A novel Cu,Zn superoxide dismutase from the fungal strain Humicola lutea 110: isolation and...

Spectrochimica Acta Part A 55 (1999) 2249–2260

A novel Cu,Zn superoxide dismutase from the fungal strainHumicola lutea 110: isolation and physico–chemical

characterization

Pavlina Dolashka-Angelova a,*, Maria Angelova b, Ljubka Genova b,Stanka Stoeva c, Wolfgang Voelter c

a Institute of Organic Chemistry, Bulgarian Academy of Sciences, 1113 Sofia, Bulgariab Institute of Microbiology, Bulgarian Academy of Sciences, 1113, Sofia Bulgaria

c Abteilung fur Physikalische Biochemie, Physiologisch-chemisches, Institut der Uni6ersitat Tubingen, Hoppe-Seyler-Straße 4,D-72076, Tubingen, Germany

Received 12 November 1998; accepted 10 February 1999

Abstract

The fungal strain Humicola lutea 110 produces a mangan- and a copper zinc-containing superoxide dismutases(SOD). In this study, the purification, N-terminal sequence and spectroscopic properties of the new Cu,Zn SOD aredescribed. The preparation of the pure metalloenzyme was achieved via treatment of the strain with acetone followedby gel and ion exchange chromatography. The protein consists of 302 amino acid residues and has a molecular massof approximately 32 kDa, as determined by PAG electrophoresis and 3100 U mg−1 protein-specific activity. It is adimeric enzyme with two identical subunits of 15 950 Da, as indicated by SDS-PAGE, mass spectroscopic and aminoacid analysis. The N-terminal sequence analysis of the Cu,Zn SOD from the fungal strain revealed a high degree ofstructural homology with enzymes from other eukaryotic sources. Conformational stability and reversibility ofunfolding of the dimeric enzyme were determined by fluorescence and circular dichroism (CD) spectroscopy. Thecritical temperature of deviation from linearity (Tc) of the Arrhenius plot ln (Q−1−1) vs. 1/T was calculated to be68°C and the respective activation energy for the thermal deactivation of the excited indole chromophores is 42 kcalmol−1. The melting temperatures (Tm) were determined by CD measurements to be 69°C for the holo- and 61°C forthe apo- enzyme. The fluorescence emission of the Cu,Zn SOD is dominated by ‘buried’ tryptophyl chromophores.Removal of the copper–dioxygen system from the active site caused a 4-fold increase of the fluorescence quantumyield and a 10 nm shift of the emission maximum position towards higher wavelength. © 1999 Elsevier Science B.V.All rights reserved.

Keywords: Superoxide dismutases; Amino acid analysis; Protein sequencing; Circular dichroism; Conformational analysis; Fungalstrain Humicola lutea

www.elsevier.nl/locate/saa

Abbre6iations: CD, circular dichroism; SOD, superoxide dismutase; PAGE, polyacrylamide gel electrophoresis; UV, ultravioletabsorption; TFA, trifluoroacetic acid; Q, quantum yield.

* Corresponding author. Tel.: +359-368200; Fax: +359-2700225.E-mail address: [email protected] (P. Dolashka-Angelova)

1386-1425/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.

PII: S1386 -1425 (99 )00036 -0

P. Dolashka-Angelo6a et al. / Spectrochimica Acta Part A 55 (1999) 2249–22602250

1. Introduction

Superoxide dismutases (SOD) (EC 1.15.1.1) areknown to play a crucial role in the defense againstoxidative cell damage. It has been well establishedthat all aerobic organisms contain SODs catalyz-ing the conversion of the superoxide anion tooxygen and hydrogen peroxide [1]. SODs are afamily of metalloproteins, as classified by theirmetal cofactor: copper–zinc, manganese and ironforms. The copper–zinc SOD is ubiquitously dis-tributed in eukaryotes, with the exception ofmixomycetes; there are only a few examples ofCu,Zn SODs isolated from protozoa or greenalgae. Cu,Zn SOD has been found in all oxygen-metabolizing organisms, e.g. in bacteria, such asPhotobacterium leiognathi, Caulobacter crescentus,Brucella abortus, Haemophilus influenzae, or Sac-charomyces cere6isiae and also in higher organ-isms up to intra- and extracellular human tissue[2–8]. The human intracellular Cu,Zn SOD con-tains 153 amino acids subunit−1, has a subunitmolecular mass of 16 kDa and is a dimer. On theother hand, human extracellular SOD is a te-trameric secreted protein in which each subunitcontains 222 amino acids and a N-linkedoligosaccharide. It is suggested that starting froma monomeric SOD precursor procaryotic and eu-caryotic Cu,Zn SODs have evolved to a dimericstructure [9], however a heat-stable monomericform of Cu,Zn SOD could be purified from theperiplasmic space of Escherichia coli [10,11]. Anovel type of SOD containing Ni in the active sitewas discovered recently in Streptomyces spp. andStreptomyces coelicolor [12], and a Cu,Co SODderivative was prepared from bovine erythrocytesand its properties were investigated by two-dimen-sional NMR [13].

Cu, Zn SODs are inherently stable enzymes, asalso demonstrated by differential scanningcalorimetry [11,14,15]. Temperatures exceeding70°C are needed for irreversible inactivation ofdismutase activity of bovine SOD. Physical un-folding occurs with transition temperatures (Tm)in the range of 80–90°C depending on experimen-tal conditions. The differential scanning calorime-try profile of human extracellular SOD consists oftwo components, probably representing the un-

folding of the oxidized and reduced forms of theenzyme, with denaturation temperatures (Tm) of74.9 and 83.6°C [16]. The role of the dimericstructure in Cu,Zn SODs was investigated, study-ing the stability to a variety of agents against themonomeric variant of the Escherichia coli enzyme.The melting temperature of the E. coli enzymewas found to be pH-dependent with holoenzymetransitions of 66°C at pH 7.8 and 79.3°C at pH6.0 [9]. Several factors contribute to the enzymestability, including the prosthetic metal ions [17],the disulfide bond [18] or the close packing in thehydrophobic interfaces between the subunits andthe two halves of the b-barrel core [19]. Structuraland functional properties of SODs produced bylower multicellular eukaryotes, such as filamen-tous fungi, have not yet been studied in detail, butamino acid compositions [20–24] and the analysisof the 3-dimensional structure of the enzyme Pho-tobacterium leiognathi have shown that prokary-otic and eukaryotic Cu,Zn SODs share aconserved active site ligand and the topology ofthe Greek key b-barrel [25].

Preliminary investigations showed the presenceof Mn and Cu,Zn SOD in the fungal strain Humi-cola lutea 110 [26,27]. In this paper the purifica-tion and characterization of a Cu,Zn-containingSODs from the filamentous fungus H. lutea 110 isdescribed.

2. Experimental

2.1. Superoxide dismutase purification procedure

The fungal strain H. lutea 110, an active argon-laser mutant, was selected in the Laboratory ofMycology at the Institute of Microbiology, Sofia[26]. The strain was kept on beer agar (pH 6.3) at4°C. The composition of the seed and productionmedia was, as described earlier [27].

The frozen mycelium was partially thawed andthen suspended in 4 volumes of 25 mM potassiumphosphate buffer, pH 7.8, containing 1 mMPMSF. The suspended material was disrupted bythe homogenizer model ULTRA-TURRAX-IKA-WERK. Cell debris were removed by filtrationthrough a Buchner funnel and the mixture was

P. Dolashka-Angelo6a et al. / Spectrochimica Acta Part A 55 (1999) 2249–2260 2251

clarified by centrifugation at 13 000×g for 20 minat 4°C. All subsequent steps were performed at4°C. The supernatant was concentrated approxi-mately three times by membrane ultrafiltration(Amicon PM-10, Oosterhout, The Netherlands).The concentrated crude extract was further frac-tionated using chilled (−20°C) acetone. Cooledacetone was slowly added to the culture filtratesto a concentration of 0.4 volume with vigorousstirring for 30 min. The mixture was incubated for2 h at (−20°C), and the precipitate was collected,centrifuged at 13 000×g for 20 min and dis-carded, while the supernatant was treated with 1.0volume of chilled acetone. The mixture was held12 h at −20°C; then the precipitate was collectedby centrifugation (13 000×g for 20 min) andsuspended in distilled H2O. The clear viscous su-pernatant was loaded on a Sephadex G-75column (2×50 cm, Pharmacia, Uppsala, Swe-den), previously equilibrated with 50 mM potas-sium phosphate buffer, pH 7.8, and the columnwas eluted with the same buffer.

Further purification was achieved by ion ex-change chromatography on a DEAE cellulose-52column (3.5×10 cm, Serva, Heidelberg, Ger-many), equilibrated in 50 mM KH2PO4 buffer,pH 7.8. The Mn-containing SOD was eluted, firstwith 60 mM NaCl in 50 mM phosphate buffer,

followed by an NaCl gradient (0–0.3 M) to elutethe Cu,Zn SOD. The fractions with lower activity,containing Cu,Zn SOD, isolated with the secondpeak, were loaded for additional purification to aFPLC system, equipped with a 10/10 Mono Qanion exchange column. The column had beenpreviously equilibrated with 50 mM phosphatebuffer, pH 7.8, and eluted with a linear sodiumchloride gradient (0–0.1 M NaCl). Fractions with3100 U mg−1 protein were further purified anddesalted on a Nucleosil RP C18 column (250×10mm, Macherey-Nagel, Duren, Germany) to deter-mine its N-terminal sequence and amino acidcomposition. The conditions used for HPLC sepa-ration were as follows: eluent A, 0.058% trifl-uoroacetic acid (TFA); eluent B, 80% acetonitrilein A. The gradient was run from 5 to 100% Bwithin 60 min at a flow rate of 1 ml min−1.

2.2. Protein and superoxide dismutase acti6ityassay.

During the purification steps, the SOD activitywas determined by the indirect method ofBeauchamp and Fridovich [28] assaying the re-duction rate of riboflavin and methionine at 560nm by superoxide radicals. Protein concentrationswere determined using the dye-binding assay ofLowry et al. [29] with bovine serum albumin as a

Fig. 1. Polyacrylamide gel electrophoresis (7.5% gel) of purified Cu,Zn SOD. Lane 1, standard mixture: (a) trypsinogen (24 kDa);(b) egg albumin (45 kDa) and (c) bovine albumin (66 kDa); lane 2: gel, stained for protein; lanes 3 and 4: gels, stained for enzymaticactivity; lane 4: MnSOD (a) and Cu,Zn SOD (b).


standard. Metal content was determined using anatomic absorption spectrophotometer equippedwith a graphite furnace.

2.3. Polyacrylamide gel electrophoresis

Purity control of the enzyme by disc gel electro-phoresis was performed on 7.5% polyacrylamidegels, as described by Davis [30], and the gels,stained for protein detection, were compared withduplicate gels, stained for SOD activity, asdescribed by Misra and Fridovich [31]. Themolecular masses of the enzyme and its subunits

were estimated by SDS-PAGE on 10% polya-crylamide gels using the discontinuous buffersystem of Laemmli [32]. The following molecularmass standards were used: (a) trypsinogen (24kDa); (b) egg albumin (45 kDa) and (c) bovinealbumin (66 kDa).

2.4. Automatic amino acid sequence analysis

N-terminal amino acid sequence analysiswas performed using an Applied Biosystemssequencer, model 473A (Weiterstadt, Ger-many).

Table 1Purification steps for Cu,Zn SOD from H. lutea 110

Specific activity (U×mg−1)Purification steps Protein (mg) Activity (U× Yield (%) Purification (fold)103)

1701161 1006830Crude extract 1.011254500 250Acetone fraction (0.4 vol.) 97.0 1.5

680 17.9Acetone fraction (1.0 vol.) 306 4.020812.313.62100Sephadex G-75 15875

2200 5.4 12.9DEAE-cellulose 52 28.6 633100 1.4 18.2FPLC-mono Q 10/10 5 16

Fig. 2. Ion exchange chromatogram (DEAE-52 cellulose column, 3.5×10 cm) of the active fractions received from gel chromatog-raphy. The column was equilibrated with 50 mM phosphate buffer, pH 7.8, and eluted with a linear NaCl gradient (0–0.3M).Fractions of 4 ml were assayed for SOD activity (------) and absorbance at 278 nm (— —).


2.5. Mass spectroscopic analysis

Mass spectra were obtained by matrix-assistedlaser desorption ionization mass spectrometry(Kratos, MALDI I equipment, Shimadzu, Eu-rope). The samples (10 pmol) were dissolved in0.1% (v/v) TFA and applied onto a target. Analy-sis was carried out in a-cyano-4-hydroxy-cinnamic acid. Solutions of chicken egg oval-bumin (M=44 400 Da) and bovine serum albu-min (M=66 430 Da) were used to calibrate themass scale.

2.6. Spectroscopic measurements

Absorption spectra of the purified enzyme wererecorded with a Shimadzu recording spectropho-tometer model 3000. CD was measured with aJasco J-720 dichrograph, equipped with a per-sonal computer IBM PC-AT, PS/2, and expressedas molecular ellipticities, defined as:

[U]=U×Mr

10× l×c

where U is the measured ellipticity in degrees, Mr

the molecular weight per mean residue, l the pathlength of the cell used for the measurements incm, and c the concentration in g ml−1.

The data of the thermal changing of the fluores-cence quantum yield were performed with aPerkin Elmer model LS5 spectrofluorimeter andanalyzed according to the equation [33]:

ln (Q−1−1)= −Ea/RT+ ln K

where Q is the fluorescence quantum yield, K thetemperature-independent rate constant for theemission of fluorescence, Ea the apparent activa-tion energy, R the gas constant and T the absolutetemperature.

The thermal denaturation study was comple-mented by CD measurements of the enzyme andcharacterized by the melting temperature (Tm).The spectra were recorded in the temperatureinterval 20–90°C and in the 200–250 nm region,reflecting the backbone conformation of theproteins.

2.7. Preparation of apo-superoxide dismutase

A sample containing 10 mg ml−1 Cu,Zn SODwas initially dialized for 24 h at 4°C against 50mM sodium acetate, pH 4.0, 2 mM EDTA andthen for 24 h against 50 mM sodium acetatebuffer, pH 4.0, 0.1 M NaCl, to remove excessEDTA. Finally the sample was dialized against 50mM phosphate buffer, pH 7.8.

3. Results and discussion

It was found by polyacrylamide gel elec-trophoresis that the crude extract of the myceliumof the fungal strain H. lutea 110 contained twoclasses of superoxide dismutases expressed by twobands [Fig. 1 (lane 4a, b)] with molecular massesof 76 and 32 kDa. Fig. 1 (lane 4a, b) shows oneslowly moving cyanide-insensitive band and onerapidly moving cyanide-sensitive band, caused by

Fig. 3. FPL chromatogram of the active fraction, collected viaion exchange chromatography. Chromatographic conditions:column, Mono Q 10/10; eluent, phosphate buffer, pH 7.8,linear NaCl gradient (0–0.1 M); flow rate, 1 ml min−1.


Fig. 4. HPL chromatogram of the purified SOD from the fungal strain Humicola lutea 110. Only the material from the majorfraction from FPL chromatography (peak No. 2) was used for further purification. Chromatographic conditions: column, NucleosilRP C 18, 250×10 mm; linear gradient elution, from A to B within 60 min (eluent A: 0.058% TFA, eluent B: 80% acetonitrile insolution A); flow rate, 1 ml min−1.

Mn SOD and Cu,Zn SOD, respectively [30,34]and corresponding to the two bands of the acry-lamide gel, stained for SOD activity [31]. TheCu,Zn SOD activity was obtained as a total activ-ity minus activity in the presence of 2 mM cyanideand the conclusion is that the ratio of Mn SOD toCu,Zn SOD is 4:1.

The results of the purification procedure areshown in Table 1 and Figures 2 to 4. The fractionfrom DEAE-cellulose 52 (tubes 20–35, Fig. 2)was concentrated and additionally purified on aMono Q 10/10 column, using a FPLC system.The fraction corresponding to peak No. 1 in Fig.3 contains Mn SOD and that corresponding to


peak No. 2 Cu,Zn SOD with an enzyme activityof about 2000 U mg−1. After rechromatographyon the same column, the enzyme shows one bandin PAG electrophorese with an activity of 3100 Umg−1 (Fig. 1, lane 2). For N-terminal sequencedetermination, amino acid analysis and massspectroscopic investigations, the Cu,Zn SOD wasfurther purified on a Nucleosil RP C18 column(Fig. 4) via elution with a linear gradient for 60min using the eluents A (100% H2O, 0.1% TFA)and B (80% CH3CN, 0.058% TFA) at a flow rateof 1 ml min−1. Every step of the enzyme purifica-tion was tested by polyacrylamide gel elec-trophoresis. Fig. 1, lanes 2 and 3, show the 7.5%acrylamide gels, stained for protein and SODactivity, respectively. One band is present only inboth gels, indicating that after the last step ofpurification, the Cu,Zn SOD is isolated as a ho-mogeneous enzyme.

The molecular weight of Cu,Zn SOD from H.lutea 110 was determined by several methods. Thesample was subjected to 7.5% PAG electrophore-sis [30] showing one band at 32 kDa (Fig. 1, lane2). Twelve percent SDS gel electrophoresis of pureCu,Zn SOD, however, indicates only one proteinband with a molecular mass of 15 kDa. By meansof matrix-assisted laser desorption ionizationmass spectrometry the mass was determined to be15 950 Da for one subunit (Fig. 5). On gel filtra-tion a single peak was obtained with an Mr of32 900 Da for the whole enzyme. From theseresults it is suggested that the investigated enzymeis a dimer made of identical subunits. Studiesconfirm the literature data that Cu,Zn SODs areusually dimeric [9], and the amino acid composi-tion of the Humicola enzyme is quite similar tothose from eukaryotic organisms, it consists of301 amino acid residues (Table 2). The amino acidanalysis was different from the values given forthe respective Cu,Zn SODs of different origin,only the content of proline and alanine beingsimilar in all species. Of special interest was theone tryptophan in Humicola enzyme.

Fe or Mn dismutases constitute a group ofproteins in which sequences are highly conservedand structures are very similar. The proteins ofthe Cu,Zn SOD family are strongly conserved asseen from Table 3. The N-terminal sequence anal-

Fig. 5. Matrix-assisted laser desorption ionization mass spec-trum of Cu,Zn SOD, isolated from Humicola lutea 110. Thesample (10 pmol) was dissolved in 0.1% (v/v) TFA and appliedonto the target. Analysis was carried out in a-cyano-4-hydrox-ycinnamic acid. Solutions of chicken egg ovalbumin (M=44 400 Da) and bovine serum albumin (M=66 430 Da) wereused to calibrate the mass scale.

ysis of Cu,Zn SOD from H. lutea 110 reveals ahigh degree of structural homology with thosefrom pro- and eukaryotic organisms (Table 3)[20–24]. The correlation between sequence ho-mologies and evolutionary time has shown thatduring the last 60×106 years the evolutionaryrate of Cu,Zn SOD constantly decreased andbecame more or less constant. The first 25residues of the Cu,Zn SOD showed more or lessthe same percentage of identity (60%) with thecorresponding regions of other Cu,Zn SODs. Ineach SOD, taken for comparison, positions 3, 4,5, 6, 9, 10, 15, 17, 19, 21 and 25 are occupied byidentical residues. The Cu,Zn SOD of the presentstudy has 95% homology with Aspergillus fumiga-tus, 76% with Neurospora crassa, 76% with Toru-laspora hansenii, 66% with Haemouchus contortusand 64% with Saccharomyces cere6isiae.

The ultraviolet (UV) and visible absorptionspectrum of the enzyme from H. lutea 110 isrepresentative for the Cu,Zn enzyme family and


Table 2Amino acid residues mol−1 of Cu,Zn SODs

Garden pea [39] Humicola lutea (this study)Human erythrocytes Neurospora crassa [6][38]

121023.2 27Lysine1511Histidine 12.8 18

6 9Arginine 7.8 103645 3536.2Aspartic acid

30 26Threonine 16.2 1814 16Serine 20.3 14

19 35.5Glutamic acid 27.4 2814 14 1611.3Proline56 39Glycine 47.1 41

20202120.6Alanine22 23Valine 29.6 21

0 0Methionine 0.1 020 13Isoleucine 16.5 1421 151119.4Leucine

2 1Tyrosine 0.1 09 6Phenylalanine 8.9 20

1Tryptophan3 4Half cystine 6.7 6

258310304 301Total amino acid residues

Fig. 6. Absorption spectrum of Cu,Zn SOD in the 270–420 nm and 420–760 nm regions.


Fig. 7. CD spectrum of Cu,Zn SOD in the 200–250 nm region. A total of 0.2 mg ml−1 SOD was dissolved in 10 mM Tris–HClbuffer, pH 7.4. The [u ] values are calculated using a mean molecular weight of 110.

to most proteins containing tyrosine and tryp-tophan residues exhibiting a peak at 280 nm witha shoulder at about 320 nm. The spectrum of thecopper chromophore of the enzyme displays aband with a maximum at 680 nm, composed bythe partial overlap of two components, due to thed–d transitions of the copper atom (Fig. 6), simi-lar to those reported from other SOD [35,7].Metal content was determined using an atomicabsorption spectrophotometer 2.490.4 ions ofCu and 2.090.4 ions of Zn molecule−1 ofenzyme.

The far-UV CD spectrum of the Cu,Zn SOD,reflecting the backbone conformation of theprotein molecule, shows two negative bands at208 and 222 nm, characteristic for the a-helixstructure (Fig. 7). Around 216 nm, the b-sheetstructures also contribute to the ellipticity of thea-helix bands. The UV CD spectrum, obtainedfor the enzyme of the present study, is close toother Cu,Zn SODs, but it is different to that ofPhotobacterium leiognathi Cu,Zn SOD, displayinga minimum at 198 nm [36]. The mean residueellipticity at 208 and 222 nm (Fig. 7) was found tobe 6800 and 5400 deg cm2 dmol−1, respectively,from which an a-helix content of 25% was calcu-lated [37].

Fig. 8. Stability of Cu,Zn SOD from Humicola lutea 110 as afunction of temperature. The purified enzyme (500 U ml−1),dissolved in 50 mM phosphate buffer, pH 7.8, was incubatedat the indicated temperatures. Aliquots were withdrawn, di-luted, and assayed for the remaining SOD activity.

The thermostability of the enzyme was exam-ined at pH 7.8, using different methods. No de-tectable loss of activity was noted at temperaturesup to 50°C after heating of the protein samplesfor 10 min (Fig. 8). Above 85°C, the protein lostactivity and the process is not reversible. The high

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Table 3Comparison of N-terminal amino acid sequences of Cu,Zn SODs Neurospora crassa [20], Aspergillus fumigatus [21], Torulaspora hansenii [22], Saccharomyces cere6isiae[23], Haemouchus contortus [24] with that from Humicola lutea 110

10 15 20 255

V L RHumicola lutea 110 GV D S K I T G T V I F E Q A N E SK A V AV V R G D S N V K G T VA INeur. Crassa F E Q E S E SVAKVV L R GAsperg. Fumigatus D S K I T G T V T F E Q A R X NV A V AV V R G D S K V Q G T VA HV F E Q E S E STorulaspora hansenii M V K AV L K G D A G V S G V V KSacchar. Cere6isiae FV E Q A S E SQ A V AV L R G D P G V T G T V WA FHaem. Contortus M S S Q D K E SN R VA


Fig. 9. Arrhenius plot, ln (Q−1−1) vs. 1/T, characterizing thethermostability of Cu,Zn SOD in 50 mM phosphate buffer,pH 7.8.

Fig. 10. Conformational stability toward thermal denaturationof the Cu,Zn SOD in 50 mM phosphate buffer, pH 7.8 at aprotein concentration of 2.3×10−4 M, using CD spec-troscopy. [holo- (—) and apo- (�—�) enzyme].

thermostability of Cu,Zn SOD from H. lutea wasconfirmed by fluorescence spectroscopy (Fig. 9).Q was calculated at the interval (25–90°C). Thethermal denaturation of Cu,Zn SOD, i.e. the un-folding process taking place after passing the crit-ical temperature, was irreversible, and for thisreason equilibrium thermodynamic parameterswere not determined. The Tc-value was used, thecritical temperature for deviation of the Arrheniusplot (ln (Q−1−1) vs. 1/T) from the linearity, tocharacterize the thermostability of this enzyme.The deviation indicates that at temperaturesabove Tc, the protein undergoes denaturation andprecipitates. A typical Arrhenius plot for theCu,Zn SOD is shown in Fig. 9. This enzyme isconformationally stable at temperatures up to65°C. The activation energy for the thermal deac-tivation of the excited protein fluorophores, Ea, is42 kcal mol−1 at pH 7.8. The activation energyfor human and bovine SOD enzymes vary from67 to 100 kcal mol−1 [16], which are more stable.Oryza SOD is not stable at higher temperatureand its activation energy is 16 kcal mol−1 [36],

about three times less than the enzyme of thepresent study.

The conformational stability toward thermaldenaturation of the enzyme was studied using CDspectroscopy (Fig. 10). The melting temperatures(Tm), are 69°C for the holo- and 57°C for the apo-enzyme. Compared to the Tm values for bovineand human SOD (74.9 and 83.6°C, respectively),a higher thermal stability of 6–14°C is found[15,16]. The results correlate with the results ob-tained for SOD from E. coli, for which 66°C forthe holo- and 53°C for the apo-SOD were deter-mined [9]. The lower value for the apo-SOD canbe explained by a stabilizing effect of the Cu andZn ions in the active site. In fact, the differencebetween the bovine holo- and apo-Cu,Zn SOD isnearly 32°C [15]. The Humicola enzyme contains 2g atoms of copper and 2 g atoms of zinc. Theresults reported above show that the metal ions

Table 4Spectroscopic properties of Cu,Zn SOD from H. lutea 110

Emission lmax (nm) excitation at Critical tempera-Quantum yield Ea (kcal Melting tempera-Cu,Zn SOD(Q) mol−1)295 nm ture (Tm)ture (Tc)

Holo- 328 0.010 6942 6168Apo- 338 0.051


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contribute to the thermal stability of the Cu,ZnSOD from H. lutea 110 to a much lower extentthan in the eukaryotic enzymes. Removal of themetal ions leads to about a 4-fold increase inquantum yield (Q= from 0.01 to 0.042), theemission maximum position shifts 10 nm towardhigher wavelength (328–338 nm) (Table 4) andloss of enzymic activity was proportional to theremoval of copper.

In conclusion, the above results show that thestrain of H. lutea 110 produces Mn SOD andCu,Zn SOD in a ratio of 4:1. The Cu,Zn-contain-ing enzyme is a novel thermostable protein, theproperties of which are more similar to Cu,ZnSODs from eukaryotic organisms. Tryptophan,included in the molecule of Humicola enzymepresents the possibility to investigate its proper-ties, using fluorescence spectroscopy.

Acknowledgements

This research was supported by grant No. X-822 from the National Science Foundation, Min-istry of Education, Science and Technologies, Bul-garia. The financial help of Fonds der Chemis-chen Industrie is greatly acknowledged.

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A novel Cu,Zn superoxide dismutase from the fungal strain Humicola lutea 110: isolation and...

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