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Biochimica et Biophysica Acta

Purification and characterization of recombinant Caulobacter crescentus

Cu,Zn superoxide dismutase

Ivana De Domenico a, Amalia Lania a, Maria Carmela Bonaccorsi di Patti b, Andrea Battistoni c,

Giovanni Musci a,*, Alessandro Desideri c

a Department of Microbiological, Genetic and Molecular Sciences, University of Messina, Salita Sperone, 31, 98166 Messina, Italyb Department of Biochemical Sciences, University of Rome La Sapienza, Rome, Italy

c Department of Biology, University of Rome Tor Vergata, Rome, Italy

Received 13 July 2005; received in revised form 20 August 2005; accepted 24 August 2005

Available online 13 September 2005

Abstract

Recombinant Cu,Zn Superoxide Dismutase from Caulobacter crescentus has been expressed in Escherichia coli and characterized. The

corresponding recombinant protein has a molecular weight typical of a homodimeric Cu,ZnSODs and an activity comparable to that of other

prokaryotic enzymes. The copper active site is characterized by a peculiar axial geometry as evidenced by its electron paramagnetic resonance

spectrum, moreover, the copper atom displays a low accessibility toward external chelating agents indicating a lower solvent accessibility when

compared to other prokaryotic enzymes. Investigation of the enzyme thermal stability through differential scanning calorimetry indicates the

occurrence of two transitions at low and higher temperature that are found to be due to the apo and holo protein, respectively, confirming that the

metals have a crucial role in the stabilization of this class of enzymes.

D 2005 Elsevier B.V. All rights reserved.

Keywords: Superoxide dismutase; Caulobacter crescentus; Copper

1. Introduction

Cu,Zn superoxide dismutase (Cu,ZnSOD) is a major

component of the antioxidant defense system that protects

cells from oxidative damage by converting superoxide radicals

into hydrogen peroxide and oxygen [1]. For a long time,

Cu,ZnSOD has been considered almost exclusively an eukary-

otic enzyme, but it is now firmly established that Cu,ZnSOD is

present in a large number of bacteria [2]. While in eukaryotes,

Cu,ZnSODs are mainly found in the cytoplasm, in bacteria,

they are exported to the periplasm or to the outer membrane.

Such extracytoplasmic location of the enzyme suggests that its

role is to protect bacteria from superoxide generated outside the

bacterium or within the periplasmic space [2].

1570-9639/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbapap.2005.08.021

Abbreviations: SOD, superoxide dismutase; DTT, dithiothreitol; EDTA,

ethylenediaminetetraacetic acid; EPR, Electron Paramagnetic Resonance; DSC,

Differential Scanning Calorimetry

* Corresponding author. Tel.: +390906765194; fax: +39090392733.

E-mail address: musci@unime.it (G. Musci).

Bacterial and eukaryotic Cu,ZnSODs share a similar

monomer fold and a conserved arrangement of the redox

center, where, in the oxidized form of the enzyme, Cu2+ is

coordinated by four histidine residues and Zn2+ is coordinated

by three histidine and one aspartate residues [3]. Despite these

similarities, which indicate that they likely derive from a

common ancestor gene, prokaryotic and eukaryotic Cu,Zn-

SODs show remarkable differences in the organization of the

active site channel and in the way subunits are assembled [2–

4]. Moreover, several prokaryotic Cu,ZnSODs show unique

sequence features, which are suggestive of significant structural

differences not only with respect to eukaryotic Cu,ZnSODs,

but also with respect to the already characterized prokaryotic

enzymes. Examples of species-specific variations include the

presence of insertions and/or deletions in the loops connecting

the elements forming the beta-barrel [3,4], mutations in

residues involved in the metal coordination [5,6] and the

presence of additional protein domains [7]. Such differences

between bacterial Cu,ZnSODs are expected to significantly

influence the structural and functional properties of individual

enzyme variants. Recent studies have also revealed that

1764 (2006) 105 – 109

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I. De Domenico et al. / Biochimica et Biophysica Acta 1764 (2006) 105–109106

bacterial Cu,ZnSOD activity and stability can be finely

modulated even by small differences in the quaternary structure

of different enzymes [8–10].

An example of bacterial Cu,ZnSOD exhibiting an unusual

primary sequence is the enzyme from Caulobacter crescentus,

a free living bacterium originally cultured from fresh water

ponds [11]. This enzyme, which has been shown to be active

and able to bind either copper and zinc [11], is characterized by

a specific pattern of insertion and deletions that could modify

either the structure of the active site or the modality of

interactions between subunits, and by the substitution of a

conserved zinc ligand [12]. These features make C. crescentus

Cu,ZnSOD a very interesting candidate for structure/function

investigations on this class of enzymes. Here, we describe the

purification and a preliminary characterization of the enzyme

produced in E. coli by recombinant DNA techniques.

2. Materials and methods

C. crescentus CB15 ATCC strain 19089 was obtained from American Type

Culture Collection. E. coli strain DH5a was a kind gift of Dr. C. Falcone

(University of Rome La Sapienza). C. crescentus was grown in nutrient broth

(peptone 1 g/l, yeast extract 0.2 g/l and MgSO4 0.2 g/l). The cells were

collected by centrifugation and genomic DNA extraction was performed

according to Chen and Kuo [13]. Oligonucleotides complementary to the amino

and the carboxyl terminals of the C. crescentus Cu,ZnSOD sequence were

synthesized and used to PCR amplify the coding region of the gene. The

amplification product was cloned into the vector pUC18 using EcoRI and XbaI.

The recombinant plasmid was transformed into E. coli DH5a. DNA sequence

analysis at Biogen-ENEA facilities was performed to verify the identity of the

cloned sequence with that of the C. crescentus Cu,ZnSOD gene present in the

GeneBank.

Recombinant clones were grown in LB medium containing 50 mg/ml

ampicillin and 1 mM CuSO4 for 18 h at 37 -C. The periplasmic fraction was

isolated as described previously [14] and dialyzed against 50 mM potassium

phosphate buffer pH 6.5 to remove sucrose. Proteins contained in the

periplasmic fraction were concentrated and subjected to (NH4)2SO4 fraction-

ation at 4 -C. At variance with what reported on the non-recombinant native

Fig. 1. Alignment of C. caulobacter Cu,ZnSOD amino acid sequence to that of o

pleuropneumoniae and E. coli. The global alignment has been deduced from optim

described [4]. The h-strand elements are indicated above amino acid sequences with

shown in bold and are indicated by C and Z, respectively. Residues involved in the d

of A. pleuropneumoniae Cu,ZnSOD, encoding a His-rich metal binding domain [7

protein expressed by C. crescentus, which was found to precipitate already at

50% (NH4)2SO4 [11], all the recombinant enzyme expressed in E. coli has

been found in the supernatant of 85% (NH4)2SO4 precipitation. The

supernatant containing Cu,ZnSOD was dialyzed against several changes of

50 mM potassium phosphate buffer pH 7.4 for 18 h, followed by 20 mM

potassium phosphate buffer pH 7.4 for 24 h. At this stage, the enzyme was

more than 90% pure and was used for subsequent studies without further

purification. Protein concentration was quantified with the bicinchoninic acid

(BCA) protein assay kit (Pierce, Rockford IL). The final yield of recombinant

Cu,ZnSOD was 8 mg of protein per liter of bacterial culture. Copper content,

evaluated by double integration of the EPR spectrum vs. a Cu–EDTA

standard complex, indicated the presence of only 1 Cu/dimer. This result was

confirmed by atomic absorption spectroscopy, carried out with a Perkin Elmer

3030 instrument equipped with a graphite furnace, that indicated the presence

of 0.9 Cu atom/protein and of 1.75 Zn atom/protein, in line with a previous

report [11].

SOD activity was visualized in non-denaturing polyacrilamide gels as an

achromatic zone that did not stain upon photochemical reduction of nitroblue

tetrazolium to formazan blue [15]. SOD activity was estimated by the inhibition

of pyrogallol autoxidation, as described previously [16]. The stability of

superoxide dismutase activity in the presence of EDTA was studied following

described procedures [10,17].

Low temperature EPR spectra were recorded on a Bruker ESP 300

spectrometer operating at 9 GHz with 100-kHz field modulation. The EPR

spectra were manipulated by using the EPR package provided by Stelar (Mede,

Italy) and simulated by using software kindly provided by Dr. Bencini

(Florence, Italy). DSC experiments were performed with a MicroCal VP-DSC

microcalorimeter (MicroCal Inc., Northampton, MA). Proteins were extensive-

ly dialyzed against 100 mM potassium phosphate buffer pH 7.8 and gently

degassed before scanning. The protein concentration was 0.2–0.3 mg/ml, and

the scan speed was 60 -C/h. No reversibility of thermal unfolding was found at

the end of the scan, therefore, the second scan was used as baseline to correct

the thermograms. Heat capacity versus temperature profiles for the thermally

induced transition were analyzed, using the Origin version 5.0 software

provided by MicroCal, to obtain values for Tm (temperature of maximum heat

capacity) and DH (heat of reaction).

Apo-Cu,Zn SOD was obtained by extensive dialysis of the enzyme (15–30

mg/ml) in 50 mM sodium acetate buffer pH 3.8 containing 1 mM EDTA for

three days at room temperature. The enzyme was then dialyzed overnight at 4

-C in the same buffer containing 100 mM NaCl to remove excess EDTA, and

finally against 50 mM acetate buffer pH 5.5 at 4 -C for 12 h. Copper removal

was monitored spectrophotometrically at 680 nm. Distilled water and glassware

ther enzymes from Gram-negative bacteria: P. leiognathi, S. typhimurium, A.

al superimposition of available bacterial Cu,ZnSOD structures, as previously

the6 symbol. The histidine residues coordinating the active site metal ions are

imer interface of P. leiognathi SOD [18] are underlined. The N-terminal region

] has been omitted.

Fig. 3. Effect of EDTA on C. crescentus and P. leiognathi SOD activity. The

proteins, 0.04 mg/ml in 10 mM phosphate buffer, pH 7.2, were incubated with

the 0.1 mM chelator for different times at 37 -C.

I. De Domenico et al. / Biochimica et Biophysica Acta 1764 (2006) 105–109 107

were made metal free by either rinsing in 10% nitric acid or treating with

Chelex-100 (Bio-Rad).

3. Results and discussion

Fig. 1 shows the amino acid sequence alignment of mature

C. crescentus Cu,ZnSOD (lacking the signal peptide sequence)

in comparison with the sequences of the four Cu,ZnSODs from

Gram-negative bacteria whose structures have been determined

by X-ray crystallography [4,8,18–20]. Although the amino

acids length of C. caulobacter Cu,ZnSOD is identical to that of

the enzyme from P. leiognathi (151 residues) and comparable

to that of the other enzyme variants included in this alignment,

its primary sequence shows several features which are

suggestive of significant modifications of its three-dimensional

structure with respect to the so far characterized bacterial

Cu,ZnSODs. In fact, while the regions forming the h-sheetelements are reasonably well conserved, there is a 7-amino acid

deletion in the disulphide bond subloop and a 8–9 residue

insertion in the Greek-key loop. The SS-subloop of bacterial

Cu,ZnSODs forms a very flexible and solvent exposed loop

containing several charged residues involved in substrate

attraction [18,19,21,22]. Despite the shorter length of this loop

in C. crescentus Cu,ZnSOD is expected to modify the

flexibility and accessibility of the active site region, it should

be noted that the SS-subloop conserves some of the charged

residues which are involved in the electrostatic guidance of

superoxide to the active site [21]. Several residues involved in

the formation of the dimer interface in bacterial Cu,ZnSODs

are provided by the h-strand 4f, close to the insertion in the

Greek key loop [3,4]. While most of the residues involved in

the formation of the dimer in the other bacterial Cu,ZnSODs

are conserved or have been substituted conservatively, suggest-

ing similar modality of interaction between the subunits, it is

likely that the amino acids insertion in the Greek key loop

extends the dimer interface area. A still more striking feature

characterizing the sequence of C. crescentus Cu,ZnSOD is the

His81Asp mutation, which leads to the substitution of one of

the histidine residues involved in zinc ligation in all the other

characterized Cu,ZnSODs, with the exception of the enzyme

from Mycobacterium tuberculosis, which, however, does not

Fig. 2. EPR spectra of P. leiognathi Cu,ZnSOD (A) and C. crescentus

Cu,ZnSOD (B). Experimental conditions: frequency 9.5 GHz; power 20 mW;

modulation 10 G; temperature 110 K. Spectrum c is a computer simulation of

spectrum b, with the following parameters: g–=2.024; g // =2.247; A–=10 G;

A// =158.5 G.

contain zinc [6]. This alignment, therefore, suggests that in C.

crescentus Cu,ZnSOD the zinc ion is coordinated in an unusual

arrangement involving two histidine and two aspartic acid

residues.

To undertake a structural characterization of C. caulobacter

Cu,ZnSOD, the enzyme has been overproduced in E. coli and

purified as described in Materials and methods. The purified

enzyme has a molecular mass of approximately 16 kDa as

evaluated by reducing SDS-PAGE, indicating that the original

leader peptide is correctly removed also in E. coli. The

recombinant enzyme was active and 50% inhibition of

pyrogallol autoxidation was achieved with 105 ng of protein

(9523 U/mg), a value that is comparable to that of the

homologous bacterial dimeric enzyme from P. leiognathi

(Battistoni, A. and Desideri, A., unpublished data). The

Fig. 4. DSC scans of C. crescentus recombinant Cu,ZnSOD. Panel A, purified

protein. Panel B, purified apoprotein. Thermodynamic data are shown in

Table 1.

Table 1

Thermodynamic parameters for recombinant C. crescentus holo- and apo

Cu,ZnSOD

Enzyme Tm(-C) DH (kcal/mol

Holo 75.8 132

Apo 50.0 67.8

I. De Domenico et al. / Biochimica et Biophysica Acta 1764 (2006) 105–109108

molecular mass of the native enzyme is 32 kDa when examined

by non-reducing SDS-PAGE, confirming that the enzyme has a

dimeric structure consisting of two identical subunits (data not

shown).

Fig. 2 shows the EPR spectrum of recombinant C.

crescentus compared to that of P. leiognathi Cu,ZnSOD [23].

The line shape, typical of a copper atom in the oxidized state, is

different in the two proteins, the spectrum of recombinant C.

crescentus Cu,Zn SOD showing an axial shape, with values of

the hyperfine splitting and of the g parameter in the parallel

region (as calculated from the simulated spectrum c) higher and

lower than that of P. leiognathi respectively (A// =158.5 G vs.

137.2 G and g// =2.247 vs. 2.267 for C. crescentus and P.

leiognathi, respectively). These parameters indicate that copper

has significantly different coordination environments in C.

crescentus vs. P. leiognathi Cu,ZnSOD, despite the fact that

the copper ligands are identical in the two proteins as evaluated

by sequence alignment. Such an effect can then be due to the

difference in the ligand coordination of the proximal zinc atom,

that in C. crescentus has a histidine in place of the conserved

aspartate, and/or to three dimensional constraints that cannot be

envisaged without the solution of the 3D structure of the

protein.

The accessibility of the copper site of Cu,ZnSOD has been

evaluated in a comparative way for C. crescentus and P.

leiognathi Cu,ZnSOD measuring the dismutase activity as a

function of time, upon incubation of the protein with 0.1 mM

EDTA in 10 mM phosphate buffer, pH 7.2. As shown in Fig. 3,

EDTA induces a loss of the enzymatic activity that can be

related to the active site copper chelation. The loss of activity is

faster in P. leiognathi than in C. crescentus enzyme, being,

after 1 h of incubation, more than 75% for P. leiognathi and

only 20% for C. crescentus. Both SODs were totally

inactivated after longer incubation times in presence of EDTA.

Previous studies have shown that the EDTA-mediated inacti-

vation is faster for the monomeric Cu,ZnSOD from E. coli than

for most dimeric bacterial enzymes of this class [24] and that

mutations at the dimer interface of P. leiognathi Cu,ZnSOD

strongly enhance the rate of copper loss in presence of EDTA

[9,10]. Such studies have suggested that differences in the rates

of inactivation by EDTA are indicative of modifications at the

dimer interface between individual bacterial Cu,ZnSODs,

which affect the conformational flexibility of the active site

region [8,10,24]. On this ground, it is possible to suggest that

C. crescentus Cu,ZnSOD is characterized by a more stable

interaction between the subunits compared to P. leiognathi

Cu,ZnSOD. Stabilization of the subunit interaction in C.

crescentus Cu,ZnSOD is likely favored by an extension of

the subunit interface area due to the insertion in the Greek key

loop.

The thermal stability of C. crescentus Cu,Zn SOD has been

assessed by differential scanning calorimetry. The DSC profile,

shown in Fig. 4A, reveals the presence of two distinct peaks

that can be analyzed in terms of two independent transitions

with melting temperature around 50 -C and 75 -C, respectively.The presence of two peaks in the DSC scan can be explained

either as the occurrence of two different denaturing steps or by

-

)

the presence of two distinct species with different stability. The

DSC scan carried out on a solution containing more than 95%

of apo Cu,ZnSOD (as estimated by atomic absorption

spectrometry) is also reported in Fig. 4B. The DSC profile of

this sample is characterized by an intense peak with melting

temperature of 50 -C. This result clearly indicates that the

peaks at low and high temperature are diagnostic for the

melting of the apo and holo form, respectively. Table 1 reports

the calculated thermodynamic parameters. The Tm of the apo

enzyme is identical to that of the apo-enzyme from E. coli and

Bos taurus [17], whereas the Tm of the metallated enzyme is

5 -C higher than that reported for the dimeric enzyme from P.

leiognathi [18]. These observations confirm that both the

metallation state and the dimeric structure contribute to the

stabilization of Cu,ZnSODs. It is likely that an extended

dimerization area in C. crescentus Cu,ZnSOD could be

responsible of the higher Tm of this enzyme compared to P.

leiognathi Cu,ZnSOD. Interestingly, as most of the contribu-

tion of metals to Cu,ZnSOD stabilization should be ascribed to

zinc [25], the high melting temperatures of both the holo

enzymes from C. crescentus and P. leiognathi suggests that the

differences in the zinc coordination environment in the two

enzymes have little influence on the stability of the zinc site.

In conclusion, the data here reported indicate that the

Cu,ZnSOD from C. crescentus displays specific characteristics

when compared to other bacterial enzymes of this class, such as

an active site with a copper atom characterized by an almost

axial geometry, a low accessibility to external chelating agents

and a high thermal stability. Although only the solution of its

3D structure will permit to formulate clear correlations between

the enzyme structure and its physicochemical properties, it is

likely that most of the specific properties of C. crescentus

Cu,ZnSOD are due to its peculiar active site metal coordination

and to an altered modality of interaction between the subunits.

Acknowledgements

We thank Dr. Anna Giartosio (Universita’ La Sapienza-

Roma) for helping with calorimetric experiments. This work

was partially supported by a Murst Cofin 2004 Project.

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