NAD-dependent isocitrate dehydrogenase (EC 1.1.1.41) in this organism, as in E. coli. In this paper, we report the molecular cloning and DNA sequencing of the gene coding for isocitrate dehydrogenase of T. thermophilus.
catalyzes the oxidative decarboxylation of threo-D.-isocitrate to 2-oxoglutarate. This enzyme has been reported as a key enzyme in the tricarboxylic acid cycle and also plays an important role in glutamate synthesis. In the tricarboxylic acid cycle, 2-oxoglutarate is formed from the condensation of acetyl coenzyme A and oxaloacetate, and then isomerization takes place through a combination of dehydration and rehydration steps. The isocitrate thus formed is subjected to oxidative decarboxylation. Likewise, 2-oxoisocaproate, from which leucine is synthesized by transamination, is formed via an analogous pathway in microorganisms and plants: i.e., condensation of acetyl coenzyme A and a 2-oxo acid, isomerization by dehydration and rehydration, and oxidative decarboxylation of a 2-hydroxy acid. In leucine biosynthesis, the enzyme corresponding to isocitrate dehydrogenase is 3-isopropylmalate dehydrogenase (threo-Dj-isopropylmalate:NAD oxidoreductase [EC 1.1.1.85]). Thorsness and Koshland (29) compared the primary structure of Escherichia coli isocitrate dehydrogenase with those of other proteins and reported that only isopropylmalate dehydrogenase showed high homology. Hurley et al. (12) found that the three-dimensional structure of E. coli isocitrate dehydrogenase is unique and significantly different from those of other members of the dehydrogenase family so far reported. We previously determined the crystal structure of isopropylmalate dehydrogenase from Thermus thermophilus (15) and found that the backbone structure of the thermophile enzyme resembles that of isocitrate dehydrogenase reported by Hurley et al. (11, 12) (unpublished data). These findings implied a close evolutionary relationship of these two dehydrogenases, which seem to have diverged from a common ancestor to create their own substrate specificities. A comparative study of these enzymes may provide the
MATERIALS AND METHODS
Materials. DNA modification enzymes were products of Takara (Kyoto, Japan) or Toyobo (Tokyo, Japan). N-TosylL-phenylalanyl chloromethyl ketone-treated trypsin was from Sigma (St. Louis, Mo.). Lysyl endopeptidase was from Wako Chemicals (Tokyo, Japan). [_y-32P]ATP (3,000 Ci/ mmol) was from Amersham (Amersham, England). All other reagents were of the purest grade available. Bacterial strains. The extremely thermophilic bacterium T. thermophilus HB8 (ATCC 27634) was grown at 75°C as described previously (20, 21). E. coli HB101 (F- hsdS20 recA13 ara-14 proA2 lacYl galK2 rpsL20 [str] xyl-5 mtl-l supE44 leuB6 thi-J) was used as a host for the T. thermo-
philus genomic library. DNA manipulations. DNA manipulations were carried out by standard methods (24). Enzyme purification and peptide sequencing. Isocitrate dehydrogenase was purified from T. thermophilus as described previously (6). The amino acid sequences of the intact protein and proteolytic fragments were determined with a gas-phase peptide sequencer (model 470A; Applied Biosystems, Foster City, Calif.). The purified protein was digested with trypsin or lysyl endopeptidase. The resulting peptides were separated by high-performance liquid chroma-
* Corresponding author. 93
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APPL. ENVIRON. MICROBIOL.
MIYAZAKI ET AL. 1 61 121 181 241 301 361 421
CCATGGGGCCGAAGGTGGCGTAGGCCAGGCCCTCCTCCCTAAAGGGCACCTTCCTTTGGG CCTCCGCCAGGGAGAGCTTCTCCACGGGGACCCCCAGGGCCTTCTGCACCCTTAGGGCCT CTTCCTGGGCCTCGGCCTGGGCCTCGGGCACCAGGAAGAGGTAGCCCGTAGCCGATAGGC GGCCTCGGGGATTTCCCGGTACTCCAGGATGGAACGGTAGGAGAGGAGGACGTTCGAGGG CTCGGAGAACTGCACCCGCACCCCGGCGGCGCTTTTCCCGGTGGACCCTTGGGCGTAGGT GGCCTCCTTCTCCAGGACCAAGACCCTAAGCCCCTTTTCCGCCAGGCGGTAGGCCGAGGC CGCCCCTACGATCCCCGCCCCCACCACGACCACCCGGGCCACGCCCCGAL CTCAAGCCCCGTGG
3STAAAGGGGGCGATTCCGCCCCCGG
GGC CCATGCC P
481 CCTGATCACCACGGAAACCGGCAAGAAGATGCACGTTCTCGAGGACGGGCGCAAGCTCAT L I T T E T G K K M H V L E D G R K L I 541 CACCGTCATCCCCGGAGACGGCATCGGGCCCGAGTGCGTGGAGGCTACCCTCAAGGTCCT T V I P G D G I G P E C V E A T L K V L 601 AGAGGCGGCCAAGGCCCCCCTGGCCTACGAGGTGCGAGAGGCGGGGGCGAGCGTCTTCCG E A A K A P L A Y E V R E A G A S V F R 661 GCGGGGCATCGCCTCGGGCGTTCCCCAGGAGACCATTGAGTCCATCCGCAAGACCCGGGT R G I A S G V P Q E T I E S I R K T R V 721 GGTCCTGAAGGGTCCCCTGGAAACCCCGGTGGGCTACGGGGAGAAGAGCGCCAACGTCAC V L K G P L E T P V G Y G E K S A N V T 781 CCTAAGGAAGCTCTTTGAGACCTACGCCAACGTCCGCCCCGTGCGGGAGTTCCCCAACGT L R K L F E T Y A N V R P V R E F P N V 841 CCCCACCCCCTATGCGGGCCGGGGCATTGACCTCGTGGTGGTGCGGGAGAACGTGGAGGA P T P Y A G R G I D L V V V R E N V E D 901 CCTCTACGCCGGGATTGAGCACATGCAGACCCCGAGCGTGGCCCAGACCCTCAAGCTCAT L Y A G I E H M Q T P S V A Q T L K L I 961 CTCCTGGAAGGGATCGGAGAAGATCGTCCGCTTCGCCTTTGAGCTGGCCCGGGCCGAGGG S W K G S E K I V R F A F E L A R A E G 1021 GCGGAAGAAGGTCCACTGCGCCACCAAGTCCAACATCATGAAGCTCGCCGAAGGACCCAA R K K V H C A T K S N I M K L A E G P K 1081 GCGGGCCTTTGAGCAGGTGGCCCAGGAGTACCCCGACATAGAAGCGGTCCACATCATCGT R A F E Q V A Q E Y P D I E A V H I I V 1141 GGACAACGCTGCCCACCAGCTGGTGAAAAGGCCCGAGCAGTTTGAGGTGATCGTCACCAC D N A A H Q L V K R P E Q F E V I V T T 1201 CAACATGAACGGAGACATCCTCTCCGACCTCACCTCGGGGCTCATTGGGGGCCTGGGCTT N M N G D I L S D L T S G L I G G L G F 1261 CGCTCCCTCGGCCAACATCGGCAACGAGGTGGCCATCTTTGAGGCCGTCCACGGTTCCGC A P S A N I G N E V A I F E A V H G S A 1321 CCCCAAGTACGCCGGGAAGAACGTCATCAACCCCACCGCGGTCCTCCTCTCGGCGGTGAT P K Y A G K N V I N .P T A V L L S A V M 1381 GATGCTCCGCTACCTGGAGGAGTTCGCCACGGCGGACCTTATAGAGAACGCCCTCCTCTA M L R Y L E E F A T A D L I E N A L L Y 1441 CACCCTCGAGGAGGGCCGGGTCCTCACGGGGGACGTGGTGGGCTACGACCGGGGGGCCAA T L E E G R V L T G D V V G Y D R G A K 1501 GACCACGGAGTACACCGAGGCCATCATCCAGAACCTGGGCAAGACCCCAAGGAAGACCCA T T E Y T E A I I Q N L G K T P R K T Q 1561 GGTGCGGGGCTACAAGCCCTTCCGCCTGCCCCAGGTGGACGGGGCCATCGCCCCCATCGT V R G Y K P F R L P Q V D G A I A P I V 1621 CCCTAGGAGCCGCCGGGTTGTGGGGGTGGACGTCTTCGTGGAAACCAACCTCCTGCCCGA P R S R R V V G V D V F V E T N L L P E 1681 GGCCCTGGGAAAGGCCCTGGAGGACCTTGCCGCGGGCACCCCCTTCCGGCTCAAGATGAT A L G K A L E D L A A G T P F R L K M I 1741 CTCCAACCGGGGCACCCAGGTCTACCCCCCCACCGGCGGGCTCACGGACCTGGTGGACCA S N R G T Q V Y P P T G G L T D L V D H 1801 CTACCGCTGCCGCTTCCTCTACACGGGGGAGGGGGAGGCTAAGGACCCGGAGATCCTGGA Y R C R F L Y T G E G E A K D P E I L D 1861 CCTCGTAAGCCGGGTGGCAAGCCGCTTCCGCTGGATGCACCTGGAGAAGCTCCAGGAATT L V S R V A S R F R W M H L E K L Q E F 1921 TGACGGCGAGCCCGGCTTCACCAAGGCCCAAGGGGAAGACTAAGGCCACCCCATGCCGGC M P X D G E P G F T K A Q G E D * 1981 TTGGGCCGGCATGGGGCCCGGCATGGCCCCGAGCCTGATGCGGCGCCTAGACCAGGGCGA W z G M G P G M A P S L M R R L D Q G E 2041 AGCGGGCCTCGAGGGCCGGAAGGTCCGCCTCCAGGAAGCGGAGGCGGGCCTCGGCGTTCA A G L E G R K V R L -Q E A E A G L G V Q 2101 GGGGAAGGGCCTTGGGCAGGGCCACCTGGGCGGAAAGCCCGAGCTC G K G L Q Q G H L G G K P E L
1
21 41
61
81 101 121 141
161 181 201 221
241
261 281 301 321
341
361 381 381 401 421
441 461
FIG. 1. Nucleotide sequence of the T. thermophilus isocitrate dehydrogenase gene, deduced amino acid sequence of the enzyme, and sequences of the 5'- and 3'-flanking regions. The deduced amino acid sequence of isocitrate dehydrogenase begins at position 479. The Shine-Dalgarno ribosome binding site is shaded. Probable -35 and -10 promoter sequences are boxed, and the terminator is marked by facing arrows. The amino acid sequence of the N terminus is overlined, tryptic fragments are underlined, and lysyl endopeptidase-digested fragments are doubly underlined.
tography with a C18 column. Several fragments were selected, and their amino acid sequences were determined. Synthesis of oligonucleotide. An oligonucleotide used as a hybridization probe was synthesized by the phosphoramidite
method with an Applied Biosystems model 381 DNA synthesizer. Genomic Southern hybridization. Taking the codon usage of a T. thermophilus gene (14) into consideration, we syn-
VOL. 58,1992
T. THERMOPHILUS ISOCITRATE DEHYDROGENASE GENE
ScX
H I
Sm Sm XSm
X
ILI II
-1-
95
.
1
I II
500 bases FIG. 2. Restriction map of the cloned HindlIl fragment from T. thermophilus and sequencing strategy. The shaded arrow indicates the T. thermophilus isocitrate dehydrogenase gene. Small arrows indicate the direction and extent of sequencing. H, HindIII; Sm, SimaI; X, XhoI; Sc, Sacl.
thesized a 50-nucleotide probe: 5'-TAC CCC GAC ATC GAG GCC GTG CAC ATC ATC GTG GAC AAC GCC GCC CAC CA-3'. The sequence was deduced from a partial amino acid sequence: Tyr-Pro-Asp-Ile-Glu-Ala-Val-HisIle-Ile-Val-Asp-Asn-Ala-Ala-His-Gln. The oligonucleotide probe was labeled with [ y-32P]ATP by use of T4 polynucleotide kinase. Chromosomal DNA isolated from T. thermophilus cells by standard procedures (23) was digested with several restriction enzymes: Hindlil, BamHI, SacI, BglII, HindIII-BamHI, HindIII-SacI, BamHI-SacI, and SaclBglII. The restriction fragments were separated on an 0.8% agarose gel and transferred to a nitrocellulose filter (Amersharn). Hybridization was carried out at 60°C for 12 h in 6x NET (1 x NET is 150 mM NaCl, 15 mM Tris-HCI, and 1 mM EDTA) containing 0.4% each polyvinylpyrrolidone, Ficoll 400, and bovine serum albumin; 0.2% sodium dodecyl sulfate (SDS); 200 F.g of sheared herring sperm DNA per ml; and labeled probe (1 x 107 to 2 x 107 cpm/ml). The filter was washed twice in 6x SSC (lx SSC is 150 mM NaCl plus 15 mM sodium citrate)-0.1% SDS at 70°C for 20 min and exposed to X-ray film with an intensifying screen at -70°C for 1 day. Colony hybridization. Total HindIII fragments were iigated into pUC118. E. coli HB101 competent cells were transformed with the resulting plasmids and used as a
genomic library. The conditions of hybridization were the same as those described above. DNA sequencing. To prevent the compression which often occurs in G+C-rich regions, we used the dideoxynucleotide chain termination method with 7-deaza-dGTP and Taq polymerase (Promega, Madison, Wis.) for sequencing (2, 25). Unidirectional deletion mutants were prepared by exonuclease III digestion. The DNA sequence of each fragment was confirmed by sequencing both strands. Sequencing was carried out with an automated DNA sequencer (Applied Biosystems Model 370A). Comparison of sequence data. The protein data bank of the National Biomedical Research Foundation (Beckman MicroGenie) was used for the computer search. The comparison of two given sequences was carried out with Genetyx software (Software Development Co., Ltd., Shibuya, Japan). RESULTS AND DISCUSSION Peptide sequencing. Automated amino acid sequencing revealed only 5 residues in the N terminus of the intact protein: Pro-Leu-Ile-Thr-Thr. To obtain more sequence data, we prepared and sequenced proteolytic fragments (Fig. 1). One of them (amino acid residues 211 to 227 [Fig. 1]) was
GENE
SEQUENCE -35
-10
SD
Init.
ICDH
KTTACAAGGCCTCAAGCCCCGTGG G
TAAAGGGGGCGATTCCGCCCCCGGAGGTGAACCCATG
TRP
ETTACdGGGAGGCCCCTCCGGG G
GAGTTGTCTTGGCGCGAGGCGCCTTTAGGGAGCGAAGCATG
SCS
1CCGTiTCCACGGCCCAAGGCCTAG
CCCTT* ACC
E. coli consensus
rTGAC3
CGGGGTCGCCCGGCAAGGGAGGTGGGTCTTG
rATGTI
FIG. 3. Comparison of the 5'-flanking regions of the T. thermophilus isocitrate dehydrogenase (ICDH) gene and the trpAB (TRP) gene (16) with the thermophile promoter sequence for the succinyl-coenzyme A synthetase operon (SCS) (19) and the E. coli consensus sequence (10). -35 and -10 regions are boxed, Shine-Dalgarno ribosome binding sites are underlined, and initiation codons are in boldface type. The transcription initiation site for the succinyl-coenzyme A synthetase operon is shaded.
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MIYAZAKI ET AL. 1
T-IPMDH T-ICDH E-ICDH
10
)EAEGLG-
PLITTETGKKMHVLEDGRK MESKVVVPAQGKKITLQAGKLNVPENP 60
T-IPMDH T-ICDH E-ICDH
EYKGE 80
70
A S--
IASGV
I
YTGEKSTQVD~
D
LERLS
QDL
E-ICDH
150
140
KZEI
PRGM-
GGI
I
YYQG ----T
ICL
HSPEL
IF
KA-AE
.SI
180
170
190
-AWNTERY4PEVE---------------------RV SVD! -------------------------KIAT Tau S -\rx LV FLREEMGVKKIRFPEHCGIGIKPCSEEGTKRL 200
FFT bF7
GX 230
220
210
R-------------------- G Lt
EVG WRKT
E-ICDH
WGY ELREEFGGELIDGGPWLKVK8PNTGKEVIKD
240
270
260
250
290
300
320
310
350
340
330
NALLYTLEEG
LEEFAT
I
5II
|EAD2iI
HAFGLVELARKVEDAVAKALLETPPPDLGGSAGT
I
E
HMGWTE A 360
+
TGDVVG
iVKGMEGAINAKT.TYDFERLM 370
380
LQEIL 280
RS AFgiGI CirAVPSL£KLRTPVF
T-IPMDH
T-ICDH E-ICDH
R--
T--T~M
FET ELD
T-IPMDH T-ICDH
T-IPMDH T-ICDH E-ICDH
~Ij~
I
160
T-IPMDH T-ICDH
KISPETGLLS§ *K
130
120
110
T-IPMDH T-ICDH E-ICDH
loo
90
AVLLGSV
EPFPEPTRKG
ID
-.
ATVLRHL T
LLKCSEIFG
390
400
T-IPMDH
T-ICDH E-ICDH
GKTPRKTQVRGYKPFRLPQVDGAIAPIVPRSRRVVGVDVFVETNLLPEALGK
4403444---------------------------------------------
410
T-IPMDH T-ICDH E-ICDH
430
440
450
460
ALEDLAAGTPFRLKMISNRGTQVYPPTGGLTDLVDHYRCRFLYTGEGEAKDPEILDLVSR ----------------__------------------------------------------
470
T-IPMDH T-ICDH E-ICDH
420
480
490
-----------------------------
VASRFRWMHLEKLQEFDGEPGFTKAQGED -----------------------------
FIG. 4. Amino acid sequence alignment of T. thermophilus isocitrate dehydrogenase (T-ICDH), T. thermophilus isopropylmalate dehydrogenase (T-IPMDH) (14), and E. coli isocitrate dehydrogenase (E-ICDH) (29). Shaded sequences represent those identical to the T. thermophilus isocitrate dehydrogenase sequence. The sequence data for T. thermophilus isopropylmalate dehydrogenase shown here were those corrected by Kirino et al. (1Sa), and the revised data are used for comparison throughout this study. The residue numbers are those for T. thermophilus isocitrate dehydrogenase.
used to synthesize
an
oligonucleotide probe to isolate the
gene.
Molecular cloning. When genomic DNA was analyzed by Southern hybridization, a 5.5-kb HindIII fragment hybridized strongly with the oligonucleotide probe described above. The Hindlll genomic library was screened with this probe, and a single clone containing the expected 5.5-kb HindIll fragment was isolated. DNA sequencing. The restriction map of the 5.5-kb HindIlI fragment and the sequencing strategy are shown in Fig. 2. The complete nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. It seems that the 50-mer
oligonucleotide probe hybridized to nucleotides 1109 to 1158 (Fig. 1); 45 of 50 nucleotides were the same in this region. The G+C content of the T. thermophilus isocitrate dehydrogenase gene was 65.6%, slightly lower than that of chromosomal DNA (69%) (18), and that of the third nucleotide of the codons was 90.3%. Codon usage was similar to that of other proteins from T. thermophilus sequenced (14). The cloned DNA contained a single open reading frame of 1,485 bp (495 amino acids). The N-terminal portion of the protein sequence suggested that the f-Met residue was removed after translation. The molecular weight of the mature protein was estimated to be
T. THERMOPHILUS ISOCITRATE DEHYDROGENASE GENE
VOL. 58, 1992
Tt EC
SC SC Tt
Yi Cu
Ba BC Bt
ICD} ICDR IPMI IPMDH
IPNDH IPDEH IP1UD] IPMDH IPNDH
IPKDH
279 293 GSAPKYAGKZIVINPT GTAPKYAGQDXVNPG GSVPD--PXNVTDPI GSAPDL-PKNHCVDPI GSAPDIAGKGIANPT GSAPDL-GKQKVNPI GSAPDL-PANXVNPI GSAPDIAG QNPF GSAPDIAGQGKANPL GSAPDIAQOiKANPI
FIG. 5. Alignment of a highly conserved sequence in isocitrate dehydrogenase, isopropylmalate isomerase, and isopropylmalate dehydrogenase. Tt ICDH, T. thermophilus isocitrate dehydrogenase; Ec ICDH, E. coli isocitrate dehydrogenase (29); Sc IPMI, Saccharocerevisiae isopropylmalate isomerase (10); Sc IPMDH, S. cerevisiae isopropylmalate dehydrogenase (1); Tt IPMDH, T. thermophilus isopropylmalate dehydrogenase (14); Yl IPMDH, Yarrowia lipolytica isopropylmalate dehydrogenase (5); Cu IPMDH, Candida utilis isopropylmalate dehydrogenase (8); Bs IPMDH, Bacillus subtilis isopropylmalate dehydrogenase (13); Bc IPMDH, B. coagulans isopropylmalate dehydrogenase (26); Bt IPMDH, B. caldotenax isopropylmalate dehydrogenase (27). The numbers indicate the residue numbers for T. thermophilus isocitrate dehydrogenase.
myces
54,189, in agreement with that determined by SDS-polyacrylamide gel electrophoresis (6). The amino acid composition calculated from the sequence was in good agreement with that obtained by amino acid analysis of the acid hydrolysate (6). Recently, the promoter sequence of the Thermus flavus succinyl-coenzyme A synthetase-malate dehydrogenase operon was reported by Nishiyama et al. (19) by an S1 nuclease mapping technique. Although they detected a sequence similar to the E. coli -10 consensus sequence, no sequence homologous to that of the E. coli promoter was found in the -35 region. In the present study, sequences highly homologous to the E. coli -35 and -10 regions were seen in the upstream flanking region of the thermophile isocitrate dehydrogenase gene. Similar sequences were also present in the upstream flanking region of the trpAB gene of T. thermophilus HB27 (16) (Fig. 3). No experimental evidence is presented to prove that the putative promoter sequences involved in the transcriptional regulation of these genes, but the observations suggest that the promoter sequences of various Thermus chromosomes are not unique. Downstream of the -10 promoter sequence, there was a Shine-Dalgarno ribosome binding site (5'-GGAGGTGA-3') which was complementary to the 3'-end sequence of T. thermophilus 16S rRNA (5'-UCACCUCC-3') (9). Two terminatorlike palindrome sequences consisting of a 12-bp stem with a 4-nucleotide loop structure (stacking energy, -41.6 kcal [ca. 174 kJ]/mol) and a 12-bp stem with a 7-nucleotide loop structure (stacking energy, -35.4 kcal [ca. 148 kJ]/mol) were located downstream of the stop codon (Fig. 1). However, a T cluster or the consensus sequence reported for the p factor binding site was not found in this region. It is possible that this region acts as an attenuator. An open reading frame starting with an ATG codon was found in the cloned DNA overlapping the first terminatorlike palindrome region. Only 174 nucleotides of this open reading frame were determined in this study, and these are shown together with the deduced amino acid sequence in Fig. 1. This partial amino acid sequence did not show any signifiwere
97
homology to those of other proteins. The predicted secondary structure was determined by the method of Chou and Fasman (4). This method predicted an a-helix (residues 1 to 7)-random coil (residues 8 to 10)-ahelix (residues 11 to 45)-random coil (residues 46 to 58) structure. The second a-helix contained 5 Leu residues (residues 15, 19, 26, 33, and 40). The last 4 Leu residues were located at intervals of 7 residues. This unique structure is known as a leucine zipper, one of the motifs of DNA binding proteins (17), suggesting that in one operon of the T. thermophilus chromosome, isocitrate dehydrogenase may be linked to a DNA binding protein. This modified peptide may be involved in the regulation of transcription of the isocitrate dehydrogenase gene. Comparison of isocitrate dehydrogenase and isopropylmalate dehydrogenase. The amino acid sequence of T. thermophilus isocitrate dehydrogenase showed significant similarity to those of E. coli isocitrate dehydrogenase (29) and T. thermophilus isopropylmalate dehydrogenase (14) (Fig. 4), although approximately 140 extra residues at the C-terminal region were found in T. thermophilus isocitrate dehydrogenase. In E. coli isocitrate dehydrogenase, two extra stretches (amino acid residues 185 to 207 and 251 to 270) were inserted into the primary structure common to these enzymes. The amino acid sequence of T. thermophilus isocitrate dehydrogenase showed 37.3 and 33.3% identities with E. coli isocitrate dehydrogenase and T. thermophilus isopropylmalate dehydrogenase, respectively, except for the extended C-terminal portion of T. thermophilus isocitrate dehydrogenase and the inserts of E. coli isocitrate dehydrogenase. On the basis of the high homology of these amino acid sequences, one can predict similarities in the threedimensional structures of these enzymes. Recently, Hurley et al. (12) determined the residues involved in the magnesium isocitrate binding site of E. coli isocitrate dehydrogenase by X-ray crystallography. As expected, 8 amino acid residues which are suggested to interact with the malate moiety (1- and 2-carboxyl groups and a 1-hydroxyl group) of isocitrate in the E. coli enzyme (Arg-119, Arg-129, Arg-153, Tyr-160, Lys-230, Asp-283, Asp-307, and Asp-311) were all conserved in both T. thermophilus isocitrate dehydrogenase (Arg-103, Arg-113, Arg-136, Tyr-143, Lys-190, Asp-222, Asp-246, and Asp-250) and T. thermophilus isopropylmalate dehydrogenase (Arg-94, Arg-104, Arg-132, Tyr-139, Lys185, Asp-217, Asp-241, and Asp-245). The 3-carboxyl group of isocitrate seems to be recognized by Ser-113 and Asn-115 in E. coli isocitrate dehydrogenase. These residues were also conserved in T. thermophilus isocitrate dehydrogenase but were missing from T. thermophilus isopropylmalate dehydrogenase. Andreadis et al. (1) reported the presence of a highly homologous region between isopropylmalate dehydrogenase and isopropylmalate isomerase (EC 4.2.1.33) and proposed that a long homologous stretch in the C-terminal halves of the enzymes play a role in the recognition of isopropylmalate. The corresponding homologous sequence was also found in T. thermophilus isocitrate dehydrogenase (this study), E. coli isocitrate dehydrogenase (29), and all isopropylmalate dehydrogenases so far reported (5, 8, 13, 14, 26, 27), as shown in Fig. 5, suggesting that a common moiety of the three substrates interacts with this part of the protein cant sequence
molecule in all three enzymes. Conservation of a Ser residue in T. thermophilus isocitrate dehydrogenase. E. coli isocitrate dehydrogenase is the first bacterial enzyme whose activity was found to be regulated by phosphorylation and dephosphorylation (7). Borthwick et
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al. (3) reported that the enzyme is inactivated by the phosphorylation of Ser-113 catalyzed by isocitrate dehydrogenase kinase-phosphatase (a dually functional enzyme). The amino acid sequence around the corresponding Ser residue (Ser-97) was conserved in T. thermophilus isocitrate dehydrogenase (Fig. 1). The conservation of this Ser residue and the ability of this organism to grow on acetate as a sole carbon source (22) imply that T. thermophilus isocitrate dehydrogenase can be phosphorylated by isocitrate dehydrogenase kinase-phosphatase. This organism may also catabolize acetate via a glyoxylate bypass by restricting the flow of carbon metabolism through the tricarboxylic acid cycle, as in E. coli. ACKNOWLEDGMENTS We thank S. Kanaya of the Protein Engineering Research Institute and Y. Fukumori and T. Fujiwara of the Tokyo Institute of Technology for peptide sequencing and K. Gearing of the Karolinska Institute for help in preparing the manuscript. This work was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japanese Government (grant 02403029), and a research grant from the Asahi Glass Foundation. REFERENCES 1. Andreadis, A., Y.-P. Hsu, M. Hermodson, G. Kohihaw, and P. Schimmel. 1984. Yeast LEU2. J. Biol. Chem. 259:8059-8062. 2. Barr, P. J., R. M. Thayer, R. C. Najarian, F. Seela, and D. R. Tolan. 1986. 7-Deaza-2'-deoxyguanosine-5'-triphosphate: enhanced resolution in M13 dideoxy sequencing. BioTechniques 4:428-432. 3. Borthwick, A. C., W. H. Holms, and H. G. Nimmo. 1984. Amino acid sequence around the site of phosphorylation in isocitrate dehydrogenase from Escherichia coli ML308. FEBS Lett. 174: 112-115. 4. Chou, P. Y., and G. D. Fasman. 1978. Empirical predictions of protein conformation. Annu. Rev. Biochem. 47:251-276. 5. Davidow, L. S., F. S. Kaczmarek, J. R. DeZeeuw, S. W. Conlon, M. R. Lauth, D. A. Pereira, and A. E. Franke. 1987. The Yarrowia lipolytica LEU2 gene. Curr. Genet. 11:377-383. 6. Eguchi, H., T. Wakagi, and T. Oshima. 1989. A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilus HB8: purification and general properties. Biochim. Biophys. Acta 990:133-137. 7. Garnak, M., and H. C. Reeves. 1979. Phosphorylation of isocitrate dehydrogenase of Escherichia coli. Science 203:11111112. 8. Hamasawa, K., K. Kobayashi, S. Harada, K. Yoda, M. Yamasaki, and G. Tamura. 1987. Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase of Candida utilis. J. Gen. Microbiol. 133:1089-1097. 9. Hartmann, R. K., and V. A. Erdmann. 1989. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit. J. Bacteriol. 171:2933-2941. 10. Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255. 11. Hurley, J. H., M. D. Anthony, J. L. Sohl, D. E. Koshland, Jr., and R. M. Stroud. 1990. Regulation of an enzyme by phosphorylation at the active site. Science 249:1012-1016. 12. Hurley, J. H., P. E. Thorsness, V. Ramalingam, N. H. Helmers, D. E. Koshland, Jr., and R. M. Stroud. 1989. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc. Natl. Acad. Sci. USA 86:8635-8639.
13. Imai, R., T. Sekiguchi, Y. Nosoh, and K. Tsuda. 1987. The nucleotide sequence of 3-isopropylmalate dehydrogenase gene from Bacillus subtilis. Nucleic Acids Res. 15:4988. 14. Kagawa, Y., H. Nojima, N. Nukiwa, M. Ishizuka, T. Nakajima, T. Yasuhara, T. Tanaka, and T. Oshima. 1984. High guanine plus cytosine content in the third letter of codons of an extreme thermophile. J. Biol. Chem. 259:2956-2960. 15. Katsube, Y., N. Tanaka, A. Takenaka, T. Yamada, and T. Oshima. 1988. Crystallization and preliminary X-ray data for 3-isopropylmalate dehydrogenase of Thermus thermophilus. J.
Biochem. 104:679-680. 15a.Kirino, H., et al. Unpublished data. 16. Koyama, Y., and K. Furukawa. 1990. Cloning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Thermus thermophilus HB27: plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T. thermophilus cells. J. Bacteriol. 172:3490-3495. 17. Landschulz, W. H., P. F. Jonson, and S. L. McKnight. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759-1764. 18. Nagahari, K., T. Koshikawa, and K. Sakaguchi. 1980. Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli. Gene 10:137-145. 19. Nishiyama, M., S. Horinouchi, and T. Beppu. 1991. Characterization of an operon encoding succinyl-CoA synthetase and malate dehydrogenase from Thermus flavus AT-62 and its expression in Escherichia coli. Mol. Gen. Genet. 226:1-9. 20. Oshima, T., and K. Imahori. 1971. Isolation of an extreme thermophile and thermostability of its transfer ribonucleic acid and ribosomes. J. Gen. Appl. Microbiol. 17:513-517. 21. Oshima, T., and K. Imahori. 1974. Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa. Int. J. Syst. Bacteriol. 24:102-112. 22. Pask-Hughes, R. A., and R. A. D. Williams. 1977. Yellowpigmented strains of Thermus spp. from Icelandic hot springs. J. Gen. Microbiol. 102:375-383. 23. Saito, H., and K. Miura. 1963. Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochim. Biophys. Acta 72:619-629. 24. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular
25. 26.
27. 28.
29.
30.
cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sanger, F., and A. R. Coulson. 1975. A rapid method for determining sequences in DNA by primer synthesis with DNA polymerase. J. Mol. Biol. 94:441-448. Sekiguchi, T., J. Ortega-Cesena, Y. Nosoh, S. Ohashi, K. Tsuda, and S. Kanaya. 1986. DNA and amino-acid sequences of 3-isopropylmalate dehydrogenase of Bacillus coagulans. Comparison with the enzymes of Saccharomyces cerevisiae and Thermus thermophilus. Biochim. Biophys. Acta 867:36-44. Sekiguchi, T., M. Suda, T. Ishii, Y. Nosoh, and K. Tsuda. 1987. The nucleotide sequence of 3-isopropylmalate dehydrogenase gene from Bacillus caldotenax. Nucleic Acids Res. 15:853. Tanaka, T., N. Kawano, and T. Oshima. 1981. Cloning of 3-isopropylmalate dehydrogenase gene of an extreme thermophile and partial purification of the gene product. J. Biochem. 89:677-682. Thorsness, P. E., and D. E. Koshland, Jr. 1987. Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J. Biol. Chem. 262:1042210425. Yamada, T., N. Akutsu, K. Miyazaki, K. Kakinuma, M. Yoshida, and T. Oshima. 1990. Purification, catalytic properties, and thermal stability of threo-Ds-3-isopropylmalate dehydrogenase coded by leuB gene from an extreme thermophile, Thermus thermophilus strain HB8. J. Biochem. 108:449-456.
Vol. 58, No. 1
0099-2240/92/010093-06$02.00/0 Copyright © 1992, American Society for Microbiology
Molecular Cloning of the Isocitrate Dehydrogenase Gene of an Extreme Thermophile, Thermus thermophilus HB8 KENTARO MIYAZAKI,* HIDETAKA EGUCHI, AKIHIKO YAMAGISHI, TAKAYOSHI WAKAGI, AND TAIRO OSHIMA Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Yokohama 227, Japan
Received 21 June 1991/Accepted 22 October 1991
The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G+C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.
The NADP-dependent threo-Ds-isocitrate dehydrogenase (threo-DS-isocitrate:NADP oxidoreductase [EC 1.1.1.42])
means to elucidate the molecular basis of the evolution of substrate specificity. We previously cloned and sequenced the isopropylmalate dehydrogenase gene of T. thermophilus (14, 28), characterized the thermophile isopropylmalate dehydrogenase (30), and also purified isocitrate dehydrogenase from the same organism (6). Only one enzyme activity was detected in the thermophile extract, suggesting the absence of an NAD-dependent isocitrate dehydrogenase (EC 1.1.1.41) in this organism, as in E. coli. In this paper, we report the molecular cloning and DNA sequencing of the gene coding for isocitrate dehydrogenase of T. thermophilus.
catalyzes the oxidative decarboxylation of threo-D.-isocitrate to 2-oxoglutarate. This enzyme has been reported as a key enzyme in the tricarboxylic acid cycle and also plays an important role in glutamate synthesis. In the tricarboxylic acid cycle, 2-oxoglutarate is formed from the condensation of acetyl coenzyme A and oxaloacetate, and then isomerization takes place through a combination of dehydration and rehydration steps. The isocitrate thus formed is subjected to oxidative decarboxylation. Likewise, 2-oxoisocaproate, from which leucine is synthesized by transamination, is formed via an analogous pathway in microorganisms and plants: i.e., condensation of acetyl coenzyme A and a 2-oxo acid, isomerization by dehydration and rehydration, and oxidative decarboxylation of a 2-hydroxy acid. In leucine biosynthesis, the enzyme corresponding to isocitrate dehydrogenase is 3-isopropylmalate dehydrogenase (threo-Dj-isopropylmalate:NAD oxidoreductase [EC 1.1.1.85]). Thorsness and Koshland (29) compared the primary structure of Escherichia coli isocitrate dehydrogenase with those of other proteins and reported that only isopropylmalate dehydrogenase showed high homology. Hurley et al. (12) found that the three-dimensional structure of E. coli isocitrate dehydrogenase is unique and significantly different from those of other members of the dehydrogenase family so far reported. We previously determined the crystal structure of isopropylmalate dehydrogenase from Thermus thermophilus (15) and found that the backbone structure of the thermophile enzyme resembles that of isocitrate dehydrogenase reported by Hurley et al. (11, 12) (unpublished data). These findings implied a close evolutionary relationship of these two dehydrogenases, which seem to have diverged from a common ancestor to create their own substrate specificities. A comparative study of these enzymes may provide the
MATERIALS AND METHODS
Materials. DNA modification enzymes were products of Takara (Kyoto, Japan) or Toyobo (Tokyo, Japan). N-TosylL-phenylalanyl chloromethyl ketone-treated trypsin was from Sigma (St. Louis, Mo.). Lysyl endopeptidase was from Wako Chemicals (Tokyo, Japan). [_y-32P]ATP (3,000 Ci/ mmol) was from Amersham (Amersham, England). All other reagents were of the purest grade available. Bacterial strains. The extremely thermophilic bacterium T. thermophilus HB8 (ATCC 27634) was grown at 75°C as described previously (20, 21). E. coli HB101 (F- hsdS20 recA13 ara-14 proA2 lacYl galK2 rpsL20 [str] xyl-5 mtl-l supE44 leuB6 thi-J) was used as a host for the T. thermo-
philus genomic library. DNA manipulations. DNA manipulations were carried out by standard methods (24). Enzyme purification and peptide sequencing. Isocitrate dehydrogenase was purified from T. thermophilus as described previously (6). The amino acid sequences of the intact protein and proteolytic fragments were determined with a gas-phase peptide sequencer (model 470A; Applied Biosystems, Foster City, Calif.). The purified protein was digested with trypsin or lysyl endopeptidase. The resulting peptides were separated by high-performance liquid chroma-
* Corresponding author. 93
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CCATGGGGCCGAAGGTGGCGTAGGCCAGGCCCTCCTCCCTAAAGGGCACCTTCCTTTGGG CCTCCGCCAGGGAGAGCTTCTCCACGGGGACCCCCAGGGCCTTCTGCACCCTTAGGGCCT CTTCCTGGGCCTCGGCCTGGGCCTCGGGCACCAGGAAGAGGTAGCCCGTAGCCGATAGGC GGCCTCGGGGATTTCCCGGTACTCCAGGATGGAACGGTAGGAGAGGAGGACGTTCGAGGG CTCGGAGAACTGCACCCGCACCCCGGCGGCGCTTTTCCCGGTGGACCCTTGGGCGTAGGT GGCCTCCTTCTCCAGGACCAAGACCCTAAGCCCCTTTTCCGCCAGGCGGTAGGCCGAGGC CGCCCCTACGATCCCCGCCCCCACCACGACCACCCGGGCCACGCCCCGAL CTCAAGCCCCGTGG
3STAAAGGGGGCGATTCCGCCCCCGG
GGC CCATGCC P
481 CCTGATCACCACGGAAACCGGCAAGAAGATGCACGTTCTCGAGGACGGGCGCAAGCTCAT L I T T E T G K K M H V L E D G R K L I 541 CACCGTCATCCCCGGAGACGGCATCGGGCCCGAGTGCGTGGAGGCTACCCTCAAGGTCCT T V I P G D G I G P E C V E A T L K V L 601 AGAGGCGGCCAAGGCCCCCCTGGCCTACGAGGTGCGAGAGGCGGGGGCGAGCGTCTTCCG E A A K A P L A Y E V R E A G A S V F R 661 GCGGGGCATCGCCTCGGGCGTTCCCCAGGAGACCATTGAGTCCATCCGCAAGACCCGGGT R G I A S G V P Q E T I E S I R K T R V 721 GGTCCTGAAGGGTCCCCTGGAAACCCCGGTGGGCTACGGGGAGAAGAGCGCCAACGTCAC V L K G P L E T P V G Y G E K S A N V T 781 CCTAAGGAAGCTCTTTGAGACCTACGCCAACGTCCGCCCCGTGCGGGAGTTCCCCAACGT L R K L F E T Y A N V R P V R E F P N V 841 CCCCACCCCCTATGCGGGCCGGGGCATTGACCTCGTGGTGGTGCGGGAGAACGTGGAGGA P T P Y A G R G I D L V V V R E N V E D 901 CCTCTACGCCGGGATTGAGCACATGCAGACCCCGAGCGTGGCCCAGACCCTCAAGCTCAT L Y A G I E H M Q T P S V A Q T L K L I 961 CTCCTGGAAGGGATCGGAGAAGATCGTCCGCTTCGCCTTTGAGCTGGCCCGGGCCGAGGG S W K G S E K I V R F A F E L A R A E G 1021 GCGGAAGAAGGTCCACTGCGCCACCAAGTCCAACATCATGAAGCTCGCCGAAGGACCCAA R K K V H C A T K S N I M K L A E G P K 1081 GCGGGCCTTTGAGCAGGTGGCCCAGGAGTACCCCGACATAGAAGCGGTCCACATCATCGT R A F E Q V A Q E Y P D I E A V H I I V 1141 GGACAACGCTGCCCACCAGCTGGTGAAAAGGCCCGAGCAGTTTGAGGTGATCGTCACCAC D N A A H Q L V K R P E Q F E V I V T T 1201 CAACATGAACGGAGACATCCTCTCCGACCTCACCTCGGGGCTCATTGGGGGCCTGGGCTT N M N G D I L S D L T S G L I G G L G F 1261 CGCTCCCTCGGCCAACATCGGCAACGAGGTGGCCATCTTTGAGGCCGTCCACGGTTCCGC A P S A N I G N E V A I F E A V H G S A 1321 CCCCAAGTACGCCGGGAAGAACGTCATCAACCCCACCGCGGTCCTCCTCTCGGCGGTGAT P K Y A G K N V I N .P T A V L L S A V M 1381 GATGCTCCGCTACCTGGAGGAGTTCGCCACGGCGGACCTTATAGAGAACGCCCTCCTCTA M L R Y L E E F A T A D L I E N A L L Y 1441 CACCCTCGAGGAGGGCCGGGTCCTCACGGGGGACGTGGTGGGCTACGACCGGGGGGCCAA T L E E G R V L T G D V V G Y D R G A K 1501 GACCACGGAGTACACCGAGGCCATCATCCAGAACCTGGGCAAGACCCCAAGGAAGACCCA T T E Y T E A I I Q N L G K T P R K T Q 1561 GGTGCGGGGCTACAAGCCCTTCCGCCTGCCCCAGGTGGACGGGGCCATCGCCCCCATCGT V R G Y K P F R L P Q V D G A I A P I V 1621 CCCTAGGAGCCGCCGGGTTGTGGGGGTGGACGTCTTCGTGGAAACCAACCTCCTGCCCGA P R S R R V V G V D V F V E T N L L P E 1681 GGCCCTGGGAAAGGCCCTGGAGGACCTTGCCGCGGGCACCCCCTTCCGGCTCAAGATGAT A L G K A L E D L A A G T P F R L K M I 1741 CTCCAACCGGGGCACCCAGGTCTACCCCCCCACCGGCGGGCTCACGGACCTGGTGGACCA S N R G T Q V Y P P T G G L T D L V D H 1801 CTACCGCTGCCGCTTCCTCTACACGGGGGAGGGGGAGGCTAAGGACCCGGAGATCCTGGA Y R C R F L Y T G E G E A K D P E I L D 1861 CCTCGTAAGCCGGGTGGCAAGCCGCTTCCGCTGGATGCACCTGGAGAAGCTCCAGGAATT L V S R V A S R F R W M H L E K L Q E F 1921 TGACGGCGAGCCCGGCTTCACCAAGGCCCAAGGGGAAGACTAAGGCCACCCCATGCCGGC M P X D G E P G F T K A Q G E D * 1981 TTGGGCCGGCATGGGGCCCGGCATGGCCCCGAGCCTGATGCGGCGCCTAGACCAGGGCGA W z G M G P G M A P S L M R R L D Q G E 2041 AGCGGGCCTCGAGGGCCGGAAGGTCCGCCTCCAGGAAGCGGAGGCGGGCCTCGGCGTTCA A G L E G R K V R L -Q E A E A G L G V Q 2101 GGGGAAGGGCCTTGGGCAGGGCCACCTGGGCGGAAAGCCCGAGCTC G K G L Q Q G H L G G K P E L
1
21 41
61
81 101 121 141
161 181 201 221
241
261 281 301 321
341
361 381 381 401 421
441 461
FIG. 1. Nucleotide sequence of the T. thermophilus isocitrate dehydrogenase gene, deduced amino acid sequence of the enzyme, and sequences of the 5'- and 3'-flanking regions. The deduced amino acid sequence of isocitrate dehydrogenase begins at position 479. The Shine-Dalgarno ribosome binding site is shaded. Probable -35 and -10 promoter sequences are boxed, and the terminator is marked by facing arrows. The amino acid sequence of the N terminus is overlined, tryptic fragments are underlined, and lysyl endopeptidase-digested fragments are doubly underlined.
tography with a C18 column. Several fragments were selected, and their amino acid sequences were determined. Synthesis of oligonucleotide. An oligonucleotide used as a hybridization probe was synthesized by the phosphoramidite
method with an Applied Biosystems model 381 DNA synthesizer. Genomic Southern hybridization. Taking the codon usage of a T. thermophilus gene (14) into consideration, we syn-
VOL. 58,1992
T. THERMOPHILUS ISOCITRATE DEHYDROGENASE GENE
ScX
H I
Sm Sm XSm
X
ILI II
-1-
95
.
1
I II
500 bases FIG. 2. Restriction map of the cloned HindlIl fragment from T. thermophilus and sequencing strategy. The shaded arrow indicates the T. thermophilus isocitrate dehydrogenase gene. Small arrows indicate the direction and extent of sequencing. H, HindIII; Sm, SimaI; X, XhoI; Sc, Sacl.
thesized a 50-nucleotide probe: 5'-TAC CCC GAC ATC GAG GCC GTG CAC ATC ATC GTG GAC AAC GCC GCC CAC CA-3'. The sequence was deduced from a partial amino acid sequence: Tyr-Pro-Asp-Ile-Glu-Ala-Val-HisIle-Ile-Val-Asp-Asn-Ala-Ala-His-Gln. The oligonucleotide probe was labeled with [ y-32P]ATP by use of T4 polynucleotide kinase. Chromosomal DNA isolated from T. thermophilus cells by standard procedures (23) was digested with several restriction enzymes: Hindlil, BamHI, SacI, BglII, HindIII-BamHI, HindIII-SacI, BamHI-SacI, and SaclBglII. The restriction fragments were separated on an 0.8% agarose gel and transferred to a nitrocellulose filter (Amersharn). Hybridization was carried out at 60°C for 12 h in 6x NET (1 x NET is 150 mM NaCl, 15 mM Tris-HCI, and 1 mM EDTA) containing 0.4% each polyvinylpyrrolidone, Ficoll 400, and bovine serum albumin; 0.2% sodium dodecyl sulfate (SDS); 200 F.g of sheared herring sperm DNA per ml; and labeled probe (1 x 107 to 2 x 107 cpm/ml). The filter was washed twice in 6x SSC (lx SSC is 150 mM NaCl plus 15 mM sodium citrate)-0.1% SDS at 70°C for 20 min and exposed to X-ray film with an intensifying screen at -70°C for 1 day. Colony hybridization. Total HindIII fragments were iigated into pUC118. E. coli HB101 competent cells were transformed with the resulting plasmids and used as a
genomic library. The conditions of hybridization were the same as those described above. DNA sequencing. To prevent the compression which often occurs in G+C-rich regions, we used the dideoxynucleotide chain termination method with 7-deaza-dGTP and Taq polymerase (Promega, Madison, Wis.) for sequencing (2, 25). Unidirectional deletion mutants were prepared by exonuclease III digestion. The DNA sequence of each fragment was confirmed by sequencing both strands. Sequencing was carried out with an automated DNA sequencer (Applied Biosystems Model 370A). Comparison of sequence data. The protein data bank of the National Biomedical Research Foundation (Beckman MicroGenie) was used for the computer search. The comparison of two given sequences was carried out with Genetyx software (Software Development Co., Ltd., Shibuya, Japan). RESULTS AND DISCUSSION Peptide sequencing. Automated amino acid sequencing revealed only 5 residues in the N terminus of the intact protein: Pro-Leu-Ile-Thr-Thr. To obtain more sequence data, we prepared and sequenced proteolytic fragments (Fig. 1). One of them (amino acid residues 211 to 227 [Fig. 1]) was
GENE
SEQUENCE -35
-10
SD
Init.
ICDH
KTTACAAGGCCTCAAGCCCCGTGG G
TAAAGGGGGCGATTCCGCCCCCGGAGGTGAACCCATG
TRP
ETTACdGGGAGGCCCCTCCGGG G
GAGTTGTCTTGGCGCGAGGCGCCTTTAGGGAGCGAAGCATG
SCS
1CCGTiTCCACGGCCCAAGGCCTAG
CCCTT* ACC
E. coli consensus
rTGAC3
CGGGGTCGCCCGGCAAGGGAGGTGGGTCTTG
rATGTI
FIG. 3. Comparison of the 5'-flanking regions of the T. thermophilus isocitrate dehydrogenase (ICDH) gene and the trpAB (TRP) gene (16) with the thermophile promoter sequence for the succinyl-coenzyme A synthetase operon (SCS) (19) and the E. coli consensus sequence (10). -35 and -10 regions are boxed, Shine-Dalgarno ribosome binding sites are underlined, and initiation codons are in boldface type. The transcription initiation site for the succinyl-coenzyme A synthetase operon is shaded.
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T-IPMDH T-ICDH E-ICDH
10
)EAEGLG-
PLITTETGKKMHVLEDGRK MESKVVVPAQGKKITLQAGKLNVPENP 60
T-IPMDH T-ICDH E-ICDH
EYKGE 80
70
A S--
IASGV
I
YTGEKSTQVD~
D
LERLS
QDL
E-ICDH
150
140
KZEI
PRGM-
GGI
I
YYQG ----T
ICL
HSPEL
IF
KA-AE
.SI
180
170
190
-AWNTERY4PEVE---------------------RV SVD! -------------------------KIAT Tau S -\rx LV FLREEMGVKKIRFPEHCGIGIKPCSEEGTKRL 200
FFT bF7
GX 230
220
210
R-------------------- G Lt
EVG WRKT
E-ICDH
WGY ELREEFGGELIDGGPWLKVK8PNTGKEVIKD
240
270
260
250
290
300
320
310
350
340
330
NALLYTLEEG
LEEFAT
I
5II
|EAD2iI
HAFGLVELARKVEDAVAKALLETPPPDLGGSAGT
I
E
HMGWTE A 360
+
TGDVVG
iVKGMEGAINAKT.TYDFERLM 370
380
LQEIL 280
RS AFgiGI CirAVPSL£KLRTPVF
T-IPMDH
T-ICDH E-ICDH
R--
T--T~M
FET ELD
T-IPMDH T-ICDH
T-IPMDH T-ICDH E-ICDH
~Ij~
I
160
T-IPMDH T-ICDH
KISPETGLLS§ *K
130
120
110
T-IPMDH T-ICDH E-ICDH
loo
90
AVLLGSV
EPFPEPTRKG
ID
-.
ATVLRHL T
LLKCSEIFG
390
400
T-IPMDH
T-ICDH E-ICDH
GKTPRKTQVRGYKPFRLPQVDGAIAPIVPRSRRVVGVDVFVETNLLPEALGK
4403444---------------------------------------------
410
T-IPMDH T-ICDH E-ICDH
430
440
450
460
ALEDLAAGTPFRLKMISNRGTQVYPPTGGLTDLVDHYRCRFLYTGEGEAKDPEILDLVSR ----------------__------------------------------------------
470
T-IPMDH T-ICDH E-ICDH
420
480
490
-----------------------------
VASRFRWMHLEKLQEFDGEPGFTKAQGED -----------------------------
FIG. 4. Amino acid sequence alignment of T. thermophilus isocitrate dehydrogenase (T-ICDH), T. thermophilus isopropylmalate dehydrogenase (T-IPMDH) (14), and E. coli isocitrate dehydrogenase (E-ICDH) (29). Shaded sequences represent those identical to the T. thermophilus isocitrate dehydrogenase sequence. The sequence data for T. thermophilus isopropylmalate dehydrogenase shown here were those corrected by Kirino et al. (1Sa), and the revised data are used for comparison throughout this study. The residue numbers are those for T. thermophilus isocitrate dehydrogenase.
used to synthesize
an
oligonucleotide probe to isolate the
gene.
Molecular cloning. When genomic DNA was analyzed by Southern hybridization, a 5.5-kb HindIII fragment hybridized strongly with the oligonucleotide probe described above. The Hindlll genomic library was screened with this probe, and a single clone containing the expected 5.5-kb HindIll fragment was isolated. DNA sequencing. The restriction map of the 5.5-kb HindIlI fragment and the sequencing strategy are shown in Fig. 2. The complete nucleotide sequence and the deduced amino acid sequence are shown in Fig. 1. It seems that the 50-mer
oligonucleotide probe hybridized to nucleotides 1109 to 1158 (Fig. 1); 45 of 50 nucleotides were the same in this region. The G+C content of the T. thermophilus isocitrate dehydrogenase gene was 65.6%, slightly lower than that of chromosomal DNA (69%) (18), and that of the third nucleotide of the codons was 90.3%. Codon usage was similar to that of other proteins from T. thermophilus sequenced (14). The cloned DNA contained a single open reading frame of 1,485 bp (495 amino acids). The N-terminal portion of the protein sequence suggested that the f-Met residue was removed after translation. The molecular weight of the mature protein was estimated to be
T. THERMOPHILUS ISOCITRATE DEHYDROGENASE GENE
VOL. 58, 1992
Tt EC
SC SC Tt
Yi Cu
Ba BC Bt
ICD} ICDR IPMI IPMDH
IPNDH IPDEH IP1UD] IPMDH IPNDH
IPKDH
279 293 GSAPKYAGKZIVINPT GTAPKYAGQDXVNPG GSVPD--PXNVTDPI GSAPDL-PKNHCVDPI GSAPDIAGKGIANPT GSAPDL-GKQKVNPI GSAPDL-PANXVNPI GSAPDIAG QNPF GSAPDIAGQGKANPL GSAPDIAQOiKANPI
FIG. 5. Alignment of a highly conserved sequence in isocitrate dehydrogenase, isopropylmalate isomerase, and isopropylmalate dehydrogenase. Tt ICDH, T. thermophilus isocitrate dehydrogenase; Ec ICDH, E. coli isocitrate dehydrogenase (29); Sc IPMI, Saccharocerevisiae isopropylmalate isomerase (10); Sc IPMDH, S. cerevisiae isopropylmalate dehydrogenase (1); Tt IPMDH, T. thermophilus isopropylmalate dehydrogenase (14); Yl IPMDH, Yarrowia lipolytica isopropylmalate dehydrogenase (5); Cu IPMDH, Candida utilis isopropylmalate dehydrogenase (8); Bs IPMDH, Bacillus subtilis isopropylmalate dehydrogenase (13); Bc IPMDH, B. coagulans isopropylmalate dehydrogenase (26); Bt IPMDH, B. caldotenax isopropylmalate dehydrogenase (27). The numbers indicate the residue numbers for T. thermophilus isocitrate dehydrogenase.
myces
54,189, in agreement with that determined by SDS-polyacrylamide gel electrophoresis (6). The amino acid composition calculated from the sequence was in good agreement with that obtained by amino acid analysis of the acid hydrolysate (6). Recently, the promoter sequence of the Thermus flavus succinyl-coenzyme A synthetase-malate dehydrogenase operon was reported by Nishiyama et al. (19) by an S1 nuclease mapping technique. Although they detected a sequence similar to the E. coli -10 consensus sequence, no sequence homologous to that of the E. coli promoter was found in the -35 region. In the present study, sequences highly homologous to the E. coli -35 and -10 regions were seen in the upstream flanking region of the thermophile isocitrate dehydrogenase gene. Similar sequences were also present in the upstream flanking region of the trpAB gene of T. thermophilus HB27 (16) (Fig. 3). No experimental evidence is presented to prove that the putative promoter sequences involved in the transcriptional regulation of these genes, but the observations suggest that the promoter sequences of various Thermus chromosomes are not unique. Downstream of the -10 promoter sequence, there was a Shine-Dalgarno ribosome binding site (5'-GGAGGTGA-3') which was complementary to the 3'-end sequence of T. thermophilus 16S rRNA (5'-UCACCUCC-3') (9). Two terminatorlike palindrome sequences consisting of a 12-bp stem with a 4-nucleotide loop structure (stacking energy, -41.6 kcal [ca. 174 kJ]/mol) and a 12-bp stem with a 7-nucleotide loop structure (stacking energy, -35.4 kcal [ca. 148 kJ]/mol) were located downstream of the stop codon (Fig. 1). However, a T cluster or the consensus sequence reported for the p factor binding site was not found in this region. It is possible that this region acts as an attenuator. An open reading frame starting with an ATG codon was found in the cloned DNA overlapping the first terminatorlike palindrome region. Only 174 nucleotides of this open reading frame were determined in this study, and these are shown together with the deduced amino acid sequence in Fig. 1. This partial amino acid sequence did not show any signifiwere
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homology to those of other proteins. The predicted secondary structure was determined by the method of Chou and Fasman (4). This method predicted an a-helix (residues 1 to 7)-random coil (residues 8 to 10)-ahelix (residues 11 to 45)-random coil (residues 46 to 58) structure. The second a-helix contained 5 Leu residues (residues 15, 19, 26, 33, and 40). The last 4 Leu residues were located at intervals of 7 residues. This unique structure is known as a leucine zipper, one of the motifs of DNA binding proteins (17), suggesting that in one operon of the T. thermophilus chromosome, isocitrate dehydrogenase may be linked to a DNA binding protein. This modified peptide may be involved in the regulation of transcription of the isocitrate dehydrogenase gene. Comparison of isocitrate dehydrogenase and isopropylmalate dehydrogenase. The amino acid sequence of T. thermophilus isocitrate dehydrogenase showed significant similarity to those of E. coli isocitrate dehydrogenase (29) and T. thermophilus isopropylmalate dehydrogenase (14) (Fig. 4), although approximately 140 extra residues at the C-terminal region were found in T. thermophilus isocitrate dehydrogenase. In E. coli isocitrate dehydrogenase, two extra stretches (amino acid residues 185 to 207 and 251 to 270) were inserted into the primary structure common to these enzymes. The amino acid sequence of T. thermophilus isocitrate dehydrogenase showed 37.3 and 33.3% identities with E. coli isocitrate dehydrogenase and T. thermophilus isopropylmalate dehydrogenase, respectively, except for the extended C-terminal portion of T. thermophilus isocitrate dehydrogenase and the inserts of E. coli isocitrate dehydrogenase. On the basis of the high homology of these amino acid sequences, one can predict similarities in the threedimensional structures of these enzymes. Recently, Hurley et al. (12) determined the residues involved in the magnesium isocitrate binding site of E. coli isocitrate dehydrogenase by X-ray crystallography. As expected, 8 amino acid residues which are suggested to interact with the malate moiety (1- and 2-carboxyl groups and a 1-hydroxyl group) of isocitrate in the E. coli enzyme (Arg-119, Arg-129, Arg-153, Tyr-160, Lys-230, Asp-283, Asp-307, and Asp-311) were all conserved in both T. thermophilus isocitrate dehydrogenase (Arg-103, Arg-113, Arg-136, Tyr-143, Lys-190, Asp-222, Asp-246, and Asp-250) and T. thermophilus isopropylmalate dehydrogenase (Arg-94, Arg-104, Arg-132, Tyr-139, Lys185, Asp-217, Asp-241, and Asp-245). The 3-carboxyl group of isocitrate seems to be recognized by Ser-113 and Asn-115 in E. coli isocitrate dehydrogenase. These residues were also conserved in T. thermophilus isocitrate dehydrogenase but were missing from T. thermophilus isopropylmalate dehydrogenase. Andreadis et al. (1) reported the presence of a highly homologous region between isopropylmalate dehydrogenase and isopropylmalate isomerase (EC 4.2.1.33) and proposed that a long homologous stretch in the C-terminal halves of the enzymes play a role in the recognition of isopropylmalate. The corresponding homologous sequence was also found in T. thermophilus isocitrate dehydrogenase (this study), E. coli isocitrate dehydrogenase (29), and all isopropylmalate dehydrogenases so far reported (5, 8, 13, 14, 26, 27), as shown in Fig. 5, suggesting that a common moiety of the three substrates interacts with this part of the protein cant sequence
molecule in all three enzymes. Conservation of a Ser residue in T. thermophilus isocitrate dehydrogenase. E. coli isocitrate dehydrogenase is the first bacterial enzyme whose activity was found to be regulated by phosphorylation and dephosphorylation (7). Borthwick et
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al. (3) reported that the enzyme is inactivated by the phosphorylation of Ser-113 catalyzed by isocitrate dehydrogenase kinase-phosphatase (a dually functional enzyme). The amino acid sequence around the corresponding Ser residue (Ser-97) was conserved in T. thermophilus isocitrate dehydrogenase (Fig. 1). The conservation of this Ser residue and the ability of this organism to grow on acetate as a sole carbon source (22) imply that T. thermophilus isocitrate dehydrogenase can be phosphorylated by isocitrate dehydrogenase kinase-phosphatase. This organism may also catabolize acetate via a glyoxylate bypass by restricting the flow of carbon metabolism through the tricarboxylic acid cycle, as in E. coli. ACKNOWLEDGMENTS We thank S. Kanaya of the Protein Engineering Research Institute and Y. Fukumori and T. Fujiwara of the Tokyo Institute of Technology for peptide sequencing and K. Gearing of the Karolinska Institute for help in preparing the manuscript. This work was partly supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture, Japanese Government (grant 02403029), and a research grant from the Asahi Glass Foundation. REFERENCES 1. Andreadis, A., Y.-P. Hsu, M. Hermodson, G. Kohihaw, and P. Schimmel. 1984. Yeast LEU2. J. Biol. Chem. 259:8059-8062. 2. Barr, P. J., R. M. Thayer, R. C. Najarian, F. Seela, and D. R. Tolan. 1986. 7-Deaza-2'-deoxyguanosine-5'-triphosphate: enhanced resolution in M13 dideoxy sequencing. BioTechniques 4:428-432. 3. Borthwick, A. C., W. H. Holms, and H. G. Nimmo. 1984. Amino acid sequence around the site of phosphorylation in isocitrate dehydrogenase from Escherichia coli ML308. FEBS Lett. 174: 112-115. 4. Chou, P. Y., and G. D. Fasman. 1978. Empirical predictions of protein conformation. Annu. Rev. Biochem. 47:251-276. 5. Davidow, L. S., F. S. Kaczmarek, J. R. DeZeeuw, S. W. Conlon, M. R. Lauth, D. A. Pereira, and A. E. Franke. 1987. The Yarrowia lipolytica LEU2 gene. Curr. Genet. 11:377-383. 6. Eguchi, H., T. Wakagi, and T. Oshima. 1989. A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilus HB8: purification and general properties. Biochim. Biophys. Acta 990:133-137. 7. Garnak, M., and H. C. Reeves. 1979. Phosphorylation of isocitrate dehydrogenase of Escherichia coli. Science 203:11111112. 8. Hamasawa, K., K. Kobayashi, S. Harada, K. Yoda, M. Yamasaki, and G. Tamura. 1987. Molecular cloning and nucleotide sequence of 3-isopropylmalate dehydrogenase of Candida utilis. J. Gen. Microbiol. 133:1089-1097. 9. Hartmann, R. K., and V. A. Erdmann. 1989. Thermus thermophilus 16S rRNA is transcribed from an isolated transcription unit. J. Bacteriol. 171:2933-2941. 10. Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 11:2237-2255. 11. Hurley, J. H., M. D. Anthony, J. L. Sohl, D. E. Koshland, Jr., and R. M. Stroud. 1990. Regulation of an enzyme by phosphorylation at the active site. Science 249:1012-1016. 12. Hurley, J. H., P. E. Thorsness, V. Ramalingam, N. H. Helmers, D. E. Koshland, Jr., and R. M. Stroud. 1989. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc. Natl. Acad. Sci. USA 86:8635-8639.
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