Eur. J. Biochem. 271, 3115–3126 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04230.x
Mitochondrial malate dehydrogenase from the thermophilic, filamentous fungus Talaromyces emersonii Purification of the native enzyme, cloning and overexpression of the corresponding gene Alan P. Maloney, Susan M. Callan, Patrick G. Murray and Maria G. Tuohy Molecular Glycobiotechnology Group, Department of Biochemistry, National University of Ireland, Galway, Ireland
Mitochondrial malate dehydrogenase (m-MDH; EC 1.1.1.37), from mycelial extracts of the thermophilic, aerobic fungus Talaromyces emersonii, was purified to homogeneity by sequential hydrophobic interaction and biospecific affinity chromatography steps. Native m-MDH was a dimer with an apparent monomer mass of 35 kDa and was most active at pH 7.5 and 52 C in the oxaloacetate reductase direction. Substrate specificity and kinetic studies demonstrated the strict specificity of this enzyme, and its closer similarity to vertebrate m-MDHs than homologs from invertebrate or mesophilic fungal sources. The fulllength m-MDH gene and its corresponding cDNA were cloned using degenerate primers derived from the N-terminal amino acid sequence of the native protein and multiple sequence alignments from conserved regions of other m-MDH genes. The m-MDH gene is the first oxidoreductase gene cloned from T. emersonii and is the first full-length
m-MDH gene isolated from a filamentous fungal species and a thermophilic eukaryote. Recombinant m-MDH was expressed in Escherichia coli, as a His-tagged protein and was purified to apparent homogeneity by metal chelate chromatography on an Ni2+-nitrilotriacetic acid matrix, at a yield of 250 mg pure protein per liter of culture. The recombinant enzyme behaved as a dimer under nondenaturing conditions. Expression of the recombinant protein was confirmed by Western blot analysis using an antibody against the His-tag. Thermal stability studies were performed with the recombinant protein to investigate if results were consistent with those obtained for the native enzyme.
Malate dehydrogenase (MDH) catalyzes the pyridine nucleotide-dependent interconversion of malate and oxaloacetate in the tricarboxylic acid cycle. It is also thought to have a role in the malate/aspartate shuttle across the inner mitochondrial membrane. In most eukaryotic cells there are two major isoenzymes of MDH, cytosolic MDH (c-MDH) and mitochondrial MDH (m-MDH) [1]. The majority of m-MDHs isolated to date from eukaryotic sources are homodimers of identical subunits, with a monomer size of 34 kDa. Mitochondrial MDH displays a complex regulatory pattern that involves
allosteric activation [2], membrane interaction [3] and formation of multienzyme complexes [4] with enzymes such as citrate synthase, aspartate aminotransferase and other mitochondrial components [5–10]. Much of the previous research has focused on the comparison of primary and three-dimensional structures of mitochondrial and cytoplasmic forms of MDH isolated from a single source, e.g. pig heart tissue [11, 12]. In contrast to bacterial, vertebrate and invertebrate m-MDH, a paucity of information exists on fungal m-MDH with significantly more known about yeast m-MDH than homologs from filamentous fungal species. Previous research by Dalhaus et al. [13] and Goward and Nicholls [14] has proposed that investigation of MDHs from mesophilic and thermophilic sources may provide valuable information on the structural basis for thermal stability, evolution and catalysis. However, to date these studies have been confined solely to prokaryotic sources. This paper reports on the isolation, molecular and biochemical characterization of a native m-MDH and its corresponding gene from a novel, thermophilic filamentous fungal species, Talaromyces emersonii. We also report the overexpression of the fungal gene in Escherichia coli and compare selected properties to those of the native enzyme. Filamentous fungi are highly efficient cell factories for the production of many important enzymes and biopharmaceuticals [15]. The prospect of high-level enzyme secretion, novel
Correspondence to M. G. Tuohy, Department of Biochemistry, National University of Ireland, Galway, Ireland. Fax: + 353 91 512504, Tel.: + 353 91 524411 ext. 2439, E-mail:
[email protected] Abbreviations: c-MDH, cytoplasmic malate dehydrogenase; HIC, hydrophobic interaction chromatography; m-MDH, mitochondrial malate dehydrogenase. Enzyme: mitochondrial malate dehydrogenase (EC 1.1.1.37). Note: The sequence reported in this paper has been deposited in GenBank under the accession numbers AF487682 (T. emersonii mitochondrial malate dehydrogenase cDNA sequence) and AF439996 (T. emersonii m-MDH chromosomal DNA sequence). (Received 3 March 2004, revised 6 May 2004, accepted 26 May 2004)
Keywords: malate dehydrogenase; filamentous fungus; bio-specific affinity chromatography; thermophilic eukaryote; Talaromyces emersonii.
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enzyme variants with high temperature optima and enhanced shelf lives has prompted the search for thermophilic sources of enzymes [16]. T. emersonii is distinguished from thermotolerant counterparts by its growth temperature range of 30–65 C and ability to thrive at growth temperatures between 45 and 55 C (data from our laboratory and [17]). Work conducted in our laboratory has revealed that T. emersonii produces a potent array of complete, multicomponent enzyme systems that degrade key biopolymers in nature, including proteins and various terrestrial and marine plant carbohydrates [18–24]. In addition, more recent studies have revealed that T. emersonii may be a Pandora’s box of other interesting glycosyl hydrolases and oxidoreductases, many of which have homologs in model eukaryotic species [25, 26]. A characteristic feature of several of the T. emersonii enzymes isolated to date is noteworthy thermal stability. This property has resulted in the investigation of this eukaryote as a source of thermozymes for key biotechnological applications, for example, the generation of high value-added products from plantrich wastes [27]. Structure–function analysis of extracellular and intracellular enzymes from this fungus will provide an understanding of the molecular and structural basis of thermal stability in T. emersonii and other eukaryotes. In addition, enzymes that mediate biological oxygen transfer, especially those with enhanced thermal stability, have potential applications as biosensors and in nanotechnology [28]. In this study, kinetic parameters and selected physicochemical properties were determined for the recombinant T. emersonii m-MDH and compared with data obtained from corresponding studies with the native enzyme. These studies, along with site-directed mutagenesis of certain residues in the amino-acid sequence, gives a valuable insight into the effect of putative post-translational and structural modifications on the stability of the native enzyme.
Materials and methods Microorganism cultivation and enzyme production T. emersonii CBS 814.70 was originally obtained from Centraal Bureau voor Schimmelcultures, Baarn, the Netherlands and cultivated as described previously [18,22]. At appropriate timed intervals, mycelia were harvested by filtration through several layers of finegrade sterilized muslin, washed with 75 mM sodium citrate, pH 7.5 and frozen immediately under liquid nitrogen. For cell disruption, 56 g wet weight of mycelia were suspended in 300 mL 50 mM potassium phosphate buffer pH 7.4 (buffer A) and homogenized in the presence of glass beads, using a pestle and mortar. Cell debris was removed by centrifugation at 5000 g in a Sorvall superspeed centrifuge, fitted with a GSE Rotor. Ammonium sulfate [(NH4)2SO4] was added slowly to the supernatant fraction, with thorough mixing, to 35% saturation and the mixture centrifuged at 8000 g for 30 min. The supernatant was recovered, brought to 85% saturation with (NH4)2SO4 and centrifuged at 10 000 g for 30 min. The resulting pellet was dissolved in 100 mL buffer A and was used as crude extract for subsequent
enzymatic analyses and purification studies. A small amount (0.l g) of thimerosal (an antimicrobial agent) was added to the crude extract to prevent microbial contamination. Assay of enzyme activity and protein content MDH activity was determined in a continuous assay by recording the decrease or increase in absorbance at 340 nm, at 30 C using a Shimadzu UV spectrophotometer. MDH activity in the direction of malate formation was assayed at pH 7.4 in buffer A containing 200 lM NADH, and suitably diluted enzyme; the reaction was initiated by the addition of 0.1 mL of 6 mM oxaloacetate solution (final assay volume: 3 mL). For the reverse reaction, i.e. the oxidation of malate, the standard reaction mixture contained buffer A, 10 mM L-malate, 1 mM NAD+ and an aliquot of enzyme solution in a total volume of 3 mL. Enzyme activity was expressed as IUÆmg)1, i.e. micromoles NADH oxidized/NAD+ reduced per minute of reaction time per milliliter of enzyme solution. Protein in crude cellular extracts, fractionated samples and standards (bovine serum albumin) was determined by the Bensadoun and Weinstein protein assay [29]. For convenience, elution of protein during chromatographic purification of m-MDH was monitored by measuring absorbance at 280 nm (A280) of collected fractions. Enzyme purification Hydrophobic interaction chromatography (HIC) was carried out on a column (18.5 · 1.75 cm) of phenyl sepharose CL-4B. The column was washed with buffer A containing 1 M NaCl, pH 7.4 (buffer B), until the conductivity and the pH of the effluent were identical to the starting buffer. The appropriate dry weight of NaCl, equivalent to a final dissolved concentration of 1 M, was added to the crude enzyme sample, after (NH4)2SO4 fractionation, and the pH was adjusted to pH 7.4 with 1 M KOH. The crude extract was applied to the matrix, and washed with five column volumes of buffer B. A decreasing linear NaCl gradient (1–0 M NaCl, i.e. equal volumes of buffer B + buffer A) was applied to the column to elute adsorbed protein (total gradient volume of 500 mL). A final wash of buffer A was applied until all MDH activity had eluted. Fractions (3.3 mL) were collected and assayed for MDH activity. Bio-specific affinity chromatography of the post-HIC, m-MDH rich pool was investigated using an S6-linked NAD+ system (sepharose CL-4B as carrier), with oxalate as the locking-on ligand [31]; the irrigating buffer was buffer A containing 0.1 M oxalate and 0.5 M KCl (buffer C). Samples were concentrated using an Amicon ultrafiltration cell fitted with the appropriate PM-10 membrane (molecular size cut-off > 10 kDa) and dialyzed overnight against buffer A. Directly before sample application, oxalate (0.1 M) and KCl (0.5 M) were added to the sample to yield a final sample concentration of both components equivalent to buffer C. Following sample application, the column was washed with three column volumes of buffer C and two column volumes of buffer C containing 10 mM
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5¢-AMP. The oxalate and 5¢-AMP were then excluded from the irrigating buffer for two further column volumes of eluent. Finally, 1 mM NADH was included with 0.5 M KCl in buffer A (buffer D) to competitively elute adsorbed dehydrogenase components from the immobilized general ligand. Fractions were collected as described earlier and 50 lL aliquots from each fraction (suitably diluted) were assayed for MDH activity. The native molecular mass of the purified MDH was determined by gel filtration on a calibrated Sephacryl S-200 superfine grade matrix (bed dimensions: 2.6 · 100.7 cm; sample application volume of 2 mL). The irrigating buffer was buffer B and the flow-rate was maintained at 13 mLÆh)1. Proteins used for calibration of the gel filtration matrix included low and high relative molecular mass (Mr) purified proteins (Amersham Biosciences Ltd, Buckinghamshire, UK).
Amplification of a DNA fragment encoding a portion of the NAD+ binding domain of the T. emersonii m-MDH was performed by the polymerase chain reaction, using degenerate primers: sense (5¢-TCCAAKGTCGCSATYC TYGGTGCMGCT-3¢) and antisense (5¢-CGSAAGC GYGGCATKACSCGNGAC-3¢). PCR products were cloned into the pGEM-T easy vector (Promega) and sequenced. The purified PCR product (320 bp) was labeled with digoxygenin (Roche Molecular Biochemicals), purified and used as a probe for Northern blot analysis.
Electrophoresis
Isolation of a full-length mMDH cDNA clone
Polyacrylamide gel electrophoresis (PAGE) was conducted under denaturing and nondenaturing conditions [18], as described by Laemmli. Isoelectric focusing (IEF) was carried out on ultrathin (1 mm) thick preprepared Ampholine PAGplates (Amersham Biosciences), over the range from pH 3.5–9.5, according to the manufacturer’s instructions (booklet no. 18-1016-67). Zymogram staining of native PAGE and IEF gels to detect m-MDHactive protein bands was performed as described by Epstein et al. [32].
Talaromyces emersonii was grown in nutrient medium described earlier [18], containing 2% (w/v) pectin as a carbon source. Total RNA was isolated from mycelia after growth for 96 h as described by Chomczynski and Sacchi [33] and used as template for RACE-PCR in a modification of the Ambion RACE-PCR protocol, as described previously [21]. 5¢-RACE primers were: outer (5¢-TTCA TACCACGCTTGCGAGGAA-3¢) and inner (5¢-AGT GACGGTGCTGTTCGTGTT-3¢); and 3¢-RACE primers were: outer (5¢-AACACGAACAGCACCGTCACT-3¢) and inner (5¢-TTCCTCGCAAGCGTGGTATGAA-3¢). Full-length m-MDH cDNA was amplified from T. emersonii first-strand cDNA, by PCR with primers corresponding to the putative 5¢- and 3¢-sequences derived from 5¢- and 3¢-RACE products (m-MDH sense primer 5¢-TCATGTTCGCTACTCGCCAGG-3¢ and m-MDH antisense primer 5¢-TGTCAAGAACAACCCCTAAGCG-3¢). PCR products were cloned and sequenced as described earlier.
Determination of selected biochemical properties Investigation of the optimum pH for m-MDH activity in the oxaloacetate reductase direction was performed with 200 lM oxaloacetate and 200 lM NADH over a wide pH range (4–12) and at 30 C. Determination of the pH optimum in the malate oxidation direction was achieved with 10 mM malate and 1 mM NAD+ as substrate and cosubstrate, respectively, over the same pH range at 30 C. The temperature optimum in the oxaloacetate reductase direction was performed with 200 lM NADH and 200 lM oxaloacetate in buffer A. Temperature stability was determined by monitoring the residual activity of buffered enzyme sample (diluted to the normal assay concentrations) at a range of temperatures including the normal assay temperature, 50 C. Preliminary evaluation of the catalytic properties of m-MDH Kinetic studies were conducted with highly purified samples of native and recombinant m-MDHs, using the assay procedure outlined earlier, in both the oxaloacetate reduction and malate oxidation directions. Km, Vmax, kcat and kcat/Km were estimated using standard formulae and from linearization of Michaelis–Menten plots. N-Terminal sequence analysis The N-terminal sequence of the native m-MDH protein was determined by Edman degradation (J. Gray, University
of Newcastle, UK). Sufficient sequence information was obtained to enable unambiguous identification of the enzyme fragment. PCR using degenerate primers and probe preparation
Northern analysis Total RNA (10 lg) from T. emersonii mycelia, cultivated on various inducing substrates, was separated electrophoretically on a 1.2% (w/v) formaldehyde-agarose gel and used for Northern blot analysis with the DIG-labeled probe described above. Mitochondrial MDH signals were normalized against 18S rRNA loading control using the TM DENSITOMETER 700 software on a Fluor-S MultiImager (Bio-Rad, UK). Nucleic acid sequence analysis Sequencing reactions were carried out by Altabisoscience Laboratories, University of Birmingham, UK (http:// www.altabio.bham.ac.uk/). Cycle sequencing reactions were carried out on a PE Biosystems 877 robotic system. Database similarity searches were performed using the National Centre for Biotechnological Information (NCBI) online program BLAST [34] against protein (BLASTP) and nucleotide (BLASTX) sequences stored in GenBank.
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Results and Discussion
Expression of m-MDH in Escherichia coli cells Primers F1, 5¢-CACCCAGCAGGCCGGCACGGCG-3¢ and R1, 5¢-TCACGAAGCGGTGAAGGTCGAGTT-3¢ corresponding to the N- and C-terminal regions of the mature protein (the signal peptide, consisting of amino acids 1–19, was removed) were used to amplify full-length m-MDH cDNA. The CACC corresponding to the GTGG overhang in the TOPO cloning vector (Invitrogen) is underlined in F1 above. The purified PCR product was ligated into the pENTR/SD/D-TOPO vector and transformed into One Shot Top10 E. coli competent cells according to the manufacturer’s instructions. An LR recombination reaction was performed between the entry clone, pE-m-MDH, and the pDEST-17 destination vector (Invitrogen) which was subsequently transformed into E. coli DH5a library efficient cells generating the expression clone with an N-terminal polyhistidine tag (His6). For expression, plasmid DNA was purified and transformed into BL21AITM (Invitrogen) competent E. coli cells. Pilot experiments indicated that m-MDH protein was expressed in the soluble supernatant fraction. The overexpressed protein was purified using a nickel-nitrilotriacetic acid purification column (Invitrogen) according to the manufacturer’s instructions. Immunoblot analysis Samples were resuspended in Laemmli’s SDS/PAGE sample buffer and boiled for 5 min. Proteins (20 lg per lane) were then resolved on 12% SDS/PAGE gels and electrophoretically transferred onto nitrocellulose for 1.5 h at 100 V. Membranes were blocked overnight in NaCl/Pi containing 0.05% (v/v) Tween 20 and 5% (w/v) nonfat dried milk. The membranes were then incubated overnight with mouse monoclonal antibody to the His6 tag (1 : 1000 dilution). This was followed by 3 h incubation with appropriate horseradish peroxidase-conjugated goat anti-(mouse IgG) Ig (1 : 10 000 dilution). Protein bands were then visualized using Supersignal West pico Western blot detection kit (Pierce). Isolation of mitochondria Mitochondria were obtained by the method according to Seebald et al. [30].
Enzyme purification and selected physicochemical properties Mitochondrial MDH was purified from T. emersonii crude extracts as summarized in Table 1. T. emersonii m-MDH was obtained at a 21.7% yield and had a final specific activity of 1325.0 IUÆmg)1. The 35–85% (NH4)2SO4 precipitation step yielded a pellet rich in MDH activity [considerable protein and particulate material had been removed by the initial 0–35% (NH4)2SO4 precipitation step], and also reduced the crude extract to a more manageable volume. However, the viscosity of the resultant resuspended pellet sample was high, which would have interfered markedly with subsequent affinity chromatography. Inclusion of a HIC step prior to bio-affinity chromatography not only reduced sample viscosity, but also minimized interfering factors and markedly decreased levels of contaminating protein, which resulted in improved adsorption characteristics and performance of the affinity matrix. In addition, HIC facilitated separation of the m-MDH component from c-MDH (Fig. 1A), which was also present in the same crude extract (verified by bioaffinity chromatography and zymogram staining of IEF gels; results not shown). The final native m-MDH preparation was adjudged pure by SDS/ PAGE and a subunit Mr of 35 kDa (Fig. 1B) was estimated. Gel filtration on a Sephacryl S-200 (superfine grade) matrix yielded a single protein (and coincidental enzyme active) peak with an estimated Mr of 70 kDa, which was also confirmed by native PAGE, which suggests that m-MDH from T. emersonii is a homodimer. This finding compares well with values reported for native m-MDHs from the yeasts Phycomyces blakesleeanus [35] and Cryptococcus neoformans [36]. Isoelectric focusing revealed the presence of three protein bands in the m-MDH sample (Fig. 1C). Zymogram staining of a replicate gel for m-MDH activity revealed that enzyme-active and protein bands were coincidental, and therefore it was concluded that the bands represent subforms of m-MDH. MDH subforms have been previously reported in the literature [37,38] and one of the main schools of thought suggests that they arise from interconversion of conformational isomers of different net molecular charge [39,40].
Table 1. Summary of the purification of m-MDH from T. emersonii. Data for the c-MDH pool obtained by HIC is given for comparative purposes.
Fraction Crude mycelial extract 35% (NH4)2SO4 fractionation (supernatant) 85% (NH4)2SO4 fractionation (resuspended pellet) Phenyl sepharose chromatography m-MDH c-MDH Immobilized S6-linked bio-affinity chromatography Ni-nitrilotriacetic acid purified His6-tagged recombinant protein
Volume (mL)
Enzyme activity (UÆmL)1)
Protein (mgÆmL)1)
Specific activity (IUÆmg)1)
Yield (%)
Purification factor
325.0 340.0
7.5 7.8
0.17 0.073
44.6 106.8
100 108.8
1.0 2.1
100.0
24.4
0.176
138.4
99.9
2.7
180.0 175.0 25.0
3.7 7.5 21.2
0.029 0.04 0.016
125.4 188.3 1325.0
27.3 53.8 21.7
2.5 3.7 26.3
2.0
402.5
0.25
1610.0
48
46
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Fig. 1. Purification of native m-MDH from T. emersonii. (A) Fractionation of (NH4)2SO4 precipitated MDH-rich pellet by HIC on phenyl sepharose CL-4B, pH 7.4. (B) Coomassie blue-stained SDS/PAGE illustrating the purity of m-MDH isolated from T. emersonii. Lane 1, protein molecular mass markers; lane 2, protein molecular mass markers; lane 3, fractionated m-MDH. (C) Isoelectric focusing gel of m-MDH from T. emersonii. Lane 1, m-MDH; pI values obtained were 5.4, 5.75 and 5.9, respectively; lane 2, pI standard proteins.
N-Terminal sequence analysis The amino acid sequence of the N-terminal fragment of the purified m-MDH was determined. Sufficient sequence information was obtained (11 amino acids) to allow unambiguous identification of the enzyme and the design of degenerate primers for PCR amplification. The sequence obtained was XKVAILGAAGGI, which has high homology to other eukaryotic and prokaryotic m-MDHs (Fig. 2). Isolation of the m-MDH gene from T. emersonii A 320 bp genomic fragment, with high homology against other eukaryotic mitochondrial malate dehydrogenases in the database, was amplified from T. emersonii chromosomal DNA. Full-length cDNA and genomic clones were obtained as described earlier; comparison of both types of
sequence data reveals the presence of five introns in the genomic sequence, all approximately 70 bases in length. Bioinformatic analysis of the cDNA sequence indicates a predicted amino acid sequence length of 339 amino acids and a predicted molecular mass of 33.3 kDa, which compares well with the monomer Mr for the native protein determined by SDS/PAGE. The amino acid sequence, deduced from the full-length cDNA sequence, had 63 and 61% identity with Saccharomyces cerevisiae and Schizosaccharomyces pombe m-MDHs, respectively; an overall homology of 53% was obtained on alignment of all three sequences using the CLUSTALW program [41] (Fig. 2). Phylogenetic analysis revealed that the T. emersonii m-MDH clusters with other eukaryotic m-MDHs, but also with E. coli MDH which would support the endosymbiont theory on mitochondrial evolution. In keeping with this, the T. emersonii m-MDH clusters less well with LDH enzymes.
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Fig. 2. CLUSTALW alignment of the cloned precursor T. emersonii mMDH (GenBank accession number AAL93265), with other eukaryotic and prokaryotic mMDH sequences. S. cerev, Saccharomyces cerevisiae (accession number NP012838); S. pombe, Schizosaccharomyces pombe (accession number NP587816); pig, Sus scrofa (accession number AF218064); mouse, Mus muluscus (accession number M16229); and E. coli (accession number NP417703). Identical residues are highlighted with asterisks; s above a residue indicates its importance for catalysis and coenzyme binding [14], and are Arg102, Arg109 and His195. This numbering scheme was established to agree with the LDH numbering system [66]. Residues in boxes indicate PROSITE MDH consensus patterns. The vertical arrow above the sequence indicates the putative cleavage site of the mitochondrial leader sequence. Lines above certain residues in the leader sequence indicate a cleavage motif conserved in most m-MDH leader sequences (i.e. Arg at )11, Phe at )8, and Ser at )7 and )5 amino acid positions upstream from the mature N-terminus) [67]. Dashes indicate gaps introduced into the sequence to maximize alignment. Underlined sequence signifies additional sequence found only in m-MDHs of fungal origin. The secondary structure elements in TeMDH are shown as block arrows for a-helices and boxes for the b-strands. Predicted secondary structure for T. emersonii m-MDH obtained using PHDSEC SECONDARY STRUCTURE PREDICTION software [48].
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Recent studies on the evolution of the malate and lactate superfamily [42] suggest that two ancestral gene duplications, and not one as originally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within this superfamily: LDH, LDH-like MDH (c-MDH) and the dimeric MDH from mitochondrial fractions (m-MDH). The latter enzyme is the focus of this study. The deduced T. emersonii m-MDH amino acid sequence contains a 22 amino acid leader sequence, which is a common feature of all eukaryotic m-MDH sequences reported to date, and is thought to be partially responsible for translocation of the unfolded preprotein from the cytosol to the mitochondrial matrix [43]. It is not thought, however, to be the only consensus sequence responsible for the import of the preprotein as the reporting of the in vivo import of a leaderless m-MDH into the mitochondrion in the yeast, S. cerevisiae [44]. Closer evaluation of the sequence shows that despite high sequence homology with other m-MDHs, the differences in amino acid distributions between this thermophilic MDH and other mesophilic eukaryotic MDHs are highly significant. While some of these differences are likely to be the outcome of phylogenetic differences, others appear to correlate with protein thermostability. Comparison of the predicted amino acid sequence of the T. emersonii m-MDH with the m-MDH homolog from the mesophilic S. cerevisiae, suggests a significant difference lies in the higher levels of proline present in the thermophilic m-MDH (21 compared with 16). The location of these proline residues suggest that they may restrict protein backbone flexibility to reduce structural vibrations at higher temperatures, and may also potentially stabilize b turns and external loops. The T. emersonii m-MDH is comprised of 21.8% charged residues, 44.8% neutral hydrophobic residues and 33.4% neutral polar residues. The relative levels of each of the three of the amino acid types remains relatively constant on comparison with levels observed for MDH and LDH enzymes from other thermophilic prokaryotic sources [45]. The number of charged lysine residues is also similar to levels observed in the prokaryotic thermophilic MDH and LDH enzymes i.e. 7.0% in T. emersonii m-MDH, 8.6% in the MDH from Methanocaldococcus jannaschii and 6.6% in the LDH from Thermotoga maritima [46]. Numbers of charged lysine residues present in the T. emersonii m-MDH (7.0%) are much higher than the corresponding amounts present in m-MDHs from mesophilic eukaryotes ( 4–5%). An increased level of Arg residues were observed in the T. emersonii m-MDH (4.4%), in comparison to mesophilic counterparts ( 2.5%), but is consistent with observations for the prokaryotic thermophilic MDHs [47]. Thermophilic proteins tend to show an increase in levels of charged amino acid residues, especially Glu, Arg and Lys. These extra charged residues are thought to be located in solvent accessible regions at the surfaces of thermophilic proteins and are generally involved in the formation of ion-pair interactions between subunits thus increasing thermal stability. Comparison of predicted secondary structural elements, such as percentage a-helix and b-strand, between MDHs from mesophilic and thermophilic eukaryotic sources, gives little insight into factors that enhance thermal stability, as
Fig. 3. Northern analysis of total RNA samples isolated after harvesting T. emersonii cultures at different timepoints during growth on the following inducing substrates. Time of cultivation is shown in parentheses: (A) Lane 1, 2% glucose (72 h); lane 2, 2% galactose (72 h); lane 3, 2% lactose (72 h); lane 4, 2% trehalose (72 h); lane 5, 2% melibiose (72 h); lane 6, 2% raffinose (72 h); lane 7, 2% xylan (72 h); lane 8, 2% citrus pectin (72 h); lane 9, 2% citrus pectin + 2% glucose (72 h). Ten micrograms of total RNA was loaded in each well. Loading control was performed by probing the membrane with 18S ribosomal RNA (lower section of panel). (B) Time-course expression of m-MDH during cultivation on 2% citrus pectin: Lane 1, 36 h; lane 2, 72 h; lane 3, 96 h; lane 4, 120 h; lane 5, 144 h 20 lg was loaded in each well. Loading control was performed by probing the membrane with 18S ribosomal RNA (lower section of panel).
relative amounts of both types of secondary structure appear to be relatively conserved between species ( 40% helix, 20% sheet and 40% loop). On closer inspection however, subtle differences emerge. The subunit–subunit interface in dimeric m-MDHs involves two sets of interactions between a helices in each subunit, aB of one subunit with aB of the other, and aC of one subunit with helices aF, aH and aI of the other (Fig. 2). On comparison of the T. emersonii (thermophilic) m-MDH with its mesophilic counterpart, pig m-MDH, two substitutions are present in these helical regions which give rise to two additional charged residues at the subunit–subunit interface (Ala173 on aF to Arg and Thr273 on aH to Asp). The potential effect of these substitutions may be to increase the number of ion–pair interactions at the subunit interface and improve the thermostability of the protein. The T. emersonii m-MDH also contained three putative N-glycosylation sites at positions 78–82 (NSTV), 116–120 (NASI) and 142– 146 (NSTV), when domain search analysis was carried out using the PROSITE database motif search [49]. However, it is unlikely that any of these sites are occupied in the native protein as it is not synthesized and processed in the secretory pathway. The sequence also contains a three amino acid (ProLeuTyr) insertion motif towards the C-terminus of the deduced protein sequence at positions 277–279, which, when compared with other known sequences, is only present in m-MDHs of fungal origin (ProLeuPhe in S. cerevisiae, ProLeuTyr in Piromyces sp. Magnaporthe grisea, Aspergillus nidulans, Neurospora crassa, Gibberella zeae, Paracoccidioides brasiliensis and Eremothecium gossypii; Fig. 2). It is unlikely however, that ProLeuTyr insertion in the T. emersonii m-MDH has a pronounced effect on thermal stability, as the same sequence in conserved in the m-MDH from the mesophile S. pombe (optimal growth temperature 30 C). Prediction of putative phosphorylation sites in the T. emersonii MDH using the NetPhos 2.0 server [50], revealed the presence of a putative eukaryotic tyrosine phosphorylation site at Tyr279, the Tyr residue present in the tripeptide insert motif. The precise function of this potential motif is currently unknown and
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Fig. 4. Purification of His-tagged TeMDH expressed in E. coli by Ni-nitrilotriacetic acid column chromatography, as described in Materials and methods. (A) Analysis of purity by SDS-PAGE. Lane 1, molecular mass markers; lane 2, crude extract; lane 3, fraction eluted with 250 mM imidazole. (B) Fractions from the 250 mM elution as described in Fig. 4A were also subjected to Western blot analysis using a monoclonal antibody against the His6 tag present on the recombinant protein. Lane 1, molecular mass standards; lane 2, fractionated recombinant T. emersonii mMDH. (C) Zymogram stained isoelectric focusing gel of recombinant m-MDH overexpressed in E. coli.
studies are underway to elucidate its function in the T. emersonii m-MDH using site-directed mutagenesis. Preliminary mutagenesis studies (data not shown; this laboratory) have shown that mutating all three amino acids to Ala results in inactivation of the enzyme. The effect of the ProLeuTyr insertion on the secondary structure of the enzyme would be to increase the size of a loop region between b strands bI and bJ, which is thought to be involved in domain–domain interactions in MDHs. Previous crystallographic studies on the malate dehydrogenase from the thermophilic prokaryote Thermus flavus, suggests that a charged Glu residue at position 275 in the corresponding loop region of TfMDH is involved in the formation of a strong ion pair with Arg149, which is thought to contribute to thermal stability by strengthening the domain–domain interaction within each subunit [47]. On alignment of the TeMDH with the TfMDH mentioned above, it can be seen that the Talaromyces emersonii sequence contains a positively charged residue (Lys163) at the same position within the sequence as the Arg149 of TfMDH (i.e. between the domain interface; aE and bF; Fig. 3). The TeMDH also contains a negatively charged Asp residue at position 274 corresponding to Glu275 in the TfMDH (the domain interface between the bI and bJ strands), suggesting a possible ion-pair interaction in the TeMDH that may confer thermal stability. The homology and phylogenetic relationship of the T. emersonii m-MDH to other known MDHs, especially eukaryotic m-MDHs, suggests that the degenerate oligonucleotide primers designed in this study may be suitable for obtaining mitochondrial MDHs from a broad range of other eukaryotes, and higher eukaryotes. Northern analysis Northern analysis was conducted on total RNA samples isolated, as outlined earlier. The list of inducing substrates and harvesting times used during cultivation is given in Fig. 4. As m-MDH is regarded to be a constitutively expressed enzyme involved primarily in the tricarboxylic acid cycle, expression was expected in all samples.
Variations in the intensity of the bands represent the effect of the inducing substrate on the expression of transcript encoding the enzyme. In general, increased expression levels were distinctly visible with mono- and disaccharides (glucose, galactose, melibiose and trehalose) as growth substrates, in contrast to the pattern noted with more complex oligosaccharides and polysaccharides such as raffinose, xylan and citrus pectin. This may reflect faster metabolic turnover by the fungus when grown on simpler carbon sources. Yet, time-course (0–144 h) expression of the m-MDH gene, with 2% w/v citrus pectin as inducing substrate, revealed a constant expression pattern (Fig. 3). Through other studies conducted in this laboratory [20,22,23], T. emersonii is known to produce an array of extracellular carbohydrate-modifying enzymes that can depolymerize pectin polysaccharides. Production of these enzymes during growth on citrus pectin may provide a continuous supply of readily metabolized sugars, which may explain the consistent expression of the m-MDH gene throughout the specified growth period. Expression of recombinant malate dehydrogenase in E. coli The TeMDH recombinant protein was expressed according to the manufacturer’s instructions under the control of the lac repressor and induced in the growing cells by the addition of L-arabinose. After growth in nutrient media for a period of 4 h, cells were harvested by centrifugation and the recombinant protein recovered under native conditions. His-tag- m-MDH preparation was adjudged pure by SDS/ PAGE and a subunit Mr of 35 kDa (Fig. 4A) was estimated. Gel filtration on a Sephacryl S-200 (superfine grade) matrix yielded a single protein peak with an estimated Mr of 70 kDa, which was also confirmed by native PAGE and suggests that the recombinant protein is a homodimer. All of these findings are in agreement with results obtained for the native enzyme. The enzyme activity of the purified protein was assayed as described earlier and the recombinant protein was found to be active. Previous research has
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m-MDH from Talaromyces emersonii (Eur. J. Biochem. 271) 3123
demonstrated the ability of chaperone proteins (groES and groEL) from E. coli to aid in vitro refolding of pig m-MDH by acting as passive binding proteins which inhibit nonproductive folding and aggregation reactions without substantially altering the productive folding pathway itself [51]. These chaperone proteins share very high homology to the cpn60 and cpn10 proteins from eukaryotic mitochondria that aid in the folding of mitochondrial proteins. It is therefore likely that these chaperones also played a vital role in the refolding of the recombinant T. emersonii m-MDH to yield active enzyme. As m-MDH is a constitutively expressed enzyme in both prokaryotic and eukaryotic systems, Western blot analysis using an anti-His6 Ig was used to confirm that the purified recombinant protein (Fig. 4B), was the His-tagged m-MDH from T. emersonii revealing a single band of the correct molecular mass (35 kDa). The pI of the recombinant protein was estimated using isoelectric focusing and a single protein band with a pI value of 6.5 was observed after zymogram staining (Fig. 4C) and Coomassie blue staining (data not shown). The presence of only one band is in stark contrast to the native enzyme, which displays a three active band isoelectric focusing pattern (Fig. 1C; pI values obtained for the native enzyme were 5.4, 5.75 and 6.0, respectively). It is therefore clear from this research, that formation of enzymatically active subforms of the native m-MDH with identical amino acid sequence, is likely to be a result of posttranslational processing event(s). Previous suggestions for MDH subform heterogeneity include deamination of glutamine and asparagine residues [52] and differences in the amounts of covalently bound phosphate [53]. Previous research [54] has also reported the in vivo phosphorylation of another 2-hydroxy acid dehydrogenase, lactate dehydrogenase, on Tyr238, which cause a change in its electrophoretic properties but has no appreciable effect on the activity of the enzyme. The presence of phospholipid and neutral lipid on the native protein surface may also have an effect on the isoelectric point by masking ionic side chains.
observed for the native enzyme (52 C), the recombinant displayed a more plateaued optimum activity profile with > 90% activity being observed between 40–56 C. In calculating the t1/2 for the recombinant enzyme, a broader temperature activity range (31–59.5 C) was observed in comparison to the native enzyme (36–56 C). Temperature stability studies at 50 C revealed that the recombinant enzyme was totally inactivated after 15 min, compared with 1 h for the native which would suggest that post-translational modification(s) of the native protein may confer some degree of thermostability. Intact mitochondria were isolated from the fungus as described in the experimental section and subjected to thermal stability analyses. Whole mitochondria were assayed for m-MDH activity over a broad time frame and the results showed that the m-MDH enzyme was much more thermostable in its native environment, being with a t1/2 11 h being noted. This may reflect the ability of heat shock proteins within the mitochondria to stabilize the enzymes therein. Previous research in this area has identified mitochondrial heat-shock proteins that associate with mitochondria and protect NADH ubiquinone oxidoreductase from heat and oxidative stress [55]. pH optima for the native m-MDH enzyme in the oxaloacetate reduction and malate oxidation directions were 7.5 and 10.0, respectively. The recombinant protein displayed an identical pH profile with pH optima values identical to those obtained for the native enzyme in both directions. These observations correspond well with data obtained by other workers for m-MDH enzymes from different sources, in particular chicken heart m-MDH which displayed pH optima of 7.8 and 10 for oxaloacetate reduction and malate oxidation, respectively [56], while a pH optimum of 7.9 (oxaloacetate reduction) was determined for m-MDH from the marine snail, Ilyanassa obsoleta [57]. It is accepted that His195 is a key residue in the mechanism of MDH, and must be protonated for oxaloacetate binding and unprotonated for malate binding [58]. The optima observed for the T. emersonii m-MDH are consistent with protonation/deprotonation of a histidine residue.
Physicochemical characterization of T. emersonii native and recombinant m-MDH
Kinetic characterization of native and recombinant m-MDH from T. emersonii
Thermal stability studies were performed on the native and recombinant enzymes to investigate if thermophilicity was retained. Native T. emersonii MDH had maximum activity observed at 52 C in the oxaloacetate reductase direction (pH 7.4). Rapid loss of activity at temperatures above the optimum value was noted, and an assay temperature of 60 C (for 10 min) renders the enzyme totally inactive. A buffered sample of the m-MDH displayed significant stability at 50 C in buffer, with an estimated half-life (t1/2) of 30 min, while < 10% of the original activity was lost over the same time period in the presence of 0.2 mgÆmL)1 bovine serum albumin as stabilizing agent. The temperature optimum and stability values observed for the T. emersonii m-MDH, in contrast to mesophilic fungal, vertebrate and invertebrate eukaryotic sources, make it one of very few eukaryotic m-MDHs isolated to date that may be considered thermostable. Slight differences were observed in temperature studies with the recombinant enzyme. While the temperature optimum was the same as
Substrate specificity studies demonstrated that native m-MDH from T. emersonii is highly specific and essentially devoid of contaminating oxidoreductase activity. Some slow oxidation of meso and D-tartaric acids was observed (¼ 2.7% of activity against L-malate), which has been noted previously for m-MDH from bovine heart [59,60]. However, unlike the bovine enzyme, the T. emersonii m-MDH displayed no activity with mesoxalate, hydroxymalonate and a-ketoglutarate [60]. Similarly, the recombinant enzyme displayed no activity against these substrate analogues. Kinetic constants were determined for each of the two substrates (oxaloacetate and malate) and two cosubstrates (NADH and NAD+) and are summarized for both the native and recombinant protein in Table 2. The lowest Km values (highest affinity) and highest Vmax, turnover number (kcat) and kcat/Km (catalytic efficiency) values were observed with oxaloacetate and NADH for both enzymes. The Km value of 0.02 mM for the native T. emersonii m-MDH in the oxaloacetate reductase
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3124 A. P. Maloney et al. (Eur. J. Biochem. 271)
Table 2. A summary of the kinetic constants and additional studies obtained for native m-MDH from T. emersonii and the recombinant m-MDH overexpressed in E. coli. All assays were carried out at 30 C.
Enzyme/substrate
Vmax (lmolÆmin)1Æmg)1)
Km (mM)
kcat (s)1)
kcat/Km (s)1ÆmM)1)
Native m-MDH Oxaloacetate ([NADH] ¼ 200 lM) NADH ([oxaloacetate] ¼ 50 lM) Malate ([NAD+] ¼ 1 mM) NAD+ ([malate] ¼ 10 mM)
3.9 4.7 1.25 1.18
· · · ·
103 103 102 102
0.02 0.025 1.25 0.12
9.1 11 2.9 2.77
· · · ·
104 104 103 103
4.5 4.4 2.32 2.3
· · · ·
106 106 103 104
Recombinant m-MDH Oxaloacetate ([NADH] ¼ 200 lM) NADH ([oxaloacetate] ¼ 50 lM) Malate ([NAD+] ¼ 1 mM) NAD+ ([malate] ¼ 10 mM)
3.1 5. 20 1.75 1.25
· · · ·
103 103 102 102
0.022 0.029 1.0 0.13
8.1 9.0 3.5 3.1
· · · ·
104 104 103 103
5.1 4.8 2.44 2.74
· · · ·
106 106 103 104
direction was identical to values determined for m-MDHs from rat liver [61] and ox heart [62], but different to reported Km values for other fungal sources, e.g. 0.08 mM and 0.04 mM for m-MDHs from the filamentous mesophilic fungus Aspergillus niger [63] and the yeast C. neoformans [36], respectively. In fact, the value obtained for the A. niger enzyme is similar to data obtained for MDH from the invertebrate Mytilus edulis [31]. Substrate inhibition of m-MDH by oxaloacetate (I0.5 ¼ 0.5 mM) in the oxaloacetate reductase direction was observed for both native and recombinant enzymes, which is a common feature of other invertebrate and vertebrate m-MDHs. I0.5 ¼ 0.5 mM values have also been reported for other fungal m-MDHs, e.g. enzymes from P. blakesleeanus [35] and C. neoformans [36]. In contrast, the m-MDH from A. niger [63] is inhibited by oxaloacetate concentrations > 1.0 mM, which resembles the inhibition pattern of vertebrate m-MDHs [56,60]. The Km value for the T. emersonii m-MDH with L-malate as substrate (1.25 mM; buffer A) is similar to data for m-MDHs from mammalian sources (e.g. 0.90 mM for chicken heart m-MDH [56]), but is markedly different to Km values for invertebrate and other fungal counterparts, e.g. 22.0 mM for the Mytilus edulis enzyme [31] and 0.25 mM for m-MDH from C. neoformans [36]. With NADH as cosubstrate, the Km value of 0.025 mM determined for the T. emersonii native m-MDH was identical to the value obtained for the recombinant and bovine heart m-MDH at pH 9.0 [62], and very similar to the value (0.02 mM) noted for m-MDH from C. neoformans [36], but markedly different to the value of 0.14 mM determined at pH 7.5 by Ma and coworkers for m-MDH from A. niger [63]. The T. emersonii native and recombinant m-MDHs yielded similar Km values with NAD+ (0.12 mM) to m-MDHs from bovine heart (0.1 mM; pH 8.4 [62]); and C. neoformans (0.11 mM [36]).
Conclusions Purification of the native m-MDH, nucleotide and predicted amino acid sequence information, as well as overexpression of the recombinant m-MDH, for the moderately thermophilic eukaryote T. emersonii, have been presented in this report. Previous investigations on this enzyme, and studies of allelic variation of MDHs, have suggested a correlation
with temperature. In the intertidal snail, Nucella lapillus, m-MDH exhibits a strong localized clinal allelic variation that correlates with intraspecific variation in morphology and physiology that is related to summer temperatures in the intertidal zone [64]. Molecular studies of allelic variation at the related LDH-B locus of Fundulus hetroclitus revealed that the clinal allelic variation could be explained by a single amino acid substitution which conferred a selective advantage under increased temperature [65]. As m-MDH is a constitutively expressed enzyme of the tricarboxylic acid cycle, playing a key role in energy metabolism, a correlation with temperature in a thermophilic source is not that surprising. Relating and comparing amino acid differences between T. emersonii and mesophilic m-MDHs may provide some insight into thermal stability of enzymes. It is thought, however, that a truer reflection of the factors conferring thermal stability may be evident in the three dimensional structure of the protein, where crystallographic analysis can give information regarding disulfide linkages, ion pair interactions and salt bridges within and between monomers of enzymes, and also on the increase in protein density upon multimerization, which is also thought to increase thermostability. The evidence in this report suggests that the ability of an enzyme to work at higher temperatures maybe dependent on the amino acid content of the protein, but the stabilization of an enzyme at higher temperatures can be partially attributed to post-translational processing by an organism and the enzyme’s native cellular conditions, all of which can also play an important role in conferring thermostability. Analysis of the three dimensional structure of an enzyme from T. emersonii would be important due to the extensive post-translational modifications, in particular glycosylation patterns that this fungus is likely to use to process proteins. The methods described in this paper for the over-production of a correctly folded mitochondrial malate dehydrogenase are ideal for the purpose of relatively large-scale purification of this thermophilic, eukaryotic protein for crystallographic study.
Acknowledgements This work was funded by a HEA PRTLI (Cycle 1) award to M.G.T.. A.M. and S.C. are grateful for junior teaching fellowships from NUI, Galway, and postgraduate scholarships from Enterprise Ireland.
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