Tài liệu Báo cáo khoa học: Crystal structure of the cambialistic superoxide dismutase from Aeropyrum pernix K1 – insights into the enzyme mechanism and stability pdf

from Aeropyrum pernix K1 – insights into the enzymemechanism and stability Tsutomu Nakamura1, Kasumi Torikai1,2, Koichi Uegaki1, Junji Morita2, Kodai Machida3,4, Atsushi Suzuki5and Yasus

from Aeropyrum pernix K1 – insights into the enzyme

mechanism and stability

Tsutomu Nakamura1, Kasumi Torikai1,2, Koichi Uegaki1, Junji Morita2, Kodai Machida3,4,

Atsushi Suzuki5and Yasushi Kawata3,4

1 National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, Japan

2 Department of Food Science and Nutrition, Faculty of Human Life and Science, Doshisha Women’s College of Liberal Arts, Kyoto, Japan

3 Department of Chemistry and Biotechnology, Graduate School of Engineering, Tottori University, Japan

4 Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science,

Tottori University, Japan

5 Power Train Material Engineering Division, Toyota Motor Corporation, Aichi, Japan

Keywords

Aeropyrum pernix; cambialistic; metal

coordination; stability; superoxide dismutase

Correspondence

T Nakamura, National Institute of Advanced

Industrial Science and Technology, 1-8-31

Midorigaoka, Ikeda, Osaka 563-8577, Japan

Fax: +81 72 751 8370

Tel: +81 72 751 9272

E-mail: nakamura-t@aist.go.jp

(Received 2 August 2010, revised 28

October 2010, accepted 2 December 2010)

doi:10.1111/j.1742-4658.2010.07977.x

Aeropyrum pernix K1, an aerobic hyperthermophilic archaeon, produces a cambialistic superoxide dismutase that is active in the presence of either of

Mn or Fe The crystal structures of the superoxide dismutase from A per-nixin the apo, Mn-bound and Fe-bound forms were determined at resolu-tions of 1.56, 1.35 and 1.48 A˚, respectively The overall structure consisted

of a compact homotetramer Analytical ultracentrifugation was used to confirm the tetrameric association in solution In the Mn-bound form, the metal was in trigonal bipyramidal coordination with five ligands: four side chain atoms and a water oxygen One aspartate and two histidine side chains ligated to the central metal on the equatorial plane In the Fe-bound form, an additional water molecule was observed between the two histidines

on the equatorial plane and the metal was in octahedral coordination with six ligands The additional water occupied the postulated superoxide bind-ing site The thermal stability of the enzyme was compared with superoxide dismutase from Thermus thermophilus, a thermophilic bacterium, which contained fewer ion pairs In aqueous solution, the stabilities of the two enzymes were almost identical but, when the solution contained ethylene glycol or ethanol, the A pernix enzyme had significantly higher thermal sta-bility than the enzyme from T thermophilus This suggests that dominant ion pairs make A pernix superoxide dismutase tolerant to organic media

Database Structural data have been deposited in the Protein Data Bank under the accession numbers 3AK1 (apo-form), 3AK2 (Mn-bound form) and 3AK3 (Fe-bound form)

Structured digital abstract

l MINT-8075688 : Superoxide dismutase (uniprotkb: Q9Y8H8 ) and Superoxide dismutase (uniprotkb: Q9Y8H8 ) bind ( MI:0407 ) by cosedimentation in solution ( MI:0028 )

l MINT-8075667 : Superoxide dismutase (uniprotkb: Q9Y8H8 ) and Superoxide dismutase (uniprotkb: Q9Y8H8 ) bind ( MI:0407 ) by x-ray crystallography ( MI:0114 )

l MINT-8075678 : Superoxide dismutase (uniprotkb: Q9Y8H8 ) and Superoxide dismutase (uniprotkb: Q9Y8H8 ) bind ( MI:0407 ) by molecular sieving ( MI:0071 )

Abbreviations

ApeSOD, superoxide dismutase from Aeropyrum pernix; SOD, superoxide dismutase; TthSOD, superoxide dismutase from

Thermus thermophilus.

Superoxide dismutases (SODs; EC 1.15.1.1) play a

pro-tective role against oxidative stress by catalyzing

dis-proportionation of the superoxide anion radical (O2Æ))

to hydrogen peroxide (H2O2) and dioxygen (O2) The

SOD-catalyzed reaction proceeds through a redox

cycle of metal ions as described by the equations [1]:

Enz Mðnþ1ÞþO2 ! Enz  MnþþO2

Enz MnþþO2þ2Hþ! Enz  Mðnþ1ÞþH2O2

where Enz and M represent the enzyme and the metal

cofactor, respectively SODs are grouped into four

classes according to their metal cofactors: copper and

zinc-containing SOD (Cu,Zn-SOD), iron-containing

SOD (Fe-SOD), manganese-containing SOD

(Mn-SOD) and nickel-containing SOD (Ni-(Mn-SOD) These

four types of SOD are divided into three groups based

on amino acid sequence homology; Fe- and Mn-SODs

are homologous [2]

Although Mn-SOD and Fe-SOD are closely related

in amino acid sequence and tertiary structure, they are

generally active only in the presence of their specific

metals For example, although the Fe-SOD and

Mn-SOD of Escherichia coli have 45% sequence identity

and can bind each other’s metals, they are inactive

when the wrong metal is incorporated at the active site

[3] However, several SODs are active in the presence

of either Fe or Mn These types of SODs are referred

as to cambialistic SODs In addition to the tertiary

structures of metal-specific SODs [4], crystal structures

of several cambialistic SODs have been reported,

including those from Porphyromonas gingivalis [5] and

Propionibacterium shermanii [6] The metal-specificity

of cambialistic SODs can be suppressed by

mutagene-sis at a site 11 A˚ away from the reaction center, as

reported for the P gingivalis SOD [7] This supports

the hypothesis that cambialism is a consequence of

multiple factors rather than the result of a unique type

of active site structure [8]

Aeropyrum pernix K1 is a strictly aerobic

hyper-thermophilic archaeon [9,10] that has been the target

of several studies investigating primitive antioxidation

mechanisms in aerobic life [11–15] A pernix K1

pro-duces a hyperthermophilic, cambialistic SOD [16] that

exhibits more activity when Mn, rather than Fe, is the

cofactor Different metals affect not only the catalytic

activity itself, but also sensitivity to inhibitors such as

sodium azide, sodium fluoride and hydrogen peroxide:

The Fe-bound SOD of A pernix K1 (ApeSOD) is

more sensitive to these inhibitors than Mn-bound

ApeSOD [16]

Despite significant characterizations of ApeSOD, the enzymological features of this enzyme have not been explained from a structural perspective because the ter-tiary structure of ApeSOD has not been elucidated

In the present study, for the first time, we describe the crystal structure of ApeSOD In particular, we focus

on the coordination of the metal cofactor in the active site as well as the changes it experiences in response to different metal cofactors Finally, by comparing Ape-SOD with the Ape-SOD from the thermophilic bacterium Thermus thermophilus, we evaluate the relationship between electrostatic interaction and the protein’s stability in an organic medium

Results

Protein preparation and incorporation

of metal ions

E coli cells harboring the expression plasmid for Ape-SOD were grown in LB medium and the enzyme was purified in the presence of EDTA An assay of the ApeSOD preparation revealed that the enzyme (referred

to as the apo-enzyme) had low activity (Table 1) This was attributed to the Mn or Fe ions incorporated into the enzyme as it accumulated in the E coli cells Indeed, the activity of the apo-enzyme was significantly lower than the metal-containing enzyme

Metal cofactors were incorporated into the enzyme; when the growth medium contained MnSO4or FeSO4, the activity of the obtained enzymes was 20-fold or six-fold higher, respectively, than that of the apo-enzyme (Table 1), indicating that the metals had suc-cessfully been incorporated during bacterial expression When the metal cofactors were added to the purified apo-enzyme and incubated at 70C, the enzyme became significantly more active (Table 1) Incubation with the metal at 37C did not raise the activity of

Table 1 SOD activity.

Enzyme

Activity (unitÆmg)1) a

Mn (molÆmol)1)

Fe (molÆmol)1)

a Values in parentheses are the calculated activities per mg of metal containing enzyme b Metal ions were added to the medium during bacterial expression. cThe enzymes were incubated at

70 C for 1 h in the presence of metal ions.

ApeSOD (data not shown); similar temperature

depen-dence has been reported for the cambialistic SOD from

Pyrobaculum aerophilium [17] This indicates that

pro-teins in solution need to have structural flexibility to

successfully incorporate metal cofactors Because the

metal contents of the reconstituted enzymes were

higher (Table 1), the crystallographic studies were

per-formed on apo- and metal-reconstituted ApeSODs

Crystallization and determination of structure

The crystals of ApeSOD were grown for 2–3 days in

the presence of polyethylene glycol The crystals

belonged to the space group P21, with four polypep-tides in the asymmetric unit After collection of diffrac-tion data, the crystal structures were refined to 1.56, 1.35 and 1.45 A˚ resolutions for apo, Mn-bound and Fe-bound ApeSODs, respectively Data collection and refinement statistics are summarized in Table 2 All three ApeSODs had essentially the same struc-ture (Fig 1A) The polypeptides consisted of seven a-helices, a three-stranded antiparallel b-sheet and loops connecting these secondary structure elements The con-tents of the a-helix and b-strand were 50% and 11%, respectively The monomer structure comprised two domains: the rod-shaped N-terminal domain consisting

Table 2 Data collection and refinement statistics.

Data collection

b = 72.25

c = 76.65

b = 90.99

a = 69.06

b = 71.78

c = 76.85

b = 91.81

a = 69.06

b = 71.76

c = 76.40

b = 91.72

Rmerge(%) a,b 7.3 (39.6) 4.8 (36.1) 7.7 (38.3)

Refinement

Rcryst(%) ⁄ R free (%) a,c,d 19.8 (33.7) ⁄ 23.6 (35.9) 18.8 (23.9) ⁄ 20.3 (25.4) 22.6 (29.5) ⁄ 25.6 (32.7)

Average B-factor

Ramachandran plot (%) e

a Values in parentheses are for the highest resolution shell b R merge ¼ P

hkl

P

j jI hkl;j <I hkl >j= P

hkl

P

i IIhkl;j, where Ihkl,jis the intensity of observation Ihkl,jand <Ihkl> is the average of symmetry-related observations of a unique reflection c Rcryst= P

||Fo| ) |F c || ⁄ P

|Fo|, where Fo and F c are observed and calculated structure factor amplitudes, respectively. dR free was calculated using a randomly-selected 5% of the dataset that was omitted from all stages of refinement e Ramachandran plots were prepared for all residues other than Gly and Pro.

of the N-terminal extended region and the following

two a-helices, and the globular, (a + b)-type

C-termi-nal domain

Oligomeric structure

The A⁄ B and C ⁄ D chains formed dimers in the crystal

packing This dimerization buried 27% of the

accessi-ble surface area of each monomer These two dimers

associated to form a tetramer in the asymmetric unit

(Fig 1B) The tetramerization buried 13% of the

accessible surface area of each dimer Neighboring

tet-ramers came into loose contact with each other in the

crystal packing Similar molecular arrangements have

been found in the crystal structures of SODs from

sev-eral sources, such as Sulfolobus solfataricus [18],

Myco-bacterium tuberculosis [19], Aquifex pyrophilus [20] and

P shermanii[6] These SODs are assumed to be

tetra-meric in solution Figure 1C illustrates the superimpo-sition of ApeSOD with the cambialistic SOD from

P shermanii The overall structures of these enzymes were similar; the rmsd of the 747 Ca atoms was 0.695 A˚

ApeSOD eluted from the gel-filtration column with the elution volume for a molecular mass of 57 kDa (Fig 2A) Because the calculated molecular mass of the ApeSOD monomer is 24 577 Da, the gel filtration results suggested a dimeric association; similar results have previously been reported for the same protein [16,21] A second gel filtration through a Superdex 200 column also suggested that ApeSOD has a dimeric structure in solution (data not shown) These findings are in contrast to the results obtained in the crystallo-graphic study (described above), which demonstrated that ApeSOD has a tetrameric structure To deter-mine whether ApeSOD polypeptide associations were

90°

90°

A

B

C

Fig 1 Crystal structure of ApeSOD.

(A) Monomer structures of apo (green),

Mn-bound (magenta) and Fe-bound (cyan)

ApeSODs are superimposed and shown as

a stereo view The metal cofactor of the

Mn-bound form is indicated by a ball.

(B) Tetramer structures of ApeSOD viewed

from two directions Chains A, B, C and D

are shown in red, yellow, green and blue,

respectively (C) ApeSOD (red)

superimposed with the SOD of P shermanii

(blue; Protein Data Bank code: 1AR5) [6].

The structure of ApeSOD is viewed from

the same directions as in (B) The Mn-bound

form is shown as the representative of

ApeSOD in (B) and (C) Prepared with PYMOL

[44].

different in crystals and in solution, we used

ultracen-trifugation to accurately measure the molecular mass

of ApeSOD in solution The result (96 265 Da) clearly

showed tetrameric assembly of ApeSOD in solution

(Fig 2B) Because ultracentrifugation is a direct,

accu-rate measurement, we conclude that ApeSOD exists as

a tetramer in solution

Active site geometry Each monomer had an independent metal-binding site

at the interface of the two domains, which consists

of four side chains: two (His31 and His79) from the N-terminal domain and two (Asp165 and His169) from the C-terminal domain (Fig 3) In Mn-reconstituted ApeSOD, the metal ion was five-coordinate in trigonal bipyramidal geometry (Fig 3A) Three of the ligands, OD2 of Asp165, NE2 of His79 and NE2 of His169, formed an equatorial plane The other protein ligand, NE2 of His31, bound to the metal, in the company of

a water oxygen, from the apical positions The manga-nese was only 0.06 A˚ out of the equatorial plane (Table 3) The angles around the metal cofactor sug-gested that the ligation form of Mn in ApeSOD is tri-gonal bipyramidal

In Fe-reconstituted ApeSOD, the metal was coordi-nated with six ligands: the five same ligands in the Mn-reconstituted enzyme and an additional water oxy-gen, which, together with the OD2 of Asp165, the NE2 of His79 and the NE2 of His169, formed an equatorial plane (Fig 3B) The metal ion and the addi-tional water oxygen were only 0.03 and 0.04 A˚, respec-tively, out of the equatorial plane defined by the other three atoms (Table 3) The angles around the metal ion indicated that Fe-bound ApeSOD contains distorted octahedral coordination around the metal cofactor

The absence of an anomalous Fourier map demon-strated that the active site in apo-ApeSOD did not contain a metal cofactor (Fig 3C) However, the side chains and apical water coordinating the metal center had the same configurations as those in the metal-bound ApeSOD This implies that the conformation around the active site of ApeSOD is independent of the presence of a metal cofactor

Figure 3D shows the superimposition of the active site structures of apo, Mn-bound and Fe-bound Ape-SODs The most significant difference among them was related to the OH of Tyr39, which shifted 1.1 A˚ toward the apical water molecule upon Fe binding (Fig 3D) The shift upon Mn binding was negligible, and no significant differences were observed in other

3 4 6

8

100

10

2

13 12 11 10 9

Elution volume (mL)

25 20

15 10

5

0

Elution volume (mL)

1.5

–0.05

0.00

0.05

0.5

1.0

A280

A280

6.95

0.0

Radius (cm)

A

B

Fig 2 Assembly of ApeSOD chains in solution (A) Representative

gel filtration chromatogram of ApeSOD and calibration curve (inset).

The standard proteins are albumin (1), ovalbumin (2),

chymotrypsin-ogen A (3) and ribonuclease A (4) The estimated molecular mass

of ApeSOD is 57.0 kDa (B) Sedimentation equilibrium distribution

of ApeSOD The line reflects the best fit of the data and indicates

that the apparent molecular mass is 96 265 Da The deviation

between empirical data and the fitted line is plotted in the upper

panel.

Fig 3 Active site structure of ApeSOD Active site structures of Mn-bound (A), Fe-bound (B) and apo (C) ApeSODs (A–C) The orange map represents the rA-weighted Fo)F c electron density map at the 3 r level, where the indicated residues are excluded from the calculation of the structure factor The blue map represents the anomalous difference map contoured at 10 r Schematic representations of coordinated atoms are shown on the right in (A–C), where oxygen, nitrogen and metal atoms are represented by red, blue and black balls, respectively (C) The anomalous difference map was not seen even when the sigma level was set to 3 (data not shown) (D) Apo (green), Mn-bound (magenta) and Fe-bound (cyan) structures are superimposed, and the residues around the active site are shown via stick models The red, magenta and cyan balls represent the active site water molecules in apo, Mn-bound and Fe-bound ApeSODs, respectively Balls labeled

‘Metal’ are Mn or Fe (E) Showing the superimposition of the active site structures of the Mn- (magenta) and Fe-bound (cyan) forms of

P shermanii SOD Water molecules and metal atoms are shown in the same way as in (D) Prepared with PYMOL [44].

E

A

B

C

H169 (NE2) D165 (OD2)

H31 (NE2) H79 (NE2)

WAT

H169 (NE2) D165 (OD2)

WAT

H31 (NE2) H79 (NE2)

WAT

H31 (NE2)

H169 (NE2) D165 (OD2)

active site residues between Mn- and Fe-bound

ApeS-ODs, although slight changes in His31, His79 and

Asp165 were observed between the metal-bound and

apo ApeSODs The shift of the conserved Tyr residue

depending on the metal cofactor does not appear to be

a common feature among cambialistic SODs because,

in the case of the cambialistic SOD from P shermanii,

no significant difference was found between the active

sites of the Mn- and Fe-bound forms (Fig 3E)

Stability in organic medium

After we elucidated the tertiary structure of ApeSOD,

we were able to compare the number of ion pairs

among thermophilic SODs Table 4 summarizes the

data obtained from two species each of archaea and

bacteria A single ApeSOD polypeptide was found to

have seven intrasubunit ion pairs, whereas 24

intersub-unit ion pairs were found in the ApeSOD tetrameric

assembly This is significantly higher than the number

of ion pairs found in the SOD from the thermophilic

bacterium T thermophilus (TthSOD) [22] Because

electrostatic interactions in proteins are more

domi-nant when the solvent polarity is lower, we hypo-thesized that ApeSOD would be more stable than TthSOD in an organic solvent

When we compared the stabilities of ApeSOD and TthSOD, we found that the two enzymes were similar

in aqueous solution at temperatures up to 85C (Fig 4A) However, when the solvent contained 40% ethylene glycol, TthSOD was inactivated by incubation

Table 4 Interactions in SODs ApSOD, SOD from A pyrophilus;

SsoSOD, SOD from S solfataricus.

Number of intrasubunit

ion pairs ⁄ monomer

Number of intrasubunit

ion pairs ⁄ residue

Number of intersubunit

ion pairs ⁄ tetramer

Number of intersubunit

ion pairs ⁄ residue

Table 3 Distances and angles around the metal ions For the

iden-tity of the atoms, see Fig 3.

Distance (A ˚ )

Metal – equatorial plane 0.06 ± 0.01 0.03 ± 0.02

Angle ()

a Water on the equatorial plane b Water at the apical position.

A

B

C

Fig 4 Thermal stability of ApeSOD and TthSOD Residual activi-ties of ApeSOD (closed circles) and TthSOD (open circles) after incubation at 85 C in the aqueous solution (A) and the solution containing 40% ethylene glycol (B) are plotted against incubation time (C) ApeSOD (closed symbols) and TthSOD (open symbols) were incubated for 1 h at 60 C (circles), 70 C (squares) or 80 C (triangles) in a solution containing various concentrations of ethanol The residual activities after the incubation are plotted against the ethanol concentration.

at 85C, whereas ApeSOD remained active (Fig 4B).

Inclusion of ethanol in the solution destabilized both

ApeSOD and TthSOD However, the extent of the

destabilization was more significant in TthSOD than in

ApeSOD (Fig 4C) In other words, ApeSOD was

more stable than TthSOD under organic conditions

This was not unexpected given the number of ion pairs

in ApeSOD and TthSOD The larger number of

elec-trostatic interactions in ApeSOD contributed to its

stability in a low-polarity solvent

Discussion

In the present study, we crystallized ApeSOD and

determined its tertiary structure The asymmetric unit

contained four polypeptides that consisted of two

homodimers Although both crystallographic study and

analytical ultracentrifugation indicated that ApeSOD

was a tetrameric protein, gel filtration suggested that

the protein was dimeric (Fig 2A); in other words, the

molecular mass estimated by gel filtration was lower

than the true value Similar results have also been

reported for other homologous SODs, such as those

from S solfataricus [23] and P shermanii [24] Ursby

et al [18] reported that the estimated molecular mass

of the S solfataricus SOD increased when the column

was recalibrated with thermophilic proteins from the

same source In cases such as these, analytical

ultra-centrifugation, rather than gel filtration, is a powerful

tool for accurately determining the oligomeric

struc-ture of SOD enzymes

We observed five-coordinate and six-coordinate

struc-tures of metal ions in Mn-bound and Fe-bound

Ape-SOD crystal structures, respectively In the

six-coordinated Fe-bound ApeSOD, an additional water

molecule was found in the equatorial plane (Fig 3B)

Although trigonal bipyramidal 5-coordinate structures

are often observed around bound metals of SODs,

the octahedral six-coordinate crystal structures are

detected only in exceptional cases; for example, in the

cryo-trapped form of E coli Mn-SOD [25], in

Fe-substi-tuted SOD of E coli [3], in peroxide-soaked

Mn-SOD of E coli [26] and in TthMn-SOD complexed with

azide, a SOD inhibitor [27] It remains unclear why the

six-coordinated structure was observed in Fe-bound

ApeSOD, but not in the Mn-bound form, because the

crystal contained neither superoxide substrate, nor

anio-nic inhibitors The answer to this question will shed light

on the reaction mechanism of this cambialistic SOD

Several enzymological properties known for

Ape-SOD [16] can be related to the structural

characteris-tics in the active site Cambialistic SODs can be

divided into two groups: one with almost the same

activity in the Mn- and Fe-forms, and the other exhib-iting low activity in the Fe-form and high activity in Mn-form ApeSOD belongs to the latter group [16] This was also confirmed by the activity assay, which demonstrated that ApeSOD was approximately 20-fold more active in its Mn-bound form than in its Fe-bound form (Table 1) We propose two possible explanations for this finding One is related to inhibi-tion of the binding of the superoxide substrate and the other to product inhibition by hydrogen peroxide There are two mechanisms proposed for the reaction cycle of Mn⁄ Fe-SOD: the 5-6-5 mechanism [27] and the associative displacement mechanism [28] In the former model, the metal is five-coordinated in the rest-ing state and six-coordinated when the substrate super-oxide is bound In the latter model, the association of superoxide is concomitant with displacement of one of the oxygen ligands In both mechanisms, the superox-ide substrate coordinates to the metal center from the equatorial plane, and the coordination site is the same

as that of the additional water molecule in the Fe-bound ApeSOD octahedral structure Thus, it can

be assumed that the coordinated water in Fe-bound ApeSOD inhibits superoxide binding

Although the polypeptide conformations of the Mn- and Fe-bound ApeSODs were almost the same (Fig 1A), a slight difference was observed in the side chain of Tyr39 (Fig 3D) This Tyr residue is conserved

in Mn- and Fe-SODs, constitutes the outer sphere of the active site and has been shown by mutational stud-ies to play a critical role in catalysis [29,30] The OH

of Tyr39 was found to have shifted toward the apical water molecule upon Fe binding (Fig 3D) This shift was analogous to that of Tyr34 in E coli Mn-SOD upon binding of hydrogen peroxide to the central metal [26] Peroxide-bound E coli Mn-SOD represents

a product-inhibited form of the reduction step from superoxide to hydrogen peroxide [26] These findings lead to the hypothesis that Fe-bound ApeSOD mimics the product-inhibited form and the shift of Tyr39 sup-presses the release of the peroxide product This may

be one of the reasons why ApeSOD is less active in its Fe-bound form It is noteworthy that this shift of the Tyr residue is not observed in the cambialistic SOD from P shermanii (Fig 3E), which exhibits almost the same activity in the presence of Mn and Fe as cofac-tors [24]

Thermophilic bacteria, as well as thermophilic archaea, produce thermophilic enzymes Comparison

of two thermophilic SODs, ApeSOD (an archaeon) and TthSOD (a bacterium) revealed that ApeSOD contained more ion pairs, especially intersubunit ion pairs, than TthSOD (Table 4) Because electrostatic

interactions are dominant under organic conditions,

we expected that the difference in number of ion pairs

would be reflected in a corresponding difference in the

stability of the proteins in organic solvents Indeed,

although the stabilities of the two SODs were

indistin-guishable in aqueous solution, ApeSOD was more

sta-ble than TthSOD in a solution containing ethylene

glycol or ethanol (Fig 4) It is reasonable to conclude

that the dominant electrostatic interaction in ApeSOD

contributed not only to heat tolerance, but also to

tolerance of the enzyme to organic environment

For enzymes to be used in industrial applications,

it is preferable that they have both structural and

func-tional stability when placed under severe conditions

Thus, because of its thermal stability and tolerance to

an organic medium, ApeSOD shows promise as a

potentially applicable enzyme

Experimental procedures

Protein expression and purification

(APE_0741) from genomic DNA of A pernix K1 (NBRC

100138, provided by National Institute of Technology and

Evaluation, Chiba, Japan) The PCR product was cloned

into an expression plasmid vector pET11 (Novagen,

Darmstadt, Germany) E coli Rosetta (DE3) cells harboring

the expression plasmid were cultivated in LB medium

expression was induced by the addition of 1 mm isopropyl

thio-b-d-galactoside (final concentration) The E coli cells

were disrupted by sonication and the soluble proteins were

subjected to streptomycin and heat treatments, as described

previously [31] The resulting solution was dialyzed in

20 mm sodium acetate buffer (pH 4.8) and applied onto

a cation exchange column (HiTrap SP; GE Healthcare,

Piscataway, NJ, USA) The protein was eluted by a linear

gradient of 0–1 m NaCl in the same buffer The fractions

containing ApeSOD were collected, concentrated and

gel-filtered with a Superdex 75 column equilibrated with

20 mm Tris-HCl (pH 8.1), with 150 mm NaCl as the final

step The purified protein was dissolved in 20 mm Tris-HCl

(pH 8.1) The protein concentration was determined from

its absorbance at 280 nm [32]

Gel filtration

Analytical gel filtration chromatography was performed

with a buffer containing 20 mm Tris-HCl (pH 8.1) and

column was calibrated using a Gel Filtration Calibration

Kit LMW (GE Healthcare)

Ultracentrifugation ApeSOD solution in 20 mm Tris-HCl (pH 8.1) containing

150 mm NaCl was subjected to a sedimentation equilibrium analysis using a Beckman Optima XL-A analytical ultracen-trifuge with an An-60 Ti rotor (Beckman Coulter, Fullerton,

CA, USA) Samples were centrifuged at 9500 g for 41 h at

from the sedimentation equilibrium plot

Incorporation of metals Metal cofactors were incorporated into the enzyme by one

of two procedures To incorporate metals during growth

to the medium [33] The metal-containing enzymes were purified using EDTA-free buffers Alternatively, the metals could be incorporated into the purified enzyme For this technique, approximately 1 mm ApeSOD in 20 mm

removed by dialysis and the enzyme was further purified by gel filtration with a Superdex75 column equilibrated with

20 mm Tris-HCl (pH 8.1) and 150 mm NaCl The metal content of the enzyme was measured by inductively coupled plasma atomic emission spectrometry using a Perkin-Elmer

MA, USA)

Activity assay The activity of SOD was assayed with a SOD Assay

manufacturer’s instructions, one unit was defined as the amount of activity that caused 50% inhibition of WST-1 reduction

Crystallization, data collection and processing ApeSOD was crystallized by hanging drop vapor diffu-sion, with a reservoir solution containing 100 mm

22.5% ethylene glycol, cooled in a nitrogen gas stream (100 K) and subjected to X-ray diffraction measurements with synchrotron radiation at SPring-8 (Harima, Japan)

molec-ular replacement with molrep in the ccp4 suite [36,37] Fe-SOD from S solfataricus [18] (Protein Data Bank code: 1WB8) was used as the search model The resulting structure was subjected to simulated annealing using cns

[38], followed by further refinement with refmac in the

The secondary structure of proteins was assigned by dssp

[40] Stereochemical analysis was performed using

Ion-pair interactions were identified using distances < 4 A˚

When we counted the interactions, we excluded all residues

involved in the binding of the metal cofactor

ApeSODs in different forms were superimposed with the

least square fit of Ca atoms of the residues in the range

10–200 ApeSOD and P shermanii SOD were superimposed

with secondary-structure matching [43]

Thermal stability

Heat treatment of ApeSOD (Mn-bound form) and TthSOD

were performed in a buffer containing 50 mm Hepes-KOH

of 0.8 lm When indicated, the buffer contained 40%

ethyl-ene glycol or various concentrations of ethanol Thermal

stability was evaluated by measuring enzyme activity after

incubation

Acknowledgements

Diffraction data were collected at the Osaka University

beamline BL44XU at SPring-8, equipped with

MX225-HE (Rayonix), which is financially supported

by Academia Sinica and National Synchrotron

Radiation Research Center (Taiwan, ROC) We

thank Ms M.Sakai (Osaka University) for performing

ultracentrifugation analysis and Mr K Mieda and

M Sakata (Tottori University) for technical assistance

in enzyme assay This study was supported by a

Grant-in-Aid for Scientific Research (21510237) from

Japan Society for the Promotion of Sciences (JSPS)

References

1 Miller AF (2004) Superoxide dismutases: active sites

that save, but a protein that kills Curr Opin Chem Biol

8, 162–168

2 Smith MW & Doolittle RF (1992) A comparison of

evolutionary rates of the two major kinds of superoxide

dismutase J Mol Evol 34, 175–184

3 Edward RA, Whittaker MM, Whittaker JW, Jameson

GB & Baker EN (1998) Distinct metal environment in

Fe-substituted manganese superoxide dismutase

pro-vides a structural basis of metal specificity J Am Chem

Soc 120, 9684–9685

4 Perry JJ, Shin DS, Getzoff ED & Tainer JA (2010) The

structural biochemistry of the superoxide dismutases

Biochim Biophys Acta 1804, 245–262

5 Sugio S, Hiraoka BY & Yamakura F (2000) Crystal structure of cambialistic superoxide dismutase from Porphyromonas gingivalis Eur J Biochem 267, 3487– 3495

6 Schmidt M, Meier B & Parak F (1996) X-ray structure

of the cambialistic superoxide dismutase from

Chem 1, 532–541

7 Yamakura F, Sugio S, Hiraoka BY, Ohmori D & Yokota T (2003) Pronounced conversion of the metal-specific activity of superoxide dismutase from

amino acid (Gly155Thr) located apart from the active site Biochemistry 42, 10790–10799

8 Tabares LC, Gatjens J & Un S (2010) Understanding the influence of the protein environment on the Mn(II) centers in superoxide dismutases using high-field elec-tron paramagnetic resonance Biochim Biophys Acta

1804, 308–317

9 Sako Y, Nomura N, Uchida A, Ishida Y, Morii H, Koga Y, Hoaki T & Maruyama T (1996) Aeropyrum

hyperthermo-philic archaeon growing at temperatures up to 100C Int J Syst Bacteriol 46, 1070–1077

10 Kawarabayasi Y, Hino Y, Horikawa H, Yamazaki S, Haikawa Y, Jin-no K, Takahashi M, Sekine M, Baba S, Ankai A et al (1999) Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum

11 Nakamura T, Yamamoto T, Inoue T, Matsumura H, Kobayashi A, Hagihara Y, Uegaki K, Ataka M, Kai Y

& Ishikawa K (2006) Crystal structure of thioredoxin peroxidase from aerobic hyperthermophilic archaeon

12 Nakamura T, Yamamoto T, Abe M, Matsumura H, Hagihara Y, Goto T, Yamaguchi T & Inoue T (2008) Oxidation of archaeal peroxiredoxin involves a hyperva-lent sulfur intermediate Proc Natl Acad Sci USA 105, 6238–6242

13 Nakamura T, Kado Y, Yamaguchi T, Matsumura H, Ishikawa K & Inoue T (2010) Crystal structure of peroxiredoxin from Aeropyrum pernix K1 complexed with its substrate, hydrogen peroxide J Biochem 147, 109–115

14 Mizohata E, Sakai H, Fusatomi E, Terada T, Muray-ama K, Shirouzu M & YokoyMuray-ama S (2005) Crystal structure of an archaeal peroxiredoxin from the aerobic hyperthermophilic crenarchaeon Aeropyrum pernix K1

J Mol Biol 354, 317–329

15 Jeon SJ & Ishikawa K (2002) Identification and charac-terization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1 Eur J Biochem 269, 5423– 5430

16 Yamano S, Sako Y, Nomura N & Maruyama T (1999)

A cambialistic SOD in a strictly aerobic