Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK
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Abstract |
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Keywords: citrate synthase/cold activity/mutagenesis/thermostability
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Introduction |
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A number of cold-active enzymes from psychrophilic bacteria have been characterized and crystal structures are now available for citrate synthase from Arthrobacter strain DS2-3R (Russell et al., 1998), triose phosphate isomerase from Vibrio marinus (Alvarez et al., 1998
),
-amylase from Alteromonas haloplanctis (Aghajari et al., 1998
) and malate dehydrogenase from Aquaspirillium arcticum (Kim et al., 1999
). High-resolution structures have also been determined for proteins from eukaryotic psychrophiles, notably trypsin and elastase from the North Atlantic salmon, Salmo salar (Smalås et al., 1994
; Berglund et al., 1995
). Comparison of these structures with their mesophilic analogues leads to the general view that cold-active enzymes need, and indeed have, an increased flexibility at low temperatures, although this in turn may lead to a decreased thermostability of the protein (Somero, 1995
; Feller et al., 1996
; Gerday et al., 1997
; Fields and Somero, 1998
; Lonhienne et al., 2000
; Sheridan et al., 2000
).
We chose the enzyme citrate synthase (CS) as a model system to study the structural basis of both cold activity and hyperthermostability (Danson and Hough, 1998). This enzyme catalyses
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Our approach has been to determine atomic structures of the enzyme from organisms spanning the biological range of temperatures and thus citrate synthase structures are now available from Pyrococcus furiosus (optimum growth temperature of 100°C) (Russell, et al., 1997), Sulfolobus solfataricus (80°C) (Bell, 1999
), Thermoplasma acidophilum (55°C) (Russell et al., 1994
), pig (37°C) (Remington et al., 1982
) and Arthrobacter strain DS2-3R (18°C) (Russell et al., 1998
). They are all dimeric enzymes and are highly homologous in overall fold and in the structural arrangement of their active site residues. Each subunit (Mr
40 000) is composed of a large and small structural domain; the large domain comprises the subunit interface, whereas the small domain is primarily concerned with catalytic activity and undergoes a rotation on the binding of substrates to convert the enzyme from its unliganded open form to the closed conformation in which catalysis takes place.
The crystal structure of the cold-active CS has been compared in detail with the hyperthermophilic P.furiosus CS, the enzyme with which it has the highest amino acid sequence identity (40%). Both enzyme structures have been solved in their closed form, with citrate and CoA bound, to a resolution of 2.1 and 1.9 Å, respectively. Structural features that might lead to an increased flexibility of the cold-active enzyme include a reduced number of proline residues in loop regions, two extended surface loops, a reduced number of inter-subunit interactions and an increase in solvent-exposed hydrophobic residues. With respect to cold activity, the Arthrobacter CS possesses a relatively more flexible small (catalytic) domain than does the P.furiosus enzyme, as judged in each enzyme by an internal comparison of the average main- and side-chain crystallographic B factors of the small with respect to the large domains. Also, the Arthrobacter CS has an increased positive electrostatic surface potential that might serve to attract its negatively charged substrates and a strikingly more open active site (Russell et al., 1998).
Interestingly, a greater active site accessibility was proposed to be a dominant feature in the cold-activity of elastase from the Antarctic fish Notothenia neglecta (Aittalab et al., 1997) Therefore, in our current study on the psychrophilic citrate synthase, we focused on the difference in accessibility of the active site of the Arthrobacter enzyme compared with that from Pyrococcus. First, we replaced two small residues at the entrance to the active site with large residues, as found in the hyperthermophilic CS, and second, we extended a loop at the entrance to the active site by three amino acids, again a change that resembles the structure found in the Pyrococcus enzyme and in the CSs from the thermophiles Thermoplasma acidophilum and Sulfolobus solfataricus. Mutant enzymes were purified and characterized with respect to their catalytic and thermostability properties and the data are discussed with respect to how enzymes from psychrophilic organisms achieve their catalytic rates at low temperatures.
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Materials and methods |
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Escherichia coli strain JM109 (Sambrook et al., 1989) and the citrate synthase-deficient strain E.coli W620 (gltA6 relA1 supE44 thi-1 pyrD36 galK30 rpsL129 Smr) were used for cloning and expression studies, respectively. The expression vector pREC7/NdeI was kindly provided by Dr L.C.Kurz (Washington University, St. Louis, MO). Plasmid pRCS12 contains the CS gene of Arthrobacter DS2-3R cloned behind the recA promoter of pREC7/NdeI (Gerike et al., 1997
).
Site-directed mutagenesis
All mutants were obtained by PCR amplification using the mutagenic oligonucleotides shown in Table I, with plasmid pRCS12 as template DNA. A 50 µl volume of incubation mixture contained 200 ng of template DNA, 5 pmol of primers, 0.2 mM each of dATP, dCTP, dGTP, dTTP, 1x Thermopol buffer and 2 U Vent polymerase (New England Biolabs, Beverly, MA). Each of the 30 cycles of the PCR consisted of a denaturation step (94°C, 45 s; first cycle 2 min), an annealing step (the temperature for which was dependent on the oligonucleotides used, 2 min) and an elongation step (72°C, 1 min). All mutants obtained were sequenced. The residue numbering corresponds to the structure-based sequence alignment of the wild-type CSs from Pyrococcus and Arthrobacter DS2-3R (Russell et al., 1998
).
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Mutants A10E and A361R/A10E. Only one PCR reaction was needed to introduce mutation A10E. Mutagenic primer 4 contains, in addition to the A10E mutation, an NdeI site at its 5' end for subcloning. Amplification using primers 4 and 5 (Table I) resulted in a 720 bp fragment, which was cloned into pRCS12 using NdeI/BstEII. The restriction marker in this case was SmI1. To construct the double mutant A361R/A10E, the PCR product was also cloned as an NdeI/BstEII fragment into the A361R mutant described above.
Mutant LoopSKG. Mutation LoopSKG was engineered in the same way as mutant A361R but using primer 6 in place of primer 2 as the mutagenic oligonucleotide. StyI was used as the restriction marker.
Mutants LoopSKG/K313L, LoopSKG/K313L/A361R and Loop SKG/K313L/A361R/A10E. To introduce K313L into the LoopSKG mutant, a 530 bp fragment was amplified from a pRCS12 template using mutagenic primer 7 and outer primer 1. The PCR product was then cloned as a BstEII/StyI fragment into mutant LoopSKG. The restriction marker in this case was PstI. The triple mutant, LoopSKG/K313L/A361R, and the quadruple mutant, LoopSKG/K313L/A361R/A10E, were obtained by subcloning the BsteII/StyI PCR fragment in mutants LoopSKG/A361R and LoopSKG/A361R/A10E, respectively. Restriction digest analysis using the marker enzymes of the respective mutation was used to identify mutant clones.
Growth and induction
E.coli W620 was transformed with the mutant plasmids. The recombinant strains were grown to OD600 = 0.5 at 37°C in Luria broth, containing 100 µg ampicillin ml and 40 µg streptomycin ml. Expression was induced by the addition of nalidixic acid (50 µg/ml). Further ampicillin (100 µg/ml) was added and the culture was then incubated at 25°C for 19 h.
Purification of recombinant enzyme
Wild-type and mutant recombinant CSs were purified from cell extracts by affinity chromatography on Dyematrex Red A (Amicon), using specific elution with 5 mM oxaloacetate and 1 mM CoA (James et al., 1994). After elution, the enzymes were concentrated to 50 µl by centrifugation using Centricon concentrators (Millipore) and residual oxaloacetate and CoA were removed by dilution with 20 mM Tris, pH 8.0, containing 100 mM KCl and 2 mM EDTA, followed by reconcentration.
Assay of citrate synthase
CS activity was assayed spectrophotometrically at 412 nm as described by Srere et al. (1963). The assay buffer (1 ml) routinely contained 50 mM HEPES, pH 8.0, 2 mM EDTA, 100 mM KCl, 0.1 mM 5,5'-dithiobis(2-nitrobenzoic acid), 0.2 mM oxaloacetate and 0.2 mM acetyl-CoA. For the determination of apparent Km and Vmax values, assays were carried out at various concentrations of one substrate whilst keeping the other substrate at near-saturating levels (10Km); the data were analysed using a direct linear plot (Eisenthal and Cornish-Bowden, 1974).
Protein determination
Protein concentrations were determined according to Bradford (1976).
Thermal inactivation
CS was added to pre-warmed buffer (50 mM potassium phosphate buffer, pH 7.0, containing 100 mM KCl) to a final protein concentration of 0.1 mg/ml. Samples were removed at known time intervals, cooled rapidly to 4°C and subsequently assayed for residual enzymic activity. The first-order rate constant of inactivation (k) was determined at a range of temperatures and the values used to construct an Arrhenius plot (lnk vs 1/temperature). The free energy of activation for the inactivation process (G°*) at a reference temperature (T) was determined from Arrhenius plots for wild-type and mutant enzymes using the equation
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Results and discussion |
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Mutations to introduce bulky side chains into the Arthrobacter CS at positions A10 and A361
The replacement of an alanine residue (A361) in Arthrobacter CS by an arginine residue, thus mimicking the situation in the Pyrococcus CS where R353 occupies the equivalent position, results in a dramatically reduced Km for acetyl-CoA with little significant change in kcat value (Table II). A comparison of the two active sites provides a rationale for this change (Figure 1A and B
). That is, R353 is one of the six residues binding the phosphate groups of acetyl-CoA in Pyrococcus CS (Km for acetyl-CoA
10 µM), whereas only four residues are involved in acetyl-CoA binding in the Arthrobacter enzyme; the mutation A361R in the latter provides an additional and strong substrate-binding interaction, resulting in the tighter binding of the acetyl-CoA.
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Interestingly, the thermostability of Arthrobacter CS A361R and A361R/A10E is increased over the wild-type enzyme, as judged by the respective rates of irreversible thermal inactivation (Figure 2). Thus, at 45°C, the rate of inactivation of the wild-type enzyme is ~1.7 times faster than that of either mutant and calculation of the free energy of activation for thermal unfolding reveals an increase of ~1.8 kJ/mol for both mutants (Table II
). The single and double mutations potentially introduce additional inter-subunit bonds; that is, if the introduced changes do indeed mirror the situation in the Pyrococcus CS (Figure 1A and B
), then in the Arthrobacter CS A361R the arginine might interact with the peptide bond carbonyl group of Lys6 on the partner subunit and in A361R/A10E an additional ionic bond could be formed between these two residues, again across the subunit interface. These extra inter-subunit interactions may then contribute to the increase in the proteins' thermostable properties.
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In a second set of experiments, three amino acids (SKG) were inserted into the loop joining helices Q and R at the entrance to the active site of Arthrobacter CS. The reduced size of this loop in the cold-active enzyme, equivalent to the removal of residues 304306 (KKG) of Pyrococcus CS (Figure 1C and D) and SKG of the CSs from the thermophiles Thermoplasma acidophilum and Sulfolobus solfataricus, creates further space at the entrance to the active site of Arthrobacter CS and may improve accessibility to the enzyme's substrates.
Another feature of the QR loop of the Pyrococcus, Sulfolobus and Thermoplasma CSs is that the central lysine residue (K305 in the Pyrococcus enzyme) binds the 3'-phosphate of acetyl-CoA (Figure 1C). The homologous residue that carries out this function in Arthrobacter CS is K313 (Figure 1D
) and so a further mutation was carried out in the Arthrobacter CS loop insertion mutant, replacing K313 by leucine, in order to avoid clashes between K313 and the lysine in the newly introduced loop (K317).
In a first step we extended the QR loop by insertion of residues SKG (loop insertion mutant) and then, in addition, K313 was mutated to leucine (Loop/K313L mutant). A kinetic comparison of the two mutants with the wild-type CS shows that the extension of loop QR by the sequence SKG results in a large increase in the Km value for acetyl-CoA (Table III). This is likely to result from the clash of K317, introduced by the loop extension, with Arthrobacter K313; consequently, after the substitution of K313 with leucine (Loop/K313L mutant), the Km value for acetyl CoA returns to a value close to that of the wild-type enzyme (Table III
).
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Combination mutants
The loop insertion mutations described above were combined with the mutations designed to introduce large residues, A361R (to create Loop/K313L/A361R) and A10E (to create Loop/K313L/A361R/A10E). To simplify the nomenclature, these two mutants will be referred to as triple and quadruple mutants, respectively. All three mutants were compared with respect to their substrate binding, cold activity (Figure 3) and thermostability (Figure 4
).
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Interestingly, the catalytic rates of all the loop mutants at 20°C are reduced by ~50% compared with the wild-type enzyme, indicating that accessibility might contribute to the high catalytic rate at low temperatures observed in the cold-active enzyme. However, at best it is only one factor as the catalytic rates observed at 20°C with the loop mutants do not drop to that observed with hyperthermophilic CSs.
As summarized in Table III, the loop mutants do not show any significant changes in thermostability as judged by their rates of irreversible thermal inactivation (Figure 4
), except perhaps for the triple mutant (Loop/K313L/A361R). Thus, the
G°* of 1.6 (±0.5) kJ/mol translates into a 1.7-fold slower rate of thermal inactivation at 45°C of this mutant enzyme compared with the native protein. However, in contrast, there were significant changes in the dependence of catalytic activity on temperature (Figure 3
), the temperature optima of three of the loop mutants being 56°C lower than that for the wild-type enzyme. Moreover, at 40°C, where the wild-type citrate synthase retains 50% of its maximum activity, all the mutants are inactive, even though at this temperature, over the time course of the assay (1 min), none of the enzymes lost significant amounts of activity through irreversible thermal inactivation.
Catalytic activity with propionyl-CoA
Arthrobacter CS possesses catalytic activity with both acetyl-CoA and the larger propionyl-CoA, the latter metabolite being condensed with oxaloacetate to form 2-methylcitrate (Gerike et al., 1998). It was of interest to learn, therefore, if the mutations that we had made at the active site of the enzyme, all of which potentially reduced the accessibility to substrates, would differentially affect the two catalytic activities. Interestingly, the effects on propionyl-CoA activity (Table IV
) are very similar to those with acetyl-CoA (Tables II and III
). That is, the Km for propionyl-CoA is reduced approximately 4-fold in the A361R mutant, but with only a small (25%) fall in the value of kcat. Similarly, with the Loop/K313L/A361R mutant, the kcat value is further reduced to approximately half of that found in the wild-type enzyme. Hence there appears to be no significant differential effect on the two enzymic activities.
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Conclusion |
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Surprisingly, the marked reductions in accessibility to substrates produced no dramatic alterations in cold activity, the maximum reductions in kcat values being ~50% (in the mutants with an introduced loop region). However, where the mutations introduced positively charged residues that are close to the acetyl-CoA binding site, then large reductions in Km for this substrate were observed, with consequent higher kcat/Km values. Additional interactions between the enzyme and the phosphate groups of the acetyl-CoA are thought to be responsible for this change in substrate affinity. Thus, rather than deleteriously affecting catalytic activity by reducing active-site accessibility, the catalytic efficiency of the Arthrobacter enzyme is increased. Moreover, the observation that the effects of the mutations were no different when the larger substrate, propionyl-CoA, was used adds further weight to the conclusion that active site accessibility is not a major factor in catalytic activity at low temperatures for this particular enzyme.
The apparent ease (A361R) with which the Km for acetyl-CoA can be reduced 5-fold with no significant reduction in kcat begs the question of why (in terms of in vivo significance) the native Arthrobacter CS has such a low affinity for this substrate (Km = 200 µM, compared with values of 550 µM for most eukaryal, archaeal and many bacterial citrate synthases). Unusually, the Arthrobacter CS possesses catalytic activity with both acetyl-CoA and propionyl-CoA, the latter giving a 3-fold higher kcat/Km than the former, although the kcat is only half of that with acetyl-CoA (Tables II and IV). Thus, it was previously thought that the poorer binding of acetyl-CoA might be a consequence of the need of the enzyme to be able to accommodate the larger propionyl substrate. However, this does not appear to be the case, the reduction in Km value for acetyl-CoA being matched with a similar reduction in that for propionyl-CoA. The poor affinity for acetyl-CoA remains unexplained with respect to the organism's physiology, even though structurally the explanation is clear.
Finally, although the effects of the mutations on thermostability and cold activity are relatively small, it is interesting that even though the two parameters are obviously connected (an enzyme cannot be active at a temperature at which it is unstable), the two parameters can be independently manipulated. That is, mutants A361R and A361/A10E have increased thermostabilities but no change in their activity temperature optima (Topt), whilst the mutant Loop/K313L/A361R has an increased thermostability yet a 56°C lower Topt value. This implies, in the dependence of enzyme activity on assay temperature, that the reduced activity at temperatures above the Topt may not be primarily due to irreversible thermal inactivation (Daniel et al., 2001). Indeed, this must be the case when one considers the thermal stability of the Arthrobacter CS with respect to the length of assay in the activitytemperature profile: over a 1 min assay, the wild-type enzyme is essentially inactive at 45°C (Figure 3
) yet it will have lost <10% of its activity through irreversible thermal inactivation. Interestingly, a similar conclusion was reached concerning thermostability and enzymic activity during a mutational analysis of the Pyrococcus CS, where chimeric mutants were constructed between the hyperstable enzyme and the less thermostable citrate synthase from Thermoplasma acidophilum (Arnott et al., 2000
). Clearly, there is still much to learn about the relationship between activity and stability and the need to consider both parameters when engineering one or the other into a protein as shown in the present study, increasing the thermostability of an enzyme does not guarantee an increased thermoactivity.
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Notes |
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Acknowledgments |
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References |
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Received December 14, 2000; revised June 8, 2001; accepted June 19, 2001.