Cytidine deaminase from two extremophilic bacteria: cloning, expression and comparison of their structural stability

Alessandra Cambi1,5, Silvia Vincenzetti2, Giampiero De Sanctis3, Jan Neuhard4, Paolo Natalini1 and Alberto Vita2,6

1 Department of Compared Morphological and Biochemical Sciences, 2 Department of Veterinary Sciences, 3 Department of MCA Biology, University of Camerino, Matelica, Italy and 4 Institute of Molecular Biology, University of Copenhagen, Copenhagen, Denmark


    Abstract
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We cloned, purified and characterized two extremophilic cytidine deaminases: CDABcald and CDABpsy, isolated from Bacillus caldolyticus (growth at 72°C) and Bacillus psychrophilus (growth at 10°C), respectively. We compared their thermostability also with the mesophilic counterpart, CDABsubt, isolated from Bacillus subtilis (growth at 37°C). The DNA fragments encoding CDABcald and CDABpsy were sequenced and the deduced amino acid sequences showed 70% identity. High sequence similarity was also found with the mesophilic CDABsubt. Both enzymes were found to be homotetramers of approximately 58 kDa. CDABcald was found to be highly thermostable, as expected, up to 65°C, whereas CDABpsy showed higher specific activity at lower temperatures and was considerably less thermostable than CDABcald. After partial denaturation at 72°C for 30 min, followed by renaturation on ice, CDABcald recovered 100% of its enzymatic activity, whereas CDABpsy as well as CDABsubt were irreversibly inactivated. Circular dichroism (CD) spectra of CDABcald and CDABpsy at temperatures ranging from 10 to 95°C showed a markedly different thermostability of their secondary structures: at 10 and 25°C the CD spectra were indistinguishable, suggesting a similar overall structure, but as temperature increases up to 50–70°C, the {alpha}-helices of CDABpsy unfolded almost completely, whereas its ß-structure and the aromatic amino acids core remained pretty stable. No significant differences were seen in the secondary structures of CDABcald with increase in temperature.

Keywords: circular dichroism/cytidine deaminase/extremophiles/thermostability


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
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Among environmental factors involved in the adaptation of organisms, temperature is crucial as it affects the structure and the stability of proteins as well as the enzymes' catalytic properties. Interestingly, the structures of homologous proteins from thermophilic, mesophilic and psychrophilic organisms are frequently quite similar with amino acid sequence identities in the range of 45–65%, and there are only few significant additions or deletions. In general, the shift of the temperature optimum among extremophilic proteins might be the result of a multitude of small changes involving the entire molecule and a comparison of extremophilic proteins with their mesophilic homologs can help to obtain more insights into the physical basis of protein stability (Lazaridis et al., 1997Go; Motono et al., 1999Go). In recent years the interest in enzymes derived from extremophilic organisms, in particular highly thermostable enzymes, has increased as they have turned out to be technologically valuable (Holst et al., 1997Go; Maloney et al., 1997Go). Cytidine deaminase (CDA, E.C.3.5.3.4) catalyses the hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively, in the salvage pathway of pyrimidines and the catalysis is mediated by a tightly bound zinc ion in the active site (Vincenzetti et al., 1996Go). In the dimeric E.coli enzyme, zinc is co-ordinated to two Cys and one His residue as revealed by X-ray crystallography (Betts et al., 1994Go), whereas the co-ordinating residues in the tetrameric human CDA were shown by site-directed mutagenesis to be three Cys residues (Cambi et al., 1998Go). The aim of this study was to characterize CDAs that differ in thermal stability in order to investigate the structural adaptations of the same enzyme to high and low temperature. The cdd genes (CDA gene) of the thermophile Bacillus caldolyticus (growth temperature 72°C) and of the psychrophile Bacillus psychrophilus (growth temperature 10°C) were cloned and their amino acid sequences compared also with the sequence of the mesophilic CDA, previously isolated from Bacillus subtilis (Song and Neuhard, 1989Go). The thermostability of the purified enzymes, the circular dichroism (CD) spectra obtained at different temperatures and the denaturant-induced unfolding were analyzed.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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Materials

Nucleosides, nucleotides, nucleobases, tris(hydroxymethyl) aminomethane (Trizma base), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and glutardialdehyde were from Sigma Chemical Co. (St. Louis, MO). Isopropyl-1-thio-ß-D-galactopyranoside (IPTG) was from Inalco (Milan, Italy). Other chemicals were from J.T.Baker Chemicals B.V. (Deventer, The Netherlands). Mono Q, Mono P and Superose 12 HR 10/30 were the products of LKB-Pharmacia (Uppsala, Sweden). Protein markers were obtained from both Bio-Rad and Boehringer (Mannheim, Germany). The pUC19 vector was from New England Biolabs (Boston, MA). The pTrc99-A vector was from Pharmacia. Oligodeoxyribonucleotide primers were synthesized by DNA Technology, ApS (Aarhus, Denmark). Restriction endonucleases were purchased from Promega Corporation (Madison, WI) and New England Biolabs.

Culture conditions

The bacterial strains used were B.caldolyticus DSM 405 (Deutsche Samlung für Mikroorganismen und Zell Kulturen), B.psychrophilus A234 (P.Nielsen, NOVO Nordic, Denmark), and the Escherichia coli K12 strains DH5{alpha} and SØ5201 (MC1061 cdd::Tn10 pyrD::Kan). DH5{alpha} was used as host for all clonings (Sambrook et al., 1989Go). For complementation tests the pyrimidine requiring CDA-negative SØ5201 was employed. The pyrimidine requirement of SØ5201 cannot be satisfied by deoxycytidine (CdR) due to a lack of cytidine deaminase activity. Escherichia coli JF611/pSO143 (Song and Neuhard, 1989Go) was used for overproduction of B.subtilis CDA. Bacillus caldolyticus and B.psychrophilus were grown in L-broth (Miller, 1972Go) containing 0.5% glucose and 5 mM CaCl2 at 60 and 25°C, respectively. Escherichia coli was grown in L-broth or phosphate-buffered AB medium (Clark and Maaløe, 1967Go) supplemented with 0.2% glucose and 0.2% vitamin-free casamino acids. When required, supplements were added at the following final concentrations (µg/ml): thiamine (1), uracil (20), deoxycytidine (40), ampicillin (100), kanamycin (30) and tetracycline (10).

Molecular cloning and DNA manipulations

The genomic libraries were constructed in the expression vectors pUC19 and pTrc99-A. Chromosomal DNA from B.caldolyticus and B.psychrophilus was digested to completion with SacI or EcoRI, respectively, and mixed with the appropriate plasmid vectors. The mixtures were ligated overnight at 14°C and constituted the genomic libraries. The libraries were transformed into the r-m+ E.coli strain SØ5201. Ampicillin resistant transformants capable of utilizing deoxycytidine as the sole pyrimidine source were selected and characterized.

Enzyme assay

Cytidine deaminase activity was determined spectrophotometrically (Cohen and Wolfenden, 1971Go) at 290 nm using deoxycytidine as substrate ({Delta}{varepsilon}290 nm = 2.1/M/cm). One unit of enzyme activity is the amount of enzyme which catalyses the deamination of 1 µmol of cytidine per minute at 37°C. The enzymatic activity at temperatures ranging from 6 to 75°C in the reaction mixture was determined by measuring the initial rate of the reaction using the continuous spectrophotometric assay described above. No pre-incubation was performed.

Expression and purification of Bacillus CDAs

Overnight cultures in LB with ampicillin (100 µg/ml) of E.coli SØ5201/pAX4(cddBcald) and E.coli JF611/pSO143(cddBsubt) grown at 37°C, and E.coli SØ5201/pBPcdd51(cddBpsy) grown at 30°C were diluted into 1 l of fresh medium. The cultures harboring pAX4 and pSO143 were grown overnight with vigorous shaking at 37°C, whereas the culture harboring pBPcdd51 was grown at 25°C for 24 h. To the E.coli SØ5201/pAX4(cddBcald) culture 0.6 mM IPTG was added to induce cdd transcription. Cells were harvested by centrifugation (6 min at 5000 g). The pellets were resuspended in 50 mM Tris–HCl, pH 7.2 (buffer A), disrupted by using a French pressure cell (15 000 p.s.i.). The purification was then based on the procedure already published (Mejlhede et al., 1999Go) with some modifications. The cell debris was removed by centrifugation at 10 000 g for 10 min and the supernatant was heat treated: CDABcald at 70°C for 10 min, CDABsubt and CDABpsy at 68°C for 10 and 5 min, respectively. The denatured proteins were removed by centrifugation (27 000 g for 5 min) and the supernatant was applied to a DE-52 column (2.5x24 cm), equilibrated with buffer A. The enzyme was eluted with a gradient of NaCl from 0 to 1 M in the same buffer. To the pooled fractions containing CDA, ammonium sulfate was added to a final concentration of 80% saturation and after incubation for 15 min on ice the precipitate was removed by centrifugation (27 000 g for 15 min). The supernatant was immediately dialyzed by ultrafiltration against 10 mM potassium phosphate, pH 7.0 (buffer B) and applied on a Mono Q HR 5/5 column connected to an FPLC system and equilibrated with buffer B. The enzymes were eluted with a gradient of KCl from 0 to 1 M in buffer B. The active fractions were collected and dialyzed by ultrafiltration against buffer B. The enzyme preparations were stored at –20°C in the presence of 10% glycerol. For CD spectral studies no glycerol was added to the enzyme preparations.

Circular dichroism spectra

CD spectra were collected with a Jasco J-710 spectropolarimeter in the far (190–240 nm) and in the near (240–320 nm) UV range. The buffer was 10 mM potassium phosphate, pH 7.0. The protein concentration was 2 µM in the far-UV range (cell length 2 mm), 10 µM in the near-UV range (cell length 10 mm) and 5 µM in the far-UV range (cell length 1 mm) for measurements of denaturant induced unfolding. Molar ellipticity [{theta}] (deg cm2/dmol) is expressed on a mean residue concentration basis in the far-UV region and on a protein concentration basis in the near-UV region. The temperature dependence of the CD spectra was explored in the range from 10 to 95°C. The enzymes were all incubated for 1 h in the cuvette at the desired temperature prior to collecting the CD spectra. The recording time was approximately 15 min (eight cycles were performed). The samples were stable for several hours, in fact the CD spectra collected at different times were overlapping. The deconvolution of CD spectra and the analysis and the estimation of protein secondary structures were carried out according to Yang et al. (Yang et al., 1986Go). The denaturant induced unfolding was performed at 25°C in 20 mM potassium phosphate, pH 7.0, in the presence of increasing guanidine hydrochloride (Gu-HCl) concentrations ranging from 0 to 4.0 M. The experimental points refer to ellipticity values obtained at 222 nm. Reversibility was checked by recording CD spectra after extensive dialysis of the guanidine-denatured samples, against 20 mM phosphate buffer, pH 7.0.

Other analytical procedures

The molecular mass was determined by gel filtration on a Superose 12 HR 10/30 (Pharmacia) connected to an FPLC system (LKB model) at room temperature and crosslinking experiments and chromatofocusing were carried out as already described (Vincenzetti et al., 1996Go). The purity of the enzymes and the molecular weight of the subunit were determined by SDS–PAGE as described by Laemmli (Laemmli, 1972Go). The native gel electrophoresis was performed omitting SDS from the solutions. Determination of the zinc content was done by inductively coupled plasma optical-emission spectrometry (ICP-OES) using a Jobin Yvon 24R model. The dye-binding method of Bradford (Bradford, 1976Go) was used in the microprotein assay using BSA as a standard.


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 Materials and methods
 Results
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Cloning and sequencing of the cdd genes from B.psychrophilus and B.caldolyticus

The cddBpsy gene encoding CDABpsy was isolated from an EcoRI library of B.psychrophilus genomic DNA fragments in pUC19 by selection for complementation of an E.coli cdd mutation. The library was transformed into E.coli SØ5201 and ampicillin resistant transformants capable of utilizing 2'-deoxycytidine as sole pyrimidine source and these were selected. From the recombinant plasmid of one such transformant the cddBpsy gene was subcloned on a 916 bp PstI–EcoRI fragment in pUC19, yielding pBPcdd51. The DNA sequence of the insert was determined on both strands and is available from EMBL databank (accession number AJ237978). It showed an open reading frame (ORF) coding for a polypeptide of 136 amino acids with a deduced molecular mass of 14 600 Da. SDS–PAGE of a lysate of SØ5201/pBPcdd51 shows a prominent band corresponding to a polypeptide of approximately 15 kDa (data not shown). The cddBcald gene from B.caldolyticus was isolated from a SacI library of B.caldolyticus genomic DNA fragments in pUC19 by selection for complementation of the cdd mutation of SØ5201, as described above for the B.psychrophilus cdd cloning. From the recombinant plasmid of one ampicillin resistant transformant capable of utilizing 2'-deoxycytidine as a sole pyrimidine source the cddBcald gene was subcloned on a 419 bp NcoI–BamHI fragment in pTrc99-A, yielding pAX4. In this construct cdd was transcribed from the plasmid-borne trc promoter, IPTG was added as inducer. The DNA sequence of the insert was determined on both strands and is available from the EMBL databank (accession number AJ237979). It contained an ORF of 399 bp encoding a 132 amino acid peptide with a deduced molecular mass of 14 237 Da. SDS–PAGE of a lysate of SØ5201/pAX4 after overnight induction with 0.6 mM IPTG shows an intense band corresponding to a polypeptide of approximately 15 kDa (data not shown). Comparison of the primary structure of the thermophilic, psychrophilic and mesophilic Bacillus CDAs (Figure 1Go) revealed 65% identity between CDABpsy and CDABsubt, 76% between CDABsubt and CDABcald, and 70% between CDABpsy and CDABcald. The overall identity between all three Bacillus CDAs was 62%. The G + C mole percentage of the coding region of cddBpsy and cddBsubt was 42.9 and 44.6%, respectively, whereas it is 56.1% for the cddBcald gene. As for other B.caldolyticus coding regions (Ghim et al., 1994Go; Jensen et al., 1997Go), the bias for G + C is particularly evident at the third codon position, where it is 67% for the cddBcald but only 40 and 33% for cddBsubt and cddBpsy, respectively. The values concerning B.caldolyticus and B.subtilis CDAs are in agreement with the data reported for the total genome, i.e. 52.3 and 44.1%, respectively (Sharp et al., 1980Go).



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Fig. 1. Multiple alignment of cytidine deaminase from B.psychrophilus (CDABpsy), B.caldolyticus (CDABcald) and B.subtilis (CDABsubt). Asterisks indicate conserved residues. Letters in bold indicate the zinc co-ordinating residues.

 
Overexpression and purification of the extremophilic CDAs

The ability of CDABpsy and CDABcald to complement the cdd-deficient E.coli strain indicated that the native folding of the proteins occurred in E.coli. SØ5201 harboring pBPcdd51 (cddBpsy) and pAX4 (cddBcald), as the recombinant enzymes were active in vivo. CDA from both sources accumulated in the cytoplasm as soluble proteins and after purification, as described under Materials and methods, they were judged more than 98% pure as estimated by SDS–PAGE gel stained by Coomassie Blue (data not shown).

Characterization of CDABpsyand CDABcald

The pure bacterial CDAs were shown to be tetrameric enzymes both by cross-linking and gel-filtration (data not shown), with a native molecular mass of approximately 57–58 kDa. Their zinc content was determined by ICP-OES and shown to be 1 mol Zn/mol subunit, in agreement with data already reported for other CDAs (Betts et al, 1994Go; Vincenzetti et al., 1996Go). The pI values, determined by chromatofocusing, were 5.3 for CDABcald and 4.4 for CDABpsy and CDABsubt, suggesting a different distribution of charged residues on the surface of these proteins. The enzymatic activities as a function of temperature are shown in Figure 2Go. CDABcald has a maximal enzymatic activity at approximately 56°C, remaining stable up to 65°C. CDABpsy and CDABsubt showed an optimal temperature for catalysis at approximately 33 and 37°C, respectively. After partial denaturation at 72°C for 30 min and following renaturation on ice, CDABcald retained its enzymatic activity, whereas CDABpsy and CDABsubt were irreversibly inactivated and showed an additional band on native gel electropherograms (data not shown), due to either enzyme aggregation or partial unfolding, since no deaminating activity was detectable after elution of these bands from the gel. The stability towards urea at different temperatures was also checked and CDABcald was found to be more resistant with respect to CDABpsy and CDABsubt (data not shown), suggesting an increased rigidity of the thermophilic protein at room temperature.



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Fig. 2. Enzymatic activities at different temperatures. CDABpsy (open circle); CDABsubt (filled square); CDABcald (filled circle). The enzyme activities were measured as described under Materials and methods at temperatures ranging from 6 to 75°C in the reaction mixture.

 
CD spectra

Figure 3Go shows the CD spectra of the three Bacillus CDAs, in the far- and near-UV range, performed at 25°C in 0.01 M phosphate buffer at pH 7.0. Only slight differences were observed in the far-UV spectra (Figure 3aGo), indicating a very similar secondary structure organization for the three enzymes. According to the spectra, CDABsubt possessed a higher content of {alpha}-helix and less ß-sheet secondary structure than the other two enzymes (see Table IGo for details). Although the aromatic pattern seemed not very indicative of a particular asymmetry center (Figure 3bGo), it may be hypothesized that similar tertiary interactions are present in the hydrophobic core of the three proteins, with a higher molar ellipticity for CDABcald. The maximum of the near-UV spectrum of CDABsubt was blue-shifted relative to the other two proteins. When CD spectra were collected at increasing temperatures different patterns were observed. Figure 4Go shows the CD spectra of the three enzymes, recorded in the far and in the near-UV region at temperatures ranging from 10 to 95°C. As expected the CDABcald was quite insensitive to temperatures up to 70°C with a drastic decrease of {alpha}-helical content and part of the ß-sheet structure only at 85–95°C (Figure 4bGo). The secondary structure organization of CDABpsy was more sensitive to heat than the thermophilic homolog, as a gradual decrease of {alpha}-helix content and part of the ß-sheet was evident from 50°C (Figure 4cGo). The same behavior was observed for CDABsubt, where the decrease of secondary structure, particularly the {alpha}-helix content, seemed even more affected by increasing temperatures (Figure 4aGo). Moreover, at 10°C the secondary structure content of CDABsubt was less than that observed from the spectrum at 25°C (see Table IGo for details). Also, in agreement with data obtained in the far-UV range, the near-UV CD spectra of CDABcald, performed at the same temperatures, were almost indistinguishable up to 70°C (Figure 4b'Go), but surprisingly, also the near-UV CD spectra of CDABpsy (Figure 4c'Go) were almost insensitive to heat, as compared to the far-UV spectra (Figure 4cGo). This result may indicate that, in spite of a relevant decrease of {alpha}-helix and part of ß-sheet, a compact core of hydrophobic interactions might be preserved in CDABpsy at increasing temperatures. The data obtained from the temperature dependence of the near-UV spectra of CDABsubt (Figure 4a'Go) were consistent with the behavior of the far-UV spectra (Figure 4aGo), which indicated that the whole protein structure seemed very sensitive to heat and that the decrease of hydrophobic tertiary interactions followed that of secondary structures. Because the thermal unfolding is not fully reversible and, as shown in Figure 4Go, it is not possible to reach a common unfolding endpoint, the reversible chemical denaturation was performed by using Gu-HCl, in order to estimate the differences in free energy of stabilization among the three proteins. In Figure 5Go are shown the unfolding profiles of the three proteins in the presence of an increasing amount of Gu-HCl, obtained at 222 nm and 25°C. The observed ellipticities are reported as a percentage of unfolding using as endpoints the values obtained in the absence, corresponding to 0% of unfolding, and in the presence of 6 M Gu-HCl, corresponding to 100% unfolding, respectively. The denaturation process was always fully reversible. From the analysis of the denaturation curves (Pace, 1975Go) the apparent free energy change {Delta}GD was calculated from the apparent denaturation equilibrium constant KD, which is determined by the expression ({theta}i{theta}obs) / ({theta}obs{theta}f), where {theta}i, {theta}f and {theta}obs represent the initial, final and observed values of ellipticity, respectively. On the assumption of a linear dependence of the {Delta}GD versus denaturant concentration, the value of {Delta}G0, i.e. the apparent free energy change in the absence of denaturant, was obtained by extrapolation to zero denaturant concentration (Figure 5Go, inset). The values obtained were: 2.5 ± 0.2 kcal/mol for CDABsubt, 4.3 ± 0.2 kcal/mol for CDABcald and 3.0 ± 0.2 kcal/mol for CDABpsy.



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Fig. 3. CD spectra in the far (a) and in the near (b) UV region at 25°C. CDA from B.subtilis (– – –), B.caldolyticus (—) and B.psychrophilus (-·-·-). The protein concentration was 2 µM and cell length 2 mm for (a) and 10 µM and 10 mm for (b). The buffer was 10 mM potassium phosphate, pH 7.0.

 

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Table I. Percentage content of secondary structures in the three Bacillus CDA
 


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Fig. 4. Temperature dependent CD spectra in the far and in the near-UV region. CDABsubt (a and a'), CDABcald (b and b') and CDABpsy (c and c'). Temperatures: 10°C (1); 25°C (2); 50°C (3); 70°C (4); 85°C (5); 95°C (6). The buffer was 10 mM potassium phosphate, pH 7.0.

 


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Fig. 5. Unfolding profiles in the presence of increasing Gu-HCl concentrations. Inset: Plots of {Delta}GD as a function of Gu-HCl concentration. CDABpsy (filled triangle); CDABsubt (filled circle); CDABcald (filled square). Data were calculated from CD measurements at 222 nm. The protein concentration was 5 µM in 20 mM phosphate buffer, pH 7.0, at 25°C, cell length 1 mm.

 

    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Cloning and expression of genes from thermophiles and psychrophiles in mesophilic hosts, such as E.coli, have clearly been shown to result in production of native proteins. Therefore, natural extremophilic conditions may not be an absolute requirement for the correct folding of intrinsically extremophilic proteins (Rentier-Delrue et al., 1998Go). Here we have presented the cloning and sequencing of CDA genes from B.psychrophilus and B.caldolyticus, and described the over-expression, purification and thermostability of the corresponding recombinant gene products, comparing them with the mesophilic counterpart isolated from B.subtilis (Song and Neuhard, 1989Go). The amino acid sequences of the three Bacillus CDAs share 62% identity and the regions considered important for the active site are highly conserved (Figure 1Go). The enzymatic activities of the three CDAs show different temperature profiles: CDABpsy and CDABsubt are inactivated at temperatures above 45 and 50–55°C, respectively, whereas CDABcald is stable up to 65°C. This is consistent with the structural changes observed in the CD spectra, collected at increasing temperatures, and is in agreement with the data of the denaturant-induced unfolding experiments. To evaluate the contribution of the various secondary structure components from the CD spectra, in particular ß-sheet, can be difficult, therefore the X-ray structure of the E.coli enzyme (Betts et al., 1994Go) and the similarities of the primary sequence between the homodimeric E.coli and the Bacillus CDAs can be useful to define the role of amino acids in maintaining the folding and the stability of the proteins. The active site domain of E.coli CDA (from residue E49 to residue K176) shows approximately 32% identity with the Bacillus CDAs and its secondary structure organization is in agreement with the percentage content of the {alpha}-helix, ß-sheet, ß-turn and random coil of Bacillus CDAs deduced from the CD spectra (Table IGo). The CD spectra in the far-UV region of the native CDAs at 25°C (Figure 3Go) are almost overlapping, suggesting that the secondary structures and tertiary interactions of these enzymes, and therefore their overall structure, are similar. The CD spectra recorded at increasing temperature showed that CDABcald maintained a high percentage of {alpha}-helix up to 70°C, with no significant alterations in ß-sheet content, whereas CDABpsy and CDABsubt showed a dramatic decrease in helix content, a partial decrease in ß-sheet structure and a parallel increase in random coils at increasing temperatures. The structural changes observed in the extremophilic CDAs are mainly due to changes in temperature. In fact, incubation of the tetrameric CDA with a low concentration of denaturant, resulted in a total dissociation of the enzyme into inactive monomers but no structural changes accompanying the dissociation were detected by CD (manuscript in preparation). Considering that at temperatures up to 50°C for the CDABpsy and CDABsubt and 75°C for the CDABcald CDA the enzymatic activity is up to 70% (see Figure 2Go), it is possible to deduce that the temperature is not able to induce a strong dissociation of the tetramer into monomers. It is reasonable to think that at very extreme temperatures, the decrease of the enzymatic activity can depend both on dissociation into monomers and protein unfolding. Since there is no extensive difference in the primary sequence, most of the amino acid substitutions might be temperature-related so that minute structural alterations could be sufficient to cope with the various extreme conditions. By comparing the amino acid compositions of the three CDAs (Figure 1Go) several features are evident. One is the lower content of uncharged polar residues (Ser, Thr, Asn and Gln), already observed in a typical (300-aa) thermophile protein (Haney et al., 1999Go) and a higher content of Glu and Pro in CDABcald relative to CDABpsy and CDABsubt. However, the proline content of CDABcald is not much higher than of CDABpsy as expected for a thermophilic protein compared to its psychrophilic homolog (Vieille and Zeikus, 1996Go). Nevertheless, the clustering of four Pro residues in CDABcald (P80, P82, P84 and P85), immediately preceding the {alpha}-helix containing the two cysteine residues co-ordinating the zinc atom in the active site (Betts et al., 1994Go; Cambi et al., 1998Go), may be important for its higher thermostability. Noteworthy is that the same region contains only two prolines (P82 and P85) in CDABpsy and three (P80, P82 and P85) in CDABsubt. Proline is known to stabilize the folded conformation of a protein by decreasing the entropy of unfolding (Matthews et al., 1987Go) and was also reported to occur frequently at the N-cap of helices. The CD spectra obtained in the far-UV region at different temperatures may help in understanding the overall folding and stability of the Bacillus CDAs. They seem to confirm the key role played by prolines in stabilizing the two {alpha}-helices of CDABcald with respect to the other two enzymes: although the CD spectra at 25°C revealed a substantial structural identity (Figure 3aGo), the behavior of the three CDAs is clearly different when CD spectra are collected at increasing temperature (Figure 4a, b, cGo). Besides the rigid constraint of the four prolines, at the beginning of the second {alpha}-helix, near the active site, the Y63 residue present only in CDABcald may indicate an aromatic–aromatic interaction with F60 that could induce a stabilization of this helix (Serrano et al., 1991Go). Further comparison of the B.psychrophilus and the B.caldolyticus CDA shows several mutations from polar amino acids to aliphatic residues (S10A, 16L, S46A, Y104I, T106A, E114V, L127A and Y132A) resulting in an aliphatic index (Ikai, 1980Go) of 93.83 for CDABcald as compared to 77.66 for CDABpsy. Also, mutations from non-polar to polar amino acids (A32T, V63Y, G81R, P102K and G123E) were observed, which may be responsible for the formation of stabilizing hydrogen bonds which cannot be formed by the corresponding non-polar residues in CDABpsy. Comparison of the amino acid sequences of CDABsubt and CDABcald showed that several polar amino acids present in the mesophilic enzyme are replaced by aliphatic residue in the thermophilic CDA (D132A, S94A, S127A, T106A, T8V, E114V and R3I), reflecting the lower aliphatic index (84.71) calculated for CDABsubt, which is intermediate between the two extremophilic counterparts (see above). The reversible denaturant-induced unfolding data show that the native state of CDABsubt is only 2.5 kcal/mol more stable than the denatured state; a slight increase is observed in CDABpsy (3 kcal/mol) and a higher increase in CDABcald (4.3 kcal/mol). The data calculated for the apparent free energy change and the behavior under thermal denaturation of the three proteins are in agreement and seem to be independent of the denaturant agent used.

Based on the CD spectra and assuming that the assignments of secondary structures are correct, ß-sheet structure seems to be more stable towards heat than {alpha}-helical structure. This might be the basis of the corresponding stability in the active site built around the two {alpha}-helices providing the zinc ligands. This would maintain an intact core in which hydrophobic interactions play the leading role for the formation and the stabilization of the secondary conformation. This can also be observed from the near-UV CD spectra recorded at different temperatures (Figure 4a', b', c'Go), mainly for CDABcald. The situation is different for CDABsubt, in which tertiary interactions seem very sensitive to temperature. A general decreased polarity and the increased presence of charged as well as aromatic amino acids may represent a general motif of stabilization of CDABcald and CDABpsy, with respect to CDABsubt. Crystallographic studies of the tetrameric CDAs in progress will hopefully provide a clearer insight into the structural differences between the three enzymes.


    Notes
 
5 Present address: Tumor Immunology Laboratory, Philips van Leydenlaan 25, NL-6525 EX Nijmegen, The Netherlands Back

6 To whom correspondence should be addressed: Dipartimento di Scienze Veterinarie, Università di Camerino, Via della Circonvallazione 93–95, 62024 Matelica (MC), Italy. E-mail: a.vita{at}cambio.unicam.it Back


    Acknowledgments
 
The authors would like to thank Mrs Natalina Cammertoni for technical assistance.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Betts, L., Xiang,S., Short,S.A., Wolfenden,R. and Carter,C.W.,Jr (1994) J. Mol. Biol., 235i, 635–656.[ISI][Medline]

Bradford,M.M. (1976) Anal. Biochem., 72, 249–254.

Cambi,A., Vincenzetti,S., Neuhard,J., Costanzi,S., Natalini,P. and Vita,A. (1998) Protein Eng., 11, 59–63.[Abstract]

Clark,D.J. and Maaløe,O. (1967) J. Mol. Biol., 23, 99–112.[ISI]

Cohen,R.M. and Wolfenden,R. (1971) J. Biol. Chem., 246, 7561–7565.[Abstract/Free Full Text]

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Received December 12, 2000; revised June 25, 2001; accepted July 10, 2001.





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