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NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli
FEMS Microbiology Letters 209 (2002) 155^160
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NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli Mo¤nica Camacho, Adoracio¤n Rodr|¤guez-Arnedo, Mar|¤a-Jose¤ Bonete
Divisio¤n de Bioqu|¤mica y Biolog|¤a Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain Received 26 November 2001; received in revised form 7 January 2002; accepted 7 January 2002 First published online 8 March 2002
Abstract A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a V EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45 837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl2 and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Isocitrate dehydrogenase; Halophile; Archaea; Haloferax volcanii
1. Introduction The NADPþ -dependent Ds-threo-isocitrate dehydrogenase (Ds-threo-isocitrate:NADPþ oxidoreductase (EC 1.1.1.42)) (ICDH) is a member of the tricarboxylic acid cycle, which catalyses the oxidative decarboxylation of Ds-threo-isocitrate to form 2-oxoglutarate, CO2 , and NADPH. 2-Oxoglutarate can be further oxidised within the cycle or reductively aminated to glutamate, and NADPH can be used for reductive biosynthetic reactions. ICDH thus supplies the cell with a key intermediate of energy metabolism, as well as with precursors and reducing power for anabolic pathways. The gene encoding isocitrate dehydrogenase has been cloned and sequenced from a variety of eukaryal and bac-
* Corresponding author. Tel. : +34 (96) 590 3524; Fax : +34 (96) 590 3880. E-mail address : [email protected] (M.-J. Bonete).
terial sources [1^5]. In addition, the crystal structure of the NADP-linked enzyme from Escherichia coli has been D in both liganded and unliganded forms solved to 2.5 A [6], and the structure has been related to the catalytic mechanism [7]. Archaea contain a single homodimeric isocitrate dehydrogenase that is either NADP-dependent or shows dual coenzyme speci¢city [8,9]. In a previous paper [9], we reported the puri¢cation, characterisation and N-terminal sequencing of isocitrate dehydrogenase from the extremely halophilic Archaeon, Haloferax volcanii, which grows at 2^4 M NaCl with isotonic concentrations of KCl internally. Here, we describe the cloning of the gene encoding this enzyme from H. volcanii. The DNA sequence was determined, and the derived amino acid sequence was compared with known ICDH sequences. The yields of E. coli-expressed enzyme were greater than those obtained by puri¢cation of the enzyme from the native organism, but the product was insoluble inclusion bodies. This required refolding of the recombinant halophilic protein, and the active enzyme was recovered, with characteristics similar to the wild-type protein.
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2. Materials and methods 2.1. Preparation of a probe by PCR For the isolation of the gene for ICDH, a probe was prepared from genomic DNA by polymerase chain reaction (PCR). The sense primer (oligonucleotide 1) had the base sequence 5P-GA(C,T)GA(C,T)GG(C,G)GA(A,G)AA(A,G)AT-3P, where the parentheses represent the degenerate sites (amino acid sequence NH2 -Asp Asp Gly Glu Lys Ile); it was based on its N-terminal amino acid sequence [9]. The antisense primer (oligonucleotide 2) had the base sequence 5P-CCCTC(C,G)GTGAACTTCAT-3P (amino acid sequence NH2 -Pro Ser Val Asn Phe Ile); it was based on a consensus sequence between E. coli (P08200) and Sulfolobus solfataricus (Q97WN0). A product of the expected size (650 bp) was detected by electrophoresis and labelled with digoxigenin-dUTP using a Nonradioactive DNA Labeling and Detection Kit (Roche Molecular Biochemicals). It was used as a probe in Southern blot and V plaque hybridisation experiments [10]. 2.2. Genomic library manipulations and DNA sequencing A genomic library of H. volcanii DNA was constructed in E. coli XL1-Blue MRA (P2) strain (Stratagene) using Sau3A-digested DNA ligated into V EMBL3 (Stratagene). V plaques were blotted onto Hybond-Nþ membranes (Amersham Pharmacia Biotech) and hybridised with the probe prepared above. Plaques that hybridised strongly with the non-radioactive probe were picked and replated to authenticate their hybridisation by a second plaques blot. Isolation and puri¢cation of the phage and subsequent DNA extraction were carried out by Lambda Maxi Kit (Qiagen). Nucleotide sequence determination was performed by the dideoxy chain termination method using a 310 DNA sequencer (Applied Biosystems). Synthesised icdh-speci¢c oligonucleotides were used as primers, developing the ‘primer walking’ strategy. Searches in the SWISS-PROT and TrEMBL databases for sequence similarities were carried out with the programs BLAST [11] and FASTA [12], and the alignment of isocitrate dehydrogenase sequences was done with PILEUP and BESTFIT by using the sequence analysis software package of the University of Wisconsin Genetics Computer Group [13]. A phylogenetic tree was constructed using CLUSTAL W [14]. The phylogenetic tree was validated by a bootstrap analysis (1000 replicates).
and BamHI to facilitate the subcloning of the H. volcanii ICDH gene for expression. Template DNA for PCR was the clone from the V EMBL3 library. The PCR product was subcloned into plasmid pCR0 2.1. After transforming E. coli Novablue cells (Novagen) with this construct, plasmid DNA was ligated into expression vector pET-3a (Novagen). The resulting clones were propagated in E. coli Novablue cells prior to transforming E. coli BL21 (DE3) (Novagen), which was used as the expression host. Cultures were grown to an OD600nm at 37‡C of 0.5^1.0 in Luria^Bertani (LB) medium containing 50 Wg ml31 ampicillin. Induction of expression was achieved by the addition of isopropyl L-D-thiogalactoside (IPTG) to a ¢nal concentration of 0.4 mM, and the cultures were shaken at 37‡C for 3 h before harvesting cells by centrifugation. 2.4. Refolding and puri¢cation of insoluble recombinant isocitrate dehydrogenase E. coli BL21 (DE3) cell pellets were resuspended in 20 mM Tris^HCl bu¡er, pH 8.0, containing 2 mM EDTA and 2 M KCl, treated with 100 Wg ml31 lysozyme and 0.1% (v/v) Triton X-100, and incubated at 30‡C for 1 h before transferring the solution onto ice for 15 min. The suspension was then sonicated and centrifuged at 10 000Ug for 10 min at 4‡C. The insoluble fraction yielded inclusion bodies together with the insoluble cell fragments, and it was dissolved in solubilisation bu¡er (20 mM Tris^ HCl pH 8.0, containing 2 mM EDTA, 8 M urea and 50 mM dithiothreitol (DTT)). Refolding was initiated by slowly diluting the solubilised pellet to a ¢nal concentration of 20 Wg ml31 into renaturation bu¡er and incubating for at least 24 h at room temperature. The renaturation bu¡er was composed of 20 mM Tris^HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2 . The solution was concentrated by ultra¢ltration on a PM 50 membrane with a stirred cell (Amicon Inc., Beverly, MA, USA) to a ¢nal volume of 10 ml. The bu¡er was exchanged to 20 mM Tris^HCl pH 8.0, containing 2.5 M (NH4 )2 SO4 , 1 mM EDTA and 10 mM MgCl2 prior to further chromatography on a DEAE-cellulose (Amersham Pharmacia Biotech) column equilibrated with the same bu¡er, and the enzyme was eluted with the renaturation bu¡er. Fractions containing isocitrate dehydrogenase activity were pooled and, ¢nally, dialysed against the same renaturation bu¡er for 24 h at 4‡C to remove the (NH4 )2 SO4 remaining, and stored at this temperature. 2.5. Enzyme and protein assays
2.3. Expression of H. volcanii isocitrate dehydrogenase in E. coli PCR techniques were used to amplify a gene fragment corresponding to the structural gene of H. volcanii ICDH with 1260 bp, including the digestion sequences of NdeI
The H. volcanii ICDH activity was assayed spectrophotometrically as previously [9] and the values of Vmax and Km were determined. Protein concentration was determined by the method of Bradford using bovine serum albumin as standard. SDS^polyacrylamide gel electropho-
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resis was performed according to the method described by Laemmli. 2.6. Thermal stability assays Thermal stability studies were carried out in the presence of salt. H. volcanii ICDH was incubated in 20 mM Tris^HCl bu¡er, pH 8.0, 1 mM EDTA, 10 mM MgCl2 containing 3 M NaCl, at temperatures in the range 60^ 80‡C for times up to several days. Samples were removed at known time intervals, rapidly cooled in ice, and the remaining activity determined by the standard assay procedure.
3. Results and discussion 3.1. PCR using oligonucleotides to conserved sequences Several ICDH sequences are known from a wide variety of bacterial and eukaryotic species. Analysis of these sequences reveals a high degree of similarity, especially in
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regions of known functional signi¢cance. Oligonucleotides 1 and 2 (sense and antisense, respectively, both described in Section 2) produced a product of about 650 bp on PCR with H. volcanii genomic DNA as target. This fragment was sequenced to con¢rm its identity and then used as a probe in order to determine the rest of the gene. 3.2. Isolation of a clone from a V EMBL3 library and sequence of the H. volcanii NADP-ICDH gene The PCR fragment was labelled with digoxigenindUTP, producing a probe that was used to screen a V EMBL3 genomic library of H. volcanii. A clone was obtained which hybridised to the probe at 65‡C. Partial sequencing of this clone revealed that it contained part of the ICDH gene. The structural analysis of the enzyme will give us abundant information in elucidating the molecular basis of the relationship between the function, such as coenzyme and substrate speci¢cities. The amino acid sequence of the coding region for the H. volcanii NADP-ICDH gene is shown in Fig. 1. The ICDH gene starts with ATG, a methionine
Fig. 1. Alignment of ICDH sequences. The derived amino acid sequence of H. volcanii ICDH was aligned with other known ICDH sequences using the program PILEUP. The alignment with the sequences from Halobacterium, B. subtilis and E. coli are shown. Asterisks indicate perfectly conserved residues and dots indicate well-conserved residues. Dashes denote gaps introduced into the sequence to achieve maximum alignment. Single-underlined residues and residues marked by double underlining are those that contact substrate and coenzyme, respectively, in the E. coli enzyme. The nucleotide sequence for H. volcanii ICDH has been submitted to the EMBL Nucleotide Sequence Database under accession No. AJ251585.
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codon, and the protein deduced from the nucleotide sequence was composed of 419 amino acids with a molecular mass of 45 837 Da, in disagreement with that determined by SDS^PAGE [9], and a calculated pI of 4.56. The amino acid sequence includes ¢ve Trp, one Cys, and six His residues, 31% hydrophobic residues (Phe+Tyr+Trp+Ile+Leu+Met+Val), and 18 and 11%, respectively, acidic and basic residues. The only cysteine residue is in position 332. ICDHs from Archaeoglobus fulgidus [5], Halobacterium (Q9HNZ7) and Thermus aquaticus [4] have also one Cys; S. solfataricus ICDH (Q97WN0) has no Cys and E. coli enzyme has six [6], which explains the decreased thermostability of the bacterial enzyme [15]. The secondary structure of the enzyme was obtained with the same GCG software package cited before. The results show 12 K-helical and 12 L-sheet conformations in the polypeptide. Like other ICDHs, in the halophilic enzyme there is no Rossman fold characteristic of the nucleotide binding, which is present in other kinds of dehydrogenases (i.e. alcohol dehydrogenases) as shown in glucose dehydrogenase from Haloferax mediterranei [16].
binding of isocitrate or coenzyme in E. coli isocitrate dehydrogenase are conserved except a few residues, which present conservative changes (Fig. 1), indicating that the enzymes probably have similar reaction mechanism and structure. The three-dimensional structure of these enzymes is only known for E. coli ICDH [6] and Bacillus subtilis [17]. A phylogenetic tree (Fig. 2) generated by comparing the most characteristic archaeal complete isocitrate dehydrogenase amino acid sequences available from sequence databases, rooted with a bacterial ICDH, revealed the great identity of the H. volcanii enzyme with that from Halobacterium (70.4%), another halophilic organism, and the sequence comparison also shows that isocitrate dehydrogenase is a very conserved enzyme [5]. ICDHs have divided into three distinct phylogenetic subfamilies: subfamily I (NAD(P)-ICDH from Archaea and Bacteria), subfamily II (NADPH-ICDH from Eucarya and Bacteria), and subfamily III (NAD-ICDH from Eucarya) [5,18]. All archaeal ICDHs characterised so far belong to subfamily I [19]. This result is con¢rmed by the presence of NADP-ICDH from H. volcanii in subfamily I.
3.3. Comparison of the coding sequence and phylogeny The amino acid sequence alignment (Fig. 1) of H. volcanii ICDH with those of several organisms con¢rms that, as shown previously [9], H. volcanii ICDH is most closely related to bacterial dimeric NADP-linked isocitrate dehydrogenases. All the amino acid residues involved in the
3.4. Expression of recombinant H. volcanii isocitrate dehydrogenase in E. coli Conditions for optimal expression were determined empirically. These included growth of cultures at 37‡C to low density and induction of the lac promoter with IPTG for
Fig. 2. Phylogenetic relationships of archaeal ICDH proteins. A phylogenetic tree was constructed using the evolutionary analysis programs of the SCI Sequence Analysis Package. Sequence alignment and the tree were constructed with the CLUSTAL W program. Coding regions for the ICDH polypeptides were derived from the sequences obtained from the SWISS-PROT and TrEMBL databases. H. volcanii is in bold type. Numbers indicate the bootstrap scores of 1000 trials.
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E. coli host enzyme prior to solubilisation, refolding and puri¢cation of the expression product [20]. 3.5. Refolding of recombinant H. volcanii isocitrate dehydrogenase
Fig. 3. SDS^gel electrophoresis of recombinant H. volcanii ICDH. A: Expression of insoluble recombinant H. volcanii ICDH. Lanes: 1, molecular mass marker proteins (with molecular mass values indicated in the left hand margin); 2, wild-type ICDH; 3, cell extract (soluble fraction); 4, cell extract (insoluble pellet). B: Puri¢cation of recombinant H. volcanii NADP-speci¢c isocitrate dehydrogenase. Lanes: 1, protein standards (with molecular mass values indicated in the left hand margin); 2, wild-type ICDH; 3, inclusion bodies ; 4, ultra¢ltration on a PM 50 membrane with a stirred cell; 5, dialysed puri¢ed isocitrate dehydrogenase in 20 mM Tris^HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2 .
3 h prior to harvesting the cells. On induction of cultures with IPTG, a protein band with a subunit molecular mass of W60 000 Da was observed in the insoluble fraction after sonication of whole cells (Fig. 3A). The expressed protein had a subunit very similar from that of the native enzyme [9]. No similar protein band was detected in the soluble fraction, and thus the majority of the expressed protein was in the form of inclusion bodies, as shown with glucose dehydrogenase from H. mediterranei [16]. Attempts were made to improve the solubility of recombinant ICDH by reducing the growth temperature to 25‡C, but this proved unsuccessful. On the other hand, the induction of cultures was performed in the presence of 0.3 M NaCl using E. coli BL21-SI competent cells (Novagen), that contain a chromosomal insertion of the T7 RNA polymerase gene under control of the proU promoter. Addition of 0.3 M salt to the growth medium could induce expression of T7 RNA polymerase, and consequently, genes cloned behind a T7 promoter. Growth was in the absence of salt; however, induction of these E. coli cells was much slower and there was no increase in the expression of soluble ICDH. The formation of inclusion bodies can be used to advantage, in that the expressed protein is resistant to cytoplasmic proteases and it can be easily puri¢ed from the cell lysate. This allows removal of the corresponding, soluble
To obtain active enzyme, the pellet was dissolved in solubilisation bu¡er, and the H. volcanii ICDH activity was restored by diluting the solution with various salts. The e⁄ciency of renaturation and restoration of enzymic activity was determined for di¡erent salts (KCl and NaCl) and di¡erent salt concentrations (0.5^4 M), and also 20% glycerol. The highest enzymic activity was obtained at 3 M NaCl. We investigated the e¡ect of a number of conditions on the refolding of ICDH (not shown), which included several temperatures, bu¡ers or variation of the MgCl2 concentration. The presence or absence of DTT was also studied. Very small or no activity was recovered with all these e¡ects. Attempts to refold the ICDH inclusion bodies after solubilisation with guanidine^HCl, combined with the renaturation bu¡er described above, failed to recover any activity. A similar e¡ect of denaturant was also observed during studies on the refolding of H. volcanii dihydrolipoamide dehydrogenase [20]. The presence of the substrate isocitrate (10 mM) did not a¡ect the refolding of the enzyme, contrary to what happens with H. volcanii dihydrolipoamide dehydrogenase [20]. 3.6. Puri¢cation of recombinant H. volcanii isocitrate dehydrogenase Renatured H. volcanii ICDH was puri¢ed as described in Section 2, and a detailed characterisation of its properties was undertaken. A yield of 72% was obtained, with a ¢nal speci¢c activity of 23.7 U mg31 (Table 1). SDS^ PAGE analysis of the puri¢ed, renatured, recombinant ICDH displayed a single protein band with a molecular mass of 60 000 Da (Fig. 3B). About 185 mg of recombinant H. volcanii ICDH were obtained from 1 l of E. coli culture. Instead, 0.25 mg of the pure enzyme can be obtained from 1 l of H. volcanii culture ; therefore, this expression system is much more useful for obtaining the pure enzyme. 3.7. Properties of the expressed enzyme The dependence of enzyme activity on the concentration
Table 1 Puri¢cation of recombinant H. volcanii ICDH from E. coli BL21 (DE3) cells Step
Total activity (U)
Total protein (mg)
Speci¢c activity (U mg31 )
Yield (%)
Puri¢cation (-fold)
Cell extract Ultra¢ltration DEAE-cellulose Dialysis
341 317 189 248
92.5 24.6 18.3 10.4
3.7 12.9 10.4 23.7
100 93 55 72
1 3.5 2.8 6.4
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of NaCl and KCl for H. volcanii ICDH, expressed in E. coli, was assayed under the standard conditions of 0.4 mM NADPþ and 1 mM D,L-isocitrate. The plot was in good agreement with the salt activity pro¢le of native ICDH [9], the activity of the expressed enzyme being maximal at 1 M NaCl or KCl. At high salt concentrations the stimulatory e¡ect of KCl was greater than that of NaCl, so this recombinant enzyme behaves similarly to the native isocitrate dehydrogenase [9] with respect to the dependence of activity on salt concentration. Kinetic analysis has also shown the puri¢ed recombinant and native enzymes to be similar [9]. For the puri¢ed recombinant enzyme, the Km values for isocitrate and NADPþ were 105 Y 16 and 93 Y 2 WM, respectively, compared to 130 Y 20 and 110 Y 20 WM for the puri¢ed native enzyme [9]. The respective Vmax values for both enzymes were found to be 77 Y 10 and 63 Y 4 U mg31 , respectively. In the presence of 3 M NaCl, the thermal stability properties of recombinant ICDH are comparable to those of native enzyme, with similar stability. At 60‡C, the enzyme has 93% of residual activity after 117 h and at 80‡C, the enzyme loses completely activity after 2.5 min. The activation energy for the inactivation process is 625 kJ mol31 , very similar to the obtained value for the native ICDH at high KCl concentration, 610 kJ mol31 [9], showing that this recombinant enzyme is equally resistant to heat denaturation as the native ICDH. The results presented show that heterologous expression in E. coli is a suitable strategy for the production of halophilic isocitrate dehydrogenase.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
Acknowledgements We thank Dr. David W. Hough and Dr. Michael J. Danson (University of Bath, UK) for their kind help. We also thank Dr. Debbie Maddox (University of Bath, UK) for the collaboration in the lambda library construction, supported by a NATO Collaborative Research Grant (DWH, MJB). Financial support is gratefully acknowledged from CICYT, PB98-0969.
[15] [16]
[17]
References [18] [1] Miyazaki, K., Eguchi, H., Yamagishi, A., Wakagi, T. and Oshima, T. (1992) Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8. Appl. Environ. Microbiol. 58, 93^98. [2] Ishii, A., Suzuki, M., Sahara, T., Takada, Y., Sasaki, S. and Fukunaga, N. (1993) Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J. Bacteriol. 175, 6873^6880. [3] Muro-Pastor, M.I. and Florencio, F.J. (1994) NADPþ -isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC
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7220 : puri¢cation and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. J. Bacteriol. 176, 2718^ 2726. Miyazaki, K. (1996) Isocitrate dehydrogenase from Thermus aquaticus YT1: puri¢cation of the enzyme and cloning, sequencing, and expression of the gene. Appl. Environ. Microbiol. 62, 4627^4631. Steen, I.H., Lien, T. and Birkeland, N.-K. (1997) Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus. Arch. Microbiol. 168, 412^420. Hurley, J.H., Dean, A.M., Koshland, D.E. and Stroud, R.M. (1991) Catalytic mechanism of NADP-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP complexes. Biochemistry 30, 8671^8678. Cherbavaz, D.B., Lee, M.E., Stroud, R.M. and Koshland Jr., D.E. D resolution (2000) Active site water molecules revealed in the 2.1 A structure of a site-directed mutant of isocitrate dehydrogenase. J. Mol. Biol. 295, 377^385. Danson, M.J. and Wood, P.A. (1984) Isocitrate dehydrogenase of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. FEBS Lett. 172, 289^293. Camacho, M.L., Brown, R.A., Bonete, M.J., Danson, M.J. and Hough, D.W. (1995) Isocitrate dehydrogenases from Haloferax volcanii and Sulfolobus solfataricus: enzyme puri¢cation, characterisation and N-terminal sequence. FEMS Microbiol. Lett. 134, 85^90. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (Eds.) (1989) Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley-Interscience, New York. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403^410. Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA 85, 2444^ 2448. Deveraux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 333^446. Thompson, J.D., Higgins, D.G. and Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-speci¢c gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673^4680. Kumar, S., Tsai, C.-J. and Nussinov, R. (2000) Factors enhancing protein thermostability. Protein Eng. 13, 179^191. Pire, C., Esclapez, J., Ferrer, J. and Bonete, M.-J. (2001) Heterologous overexpression of glucose dehydrogenase from the halophilic Archaeon Haloferax mediterranei, an enzyme of the medium chain dehydrogenase/reductase family. FEMS Microbiol. Lett. 200, 221^ 227. Singh, S.K., Matsuno, K., LaPorte, D.C. and Banaszak, L.J. (2001) Crystal structure of Bacillus subtilis isocitrate dehydrogenase at 1.55 D . Insights into the nature of substrate speci¢city exhibited by EsA cherichia coli isocitrate dehydrogenase kinase/phosphatase. J. Biol. Chem. 276, 26154^26163. Dean, A.M. and Golding, G.B. (1997) Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase. Proc. Natl. Acad. Sci. USA 94, 3104^3109. Steen, I.H., Madern, D., Karlstro«m, M., Lien, T., Ladenstein, R. and Birkeland, N.-K. (2001) Comparison of isocitrate dehydrogenase from three hyperthermophiles reveals di¡erences in thermostability, cofactor speci¢city, oligomeric state, and phylogenetic a⁄liation. J. Biol. Chem. 276, 43924^43931. Connaris, H., Chaudhuri, J.B., Danson, M.J. and Hough, D.W. (1999) Expression, reactivation, and puri¢cation of enzymes from Haloferax volcanii in Escherichia coli. Biotechnol. Bioeng. 64, 38^45.
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NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning, sequence determination and overexpression in Escherichia coli Mo¤nica Camacho, Adoracio¤n Rodr|¤guez-Arnedo, Mar|¤a-Jose¤ Bonete
Divisio¤n de Bioqu|¤mica y Biolog|¤a Molecular, Facultad de Ciencias, Universidad de Alicante, Ap. 99, E-03080 Alicante, Spain Received 26 November 2001; received in revised form 7 January 2002; accepted 7 January 2002 First published online 8 March 2002
Abstract A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a V EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45 837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl2 and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature. 9 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Isocitrate dehydrogenase; Halophile; Archaea; Haloferax volcanii
1. Introduction The NADPþ -dependent Ds-threo-isocitrate dehydrogenase (Ds-threo-isocitrate:NADPþ oxidoreductase (EC 1.1.1.42)) (ICDH) is a member of the tricarboxylic acid cycle, which catalyses the oxidative decarboxylation of Ds-threo-isocitrate to form 2-oxoglutarate, CO2 , and NADPH. 2-Oxoglutarate can be further oxidised within the cycle or reductively aminated to glutamate, and NADPH can be used for reductive biosynthetic reactions. ICDH thus supplies the cell with a key intermediate of energy metabolism, as well as with precursors and reducing power for anabolic pathways. The gene encoding isocitrate dehydrogenase has been cloned and sequenced from a variety of eukaryal and bac-
* Corresponding author. Tel. : +34 (96) 590 3524; Fax : +34 (96) 590 3880. E-mail address : [email protected] (M.-J. Bonete).
terial sources [1^5]. In addition, the crystal structure of the NADP-linked enzyme from Escherichia coli has been D in both liganded and unliganded forms solved to 2.5 A [6], and the structure has been related to the catalytic mechanism [7]. Archaea contain a single homodimeric isocitrate dehydrogenase that is either NADP-dependent or shows dual coenzyme speci¢city [8,9]. In a previous paper [9], we reported the puri¢cation, characterisation and N-terminal sequencing of isocitrate dehydrogenase from the extremely halophilic Archaeon, Haloferax volcanii, which grows at 2^4 M NaCl with isotonic concentrations of KCl internally. Here, we describe the cloning of the gene encoding this enzyme from H. volcanii. The DNA sequence was determined, and the derived amino acid sequence was compared with known ICDH sequences. The yields of E. coli-expressed enzyme were greater than those obtained by puri¢cation of the enzyme from the native organism, but the product was insoluble inclusion bodies. This required refolding of the recombinant halophilic protein, and the active enzyme was recovered, with characteristics similar to the wild-type protein.
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2. Materials and methods 2.1. Preparation of a probe by PCR For the isolation of the gene for ICDH, a probe was prepared from genomic DNA by polymerase chain reaction (PCR). The sense primer (oligonucleotide 1) had the base sequence 5P-GA(C,T)GA(C,T)GG(C,G)GA(A,G)AA(A,G)AT-3P, where the parentheses represent the degenerate sites (amino acid sequence NH2 -Asp Asp Gly Glu Lys Ile); it was based on its N-terminal amino acid sequence [9]. The antisense primer (oligonucleotide 2) had the base sequence 5P-CCCTC(C,G)GTGAACTTCAT-3P (amino acid sequence NH2 -Pro Ser Val Asn Phe Ile); it was based on a consensus sequence between E. coli (P08200) and Sulfolobus solfataricus (Q97WN0). A product of the expected size (650 bp) was detected by electrophoresis and labelled with digoxigenin-dUTP using a Nonradioactive DNA Labeling and Detection Kit (Roche Molecular Biochemicals). It was used as a probe in Southern blot and V plaque hybridisation experiments [10]. 2.2. Genomic library manipulations and DNA sequencing A genomic library of H. volcanii DNA was constructed in E. coli XL1-Blue MRA (P2) strain (Stratagene) using Sau3A-digested DNA ligated into V EMBL3 (Stratagene). V plaques were blotted onto Hybond-Nþ membranes (Amersham Pharmacia Biotech) and hybridised with the probe prepared above. Plaques that hybridised strongly with the non-radioactive probe were picked and replated to authenticate their hybridisation by a second plaques blot. Isolation and puri¢cation of the phage and subsequent DNA extraction were carried out by Lambda Maxi Kit (Qiagen). Nucleotide sequence determination was performed by the dideoxy chain termination method using a 310 DNA sequencer (Applied Biosystems). Synthesised icdh-speci¢c oligonucleotides were used as primers, developing the ‘primer walking’ strategy. Searches in the SWISS-PROT and TrEMBL databases for sequence similarities were carried out with the programs BLAST [11] and FASTA [12], and the alignment of isocitrate dehydrogenase sequences was done with PILEUP and BESTFIT by using the sequence analysis software package of the University of Wisconsin Genetics Computer Group [13]. A phylogenetic tree was constructed using CLUSTAL W [14]. The phylogenetic tree was validated by a bootstrap analysis (1000 replicates).
and BamHI to facilitate the subcloning of the H. volcanii ICDH gene for expression. Template DNA for PCR was the clone from the V EMBL3 library. The PCR product was subcloned into plasmid pCR0 2.1. After transforming E. coli Novablue cells (Novagen) with this construct, plasmid DNA was ligated into expression vector pET-3a (Novagen). The resulting clones were propagated in E. coli Novablue cells prior to transforming E. coli BL21 (DE3) (Novagen), which was used as the expression host. Cultures were grown to an OD600nm at 37‡C of 0.5^1.0 in Luria^Bertani (LB) medium containing 50 Wg ml31 ampicillin. Induction of expression was achieved by the addition of isopropyl L-D-thiogalactoside (IPTG) to a ¢nal concentration of 0.4 mM, and the cultures were shaken at 37‡C for 3 h before harvesting cells by centrifugation. 2.4. Refolding and puri¢cation of insoluble recombinant isocitrate dehydrogenase E. coli BL21 (DE3) cell pellets were resuspended in 20 mM Tris^HCl bu¡er, pH 8.0, containing 2 mM EDTA and 2 M KCl, treated with 100 Wg ml31 lysozyme and 0.1% (v/v) Triton X-100, and incubated at 30‡C for 1 h before transferring the solution onto ice for 15 min. The suspension was then sonicated and centrifuged at 10 000Ug for 10 min at 4‡C. The insoluble fraction yielded inclusion bodies together with the insoluble cell fragments, and it was dissolved in solubilisation bu¡er (20 mM Tris^ HCl pH 8.0, containing 2 mM EDTA, 8 M urea and 50 mM dithiothreitol (DTT)). Refolding was initiated by slowly diluting the solubilised pellet to a ¢nal concentration of 20 Wg ml31 into renaturation bu¡er and incubating for at least 24 h at room temperature. The renaturation bu¡er was composed of 20 mM Tris^HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2 . The solution was concentrated by ultra¢ltration on a PM 50 membrane with a stirred cell (Amicon Inc., Beverly, MA, USA) to a ¢nal volume of 10 ml. The bu¡er was exchanged to 20 mM Tris^HCl pH 8.0, containing 2.5 M (NH4 )2 SO4 , 1 mM EDTA and 10 mM MgCl2 prior to further chromatography on a DEAE-cellulose (Amersham Pharmacia Biotech) column equilibrated with the same bu¡er, and the enzyme was eluted with the renaturation bu¡er. Fractions containing isocitrate dehydrogenase activity were pooled and, ¢nally, dialysed against the same renaturation bu¡er for 24 h at 4‡C to remove the (NH4 )2 SO4 remaining, and stored at this temperature. 2.5. Enzyme and protein assays
2.3. Expression of H. volcanii isocitrate dehydrogenase in E. coli PCR techniques were used to amplify a gene fragment corresponding to the structural gene of H. volcanii ICDH with 1260 bp, including the digestion sequences of NdeI
The H. volcanii ICDH activity was assayed spectrophotometrically as previously [9] and the values of Vmax and Km were determined. Protein concentration was determined by the method of Bradford using bovine serum albumin as standard. SDS^polyacrylamide gel electropho-
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resis was performed according to the method described by Laemmli. 2.6. Thermal stability assays Thermal stability studies were carried out in the presence of salt. H. volcanii ICDH was incubated in 20 mM Tris^HCl bu¡er, pH 8.0, 1 mM EDTA, 10 mM MgCl2 containing 3 M NaCl, at temperatures in the range 60^ 80‡C for times up to several days. Samples were removed at known time intervals, rapidly cooled in ice, and the remaining activity determined by the standard assay procedure.
3. Results and discussion 3.1. PCR using oligonucleotides to conserved sequences Several ICDH sequences are known from a wide variety of bacterial and eukaryotic species. Analysis of these sequences reveals a high degree of similarity, especially in
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regions of known functional signi¢cance. Oligonucleotides 1 and 2 (sense and antisense, respectively, both described in Section 2) produced a product of about 650 bp on PCR with H. volcanii genomic DNA as target. This fragment was sequenced to con¢rm its identity and then used as a probe in order to determine the rest of the gene. 3.2. Isolation of a clone from a V EMBL3 library and sequence of the H. volcanii NADP-ICDH gene The PCR fragment was labelled with digoxigenindUTP, producing a probe that was used to screen a V EMBL3 genomic library of H. volcanii. A clone was obtained which hybridised to the probe at 65‡C. Partial sequencing of this clone revealed that it contained part of the ICDH gene. The structural analysis of the enzyme will give us abundant information in elucidating the molecular basis of the relationship between the function, such as coenzyme and substrate speci¢cities. The amino acid sequence of the coding region for the H. volcanii NADP-ICDH gene is shown in Fig. 1. The ICDH gene starts with ATG, a methionine
Fig. 1. Alignment of ICDH sequences. The derived amino acid sequence of H. volcanii ICDH was aligned with other known ICDH sequences using the program PILEUP. The alignment with the sequences from Halobacterium, B. subtilis and E. coli are shown. Asterisks indicate perfectly conserved residues and dots indicate well-conserved residues. Dashes denote gaps introduced into the sequence to achieve maximum alignment. Single-underlined residues and residues marked by double underlining are those that contact substrate and coenzyme, respectively, in the E. coli enzyme. The nucleotide sequence for H. volcanii ICDH has been submitted to the EMBL Nucleotide Sequence Database under accession No. AJ251585.
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codon, and the protein deduced from the nucleotide sequence was composed of 419 amino acids with a molecular mass of 45 837 Da, in disagreement with that determined by SDS^PAGE [9], and a calculated pI of 4.56. The amino acid sequence includes ¢ve Trp, one Cys, and six His residues, 31% hydrophobic residues (Phe+Tyr+Trp+Ile+Leu+Met+Val), and 18 and 11%, respectively, acidic and basic residues. The only cysteine residue is in position 332. ICDHs from Archaeoglobus fulgidus [5], Halobacterium (Q9HNZ7) and Thermus aquaticus [4] have also one Cys; S. solfataricus ICDH (Q97WN0) has no Cys and E. coli enzyme has six [6], which explains the decreased thermostability of the bacterial enzyme [15]. The secondary structure of the enzyme was obtained with the same GCG software package cited before. The results show 12 K-helical and 12 L-sheet conformations in the polypeptide. Like other ICDHs, in the halophilic enzyme there is no Rossman fold characteristic of the nucleotide binding, which is present in other kinds of dehydrogenases (i.e. alcohol dehydrogenases) as shown in glucose dehydrogenase from Haloferax mediterranei [16].
binding of isocitrate or coenzyme in E. coli isocitrate dehydrogenase are conserved except a few residues, which present conservative changes (Fig. 1), indicating that the enzymes probably have similar reaction mechanism and structure. The three-dimensional structure of these enzymes is only known for E. coli ICDH [6] and Bacillus subtilis [17]. A phylogenetic tree (Fig. 2) generated by comparing the most characteristic archaeal complete isocitrate dehydrogenase amino acid sequences available from sequence databases, rooted with a bacterial ICDH, revealed the great identity of the H. volcanii enzyme with that from Halobacterium (70.4%), another halophilic organism, and the sequence comparison also shows that isocitrate dehydrogenase is a very conserved enzyme [5]. ICDHs have divided into three distinct phylogenetic subfamilies: subfamily I (NAD(P)-ICDH from Archaea and Bacteria), subfamily II (NADPH-ICDH from Eucarya and Bacteria), and subfamily III (NAD-ICDH from Eucarya) [5,18]. All archaeal ICDHs characterised so far belong to subfamily I [19]. This result is con¢rmed by the presence of NADP-ICDH from H. volcanii in subfamily I.
3.3. Comparison of the coding sequence and phylogeny The amino acid sequence alignment (Fig. 1) of H. volcanii ICDH with those of several organisms con¢rms that, as shown previously [9], H. volcanii ICDH is most closely related to bacterial dimeric NADP-linked isocitrate dehydrogenases. All the amino acid residues involved in the
3.4. Expression of recombinant H. volcanii isocitrate dehydrogenase in E. coli Conditions for optimal expression were determined empirically. These included growth of cultures at 37‡C to low density and induction of the lac promoter with IPTG for
Fig. 2. Phylogenetic relationships of archaeal ICDH proteins. A phylogenetic tree was constructed using the evolutionary analysis programs of the SCI Sequence Analysis Package. Sequence alignment and the tree were constructed with the CLUSTAL W program. Coding regions for the ICDH polypeptides were derived from the sequences obtained from the SWISS-PROT and TrEMBL databases. H. volcanii is in bold type. Numbers indicate the bootstrap scores of 1000 trials.
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E. coli host enzyme prior to solubilisation, refolding and puri¢cation of the expression product [20]. 3.5. Refolding of recombinant H. volcanii isocitrate dehydrogenase
Fig. 3. SDS^gel electrophoresis of recombinant H. volcanii ICDH. A: Expression of insoluble recombinant H. volcanii ICDH. Lanes: 1, molecular mass marker proteins (with molecular mass values indicated in the left hand margin); 2, wild-type ICDH; 3, cell extract (soluble fraction); 4, cell extract (insoluble pellet). B: Puri¢cation of recombinant H. volcanii NADP-speci¢c isocitrate dehydrogenase. Lanes: 1, protein standards (with molecular mass values indicated in the left hand margin); 2, wild-type ICDH; 3, inclusion bodies ; 4, ultra¢ltration on a PM 50 membrane with a stirred cell; 5, dialysed puri¢ed isocitrate dehydrogenase in 20 mM Tris^HCl pH 8.0, containing 3 M NaCl, 1 mM EDTA and 10 mM MgCl2 .
3 h prior to harvesting the cells. On induction of cultures with IPTG, a protein band with a subunit molecular mass of W60 000 Da was observed in the insoluble fraction after sonication of whole cells (Fig. 3A). The expressed protein had a subunit very similar from that of the native enzyme [9]. No similar protein band was detected in the soluble fraction, and thus the majority of the expressed protein was in the form of inclusion bodies, as shown with glucose dehydrogenase from H. mediterranei [16]. Attempts were made to improve the solubility of recombinant ICDH by reducing the growth temperature to 25‡C, but this proved unsuccessful. On the other hand, the induction of cultures was performed in the presence of 0.3 M NaCl using E. coli BL21-SI competent cells (Novagen), that contain a chromosomal insertion of the T7 RNA polymerase gene under control of the proU promoter. Addition of 0.3 M salt to the growth medium could induce expression of T7 RNA polymerase, and consequently, genes cloned behind a T7 promoter. Growth was in the absence of salt; however, induction of these E. coli cells was much slower and there was no increase in the expression of soluble ICDH. The formation of inclusion bodies can be used to advantage, in that the expressed protein is resistant to cytoplasmic proteases and it can be easily puri¢ed from the cell lysate. This allows removal of the corresponding, soluble
To obtain active enzyme, the pellet was dissolved in solubilisation bu¡er, and the H. volcanii ICDH activity was restored by diluting the solution with various salts. The e⁄ciency of renaturation and restoration of enzymic activity was determined for di¡erent salts (KCl and NaCl) and di¡erent salt concentrations (0.5^4 M), and also 20% glycerol. The highest enzymic activity was obtained at 3 M NaCl. We investigated the e¡ect of a number of conditions on the refolding of ICDH (not shown), which included several temperatures, bu¡ers or variation of the MgCl2 concentration. The presence or absence of DTT was also studied. Very small or no activity was recovered with all these e¡ects. Attempts to refold the ICDH inclusion bodies after solubilisation with guanidine^HCl, combined with the renaturation bu¡er described above, failed to recover any activity. A similar e¡ect of denaturant was also observed during studies on the refolding of H. volcanii dihydrolipoamide dehydrogenase [20]. The presence of the substrate isocitrate (10 mM) did not a¡ect the refolding of the enzyme, contrary to what happens with H. volcanii dihydrolipoamide dehydrogenase [20]. 3.6. Puri¢cation of recombinant H. volcanii isocitrate dehydrogenase Renatured H. volcanii ICDH was puri¢ed as described in Section 2, and a detailed characterisation of its properties was undertaken. A yield of 72% was obtained, with a ¢nal speci¢c activity of 23.7 U mg31 (Table 1). SDS^ PAGE analysis of the puri¢ed, renatured, recombinant ICDH displayed a single protein band with a molecular mass of 60 000 Da (Fig. 3B). About 185 mg of recombinant H. volcanii ICDH were obtained from 1 l of E. coli culture. Instead, 0.25 mg of the pure enzyme can be obtained from 1 l of H. volcanii culture ; therefore, this expression system is much more useful for obtaining the pure enzyme. 3.7. Properties of the expressed enzyme The dependence of enzyme activity on the concentration
Table 1 Puri¢cation of recombinant H. volcanii ICDH from E. coli BL21 (DE3) cells Step
Total activity (U)
Total protein (mg)
Speci¢c activity (U mg31 )
Yield (%)
Puri¢cation (-fold)
Cell extract Ultra¢ltration DEAE-cellulose Dialysis
341 317 189 248
92.5 24.6 18.3 10.4
3.7 12.9 10.4 23.7
100 93 55 72
1 3.5 2.8 6.4
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of NaCl and KCl for H. volcanii ICDH, expressed in E. coli, was assayed under the standard conditions of 0.4 mM NADPþ and 1 mM D,L-isocitrate. The plot was in good agreement with the salt activity pro¢le of native ICDH [9], the activity of the expressed enzyme being maximal at 1 M NaCl or KCl. At high salt concentrations the stimulatory e¡ect of KCl was greater than that of NaCl, so this recombinant enzyme behaves similarly to the native isocitrate dehydrogenase [9] with respect to the dependence of activity on salt concentration. Kinetic analysis has also shown the puri¢ed recombinant and native enzymes to be similar [9]. For the puri¢ed recombinant enzyme, the Km values for isocitrate and NADPþ were 105 Y 16 and 93 Y 2 WM, respectively, compared to 130 Y 20 and 110 Y 20 WM for the puri¢ed native enzyme [9]. The respective Vmax values for both enzymes were found to be 77 Y 10 and 63 Y 4 U mg31 , respectively. In the presence of 3 M NaCl, the thermal stability properties of recombinant ICDH are comparable to those of native enzyme, with similar stability. At 60‡C, the enzyme has 93% of residual activity after 117 h and at 80‡C, the enzyme loses completely activity after 2.5 min. The activation energy for the inactivation process is 625 kJ mol31 , very similar to the obtained value for the native ICDH at high KCl concentration, 610 kJ mol31 [9], showing that this recombinant enzyme is equally resistant to heat denaturation as the native ICDH. The results presented show that heterologous expression in E. coli is a suitable strategy for the production of halophilic isocitrate dehydrogenase.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
Acknowledgements We thank Dr. David W. Hough and Dr. Michael J. Danson (University of Bath, UK) for their kind help. We also thank Dr. Debbie Maddox (University of Bath, UK) for the collaboration in the lambda library construction, supported by a NATO Collaborative Research Grant (DWH, MJB). Financial support is gratefully acknowledged from CICYT, PB98-0969.
[15] [16]
[17]
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