Hunter1 1 Department of Physiology and Biochemistry, University of Malta, Msida, Malta;2Cranfield Biotechnology Centre, Institute of BioScience and Technology, Cranfield University, Silsoe
Trang 1Thermostability of manganese- and iron-superoxide dismutases
of a glutamine residue
The´re`se Hunter1, Joe V Bannister1,2and Gary J Hunter1
1
Department of Physiology and Biochemistry, University of Malta, Msida, Malta;2Cranfield Biotechnology Centre, Institute of BioScience and Technology, Cranfield University, Silsoe, Bedfordshire, UK
The structurally homologous mononuclear iron and
man-ganese superoxide dismutases (FeSOD and MnSOD,
respectively) contain a highly conserved glutamine residue in
the active site which projects toward the active-site metal
centre and participates in an extensive hydrogen bonding
network The position of this residue is different for each
SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of
Escherichia coli) Although site-directed mutant enzymes
lacking this glutamine residue (FeSOD[Q69G] and
MnSOD[Q146A]) demonstrated a higher degree of
selec-tivity for their respective metal, they showed little or no
activity compared with wild types FeSOD double mutants
(FeSOD[Q69G/A141Q]), which mimic the glutamine
posi-tion in MnSOD, elicited 25% the activity of wild-type
FeSOD while the activity of the corresponding MnSOD
double mutant (MnSOD[G77Q/Q146A]) increased to 150%
(relative to wild-type MnSOD) Both double mutants
showed reduced selectivity toward their metal Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine resi-dues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile Significantly, the MnSOD double mutant exhibited a thermal-inactivation profile similar to that of wild-type FeSOD while that of the FeSOD double mutant was similar to wild-type MnSOD We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability
Keywords: superoxide dismutase; site-directed mutagenesis; metal specificity; thermostability
Iron superoxide dismutases (FeSOD, E.C.1.15.1.1) and
manganese superoxide dismutases (MnSOD) constitute a
class of structurally equivalent metalloenzymes prevalent in
prokaryotes and in eukaryotic mitochondria, respectively
They exhibit a very high degree of homology in both
sequence and structure (Fig 1) The SODs are active only in
dimeric association and all share structural homology in this
respect [1] The metal cofactors are required to catalyse the
disproportionation of the superoxide radical into hydrogen peroxide and molecular oxygen [2] in a cyclic, two-stage oxidation-reduction mechanism:
M3þþ O2 ! M2þþ O2 ð1Þ
M2þþ O
2 þ 2Hþ! M3þþ H2O2 ð2Þ where M represents either iron or manganese
Selectivity of the proteins for their metal cofactor has been demonstrated in vivo [3] and although apoenzymes of each type of SOD may be reconstituted by the addition of metals, the resulting enzyme is active only with the authentic metal at its active centre [4–7] A small number of cambialistic SODs have been shown to be active with either iron or manganese, though only those of Propionibacterium shermanii [8], Bacteroides gingivalis [9] and Bacteroides fragilis [10] demonstrate similar specific activities with either metal
In all structures solved for the mononuclear SODs, the metal ion is held in place by an absolutely conserved quartet of residues comprising three histidines and one aspartic acid which act as ligands to the metal (H26, H81, D167 and H171 for Escherichia coli MnSOD, Fig 1B This numbering will be used throughout except where indicated) [11–21] This conservation is also reflected in all sequences elucidated for this large group of ubiquitous enzymes A fifth metal ligand, either water or hydroxide, present in all structures produces a trigonal-pyramidal geometry at the active site A distorted-octahedral geo-metry is assumed during catalytic turnover or inhibition,
Correspondence to G J Hunter, Department of Physiology and
Biochemistry, University of Malta, Msida, MSD 06, Malta.
Fax: + 356 21310577, Tel.: + 356 21316655,
E-mail: gary.hunter@um.edu.mt
Abbreviations: Fe[Q69G], Fe[A141Q] and Fe[Q69G/A141Q],
Escherichia coli FeSOD mutated at glutamine 69 to glycine, alanine
141 to glutamine, or both, respectively; FeSOD, MnSOD, the iron- or
manganese-containing SOD, respectively; FXa, active form of
restriction protease factor X; GSH, reduced glutathione; GSt,
gluta-thione S-transferase; IPTG, isopropyl thio-b- D -galactoside;
Mn[G77Q], Mn[Q146A] and Mn[G77Q/Q146A], E coli MnSOD
mutated at glycine 77 to glutamine, glutamine 146 to alanine, or both,
respectively; SOD, superoxide dismutase; wt, wild type.
Enzymes: iron superoxide dismutase from E coli; SODF_ECOLI,
manganese superoxide dismutase from E coli; SODM_ECOLI
(E.C 1.15.1.1).
(Received 11 March 2002, revised 10 July 2002,
accepted 22 August 2002)
Trang 2where a sixth ligand, presumed to be hydroxide, is bound
to the metal [11,22,23]
Beyond the metal ligand residues and within 10 A˚ of the
metal there are fewsignificant differences between iron- and
manganese-containing SODs During catalysis substrate
and products must pass through a funnel made up of
residues from each subunit [11,24] and include so-called
gateway residues His30, Tyr34, Trp169 and Glu170, the
latter from the second subunit of the functional dimer [25]
Studies of the highly conserved residues within this outer
sphere have revealed structural or chemical roles for these
residues and highlighted the importance of a
hydrogen-bonded network between various residues and the water (or
hydroxide) coordinated to the metal ion (participating
residues are shown in Fig 1B) Site-directed mutations of
Y34 in E coli FeSOD [24,26], MnSOD [27] and human
MnSOD [28] showthat the phenolic hydroxyl is not
necessary for maximal activity and mutants display no
overall change in structure Importantly, Y34 can not be the
sole source of protons for the dismutation reaction,
although this residue has been shown to be a source of
the pH dependence of FeSOD activity [26] Moreover, these
mutants showan increased sensitivity to the inhibitor azide
[24] and also, in the case of human MnSOD, to product
inhibition [28], processes thought to be analogous Y34 is
hydrogen bonded to Q146 and thus forms part of an extensive hydrogen-bonding network involving various residues as well as the coordinated solvent
Q146 is of interest as it represents one of two residues originally identified to distinguish between FeSOD and MnSOD [7] The position of the glutamine residue, which is structurally equivalent in the enzymes, is contributed by the
N domain of FeSODs (Q69) and the C domain of MnSODs (Q146) Exceptions to this scheme include the substitution
of Gln by His in two MnSODs and some FeSODs which have substituted His at the equivalent position of A141 for the glutamine at position 69 of most other FeSODs Substitution of Q143 (equivalent to Q146) in human MnSOD with Asn drastically reduced enzymatic activity and effectively opened the active site of the enzyme by reducing the occupied internal volume, allowing the intro-duction of an extra water molecule [29] Replacement of the same residue by Ala similarly maintains the same charac-teristic enzyme fold but with a concomitant introduction of further water molecules, in this case two solvent molecules occupy positions equivalent to the Oe1 and Ne2 of the missing Gln30 Interestingly, replacement of this residue by Glu had little effect but replacement by Lys yielded an enzyme too unstable to purify [30] In E coli, MnSOD mutation of Q146 to Glu generated an apoprotein only,
Fig 1 Comparison of E coli mononuclear superoxide dismutase molecular features (Top) Stereo viewof the superposition of the backbone peptide chain of one subunit of FeSOD (black) and MnSOD (grey) of E coli (coordinates taken from database entries 1ISB (10), chain A, and 1VEW [18], chain C, respectively) The iron ion is shown as a black sphere and amino acid sidechains are shown in ball and stick for FeSOD residues Q69 and A141, relevant to this study The corresponding sites in MnSOD are occupied by G77 and Q146, respectively (not shown) Superposition was calculated using the combinatorial extension method to maximize backbone contacts [42] Labels indicate the positions of the N- and C-termini, the iron ion and residues Q69 and A141 in FeSOD (Bottom) Stereo viewof selected residues around the metal centres Superposition, orientation and colour are the same as above Metal and hydroxyl ions are shown as light coloured spheres (only those of FeSOD are labelled) and sidechains of residues relevant to mutation studies here are shown as ball and stick (FeSOD Q69, A141 and MnSOD G77, Q146) Hydrogen bonds and metal contacts between residues of FeSOD are shown as dashed lines.
Trang 3whereas mutation to Leu or His reduced activity to less than
10% with little or no structural changes in the mutants [27]
Although low with either metal, the Q146H mutation was
reported to give similar activities with either iron or
manganese in the reconstituted enzyme [27]
Double mutations have been introduced into P
gingi-valisand E coli SODs with the intention of altering their
metal specificities In the former cambialistic enzyme,
mutations Q70G/A142Q reduced the iron-supported
activity of the enzyme and altered the ratio of Mn : Fe
from 1.4 to 3.5 [31] In the latter MnSOD, the equivalent
mutation G77Q/Q146A was demonstrated to reduce
specific activity to 71% and introduce an iron-supported
SOD activity which did not exist in the wild-type enzyme,
though this was only 6% of manganese-supported activity
in the wild type [32]
Here we present data for FeSOD mutations Q69G and
A141Q, both single and double mutations, and a
compari-son with data on equivalent mutations in MnSOD (G77Q
and Q146A), which change in vivo metal selectivity, specific
activity and thermal stability of these isoenzymes
M A T E R I A L S A N D M E T H O D S
Chemicals and enzymes
Superoxide dismutases (iron-containing or
manganese-containing enzymes from E coli) were purchased from
Sigma (Poole, Dorset, UK) Xanthine oxidase (from cow
milk), all restriction endonucleases (used according to the
manufacturers instructions in the buffers provided) and
protease factor Xa cleavage and removal kit, were
pur-chased from Boehringer Mannheim (Mannheim, Germany)
Nitro Blue tetrazolium and isopropyl thio-b-D-galactoside
(IPTG) were obtained from United States Biochemicals
(Cleveland, Ohio USA) T4 DNA ligase (FPLCpure) and
reduced glutathione (GSH)-sepharose were purchased from
Pharmacia Biotech (Vienna, Austria)
Visualization and analysis of molecular structures
Molecular structures whose coordinates were obtained
from the RCSB database were visualized using the
programs MOLMOL [33] or GeneMine [34]
Addition-ally, POVRAY (http://www.povray.org) was used to
produce Fig 1 Structural alignment of SODs was
maximized using the combinatorial extension program
CE [35] while mutational analyses were carried out using
the CARA and ENCAD algorithms included in the
GENEMINE program
Bacterial strains and vectors
The mutagenesis and expression phagemid, pGHX(–) was
produced in our laboratory and is described elsewhere [36]
E coliK12 strain TG1 [sup E, hsd D5, thi, D(lac–proAB),
F¢(tra D36 pro A+B+ lacIq lacZDM15)], was supplied
with the Sculptor Oligonucleotide-Directed In Vitro
Muta-genesis System kit obtained from Amersham International,
UK, which was used to generate site-directed mutations
E coli OX326A (DsodA DsodB) was kindly supplied by
H Steinman, Albert Einstein College of Medicine, New
York, USA
Oligonucleotide synthesis Oligonucleotides were synthesized on an Applied Biosys-tems model 392 DNA synthesizer and purified by prepar-ative gel electrophoresis in 20% polyacrylamide gel containing 7M urea Before use in mutagenesis protocols the oligonucleotides were first used as primers in dideoxy sequencing [37] to confirm the position of their unique binding site within the sodA or sodB gene (see below) Dideoxy DNA sequencing
DNA sequencing was carried out by the dideoxy method [37] using ABI prism dye-terminator DNA sequencing reagents and an Applied Biosystems model 800 Catalyst sequencing station followed by detection and analysis on
an Applied Biosystems model 373A automated DNA sequencer
The cloned wild-type and mutant sod genes were fully sequenced in both directions using the sequencing primers PGEXPLUS, d(5¢-GTTTGGTGGTGGCGACCATCCT) and PGEXMINUS, d(5¢-GAGGCAGATCGTCAGCAG TCA) and various mutagenic primers (see below)
Construction of pGH-MnSOD and pGH-FeSOD Wild-type sodA and sodB genes were isolated by PCR FeSOD was cloned using the primers ECF-5¢ d(5¢-TCATT CGAATTACCTGCACTAC) and ECF-3¢ d(5¢-TTATGC AGCGAGATTTTTCGCT) and the sodB plasmid, pHS1-8 (supplied by D Touati, Institut Jacques Monod, Paris, France) as template DNA MnSOD was cloned using the primers ECM-5¢ d(5¢-AGCTATACCCTGCCATCCCTG) and ECM-3¢ d(5¢-TTATTTTTTCGCCGCAAAACGTG) and E coli genomic DNA as template PCR was carried out using Amplitaq enzyme according to the manufacturer’s instructions (Perkin-Elmer corporation) although the exten-sion reaction was omitted Instead, the DNA products were incubated in the presence of 1 Unit of KlenowDNA polymerase enzyme for 30 min at 30C This step greatly improved the efficiency of blunt-end cloning of the PCR products into the vector [36] Constructs were designated pGH-FeSOD and pGH-MnSOD
In vitro site-directed mutagenesis Oligonucleotide site-directed mutagenesis was carried out
by the phosphorothioate DNA method of Eckstein [38] utilized in the Sculptor in vitro mutagenesis kit from Amersham International, UK Single-stranded DNA tem-plate was produced from the pGH-SOD constructs using VCS-M13 helper phage (Stratagene) One microgram was utilized in mutagenesis reactions together with oligonucleo-tides ECF-Q69G d(5¢-AACAACGCAGCTGGGCTCTG GAACCAT), ECF-A141Q d(5¢-TCAACCTCTAACCAG GCTACTCCGCTG) ECM-G77Q d(5¢-AACAACGCTGG CCAGCACGCTAACCAC) and ECM-Q146A d(5¢-TCT ACTGCTAACGCGGATTCTCCGCTG) following the manufacturer’s instructions (mutagenic nucleotide are underlined) Mutated plasmids were designated pGH-FeSOD[Q69G], pGH-FeSOD[A141Q], pGH-MnSOD [G77Q] and pGH-MnSOD[Q146A] ssDNA produced from cells harbouring pGH-FeSOD[Q69G] and
Trang 4pGH-MnSOD[G77Q] was used as the template for the
production of the double mutants using oligonucleotides
ECF-A141Q and ECM-Q146A, respectively
Induced expression of SOD
E coliOX326A (DsodA DsodB) harbouring the pGH-SOD
plasmids was grown at 30C with shaking in 2 L culture
flasks containing 500 mL 2TY medium (1.6% tryptone, 1%
yeast extract and 0.5% sodium chloride) supplemented with
100 lgÆmL)1ampicillin, sodium salt, 50 lMiron(III) sulfate
and 50 lMmanganese sulfate When the D600of the culture
reached a value of 0.4, IPTG was added to a final
concentration of 10 mM and the culture incubated for a
further 6 h
Protein purification
Cells from IPTG-induced cultures were harvested by
low-speed centrifugation and resuspended in approximately
35 mL of NaCl/Pibuffer (20 mMsodium phosphate buffer
pH 7.2, 150 mMsodium chloride) containing SDS (0.03%)
and Triton X-100 (1%) All sample volumes were then
adjusted to give an equal D600 Resuspended cells were lysed
by passage through a French pressure cell (Amicon) at
16 000 psi The cell lysates were clarified by centrifugation at
10 000 r.p.m (SS-34 rotor, Sorval RC-5C centrifuge) for
20 min and supernatants were mixed by gentle shaking in
batch at 4C overnight with 3–5 mL of GSH-sepharose
resin prewashed with buffer The resin was then packed into
columns and the unbound protein washed through the
column with 25 mL NaCl/Pi followed by 2 mL GSH
(10 mMin 50 mMTris/HCl pH 8.0) GSH was used to elute
the bound fusion protein which usually eluted in the first 6–
10 mL Buffer-exchange using KP buffer (50 mMpotassium
phosphate, 0.1 mMEDTA, pH 7.8) and concentration was
carried out using Microcon 30 (Amicon) centrifugal
concentrators and recovered proteins were stored at)80 C
To obtain pure SOD enzymes with authentic N-termini,
the glutathione S-transferase (GST)-fusion proteins (50 lg)
were diluted into Tris/HCl buffer (50 mM, pH 8.3)
contain-ing calcium chloride (2 mM) and incubated overnight with
the active form of restriction protease factor X (FXa; 6 lg,
biotinylated) at 4–22C in a final volume of 100 lL The
digest was subjected to a further round of GSH-sepharose
affinity chromatography after addition of protein A-agarose
(50 lL) to remove FXa, the purified SOD being present in
the through-wash Buffer exchange and concentration was
performed as described above
Assay for superoxide dismutase activity
The specific activity of SOD was measured
spectrophoto-metrically by its inhibitory action on the
superoxide-dependent reduction of cytochrome c by xanthine-xanthine
oxidase as described by McCord and Fridovich [39] and
Ysebaert-Vanneste and Vanneste [40] The reduction of
cytochrome c was followed at a wavelength of 550 nm using
a Beckman Diode Array DU7500 spectrophotometer in KP
buffer at 25C and a final volume of 1 mL A blank
measurement was recorded in the absence of sample over
1 min (Vb) SOD proteins were diluted 200- to 6000-fold
depending on the activity of the enzyme and cytochrome c
reduction was followed over 1 min (Vs) for a range of sample dilutions (200 lL sample per reaction) The slope of
a plot of the reciprocal of the sample volume against Vb/Vs was used to calculate SOD activity [40] All assay constit-uents were dissolved in KP buffer before use and the amount of xanthine oxidase required was adjusted to give a blank value (Vb) of approximately 0.025 DAÆmin)1 All spectrophotometric measurements were used after subtrac-tion of a blank containing SOD sample but no xanthine oxidase to ensure lack of interference with the assay constituents by mutant proteins
For activity measurements at different temperatures or in the presence of sodium azide, an initial dilution of SOD was adjusted to give Vsequal to half Vb(equal to 1 unit of SOD activity under standard conditions) After incubation of aliquots at the required temperature or after addition of sodium azide at the required concentration, Vswas meas-ured again Activities were expressed as a percentage of SOD activity at 25C without azide
For measurements of hydrogen peroxide inactivation, the SOD sample (1.6 mL) was incubated at 23C with 0.25 mM (FeSODs) or 5 mM(MnSODs) hydrogen peroxide Aliqu-ots (200 lL) were added to catalase (100 U, 1 lL) and then used to measure SOD activity as described above, SOD concentrations having been calculated to yield 1 U SOD activity in a standard 1 mL assay Activities were expressed
as a percentage of SOD activity at 25C without hydrogen peroxide Blanks were performed with hydrogen peroxide and catalase to ensure there were no adverse effects on the SOD assay
Polyacrylamide gel electrophoresis (PAGE) Native 8% polyacrylamide gels (acrylamide : N,N¢-methyl-enebisacrylamide, 29 : 1, w/w) containing NaCl/Tris,
pH 8.8 utilized a Tris/glycine electrophoresis buffer system consisting of 25 mMTris, 250 mMglycine, pH 8.3 Samples contained 50 mM Tris/HCl, pH 6.8, 0.1% bromophenol blue and 10% glycerol prior to gel application Denaturing polyacrylamide gel electrophoresis (SDS/PAGE) was carried out in 15% polyacrylamide gels essentially by the procedure
of Laemmli [41] utilizing a 5% stacking gel Samples were pretreated by boiling for 4 min in 100 mMTris/HCl, pH 6.8,
100 mMdithiothreitol, 2% SDS, 0.1% bromophenol blue and 10% glycerol prior to application to the gel
Superoxide dismutase activity stain Native PAGE (8%) gels were stained for SOD activity
by the Nitro Blue tetrazolium reaction as described by Beauchamp and Fridovich [42]
Protein concentration Estimation of the concentration of purified protein or in the lysates was by the method of Bradford using BSA as standard [43]
Protection against paraquat-induced stress Overnight cultures (5 mL) of E coli OX326A transformed with the appropriate plasmid were diluted 1 : 100 in 2TY medium to a final volume of 5 mL, grown with shaking at
Trang 537C for 1–2 h and used to inoculate 50 mL of 2TY media
containing 100 lgÆmL)1 ampicillin, 50 lM ferric sulfate,
50 lM manganese sulfate, 250 lM paraquat and 0.1 mM
IPTG to an initial D600 of 0.03 Cultures were grown in
250 mL flasks with shaking at 37C and 1 mL aliquots
were removed regularly to measure optical density
Metal analysis
Concentrations of iron and manganese in protein samples
were determined by atomic absorbance with a Hitachi
Z-9000 atomic spectrophotometer (Showa Woman’s
University, Japan) by Professor F Yamakura (Juntendo
University School of Medicine, Japan) and Professor
T Matsumoto (Showa Woman’s University)
R E S U L T S
Mutation of FeSOD and MnSOD
The sodB and sodA genes of E coli were amplified by PCR
from either the sodB-containing plasmid pHS1-8 or E coli
genomic DNA, respectively, and subcloned (by blunt-end
ligation) into the novel expression vector pGHX(–) [36]
Single-stranded DNA was produced from pGH-SOD
clones and used to carry out site-directed mutagenesis
(Materials and methods) The introduction of the correct
unique mutations was confirmed by dideoxy DNA
sequen-cing of the entire SOD-coding region of each expression
construct (results not shown)
Expression of pGH-SOD derivatives
In pGHX(–), SOD gene expression is under the control of
the powerful tac promoter [44] We developed this vector
specifically to be able to purify authentic SOD proteins via a
GST fusion protein intermediate similar to that reported
previously [24] Authenticity of the N-terminal amino acid
residues is ensured by the inclusion of a factor Xa cleavage
site appropriately situated with respect to the SalI cloning
site in this vector Our oligonucleotide primers utilized for
PCR were designed to encode the SODs from the second
codon (i.e after the ATG start codon) When cloned into
pGHX(–) as described these SOD genes are rendered
in-frame with the GST gene, separated by the FXa
recognition sequence
Expression of GST-SOD proteins was high,
correspond-ing to approximately 40% of the total cell protein (results
not shown) Single column purification on
glutathione-sepharose yielded proteins of the expected size (47 300 Da
for GST-FeSOD and 49 000 Da for MnSOD, Fig 2A)
Purity of the fusion proteins was estimated to be 98% as
measured by laser densitometry of Coomassie-stained SDS/
PAGE gels (results not shown) Perhaps surprisingly, the
GST-SOD fusion proteins exhibited SOD activity on native
PAGE (Fig 2B), although only GST-FeSOD[wt] (wild
type), GST-Mn[wt] and GST-Mn[G77Q/Q146A] showed
any appreciable activity on native gels and higher protein
concentrations were required to visualize SOD activity of
GST-Fe[A141Q] and GST-Mn[G77Q] (Fig 2B) This result
suggests the formation of active dimers between the SOD
domains of the fusion proteins products Slower-migrating
bands also visualized by SOD activity staining may have
been derived from further interactions of the fusion protein GST domains (Fig 2B) Although not visualized in these zymograms, all of the fusion proteins had detectable SOD activity in a spectrophotometric assay (Table 1)
Purification of authentic SOD Utilization of the pGHX(–) vector enabled the purification
of SOD proteins containing authentic N-terminal amino acid residues SOD is released from GST-SOD fusion proteins by cleavage with FXa (Fig 2C) Although this method is less efficient than cleavage by thrombin [24] the released SOD proteins contain no additional N-terminal
Fig 2 Purification and superoxide dismutase activity of FeSOD and MnSOD mutants (A) SDS/PAGE (15%) of GST-SOD fusion pro-teins from single-column affinity purification Lanes 1–8 are Fe[wt], Fe[Q69G], Fe[A141Q], Fe[Q69G/A141Q], Mn[wt], Mn[G77Q], Mn[Q146A] and Mn[G77Q/Q146A], respectively (B) Native-PAGE (8%) stained for superoxide dismutase activity Lanes 1–8 as for A (8 lg per lane), lanes 9–12 are Fe[A141Q], Fe[Q69G/A141Q], Mn[G77Q] and Mn[G77Q/Q146A] (20 lg per lane) (C) Cleavage and purification of authentic SODs SDS/PAGE (15%) of GST-SOD fusion proteins (examples shown are Fe[A141Q], Fe[Q69G/A141Q], Mn[G77Q] and Mn[G77Q/Q146A] in lanes 1–4, respectively) were cleaved by treatment with the restriction protease FXa (lanes 5–8, samples as for lanes 1–4 after FXa treatment), Samples subjected to further affinity chromatography to remove uncut fusion protein and released GST, to leave pure, authentic SOD (lanes 9–12 as for lanes 1–
4, 9 and 10, 1 lg each, 11 and 12, 5 lg each) Lane 13 contains FeSOD and MnSOD (from Sigma, 4 lg each) (D) Native-PAGE (8%) of purified SOD samples stained for SOD activity Lane 1 contains FeSOD and MnSOD markers (from Sigma, arrowed, 4 lg each), lanes 2–9 as for lanes A1 to A8, lanes 10–13 as for lanes C1 to C4.
Trang 6amino acid residues sometimes present due to cloning [36].
The efficiency of FXa cleavage varied between 50% and
70% as measured by laser densitometry (results not shown)
Biotinylated FXa was removed by the addition of protein
A-agarose to the digest prior to further affinity
chromato-graphy on GSH-sepharose which then removes both
undigested GST-SOD and released GST proteins (Fig 2C)
All purified SOD mutants comigrated on SDS/PAGE with
authentic FeSOD or MnSOD obtained from Sigma
Chemical Company, Poole, UK (Fig 2C and results not
shown)
Samples of pure SOD exhibited a similar pattern of SOD
activity on native-PAGE as the GST-SOD fusion proteins
(Fig 2D) Higher protein concentrations were necessary to
visualize Fe[A141Q], Fe[Q69G/A141Q] and Mn[G77Q],
however, both Fe[A141Q] and Mn[G77Q] were observed to
stain a light red colour rather than achromatically as is the
case for SOD in this system (Fig 2D) This aberrant
staining has been observed before with high protein
concentrations in the system used [24]
Enzyme activity and metal content
The specific activity of superoxide dismutase mutants was
measured in a spectrophotometric assay Both fusion and
purified proteins reflect the same differences in activity
between the mutant enzymes Assay results for GST-SOD
fusion derivatives are lower than for pure SOD proteins but
are proportional to the difference in size between the
proteins (Table 1) Wild-type SODs showa similar level of
specific activity (per mg protein basis) while mutants lacking
a glutamine at the active site (Fe[Q69G] and Mn[Q146A])
exhibit a large reduction, the manganese enzyme being
reduced to an undetectable level (Table 1) The addition of a
second glutamine to the active site location of the iron
enzyme (Fe[A141Q]) has a very similar effect to removal of
the existing glutamine and SOD activities are reasonably
similar between the two mutants In contrast, however,
addition of a second glutamine residue to the manganese
enzyme (Mn[G77Q]) leaves the mutant with about half the
specific activity of the wild-type enzyme A final contrast can
be seen between the double mutant enzymes where the
glutamine residue has been removed from the wild-type location and replaced by a glutamine at the location corresponding structurally to the position found in its isoenzyme (Fe[Q69G/A141Q] and Mn[G77Q/Q146A]) In this case the FeSOD demonstrates a reduction in activity to about a quarter of its wild-type level but the MnSOD increases by half as much again (Table 1)
We measured the metal content of the mutant proteins by atomic absorbance spectroscopy The results of this analysis are presented in Table 2, with both iron and manganese levels reported for each mutant as the number of metal ions present per subunit of protein In wild-type enzymes, the metal sites of the purified proteins are either completely (FeSOD, 1.17) or nearly fully occupied by the metal ion from which the SOD derives, and very little of the contrary metal is present (Table 2) This result demonstrates the selectivity of each of the wild-type enzymes for its respective
Table 1 Specific activities of mutant superoxide dismutases Results are expressed as the mean from at least three individual measurements (duplicate measurements were within 5% of each other) Mn[Q146A] gave no discernible activity even at high protein concentrations.
SOD expressed GST-SOD activitya
SOD activity a
(unitsÆmg)1)
SOD activity b
(unitsÆmetal ion)1) Iron superoxide dismutase mutants
Manganese superoxide dismutase mutants
a
Activity of SOD as measured by the xanthine-xanthine oxidase assay with percentage activity relative to wild type in parentheses.
b
Activity of SOD expressed on a per-iron metal basis (iron superoxide dismutase mutants) and per-manganese metal basis (manganese superoxide dismutase mutants).
Table 2 Metal contents of mutant superoxide dismutases Metal con-tent was measured by atomic absorbance and is given as the number of metal ions per subunit protein.
SOD
Metal content expressed (mol metalÆmol SOD)1)
Ratio a
Iron Manganese Iron superoxide dismutase mutants
Fe[Q69G/A141Q] 0.83 0.16 5 Manganese superoxide dismutase mutants
Mn[Q146A] 0.00 0.50 100 Mn[G77Q/Q146A] 0.23 0.87 3
a Ratio is given as Fe : Mn content (iron superoxide dismutase mutants) and Mn : Fe content (manganese superoxide dismutase mutants).
Trang 7metal in vivo and corresponds to a ratio around 20 times in
favour of the correct metal This ratio of correct to
incorrect metal in the active site of the enzyme is greatly
increased by more than tw o fold to greater than 50 times
when the active-site glutamine residue is removed
(Fe[Q69G] and Mn[Q146A], Table 2) Although all of the
sites are metallated in the Fe[Q69G] mutant, only half of the
sites appear to be occupied in the Mn[Q146A] mutant
Selectivity for metal is somewhat reduced in Mn[G77Q] and
similar to wild type in Fe[A141Q] but is greatly reduced in
the double mutants Fe[Q69G/A141Q] and Mn[G77Q/
Q146A] where the ratio of correct to incorrect metal is
lower than 5 (Table 2)
As SODs are active only when a metal ion is present in at
least one active site of the dimeric enzyme, we recalculated
the specific activity of each mutant enzyme on a per metal
ion basis using values obtained for the correct metal
Relative results do not vary significantly from the specific
activities on a per mg protein basis, except for Mn[G77Q]
which was not very well metallated and its specific activity
becomes very similar to that of the wild-type enzyme
(Table 1)
Protection against paraquat-induced stress
As the GST-SOD fusion proteins are active SODs, we
tested the ability of the mutant enzymes to protect SOD
minus E coli cells from the effects of oxidative stress The
herbicide paraquat was used to induce oxidative stress in
E coli and acts via the electron transport chain to
produce superoxide anions intracellularly [45] Both stress
and protein expression were induced simultaneously after
inoculation of media with cultures grown to exponential
phase in the absence of paraquat and IPTG (Materials
and methods [24]) The final concentration of paraquat
and IPTG used for the experiment presented were chosen
empirically to give differential growth rates between the
mutant enzymes Expression of each mutant SOD was
similar (approximately 40% of total protein) and did not
change throughout the time course of the experiment,
being similar to expression levels observed in overnight cultures (results not shown) As illustrated in Fig 3A,B, growth rates of OX326A (DsodA DsodB) cells harbouring the expression vector are very slowunder the selected conditions As can be seen by comparison of Fig 3A with Fig 3B, increases in growth rates of the induced cultures varies similarly between the different mutants of FeSOD and MnSOD, although absolute growth rates are some-what different (results not shown) Cells harbouring the double mutants (Fe[Q69G/A141Q] and Mn[G77Q/ Q146A]) grow with the fastest rates (circles, Fig 3), with wild-type enzymes and SODs lacking a glutamine residue (Fe[Q69G] and Mn[Q146A]) demonstrating equivalent growth rates representative of the slowest of the SOD-containing cells (squares and diamonds, respectively, Fig 3) SOD mutants which contain an extra glutamine (Fe[A141Q] and Mn[G77Q]) exhibit growth rates between wild-type and double mutants (triangles, Fig 3) This is a somewhat surprising result as we had previously shown that under equivalent conditions, cellular growth rates are proportional to the specific activity of the SOD being expressed by the cell type [24] In this experiment, this is clearly not the case Several repeated experiments under slightly different conditions lead to the same conclusive result
Effect of hydrogen peroxide on enzyme activity Hydrogen peroxide, the product of the SOD reaction, is
an inhibitor of SODs and an inactivator of FeSOD It is often used to distinguish between the two types of isoenzyme, particularly in conjunction with native PAGE gels stained for SOD activity, where 5 mMconcentrations inhibit FeSODs but leave MnSODs virtually unaffected [46] We found that there was very little difference between any of three active MnSOD types when incubated with hydrogen peroxide at a concentration of 5 mM prior to spectrophotometric assay of SOD activity (Materials and methods and Fig 4) FeSOD mutants, however, could be distinguished by their different sensitivities to 0.25 m
0.0 0.2 0.4 0.6 0.8
0.0 2.5 5.0
A
7.5 10.0 Time (Hr) 0.0 2.5 5.0 7.5 10.0
B
Fig 3 Effect of GST-SODs expressed in E coli OX326A DsodA, DsodB cells under paraquat-induced oxidative stress E coli OX326A (DsodA, DsodB) cells harbouring the appropriate plasmid were grown to exponential phase and used to inoculate media containing IPTG (0.1 m M ) and paraquat (250 l M ) Cell growth was then followed by measuring the optical density at 600 nm (A) FeSOD samples Cells expressing GST-SOD fusion proteins for GST-Fe[wt] (h), GST-Fe[A141Q] (n), GST-Fe[Q69G] (e) and GST-Fe[Q69G/A141Q] (s) or the pGHX(–) vector alone (·) (B) MnSOD samples Cells expressing GST-SOD fusion proteins for Mn[wt] (j), Mn[G77Q] (m), Mn[Q146A] (r) and Mn[G77Q/Q146A] (d) or the pGHX(–) vector alone (·).
Trang 8hydrogen peroxide (Fig 4) Most sensitive was the mutant
containing two glutamines, Fe[A141Q] (50% inhibited
after 2 min), wild-type FeSOD was similarly inhibited to
50% after 5 min, the double mutant Fe[Q69G/A141Q]
after 9 min and the least sensitive was Fe[Q69G] (50%
inhibited after 11 min) (Fig 4)
Hydrogen peroxide inactivates FeSODs via a Fenton
reaction with the iron metal at the active site The presence
of iron is therefore a prerequisite for its effect and suggests
that the absence of reactivity with the MnSOD mutants is
due to the lack of activity in these mutants with iron at the
active site, despite there being some 20% iron in the
Mn[G77Q/Q146A] mutant (Tables 1 and 2) The
differen-tial effects of hydrogen peroxide on the FeSOD mutants
may be explained by an alteration of reactivity induced by
the electronic configuration of active site residues, in this
case glutamine 69 (or 141) Sensitivity to hydrogen peroxide
was observed to increase in the order: no glutamine
< Q141 < Q69 < tw o glutamines
Effect of azide on enzyme activity
Azide has similarly been used to discriminate between
different SOD types [47], FeSOD exhibiting a higher
sensitivity than MnSODs In our assay procedure
(Mate-rials and Methods) there appeared to be little to distinguish
any of the FeSOD mutants from the wild-type enzyme,
exhibiting a Kiof between 1.0 and 2.0 mM(Fig 5) All the
FeSOD mutants, however, appeared to be more inhibited at
higher azide concentrations than did wild type (Fig 5) Fe[Q69G] was inhibited to a greater degree at lower azide concentrations than other FeSODs, but was not affected to the same extent at 10 mM azide, a concentration which virtually eliminated any activity from Fe[A141Q] or Fe[Q69G/A141Q] (Fig 5) Although not affected to the same extent as FeSOD derivatives, the Mn[G77Q/Q146A] mutant showed a similar sensitivity to azide as Mn[wt] (Ki approx 12.0 mM), and Mn[Q146A] was apparently the least sensitive of all SODs tested (Kiapprox 40 mM, results not shown and Fig 5)
Effect of temperature on enzyme activity The thermostability of the enzymatic activity of the SOD mutants was investigated at 50C (Fig 5) Mn[wt] is inherently less thermostable than Fe[wt], as shown in Fig 6, where the relative activity of Fe[wt] takes 50 min
to reduce to 50% while Mn[wt] takes only 9 min The most dramatic differences in thermostability were observed for those enzymes that have gained a second glutamine residue Mn[G77Q] was found to be the most thermostable of the enzymes studied, and maintained almost full activity for over one hour (Fig 6) In contrast, Fe[A141Q] was found to be the most thermolabile of the enzymes Its SOD activity was reduced by half within
4 min of incubation at 50C and reducing to zero within
20 min (Fig 6) The Fe[Q69G] mutation demonstrated a thermal stability profile very similar to that of Fe[wt], and
0
20
40
60
80
100
Time (min.) 10
Fig 4 Effect of hydrogen peroxide on the activity of SODs Samples of
purified SOD were incubated with hydrogen peroxide (0.25 m M for
FeSODs and 5 m M for MnSODs) at 22 C At the indicated times,
aliquots were removed for analysis These were calculated to give 1 unit
of SOD activity in the standard SOD assay conditions used and all
values were normalized to this At least three independent
measure-ments were made for each data point Samples shown are Fe[wt] (h),
Fe[A141Q] (n), Fe[Q69G] (e) Fe[Q69G/A141Q] (s), Mn[wt] (j),
Mn[G77Q] (m) and Mn[G77Q/Q146A] (d) Mn[Q146A] is not
rep-resented due to its lack of measurable activity even at high protein
concentration.
0 20 40 60 80 100
Sodium Azide (mM)
Fig 5 Effect of azide on the activity of SOD Samples of purified SODs were adjusted to give 1 unit of SOD activity under standard assay conditions Changes in the observed activity in the presence of azide were normalized to this as a percentage Aliquots of each SOD were added to sodium azide at the appropriate concentration to yield the required concentration of azide in the complete assay solution, and assayed immediately At least three independent measurements were made for each data point Samples shown are Fe[wt] (h), Fe[A141Q] (n), Fe[Q69G] (e), Fe[Q69G/A141Q] (s), Mn[wt] (j), Mn[G77Q] (m) and Mn [G77Q/Q146A] (d) Mn[Q146A] is not represented due to its lack of measurable activity even at high protein concentrations.
Trang 9the Mn[Q146A] mutant could not be assayed due to its
lack of activity
Most significantly, however, the double mutant enzymes
demonstrated thermal profiles similar to their isoenzyme
counterpart Fe[Q69G/A141Q] exhibited a half-time of
9 min similar to Mn[wt], and Mn[G77Q/Q146A] exhibited a
half-time of 50 min similar to that of Fe[wt] (Fig 6) These
results suggest a structural role for these glutamine residues
in the mononuclear SODs
D I S C U S S I O N
The basis for metal cofactor selectivity in vivo and metal ion
reaction specificity in the homologous FeSODs and
MnSODs has been difficult to ascertain by comparative
analyses of sequence and/or structural information Several
MnSODs and FeSODs have been crystallized [11–21],
MnSOD has been crystallized containing an iron ion [48] and
cambialistic SOD holoenzyme structures have been
com-pared reconstituted with different metals [21] Furthermore,
a plethora of SOD sequences is available from the DNA
and protein sequence databases There appears, therefore
to be no obvious sequence or structural signal that
determines selectivity or specificity in this important class
of metalloenzymes
Despite recent reports of changes in metal specificity in
engineered SOD enzymes [31,32], the results have been
unspectacular, resulting in small increases in enzyme activity
in SOD holoenzymes reconstituted with the wrong metal
Unfortunately, the H145E mutation of Mycobacterium
tuberculosisFeSOD which produces an active MnSOD [49] bears no relevance to the central question of specificity as no other SODs with this mutation exist naturally The nature
of this phenomenon still remains elusive
Metal specificity may be considered as consisting of two, presumably distinct, stages First, selectivity in vivo of the proteins for their metal cofactor is a prerequisite, as the proteins must fold de novo about their corresponding metal ion Even at this stage it is unclear as to whether the enzymes utilize a divalent or trivalent metal ion during folding, or whether, like their copper- and zinc-containing SOD counterparts, they utilize a chaperone to transfer only the correct metal [50] Mononuclear SODs appear to fold with only the correct metal when presented with both, whether
in vivoor in vitro [3,4] Second, specificity of the reaction is determined by the metal ion: the enzyme is active only with the correct metal Although, in the complete absence of the
correct metal, each type of SOD may be forced to fold with the incorrect metal at its active centre, the resulting enzyme
is no longer an active SOD [3,4]
Here we report differences in metal selectivity in muta-tions that affect residues which contribute to the hydrogen bonding network around the metal and metal-ligand residues (Table 2) Selectivity appears to followa pattern dependent upon the presence and orientation of the active-site Q69 (FeSOD) or Q146 (MnSOD) The ratio of metals incorporated into SOD mutants examined showed a reduction in selectivity in the order: no glutamine > one glutamine[wt] > two glutamines > one glutamine (the double mutants) Thus the presence and position of this residue affects in vivo, the selectivity for metal ion (Table 2) Changes in metal selectivity were not apparently related
to the activity of the mutant enzymes (Table 1) Mutants lacking a glutamine residue showed the highest selectivity but either lowor no discernible SOD activity Double mutants showed reasonable levels of activity, with the Mn[G77Q/Q146A] mutant actually exceeding that of wild-type MnSOD to 150% (Table 1) This is in contrast to the activity of the same mutant reported previously as 75% [32] However, methodological differences exist in these two studies which make accurate and direct comparisons extremely unreliable Also, these authors observed an iron-supported activity in this mutant of some 7% We have observed no changes in the hydrogen peroxide sensitivity of this mutant compared to wild type (Fig 3), though the high manganese-supported activity may mask any changes in our experiments Furthermore, in vivo experiments which produced iron-containing enzymes Fe[wt], Fe[Q69G/A141Q], Mn[wt] and Mn[G77Q/Q146A] using anaerobic culture conditions in the presence of iron and absence of manganese salts [3], generated only active Fe[wt] and Fe[Q69G/A141Q] enzymes (results not shown) Both Mn[wt] and Mn[G77Q/Q146A] were inactive when cultured under these conditions which favour the insertion
of iron into the active site of SODs [3] (results not shown) Hydrogen peroxide inhibits MnSOD as it is the product
of the enzymatic dismutation of the superoxide radical In addition to this, however, FeSODs are inactivated by hydrogen peroxide [46] A Fenton reaction with the coordinated iron produces a peroxide radical which then attacks critical residues near the iron active centre This residue has been shown to be a tryptophan in other SODs [46,51–53] Reduction of SOD activity at relatively low
0
20
40
60
80
100
Time (min.)
Fig 6 Effect of temperature on SOD activity Samples of SOD at the
appropriate concentrations were incubated at 50 C for the indicated
times Aliquots were removed for analysis of SOD activity and were
calculated to give 1 unit of SOD activity in the standard SOD assay
conditions used and all values were normalized to this At least three
independent measurements were made for each data point Samples
shown are Fe[wt] (h), Fe[A141Q] (n), Fe[Q69G] (e) Fe[Q69G/
A141Q] (s), Mn[wt] (j), Mn[G77Q] (m) and Mn[G77Q/Q146A] (d).
Mn[Q146A] is not represented due to its lack of measurable activity
even at high protein concentration.
Trang 10levels of hydrogen peroxide has therefore been used as an
indicative marker for the presence of iron at the active site
and, indeed, is frequently used to determine the type of SOD
isoenzyme present in a sample [49] Both wild-type and
mutant MnSODs were affected to some extent by hydrogen
peroxide but showed no significant differences (Fig 3)
FeSODs were affected to differing degrees, but in general all
demonstrated a similar inactivation to wild type The least
affected was Fe[Q69G] followed by Fe[Q69G/A141Q],
Fe[wt] with Fe[A141Q] being the most affected (Fig 3)
Although steric effects may need to be considered, the
glutamine appears to play a role in hydrogen peroxide
sensitivity (the presence of two glutamine residues generates
a more sensitive enzyme, whereas the absence of any
glutamine residue in the active site renders the enzyme more
sensitive) The reason for this is unclear
Azide, like hydrogen peroxide, is also often used to
discriminate between FeSOD and MnSODs as the latter are
usually much less sensitive to its inhibitory action [47] The
effect of azide, a substrate analogue, showed no prominently
different behaviour between mutant SODs, though
Mn[G77Q/Q146A] was significantly less sensitive to azide
than wild type or Mn[G77Q] In addition, all FeSOD
mutants were significantly, though only slightly, more
sensitive to azide than wild type The order in which azide
effects the FeSOD mutant proteins may reflect the
acces-sibility of the active site to this compound (Fe[wt] <
Fe[Q69G/A141Q] < Fe[A141Q] < Fe[Q69G], Fig 4) as
this trend matches the degree of steric hindrance to be
expected from these mutations, if azide were to approach
the active site via the substrate funnel The orientation of
azide bound in the active site appears to be different for
FeSOD and MnSOD enzymes [11] Bound azide is
contained within a pocket formed by no less than six
residues all within 4 A˚ (azide N1 comes within 2.1 A˚ of the
metal ion) Azide binding residues include the three His
metal ligands (H26, H81 and H171) and gateway residues
H30 and H34 A further gateway residue, H31, is involved
in FeSOD but not in MnSOD Only in MnSOD is the active
site glutamine (Q146) also in proximity to the bound azide
Azide binding could therefore be affected by electrostatic
interactions as well as steric interference by the mutational
changes studied
The most striking change in physical characteristics of the
mutant SODs was revealed in temperature-sensitivity
stud-ies (Fig 5) These showed that double mutations in each
enzyme (Fe[Q69G/A141Q] and Mn[G77Q/Q146A])
endowed the mutant with a similar
temperature-deactiva-tion profile to that of its opposing wild-type enzyme
(Mn[wt] and Fe[wt], respectively) We show therefore that
the position of this glutamine residue in the active site is
responsible for this behaviour, presumably primarily
through its participation in a hydrogen-bonding network
involving other residues and solvent molecules An
unex-pected result was the extremely high thermostability of
Mn[G77Q] and the very lowthermostability of Fe[A141Q]
(Fig 5) Each of these mutants has two glutamine residues
in their active sites; one from the naturally occurring
residue, the other engineered to mimic its counterpart
Molecular modelling [34] indicates that the active sites of
both MnSOD and FeSOD would most probably not be
able to accommodate a second glutamine residue (results
not shown) The most stable conformations of these
mutants leave one glutamine extended away from the active site and stabilized by hydrogen bonding to an Asn residue Q77 is capable of hydrogen bonding with N74 or Q146 with N73 As the numbering indicates, N73 and N74 lie adjacent
to each other, and both are conserved in FeSOD and MnSOD sequences Simulation suggests that Q77 bonding
to N74 also disrupts the active site residues, causing a shift in the position of Y34 and a twist in the ring of the metal-binding H81 Thermostability is not a newphenomenon amongst SODs; those isolated from hyperthermophilic organisms, for example, exhibit similarly high thermosta-bility [16,17,54,55] Recent mutational studies of the FeSOD from Aquifex pyrophilus demonstrate the importance of hydrogen bonding patterns on thermal stability [54] It is not possible to predict which of the glutamine residues will be distorted out of the active site, or for which of the mutants, but it may be more likely that the FeSOD[A141Q] with lowest activity has the largest disruption around the active site
Although hydrogen bond pairs and bond strengths may have been altered in the mutations discussed, other effects due to repackaging of the active site around the hydropho-bic stem of this residue and the corresponding alanine replacement also should not be ignored Nor too should the possible inclusion of water molecules into the active site as has been reported for human MnSOD[Q143A] [30] Exten-sion of these effects through the dimer interface via local residues may also be important, though all these possibilities remain highly speculative at this time Elucidation of the structures of the mutants presented here should help to explain the chemical nature of the physical effects observed
It seems certain, however, that metal selectivity and specificity in the mononuclear SODs is not governed by one or even two residues, but is most likely accomplished by the concerted effects of a combination of key residues Further analysis of these and other mutant SODs is currently underway
A C K N O W L E D G E M E N T S
We are indebted to G Peplow, F Yamakura and T Matsumoto for the analyses of iron and manganese in protein samples We also wish to thank H Steinman for the gift of E coli OX326A We finally thank Mr
M Farrugia for photographic assistance.
R E F E R E N C E S
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2 Bannister, J.V., Bannister, W.H & Rotilio, G (1987) Aspects of the structure, function, and applications od superoxide dismutase Crit Rev Biochem 22, 111–180.
3 Beyer, W.F & Fridovich, I (1991) In vivo competition between iron and manganese for the occupancy of the active site region of the manganese-superoxide dismutase of Escherichia coli J Biol Chem 266, 303–308.
4 Ose, D.E & Fridovich, I (1979) Manganese-containing super-oxide dismutase from Escherichia coli: reversible resolution and metal replacements Arch Biochem Biophys 194, 360–364.
5 Brock, C.J & Harris, J.L (1977) Superoxide dismutase from Bacillus stearothermophilus: reversible removal of manganese and its replacement by other metals Biochem Soc Trans 5, 1533– 1539.