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
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Abstract |
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Keywords: circular dichroism/cytidine deaminase/extremophiles/thermostability
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Introduction |
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Materials and methods |
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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 and SØ5201 (MC1061 cdd::Tn10 pyrD::Kan). DH5
was used as host for all clonings (Sambrook et al., 1989
). 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, 1989
) was used for overproduction of B.subtilis CDA. Bacillus caldolyticus and B.psychrophilus were grown in L-broth (Miller, 1972
) 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, 1967
) 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, 1971) at 290 nm using deoxycytidine as substrate (
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 TrisHCl, 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., 1999) 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 (190240 nm) and in the near (240320 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 [] (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., 1986
). 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., 1996). The purity of the enzymes and the molecular weight of the subunit were determined by SDSPAGE as described by Laemmli (Laemmli, 1972
). 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, 1976
) was used in the microprotein assay using BSA as a standard.
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Results |
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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 PstIEcoRI 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. SDSPAGE 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 NcoIBamHI 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. SDSPAGE 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 1) 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., 1994
; Jensen et al., 1997
), 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., 1980
).
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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 SDSPAGE 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 5758 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, 1994; Vincenzetti et al., 1996
). 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 2
. 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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Figure 3 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 3a
), indicating a very similar secondary structure organization for the three enzymes. According to the spectra, CDABsubt possessed a higher content of
-helix and less ß-sheet secondary structure than the other two enzymes (see Table I
for details). Although the aromatic pattern seemed not very indicative of a particular asymmetry center (Figure 3b
), 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 4
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
-helical content and part of the ß-sheet structure only at 8595°C (Figure 4b
). The secondary structure organization of CDABpsy was more sensitive to heat than the thermophilic homolog, as a gradual decrease of
-helix content and part of the ß-sheet was evident from 50°C (Figure 4c
). The same behavior was observed for CDABsubt, where the decrease of secondary structure, particularly the
-helix content, seemed even more affected by increasing temperatures (Figure 4a
). Moreover, at 10°C the secondary structure content of CDABsubt was less than that observed from the spectrum at 25°C (see Table I
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'
), but surprisingly, also the near-UV CD spectra of CDABpsy (Figure 4c'
) were almost insensitive to heat, as compared to the far-UV spectra (Figure 4c
). This result may indicate that, in spite of a relevant decrease of
-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'
) were consistent with the behavior of the far-UV spectra (Figure 4a
), 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 4
, 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 5
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, 1975
) the apparent free energy change
GD was calculated from the apparent denaturation equilibrium constant KD, which is determined by the expression (
i
obs) / (
obs
f), where
i,
f and
obs represent the initial, final and observed values of ellipticity, respectively. On the assumption of a linear dependence of the
GD versus denaturant concentration, the value of
G0, i.e. the apparent free energy change in the absence of denaturant, was obtained by extrapolation to zero denaturant concentration (Figure 5
, 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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Discussion |
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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 -helical structure. This might be the basis of the corresponding stability in the active site built around the two
-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'
), 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.
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Notes |
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6 To whom correspondence should be addressed: Dipartimento di Scienze Veterinarie, Università di Camerino, Via della Circonvallazione 9395, 62024 Matelica (MC), Italy. E-mail: a.vita{at}cambio.unicam.it
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Acknowledgments |
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References |
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Received December 12, 2000; revised June 25, 2001; accepted July 10, 2001.