Heegaard2, Marcus O¨hman1 and Thomas Nilsson1 1 Department of Chemistry, Karlstad University, Sweden;2Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark A detailed
Trang 1Comparison of native and recombinant chlorite dismutase
Helena Danielsson Thorell1, Natascha H Beyer2, Niels H H Heegaard2, Marcus O¨hman1
and Thomas Nilsson1
1
Department of Chemistry, Karlstad University, Sweden;2Department of Autoimmunology, Statens Serum Institut, Copenhagen, Denmark
A detailed comparison between native chlorite dismutase
from Ideonella dechloratans, and the recombinant version
of the protein produced in Escherichia coli, suggests the
presence of a covalent modification in the native enzyme
Although the native and recombinant N- and C-terminal
sequences are identical, the enzymes display different
electrophoretic mobilities, and produce different peptide
maps upon digestion with trypsin and separation of
fragments using capillary electrophoresis Comparison of
MALDI mass spectra of tryptic peptides from the native
and recombinant enzymes suggests two locations for
modification in the native protein Mass spectrometric
analysis of isolated peptides from a tryptic digest of the
native enzyme identifies a possible cross-linked dipeptide,
suggesting an intrachain cross-link in the parent protein
Spectrophotometric titration of the native enzyme in the denatured state reveals two titrating components absorb-ing at 295 nm, suggestabsorb-ing the presence of about one tyrosine residue per subunit with an anomalously low
pKa The EPR spectrum for the recombinant enzyme is different from that of the native enzyme, and contains a substantial contribution of a low-spin species with the characteristics of bis-histidine coordination These results are discussed in terms of a covalent cross-link between a histidine and a tyrosine sidechain, similar to those found
in other heme enzymes operating under highly oxidizing conditions
Keywords: chlorate; chlorite dismutase; recombinant chlorite dismutase; post-translational modification
Chlorate- and perchlorate-respiring bacteria have attracted
interest due to their potential use in the treatment of soil and
water contaminated by oxyanions of chlorine Perchlorate,
chlorate, and chlorite are recognized as potential health and
environmental hazards [1–3] In general, these compounds
are not formed naturally Rather, their appearance in the
natural environment is due to their manufacture and use as
bleaching agents, disinfectants, herbicides, and components
of explosives and rocket propellants [4–8] The microbial
decomposition of oxochlorates is important in the treatment
of pulp bleaching effluents [9], as well as in the degradation
of oxochlorates released into the environment by other
routes [10] Despite the fact that oxochlorates are not
formed naturally, chlorate-respiring bacteria are quite
ubiquitous [11,12]
Ideonella dechloratans is a well-characterized species
capable of chlorate respiration [13] Chlorate is first
converted to chlorite by a periplasmic chlorate reductase
[14] In the second step, chlorite is decomposed to chloride
and molecular oxygen by chlorite dismutase [15] The
presence of chlorite dismutase is a prerequisite for bacterial
growth as chlorite is toxic due to its high reactivity The
oxygen produced is utilized by a cytochrome c oxidase [13]
Chlorite dismutase has been purified, initially from strain GR-1 [16,17], and subsequently from strain CKB [18], and from I dechloratans [15] Chlorite dismutases isolated from the different species appear quite similar, being homotetra-meric heme proteins with molecular masses around
100 kDa The gene encoding chlorite dismutase has been cloned and sequenced from two different species, I dechlo-ratans [19] and Dechloromonas agitata [20] The latter reference also describes a homologous gene in the genome
of Magnetospirillum magnetotacticum, but in this case no expression of chlorite dismutase has been observed The I dechloratans chlorite dismutase gene has been expressed in Escherichia coli, and the resulting recombinant enzyme has been partially characterized [19] In the present study, we present a more detailed characterization of recombinant chlorite dismutase, and a comparison with the native enzyme Our results suggest the presence of
a post-translational modification, possibly an intrachain covalent cross-link, in the enzyme produced in the natural host
Materials and methods
Protein purification Native chlorite dismutase was purified from I dechloratans (ATCC 51718) as previously described [15] Recombinant chlorite dismutase was expressed and purified from E coli
as described in [19], except that the cells were homogenized
by a Bead-Beater (Biospec Products, Bartlesville, USA)
Correspondence to T Nilsson, Karlstad University, Department of
Chemistry, SE 651 88 Karlstad, Sweden Fax: + 46 54 7001457,
Tel.: + 46 54 7001776, E-mail: thomas.nilsson@kau.se
(Received 6 May 2004, revised 8 July 2004, accepted 14 July 2004)
Trang 2polydimethyl acrylamide coated fused silica capillary as
described in [22]
Peptide mass mapping
For in-gel digestion and sample preparation, gel plugs from
SDS/PAGE stained with Coomassie brilliant blue were
excised In-gel digestion was carried out according to the
protocol for silver stained bands in [23] and modified as
described in [24] Micropurification was performed
accord-ing to Kussmann et al [25] and Gobom et al [26] Samples
were eluted directly onto a polished steel target plate with
0.8 lL a-cyano-4-hydroxycinnamic acid, 6 mgÆmL)1 in
0.1% trifluoroacetic acid, 30% methanol, and 30%
aceto-nitrile (premade from Agilent Technologies, Palo Alto,
USA), and left to air-dry
For peptide separation by RP-HPLC, the purified native
enzyme was also digested by trypsin in solution The
peptides were separated by HPLC and peak fractions were
analyzed by MALDI-MS Native chlorite dismutase (20 lL
at 7 mgÆmL)1) was precipitated with 3 lL trichloroacetic
acid (100%), left 30 min on ice, and centrifuged at 10 000 g,
15 min The precipitate was washed with cold acetone,
vortexed and centrifuged at 10 000 g, 15 min and then
resuspended in 20 lL of 8M urea in 0.4M NH4HCO3,
pH 8 Water was added to a volume of 80 lL Trypsin
(4 lg) was added, and the sample was incubated with
shaking at 37°C, 52 h in an Eppendorf Thermomixer The
digest was fractioned on a Vydac C18 peptide column, with
a gradient of 3–97% buffer A (70% acetonitrile in 0.1%
trifluoroacetic acid, v/v), 1 mLÆmin)1, over 1 h Fractions
were collected manually, subsequently dried in a speed-vac
and resuspended in 10 lL of 0.1% (v/v) trifluoroacetic acid
One microliter was applied to the polished steel target
(Scout 384) with 0.5 lL a-cyano-4-hydroxycinnamic acid
(Agilent) and allowed to dry (dried droplet)
Peptide mass spectra were recorded on a Bruker
UltraFlex TOF reflector mass spectrometer (Bruker
Dal-tonics, Bremen, Germany), equipped with a nitrogen laser
(k¼ 337 nm) The spectra were recorded in the positive
mode, using the reflector mass analyzer Calibration was
initially performed by external calibration using the
Bruker Peptide Standard Whenever possible, internal
mass calibration was subsequently carried out on the
in-gel digestion spectra using the porcine trypsin
auto-digestion products (m/z 841.502 and 2210.096) Data
analysis was carried out by M/Z)FREEWARE, edition
2001.08.14 (Proteomics, New York, NY, USA) Database
searches were carried out using (Proteomics),
Spectrophotometric titration Native chlorite dismutase, 6 lM(monomer), was diluted in
6Mguanidinium chloride, 10 mMborate, 10 mMTris/HCl,
pH 6 Aliquots of 1Msodium hydroxide were added to the solution At each pH value, the UV/visible spectrum was recorded using a Shimadzu UV2101 spectrophotometer Fitting of theoretical titration curves to data was carried out using IGOR(Wavemetrics, Portland, OR, USA)
Electron paramagnetic resonance (EPR) spectroscopy EPR spectra were acquired on a Bruker ER-200D-SCR spectrometer equipped with an Oxford Instruments ESR-9 helium cryostat The concentrations of species giving rise to high- and low-spin signals were estimated as described in [27] and [28], respectively
Results
Electrophoresis of proteins and tryptic peptides
We have previously reported different electrophoretic mobilities for the native and recombinant chlorite dismu-tases when examined by SDS/PAGE [19] The recombinant enzyme migrates with a mobility close to that predicted by the amino acid sequence (corresponding to a molecular mass of 28 kDa), whereas the native enzyme migrates faster (corresponding to a molecular mass of 25 kDa) The molecular mass, calculated from the DNA sequence, of the mature protein is 27.8 kDa The recombinant protein contains an extra N-terminal methionine and its predicted molecular mass is 27.9 kDa As we have suggested [19], a possible explanation of the different mobilities is post-translational processing of chlorite dismutase in I dechlo-ratans Proteolytic processing at the N-terminus, however, can be excluded from the N-terminal sequencing reported in our earlier work [15] In the present work, the C-terminal sequence was also investigated, and found to be that predicted from the gene (see below) These results exclude proteolytic processing as an explanation for the different mobilities of the native and recombinant enzymes
To investigate other covalent modifications that could affect the electrophoretic mobility, tryptic peptide maps of native and recombinant enzymes were prepared During the course of this work we found that the recombinant enzyme was less stable than the native enzyme during the latter stages of the purification procedure, and was only possible obtain in about 70% purity Tryptic digests were therefore prepared from proteins blotted from SDS gels to
Trang 3polyvinylidene difluoride membranes The digests were
analyzed by capillary electrophoresis using a coated
capillary Figure 1 shows electropherograms of tryptic
digests from the native and recombinant enzymes
Migra-tion times in this type of analysis are prone to variability
[29], but most of the peptide peaks seen in the
electro-pherogram of the native enzyme are also found in that
obtained from the recombinant enzyme However, there
are clear differences, particularly at later migration times
(marked in the figure), which are not due to migration
time shifts Thus, two peaks (denoted by arrows in Fig 1)
in the electropherogram of the native enzyme are missing
in the electropherogram of the recombinant enzyme
There are also two peaks in the electropherogram of the
recombinant enzyme, which do not appear to have
counterparts in the native enzyme Our finding that
different peptide maps are obtained from the native and
recombinant enzymes suggests a difference between their
covalent structures Although the nature of such a
difference cannot be inferred from these results, we note
that anomalously high electrophoretic mobilities in SDS/
PAGE analyses have been observed in proteins containing
covalent cross-links, such as disulfide bonds [30,31] or
cross-links caused by oxidative coupling of sidechains
[32,33] The higher electrophoretic mobility in these
proteins is probably due to the smaller hydrodynamic
radius caused by the cross-link
Mass spectrometry
Detailed investigations of possible differences between
native and recombinant chlorite dismutase covalent
structure were carried out using MALDI-TOF mass
spectrometry Tryptic peptide mass maps of the native
enzyme, from in-gel digestion and digestion in solution,
were analyzed Masses covering most of the predicted
amino acid sequence of the enzyme could be identified in
these spectra, when allowing four missed cleavages in the
tryptic in silico digestion The sequence coverage based
on the mass spectra, and on C-terminal sequencing
of the native enzyme, is shown in Fig 2A Four fragments, corresponding to HK(52–53), RK(180–181), VPENKYHVR(215–223) and T(242) (bold) were not covered
To compare the native and recombinant enzymes, peptides were generated by in-gel trypsin digestion and subject to mass analysis using as above For the native enzyme, we obtained basically the same sequence coverage
as above The recombinant enzyme produced, however, a prominent peak at a mass of 1571.7, which is completely absent in the native enzyme A comparison of the mass spectra obtained from the native and recombinant enzymes
is shown in Fig 3 Analysis of the sequence reveals the fragment HKEKVIVDAYLTR(52–64) (Fig 2B) as the probable origin of this peak This fragment includes HK(52–53), which is missing in the sequence coverage of the native enzyme This result implicates HK(52–53) as a possible location for a covalent modification The fragment VPENKYHVR(215–223), also missing in the mass spectra,
is another possible location In the mass spectrum of recombinant enzyme, VPENK(215–219) was absent, whereas YHVR(220–223) was observed as a part of fragment (220–241)
To identify modified fragments, tryptic peptides from the native enzyme were separated by HPLC and individually analyzed by MS Matching sequence coverage was obtained after analysis of the mass spectra of the individual peptide fractions One peptide fraction from the chromatographic separation produced a mass spectrum containing a peak (m/z¼ 1679.8) (Fig 4), corresponding to the sum (minus one hydrogen) of the fragments containing HKEK(52–55) and VPENKYHVR(215–223) (Fig 2C) We could not, however, detect a fragment at m/z¼ 1426 corresponding to fragment (52–53) combined with fragment (215–223) Localization of a modification to fragment (52–53) is therefore tentative
Fig 1 Separation of tryptic peptides of native
(A) and recombinant (B) chlorite dismutase by
capillary electrophoresis with a polydimethyl
acrylamide-coated fused silica capillary.
Dashed arrows indicate correspondence, and
solid arrows denote peaks that do not have
counterparts in the other electropherogram.
Trang 4native enzyme, completely denatured in 6Mguanidinium chloride, was carried out Figure 5 shows the absorbance at
295 nm (the absorption maximum of the tyrosinate ion [37]
as a function of pH A curve fit of a single titration curve (Fig 5A) did not yield a satisfactory fit, suggesting the presence of more than one titrating component This is not expected when the enzyme is completely denatured, as all tyrosines should be in the same chemical environment A curve fit with two titrating components gave a better fit (Fig 5B) The major component, accounting for 92% of the total amplitude, titrated with a pKavalue of 10.15 ± 0.03,
in accordance with the pKavalue of 10.1 for tyrosine [35] For the minor component, accounting for 8% of the total amplitude, a pKa value of 8.35 ± 0.3 was found This is similar to the value found for the histidine methyl ester derivative studied in [35] Chlorite dismutase contains 12 tyrosine residues per subunit We note that the fraction of the minor component corresponds to about one of the 12 tyrosines titrating with the lower pKa value
EPR The EPR spectrum of the recombinant chlorite dismutase
at pH 7 is shown in Fig 6 In contrast to the EPR spectrum of the native enzyme at neutral pH (trace A; see
Fig 3 Mass analyses of tryptic peptides from native (A) and recombinant (B) chlorite dismutase Only the 1558–1615 mass range is shown.
Fig 2 Sequences for the complete protein and for detected fragments
of chlorite dismutase (A) The native chlorite dismutase amino acid
sequence with the coverage obtained by using MALDI-MS The bold
and italic sequences were not detected The sequence in italics is that
obtained in C-terminal sequencing (B) The calculated monoisotopic
mass [MH]+of the peptides are shown (C) The monoisotopic size of
the peptides that would result from histidine–tyrosine covalent linkage
(1679.92 Da).
Trang 5also [15]), which contains only high-spin heme, the
spectrum of the recombinant enzyme (trace B) is a
mixture of contributions from high- and low-spin species
The high-spin heme component in the spectrum consists
of both a rhombic and an axial species with a total
concentration of 58 lM The majority of the high-spin
heme has the characteristics of a rhombically distorted
heme From the spectrum, the g-values 6.31 and 5.47 are
obtained Essentially the same g-values are found in the
EPR spectrum of native chlorite dismutase at neutral pH
A minor part of the high-spin heme is axial with a g-value
at 5.9 This axial high-spin heme is not found in the spectrum of the native enzyme from I dechloratans but a similar component was observed the in EPR spectra of chlorite dismutase from strain GR-1 recorded at neutral
pH [38] For the low-spin component, the, g-values at 3.04, 2.25, and 1.52 are obtained The integrated ampli-tude for this signal corresponds to a concentration of
43 lM, which is little less than half of the total heme concentration
Fig 4 MALDI mass spectrum of at HPLC
fraction of tryptic digest of native chlorite
dismutase The 1679.8 Da mass fragment is
denoted by an arrow The inset shows an
expanded view of the 1600–1700 mass range.
Fig 5 Spectrophotometric titration of tyrosine
residues in the denatured native chlorite
dismu-tase The titration was monitored at 295 nm at
which only tyrosinate absorbs The protein
contains 12 tyrosine residues per subunit (A)
The solid line is the result of curve fitting with
A tot ¼ 0.216 and pK a ¼ 10.1 (B) The solid
line is the result of curve fitting with A tot1 ¼
0.206, pK a 1 ¼ 10.15, A tot2 ¼ 0.017, pK a 2 ¼
8.35.
Trang 6The detailed characterization of recombinant I
dechlora-tans chlorite dismutase, and comparison with the native
enzyme carried out here, suggest the presence of a covalent
modification in chlorite dismutase produced in the natural
host, but not in the recombinant version of the enzyme
Comparison of mass spectra for tryptic peptides obtained
from the native and recombinant enzymes suggest HK(52–
53) and YHVR(220–223) as sites of modification
Further-more, a fragment, isolated by HPLC, in the tryptic digest
of the native enzyme could be identified as a possible
product of cross-linking between HKEK(52–55) and
VPENKYHVR(215–223) (Fig 2C) Cross-linking is an
attractive candidate for covalent modification, as it would
account also for the higher electrophoretic mobility (due to
the smaller hydrodynamic radius) observed for the native
enzyme, and for the different peptide maps observed after
tryptic cleavage and separation by capillary electrophoresis
The nonenzymatic formation of covalently or
oxida-tively modified amino acids has been demonstrated
[39–41], and several cases of cross-links including histidine
and tyrosine residues in oxidative enzymes have been
reported recently [34] The crystal structure of galactose
oxidase revealed that the enzyme contained a modified
active site tyrosine covalently cross-linked to a cysteine at
the ortho-position to the phenolic oxygen [42] More
recently, cytochrome c oxidase has also been found to
contain a modified tyrosine, with the crystal structures
showing a covalent link between the active site tyrosine
(at the ortho-position) to the imidazole Ne of a histidine
[43,44] A different type of histidine–tyrosine cross-link
was discovered in the crystal structure of catalase HPII
tyrosine in chlorite dismutase The result of the spectro-photometric titration, together with the mass spectrometric data implicating the tyrosine-containing fragment VPEN-KYHVR(215–223) as a part of a cross-link, is consistent the participation of tyrosine in cross-linking From the low
pKavalue of 8.3 found in the spectrophotometric titration, the catalase HPII variant of cross-link is less likely as substitution at the Cbis not expected to affect the phenolic
pKavalue
An histidine–tyrosine bond may be somewhat labile [45] and this, in addition to ionic suppression, could explain the rather low yield of the dipeptide fragment mass in the MS analyses The fragmented dipeptide would not necessarily yield its constituent two tryptic fragment peptide masses as fragmentation may involve various parts of the molecule and sidechains may be derivatized Also, the small mole-cular mass part of the dipeptide would be prone to be obscured in the area of the mass spectrum dominated by signals from matrix components
The environment of the heme group in the recombinant enzyme was investigated using EPR spectroscopy In contrast to what is observed in the native enzyme, the EPR spectrum shows the presence of several species The major components are a high-spin species with a spectrum similar to that observed in the native enzyme, and a low-spin species An earlier characterization using optical spectroscopy [19] also revealed two components, one with
a native-like spectrum and one with absorption maxima at
405 and 525 nm in the oxidized state The later species could not be reduced by dithionite This species is probably the same as the one displaying the low-spin EPR signal The g-values of this component are different from those found for the low-spin component in the EPR spectrum of native chlorite dismutase at high pH (2.56, 2.19, and 1.87) [15], and they are also distinct from those found in other hydroxide-coordinated systems [47] The g-values are more similar to those observed for bis-histidine coordinated heme [47] Moreover, a similar EPR spectrum was observed in [38] after addition of imidazole
to chlorite dismutase from GR-1 Therefore, the heme group is probably coordinated by two histidine sidechains
in the low-spin component of the recombinant chlorite dismutase These results suggest a difference in structure of the heme pocket in the native and recombinant enzymes, with a histidine sidechain being more accessible for heme coordination from the distal side in the recombinant enzyme The difference between the heme environments in the native and recombinant enzymes is probably due to structural differences caused by covalent cross-linking
Fig 6 EPR spectra of native and of recombinant chlorite dismutase at
neutral pH (A) Native chlorite dismutase (B) Recombinant chlorite
dismutase Protein concentrations were about 100 l M (hem) EPR
conditions: temperature 10 K; microwave power, 2 mW; microwave
frequency, 9.449 GHz; modulation amplitude, 20 G.
Trang 7As discussed above, the histidine residue in fragment (52–
53) could be involved in cross-linking, and an interesting
possibility is that this residue is available for coordination
in the recombinant enzyme where a cross-link is not
present
Cross-links involving oxidatively coupled sidechains have
been found in enzymes operating under highly oxidizing
condition, and have been suggested to originate from
radicals formed in the reaction of the heme group with
oxidants In cytochrome c oxidase, a tyrosyl radical is
formed during the reaction of the mixed-valence state of the
enzyme with oxygen [48] MacMillan et al [49] have
reported the EPR signal of a radical generated in
cyto-chrome c oxidase This signal was proposed to originate
from the cross-linked tyrosine In catalase, the compound I,
containing Fe(IV) and a porphyrin radical is produced after
the reaction with one equivalent of hydrogen peroxide For
catalase HPII, which contains a histidine–tyrosine
cross-link, it has been proposed that compound I is the species in
which the post-translational modification takes place
[45,50,51] Although the catalytic mechanism of chlorite
dismutase is not known, the formation of similar
interme-diates appears likely, given the nature of the reactant The
reaction of chlorite with other heme enzymes, horseradish
peroxidase and chloroperoxidase, has been shown to
produce the highly oxidized compound I [52] Moreover,
a radical signal is present in the EPR spectrum of chlorite
dismutase from strain GR-1 [38] The formation of a
cross-link in chlorite dismutase by oxidative coupling, similar to
the mechanisms suggested for cytochrome c oxidase
[41,48,49] and catalase HPII [45,50,51], therefore appears
possible
Cross-linking is expected to increase the stability of a
protein, and it absence in the recombinant enzyme could
account for the lower stability during the latter stages of
its purification The catalytic properties of the recombinant
enzyme are, however, similar to those of the native
enzyme, suggesting cross-linking is not important for
catalysis This would be similar to cytochrome c oxidase,
where the histidine–tyrosine cross-link has been suggested
to play role in preserving the binuclear site architecture
[40,41,53,54]
In conclusion, our comparison between the native and
recombinant I dechloratans chlorite dismutase suggests
that the enzyme produced in the natural host contains a
covalent modification, probably an intrachain cross-link
involving a residue in the 52–55 region and a residue in
the 215–223 region A tyrosine–histidine cross-link
appears possible, and could account for EPR differences
between the native and recombinant enzymes as well as
the spectrophotometric titration of the native enzyme
More work is, however, needed to establish the nature of
the modification
Acknowledgements
We thank Roland Aasa (Chalmers University of Technology, Sweden)
for recording the EPR spectrum and for helpful suggestions regarding
its interpretation We also thank Annika Norin and Ella Cederlund
(Karolinska institutet, Sweden) for C-terminal amino acid sequencing
of the native enzyme, and Justyna M Czarna for help with the mass
spectrometric analyses.
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