EDUARD TORRENTS, MARIANN WESTMAN, MARGARETA SAHLIN AND BRITT-MARIE SJÖBERG1
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JBC Papers in Press. Published on July 7, 2006 as Manuscript M601794200
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M601794200
RIBONUCLEOTIDE REDUCTASE MODULARITY: ATYPICAL DUPLICATION OF THE
ATP-CONE DOMAIN IN PSEUDOMONAS AERUGINOSA
Eduard Torrents, MariAnn Westman, Margareta Sahlin and Britt-Marie Sjöberg1
Department of Molecular Biology and Functional Genomics, Arrhenius Laboratories for
Natural Sciences, Stockholm University, SE-10691 Stockholm, Sweden.
Running title: Pseudomonas aeruginosa class Ia ribonucleotide reductase.
Keywords: protein evolution, allosteric regulation, protein modularity, ATP-cone, tyrosyl radical,
diiron-oxo center
1
Corresponding author. Mailing address: Department of Molecular Biology and Functional Genomics,
Stockholm University, Svante Arrhenius väg 16-18 F3, SE-106 91 Stockholm, Sweden.
E-mail: britt-marie.sjoberg@molbio.su.se
The opportunistic pathogen that protein modularity built on the common
Pseudomonas aeruginosa causing serious catalytic core of all RNRs plays an important
nosocomial infections is a γ-proteobacterium role in class diversification within the RNR
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that can live in many different environments. family.
Interestingly, P. aeruginosa encodes three
ribonucleotide reductases (RNRs) that all Ribonucleotide reductase (RNR) is an
differ from other well-known RNRs. The essential constituent of all living cells and
RNR enzymes are central for de novo organisms. It catalyzes the production of
synthesis of deoxyribonucleotides and deoxyribonucleotides for DNA synthesis from
essential to all living cells. The RNR of this their corresponding ribonucleotides and thereby
study (class Ia) is a complex of the NrdA controls a key step for cell growth and
protein harboring the active site and the proliferation. In recent years it has also been
allosteric sites, and the NrdB protein recognized that RNR played a key role in the
harboring a tyrosyl radical necessary to transition from an RNA-world to a DNA-world.
initiate catalysis. P. aeruginosa NrdA Surprisingly, there are at least three major
contains an atypical duplication of the N- classes of RNRs in contemporary organisms,
terminal ATP-cone, an allosteric domain differing primarily in cofactor requirements and
that can bind either ATP or dATP and quaternary structures (1-4). Yet, a common
regulates the overall enzyme activity. Here theme among all three classes is a radical-based
we characterized the wild type NrdA and reaction mechanism. Recent structural studies
two truncated NrdA variants with precise N- have shown that the catalytic core regions of
terminal deletions. The N-terminal ATP- the three RNR classes are essentially identical
cone (ATP-c1) is allosterically functional, (5-8), strongly suggesting that all RNR classes
whereas the internal ATP-cone lacks have a common evolutionary origin.
allosteric activity. The P. aeruginosa NrdB is One signature of all RNRs is an
also atypical with an unusually short-lived elaborate allosteric regulation controlling the
tyrosyl radical, that is efficiently regenerated enzymes’ substrate specificities. The net result
in presence of oxygen as the iron ions remain of the allosteric regulation is a balanced
tightly bound to the protein. The P. production of the four different
aeruginosa wild type NrdA and NrdB deoxyribonucleotide products. Carefully
proteins form an extraordinary tight equilibrated concentrations of the four dNTPs
complex with a suggested α 4β 4 composition. are required by all cells to avoid errors during
An α 2 β 2 composition is suggested for the DNA replication and repair (9). Binding sites
complex of truncated NrdA (lacking ATP-c1) for the allosteric effectors which are ATP and
and wild type NrdB. Duplication or several dNTPs have been resolved in a number
triplication of the ATP-cone is found in some of RNR crystal structures (10-12) and are
other bacterial class Ia RNRs. We suggest comparatively close to the active site regions.
Specific side chain and backbone movements
1
Copyright 2006 by The American Society for Biochemistry and Molecular Biology, Inc.may explain the general molecular basis for the of the allosteric activity site is present in P.
allosteric regulation of substrate specificity. aeruginosa NrdA (Fig. 1a) and to study its
Another allosteric fine-tuning of RNRs possible role in the allosteric and biochemical
concerns the overall activity of ribonucleotide properties of the pure enzyme system.
reduction and presence of this type of control
differs between the different RNR classes and Material and Methods
also within the classes. The overall activity
control was first localized to the N-terminal Bacterial strains, plasmids and materials - E.
region of the E. coli R1 (or NrdA) component coli DH5α (Clontech) was used for cloning and
and forms a separate globular domain propagation of plasmids. E. coli Rosetta (DE3)
consisting of four α−helices and three (Novagen) was used for protein over-
β−strands (10). This domain binds ATP as a expression. Wild type P. aeruginosa PAO1
positive allosteric effector and dATP as a strain (ATCC15692/CECT4122) obtained from
negative effector. Recently, similar domains the Spanish National Collection of Type
were found in several different proteins and the Cultures was used for genomic DNA
motif was termed ATP-cone (13). Most of the purification. All strains were grown at 37ºC in
conserved residues of the ATP-cone are LB medium. Plasmid vectors used were pGEM-
involved in stabilizing the secondary structure T Easy (Promega) for cloning of PCR amplified
elements, which indicates that all sequences fragments and pET22b (Novagen) for protein
adopt the same structure and presumably bind over-expression. Oligonucleotide primers were
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ATP. from Invitrogen (England). Restriction
The presence or absence of an ATP- endonucleases and other enzymes were from
cone in the N-terminal region of RNR is one Fermentas (Germany).
basis for dividing the class I RNRs into
subclasses Ia (with the ATP-cone) and Ib General DNA and protein techniques -
(lacking the ATP-cone). Many class II RNRs Molecular cloning techniques were by standard
lack the ATP-cone, whereas most class III procedures (17). Genomic DNA of P.
RNRs include an ATP-cone. In addition, groups aeruginosa PAO1 was isolated using the
of organisms have RNRs that diverge from this DNeasy Tissue Kit (Quiagen) according to the
general presence or absence of ATP-cone, e.g. manufacturer’s specifications.
the herpesviridae carry an ATP-cone that has
lost its capacity to bind nucleotides. Among the Construction of overproduction strains - All
eubacterial species there are a few examples of constructs were cloned into the over-expression
organisms that carry more than one ATP-cone. plasmid pET22b. The entire wild type nrdA
The most striking example is that all known gene was amplified from genomic DNA by
Chlamydiaceae have an N-terminal triplication PCR using the following primers; forward OP-
of their ATP-cone (14), and some NrdA1up, 5’-
proteobacteria have a duplication of the ATP- ACATATGCATACCGACACCACACG-3’
cone. and reverse OP-NrdAlw, 5’-
One example of an ATP-cone TAAGCTTGCCCGGCCGCGACACGGAC-3’.
duplication is found in Pseudomonas The primers were designed to generate an NdeI
aeruginosa, a γ−proteobacterium that encodes and HindIII restriction site (underlined),
three different classes of RNR, Ia, II and III respectively, at the start and end of the resulting
(15). We have earlier shown that all three RNR amplified fragment. The 2937-bp PCR product
classes in P. aeruginosa are expressed, albeit at was cloned in the pGEM-T Easy vector. After
different growth conditions, and are digestion with NdeI and HindIII, the nrdA
enzymatically functional (15,16). All class Ia fragment was ligated into pET22b generating
RNRs consist of two different components, plasmid pETS141. The following primers
NrdA and NrdB. The active site region where (underlining shows the NdeI restriction site)
the substrates bind is found in the NrdA were used for PCR amplification of the
component, whereas the NrdB component different nrdA fragments and the nrdB gene; for
harbors a stable tyrosyl radical that is needed to nrdAΔ147 forward OP-NrdA2up, 5’-
initiate the radical-based catalysis. The aim of ACATATGCGCATCACCCGCGCCG-3’ and
this work was to understand why a duplication reverse OP-NrdAlw, for nrdAΔ182 forward
2OP-NrdA3up, 5’- DuoFlow System fast protein liquid
ACATATGATCGAGCGCGAGACCC-3’ and chromatography instrument (BioRad)
reverse OP-NrdA-lw, and for nrdB forward OP- previously equilibrated with 10 volumes of
NrdBup, 5’- buffer A. The different RNR components (wild
GAGCCATATGCTGAGCTGGGACG-3’ and type NrdA and NrdB, truncated NrdA proteins)
reverse OP-NrdBlw, 5’- were eluted with a linear gradient of ammonium
AGGATCCTAAATCGAGCATCTCAATCC- sulfate (0.75 to 0 M, 1 ml/min) in buffer B (50
3’ (this underlining shows a BamHI restriction mM Tris-HCl, pH 7.5, 2 mM DTT). After SDS-
site). All amplified fragments were cloned into PAGE fractions with the highest purity were
the pGEM-T Easy vector as described concentrated using Centricon (Millipore).
previously. The NdeI-HindIII digested DNA NrdB protein was further purified by
fragments nrdAΔ147 (2499 bp) and nrdAΔ182 ion exchange chromatography. Pooled fractions
(2385 bp) were cloned into the pET22b were diluted to 5 mg/ml protein concentration
generating pETS142 and pETS143, and loaded on a HiLoad™ 16/10 Q-Sepharose®
respectively. The NdeI-BamHI digested nrdB High Performance (GE Healthcare) pre-
DNA fragment (1268 bp) was cloned into equilibrated with buffer B plus 30 mM KCl.
pET22b generating plasmid pETS144. All four NrdB protein was eluted with a linear gradient
pETS clones were sequenced in both directions of KCl (30 to 300 mM, 3 ml/min) in buffer B.
to assure the lack of mutations during the PCR Fractions containing the NrdB protein were
amplification and cloning steps. pooled, concentrated using Centricon 30
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(Millipore) and step-by-step the buffer was
Overproduction and purification - The E. coli exchanged to buffer B.
Rosetta (DE3) cells containing any of the NrdAΔ182 protein was purified as
pETS141, pETS142, pETS143 and pETS144 described for the NrdB protein followed by a
were grown in LB medium (Difco) with 100 gel filtration chromatography. Pooled fractions
µg/ml ampicillin and 17 µg/ml from the ion exchange purification were loaded
chloramphenicol. The cultures were grown with at 0.5 ml/min on a 24 ml Superdex-75
vigorous shaking (250 rpm) at 30ºC. When cells column equilibrated and eluted with buffer B
reached an OD550≈0.5 expression of the cloned plus 200 mM KCl. Each fraction was analyzed
gene was induced by the addition of IPTG to a by Phastgel electrophoresis (GE Healthcare)
final concentration of 0.5 mM. After four hours and fractions with the highest purity were
of induction the cells were harvested by pooled, concentrated in a Centriprep-50
centrifugation and cell pellets were frozen on (Millipore) and finally freed from KCl by
dry ice and stored at –80ºC. washing with buffer B.
All the protein purification steps were
carried out at 4ºC. All RNR proteins used in Iron reconstitution of P. aeruginosa NrdB for
this study were purified using the same enzymatic assays - NrdB protein was
procedure. Frozen cells were disintegrated by reconstituted at 25ºC by reaction with anaerobic
X-press (Biox AB), homogenized and extracted ferrous iron ascorbate solution under aerobic
with 4 volumes (v/w) of buffer containing 50 conditions as previously described (18) with a
mM Tris-HCl, pH 7.5, 30 mM KCl, 10 mM molar ratio of 3 irons per NrdB polypeptide.
DTT and Complete protease cocktail inhibitor After 5 minutes the reaction mixture was
(Roche), and centrifuged for one hour at desalted on a NAP-10 column (GE-Healthcare)
40 000xg. Nucleic acids were precipitated by equilibrated with buffer B and concentrated
addition of streptomycin sulfate to a final with Centricon 30 (Millipore).
concentration of 1% and removed by
centrifugation. The supernatant was precipitated Gel filtration in presence of effector nucleotides
by slow addition of ammonium sulfate to 45% - A 2 ml column of Superdex-200 (GE-
final saturation. After centrifugation the Healthcare) was used to estimate the molecular
precipitate was dissolved in 5 ml of buffer A mass of the NrdA, NrdAΔ147 and NrdB
(0.75 M ammonium sulfate, 5 mM DTT). proteins alone or in combination. Proteins at
Around 200 mg of total protein was applied to a 1.25, 2.5 and 5 mg/ml were chromatographed at
HiLoad16/10 PhenylSepharose High 0.1 ml/min in buffer containing 50 mM Tris-
Performance (GE Healthcare) on a BioLogic HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, 100
3mM NaCl, and 5 mM ATP. The column had addition of Fe2+ and the formation and decay of
been standardized with thyroglobulin (669 absorption bands were observed between 300-
kDa), apoferritin (443 kDa), β−amylase (200 500 nm in cycles of 90 s with a scanning rate of
kDa) and alcohol dehydrogenase (150 kDa), all 240 nm/min. The tyrosyl radical concentration
supplied from Sigma. at different time points was determined by
To estimate the relative proportions of subtracting an “endpoint” spectrum at ca 30
NrdA versus NrdB components in the min after iron addition from the spectrum
holoenzyme complexes, eluted protein peaks immediately after iron addition, giving a tyrosyl
were analyzed by SDS-PAGE followed by radical absorption at 413 nm (Supplement, Fig.
densitometric analyses. 1) that was quantified using the extinction
coefficient 3.25 mM–1cm–1 (23).
Enzymatic assay and other analytical For experiments measuring iron content
techniques - Standard conditions implied the during radical decay 1.2 ml of apoNrdB was
use of 0.7 mM 3H-CDP (10 775 cpm/pmol), 5 reconstituted with 1.5 Fe per polypeptide, since
mM ATP, 30 mM DTT, 30 mM magnesium higher iron-to-protein ratio gave less well-
acetate, 1 mg/ml BSA, 30 mM Tris-HCl, pH resolved spectra. After 1, 2, 5, 9 and 15 scans
7.5 and 30 nmoles of NrdA and 60 nmoles of (i.e. after 70, 190, 560, 1040 and 1500 s) 110 µl
NrdB in a final volume of 50 µl. Incubation was was withdrawn, mixed with 2 µl 15 mM EDTA,
at 25ºC for 20 min and the amount of dCDP desalted on a NAP-5 column equilibrated with
formed was determined by the standard method 50 mM Tris-HCl, pH 7.6, and used for analyses
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as previously described (19). One unit of of iron and protein concentrations. The
enzyme corresponds to 1 nmol of product registered spectra (Fig. 3 inset) were used to
formed per min, and the specific activity is calculate the tyrosyl radical after subtraction as
expressed as units per mg total protein. KL, KD described above.
and Vmax values were obtained by direct curve
fitting as described before (20,21) using the EPR Spectroscopy - EPR spectra were
KaleidaGraph software (Synergy Software). registered on an ESP 300 X-band spectrometer
Standard errors of mean shown in tables 3-4 (Bruker) equipped with a liquid nitrogen flow
and in figures 5-6 are not repeated in the text. system for temperatures ≥90K. Radical
Protein concentrations were determined concentrations were determined using a B.
by the Bradford assay, with crystalline bovine anthracis NrdF sample and an E. coli NrdB
serum albumin as a standard. Iron analyses sample with known tyrosyl radical
were determined as described by Sahlin & al. concentrations as secondary standards. The
(22). error limits for radical quantitations are ≤±10%.
UV-vis absorption spectroscopy and formation Nucleotide binding assay - Nucleotide binding
and decay of radical-diiron site - Stopped flow was performed according to the method of
spectroscopy was applied to study formation of direct partition through ultrafiltration (24). The
the radical-diiron site. Kinetic traces were experiments were carried out at 25ºC, using a
registered every 5th nm in the region 440-350 range of 0.5 to 100 µM NrdA and NrdAΔ147
nm using a DX 18MV Biosequential stopped polypeptide and 0.5 to 100 µM dATP/ATP.
flow ASVD spectrometer (Applied
Photophysics) and a split time base of 2/20s. RESULTS
Equal volumes of 80 µM apo-NrdB polypeptide
(aerobic) and 80 µM Fe2+ (anaerobic) were Atypical class Ia RNR in P. aeruginosa
mixed at 25°C, and 55 µl of each reactant were The nrdA and nrdB genes code for the
used per shot. class Ia RNR that is active only during aerobic
Scanning spectra were registered on a conditions (15,16). The nrdA gene (2892 bp)
Perkin Elmer Lambda2 spectrometer at 25° C. encodes a protein of 963 amino acid residues
Typically 20 µM NrdB polypeptide in 50 mM with an expected molecular mass of 107 kDa.
Tris-HCl, pH 7.6 was used. For reconstitution The nrdB gene (1248 bp) encodes a protein of
1-6 µl of 5 mM Fe2+ in 0.1 M HCl was added to 415 amino acid residues with an expected
500 µl protein solution giving 0.5-3 Fe/NrdB molecular mass of 47 kDa. Whereas the NrdB
polypeptide. Spectra were registered before protein is overall similar to other NrdB
4proteins, the NrdA protein revealed interesting were carried out for each protein (see Material
differences compared to other well-known class and Methods) yielding approximately 8.5 mg of
Ia RNRs. The deduced amino-acid sequence of pure protein per liter of culture for the NrdA
P. aeruginosa NrdA is 202 residues longer than wild type, 17.6 mg per liter for the NrdAΔ147
the 761-residues E. coli class Ia prototype. and 22.5 mg per liter for the NrdB purification.
Alignment of these two proteins shows that After several purification steps for the
most of the extra amino acids (around 155 NrdAΔ182 protein we obtained only a partially
residues) are located at the N-terminus of the purified fraction (around 60% purity) with a
protein (Fig. 1a). The remaining 47 residues are yield of 2.5 mg per liter of culture. SDS-PAGE
found in the C-terminal part of the protein. All of the purified proteins gave only one band for
the essential cysteine residues known to be NrdA wild type, NrdAΔ147 and NrdB with
involved in catalysis and enzyme turnover and apparent polypeptide sizes similar to the
other residues that are functionally important in theoretical molecular masses of the cloned
class I proteins are present in the deduced P. genes (107, 91 and 47 kDa respectively; Fig 2).
aeruginosa NrdA protein (data not shown). One of the two prominent bands in the
Comparison of the NrdA against the Pfam NrdA∆182 preparation also corresponds to its
database (25) revealed high similarity to the theoretical molecular mass (88 kDa, Fig. 2, lane
RNR central core (residues 293-910, 3). Western-blot analysis using polyclonal
Pfam02867; E value 5*e–187) and to an ATP- antibodies against the wild type P. aeruginosa
cone domain (residues 32-132 (denoted ATP- NrdA protein confirmed that only the 88-kDa
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c1), Pfam03477; E value 3*e–28) previously band is a NrdA protein product (data not
described as the allosteric site regulating the shown).
overall enzyme activity of the enzyme (10).
With a lower similarity (E value 5*e–3) we General biochemical properties of the purified
found another ATP-cone structure between the NrdA proteins
two structures previously identified (residues None of the purified proteins had any
151-250, denoted ATP-c2). RNR activity on their own (data not shown).
The amino acid sequence of the N- Combinations of either NrdA wild type or
terminal region of P. aeruginosa NrdA (Fig. NrdAΔ147 proteins with reconstituted NrdB
1b) implies a duplication of the residues for a protein (see below and Table 1) exhibited
putative activity site domain. As shown in specific class Ia RNR enzyme activity. Using
figure 1b, several residues responsible for the ATP as positive allosteric effector and DTT as
nucleotide binding (10) are present in both an artificial electron donor for this system we
putative allosteric domains (ATP-c1 and ATP- obtained a specific activity of 88 U/mg for the
c2). To study whether both domains can bind NrdA wild type and 96 U/mg for the
nucleotides and regulate the overall activity of NrdAΔ147. The preferred substrate for this
the enzyme two different constructions were system is nucleoside diphosphates with a KM
made (Fig. 1a) and their biochemical and for CDP of 27±8 µM for both NrdA wild type
allosteric properties were studied.
and NrdAΔ147. A calculated half maximal
activity was obtained at 7.9 mM DTT for the
Purification of wild type P. aeruginosa proteins
NrdA wild type protein and at 3.6 mM for
and truncated NrdA proteins
NrdAΔ147.
The full-length P. aeruginosa nrdA
No activity was detected when
gene product and two engineered variants of
NrdAΔ182 was assayed together with the
truncated nrdA, designated NrdAΔ147 and
reconstituted NrdB protein. Competition assays
NrdAΔ182, were overproduced in E. coli. The
of the wild type NrdA protein with different
P. aeruginosa NrdAΔ147 protein (816 residues)
amounts of NrdAΔ182 showed no inhibition of
lacks the first activity site (ATP-c1). The
the wild type protein activity (data not shown)
NrdAΔ182 (781 residues) lacks the first and
suggesting that the NrdA∆182 protein cannot
part of the second activity site domain (ATP-
bind to the NrdB protein. Due to the lack of
c2) and corresponds in size and protein
enzymatic activity with the NrdAΔ182 protein
sequence to the class Ib NrdE proteins (Fig. 1a).
no further experiments were carried out with
The P. aeruginosa nrdB gene was also
this protein.
expressed in E. coli. Different purification steps
5The tyrosyl radical in P. aeruginosa NrdB components (Fig. 4b). However, the radical
protein forms and decays rapidly content is lower than normally observed when
The purified NrdB protein contained no recombinant NrdB proteins are expressed and
tyrosyl radical and no diiron site as determined analyzed under similar conditions (e.g. Bacillus
from EPR, light absorption and iron analysis. anthracis NrdF with ca 45 µM radical content,
Reconstitution of purified NrdB apoprotein Supplement, Fig. 3), suggesting that the P.
with Fe2+/O2 gives a spectrum with aeruginosa tyrosyl radical decays also under
characteristic diiron-oxo charge transfer bands these conditions.
at 325 and 370 nm and a prominent tyrosyl
radical band at 413 nm (top spectrum, inset Fig. Recombinant NrdB protein requires continuous
3). Stopped flow experiments showed that the oxygen-dependent reactivation similar to the
major fraction of the radical and the diiron site native P. aeruginosa system
formed with a rapid rate of ca 4 s-1 and a minor As expected, the enzyme activity of the
fraction (relative amplitude 0.13-0.17 of the reconstituted P. aeruginosa NrdB protein
faster rate) with a rate of ca 0.15 s-1 followed the radical content when assayed
(Supplement, Fig. 2). This means that the immediately after addition of iron (Table 1), but
radical and diiron sites were essentially formed what happens to the diiron site with time and
after 2 s. Increasing amounts of added iron during assay conditions? We have shown that
resulted in increasing amount of radical up to a the radical and diiron-oxo features have
ratio of 2 Fe/polypeptide when it leveled off (cf. disappeared after long incubation times (lowest
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Table 1). Addition of more iron did not increase spectrum, inset Fig. 3), but that iron still
the amount of formed radical, instead it remains bound to the protein (Fig. 3). Notably,
increased background absorbance and hence a reconstituted NrdB sample that was desalted
disturbed the resolution of diiron-oxo charge and concentrated as the samples used for the
transfer bands in the spectra. activity assays described above, displayed no
Surprisingly, the formed spectra radical band but had almost full enzyme activity
immediately began to decay, losing both radical (Table 1, lowest line). This experiment
and diiron-oxo bands concomitantly (Fig. 3). convincingly shows that the radical is
The decay of the tyrosyl radical can be fitted efficiently regenerated during the normal
with two rate constants of 0.016±0.005 s-1 and enzyme activity measurements.
0.0011±0.0001 s-1 (relative amplitude 2.3 of the Class Ia RNR purified from growing P.
faster decay). Tyrosyl radical decay was not aeruginosa cells was previously found to
prevented by reconstitution in the presence of 1 require continuous supply of oxygen during
mg/ml BSA (as used in activity measurements) enzyme activity measurements (15). To
and/or an excess of NrdA (data not shown). establish if this was also the case for the
Instead, we observed that the iron ions recombinant P. aeruginosa class Ia RNR
remained tightly bound to the protein (Fig. 3) system used in this study we performed
despite the fact that the typical diiron-oxo experiments where the enzyme mixtures were
charge transfer bands decayed concomitantly preincubated anaerobically or aerobically for 40
with the decay of the radical band (Fig. 3 inset). min and then assayed for enzyme activity
Rapid freezing of a sample 4 s after anaerobically or aerobically. The recombinant
addition of 2.5 Fe per NrdB polypeptide yielded P. aeruginosa class Ia RNR system behaves as
0.29 radical/monomer. The tyrosyl radical in P. the native system and has a prominent
aeruginosa NrdB as observed by EPR is a requirement for continuous oxygen supply
typical class Ia radical in line shape (Fig. 4a). It during enzyme activity measurements (Table
differs from the E. coli radical (Fig. 4c) in the 2). In a parallel set of assays we added EDTA
resolution of the spectrum, and the 0.7 mT to the samples to chelate any iron that was not
hyperfine splittings (26) from the 3,5 ring bound to protein. The results in Table 2 show
protons of the P. aeruginosa NrdB tyrosyl that the enzyme activity is the same in presence
radical are well resolved in the 93K spectrum. as in absence of EDTA, again demonstrating
The P. aeruginosa NrdB tyrosyl radical content that the iron ions are tightly bound to the
of ca 5 µM in overproducing E. coli cells shows protein and that continuous regeneration of the
that the P. aeruginosa radical can be formed in radical requires oxygen.
the absence of any additional P. aeruginosa
6Only one allosteric overall activity site in P. the wild type protein and 16±3 µM for the
aeruginosa NrdA is fully functional. NrdAΔ147 protein. In general the Vmax values
As shown in figure 1b two putative for both proteins were considerably lower with
allosteric activity sites (ATP-c1 and ATP-c2) dTTP as effector (Fig. 6b, starting values)
were found in the wild type NrdA protein. To compared to those obtained with ATP (Fig. 5a)
test whether these sites are fully functional we or dATP. Binding of dTTP to the allosteric
studied the behavior of the wild type protein specificity site was efficiently displaced by
and the truncated protein NrdAΔ147 against increasing concentrations of dATP, but with
different concentrations of ATP and dATP. In diametrically opposite results for NrdA∆147
an ATP curve both proteins showed similar and NrdA wild type (Fig. 6b). In NrdA∆147,
behavior (Fig. 5a) and KL values (2.9 mM for dATP amplified the dTTP-stimulated reaction
the NrdA wild type and 2.7 mM for more than 5-fold (Fig. 6b), as expected when
NrdAΔ147). The NrdAΔ147 protein had a two positive effector nucleotides compete for
slightly higher calculated Vmax (118 U/mg) the specificity site. The apparent KL of about 33
compared to the wild type (95 U/mg). µM for the dATP stimulation in this
At low concentrations dATP works as a competition experiment compares well with the
positive effector for class Ia RNR by binding to KL of 17.3 µM for dATP’s allosteric effect on
the allosteric specificity site, but at high the NrdA∆147 protein. In stark contrast, dATP
concentrations it inhibits the enzyme activity by inhibited the dTTP-stimulated reaction in the
binding to the allosteric overall activity site. wild type NrdA protein approximately 4-fold,
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Titration curves of the P. aeruginosa NrdA as expected if dATP binds both competitively
wild type and NrdA∆147 proteins with different with dTTP to the specificity site and with
concentrations of dATP highlighted drastic negative allosteric effect to the overall activity
differences between the two proteins (Fig 5b). site. This experiment shows conclusively that
As expected for a typical NrdA protein the P. the truncated NrdA∆147 protein retains only
aeruginosa wild type protein was activated by the allosteric specificity site and lacks the
low concentrations of dATP with a KL1 allosteric overall activity site, confirming that
(specificity site binding) of 3.2 µM and the ATP-c2 in P. aeruginosa NrdA has lost its
inhibited by higher dATP concentration with a function as an allosteric site.
KL2 (activity site binding) of 17.3 µM.
Contrarywise, the NrdAΔ147 protein was The wild type P. aeruginosa class Ia RNR is a
completely resistant to high concentrations of high molecular mass complex
dATP and remained fully active even at 1 mM To estimate the apparent molecular
dATP (Fig. 5b) with a KL1 value of 17.1 µM. A weight of the complex formed by the wild type
likely explanation for the observed difference in NrdA or NrdAΔ147 with the NrdB, mixtures of
the dATP response of the two proteins could be proteins at different concentrations (1.25, 2.5, 5
in the ability to bind the nucleotide. Binding mg/ml) were chromatographed on a
assays confirmed that the NrdA wild type Superdex-200 column. The Ve/Vo (elution
protein binds 2.1 molecules of dATP per volume/void volume) was calculated and
polypeptide chain (KD=15 µM, Fig 6a); translated to molecular mass for all the samples
plausibly one dATP bound at the allosteric (Table 3). NrdB was resolved in a single peak
specificity site and one at the allosteric overall with an estimated molecular mass of 184 kDa,
activity site, as is typically found for other class which corresponds to around four NrdB
Ia NrdAs (4). On the other hand the NrdAΔ147 subunits (β4). The NrdA wild type protein
protein binds only half as much dATP, 0.9 eluted in a major peak (estimated molecular
molecules per polypeptide chain (KD=26 µM, masses of 528 kDa) and a minor peak
Fig 6a), in this case only to the specificity site. (estimated molecular mass ca. 120 kDa)
These experiments suggest that the second corresponding to oligomerization of four NrdA
putative nucleotide-binding site (ATP-c2) is not wild type subunits (α4-6) and to a single NrdA
allosterically functional. wild type protein (α). NrdAΔ147 eluted as a
The allosteric effector dTTP that is single peak corresponding an estimated
known to bind only to the specificity site homodimeric (α2) molecular mass of 202 kDa.
stimulated in our case the reduction of CDP in Equimolar mixtures (at 1.25, 2.5 and 5
both proteins with a KL value of 19±3 µM for mg/ml) of NrdA wild type or NrdAΔ147 with
7NrdB protein in the presence of ATP as a emphasize that P. aeruginosa class Ia RNR
positive effector were used to detect oligomeric differs in several aspects from the well-
forms of active complexes (Table 3). For the characterized class Ia RNRs of E. coli,
first time we could observe distinct complexes bacteriophage T4 and mouse (29).
in gel filtration chromatographies compared to Firstly, the NrdA component of P.
other RNRs studied so far (27,28). Wild type aeruginosa class Ia RNR contains in its N-
complexes eluted in two peaks with molecular terminus a duplication of the allosteric activity
masses estimated to be 634 and 217. Analysis domain, which is a member of the ATP-cone
of the high molecular mass peak showed an family (13). We engineered a precise truncation
equimolar amount of NrdA and NrdB of the N-terminal ATP-cone (NrdA∆147) and
polypeptides and the molecular mass indicated measured the allosteric regulation of the
that four subunits each of NrdA wild type and NrdA∆147 enzyme activity as well as its
NrdB proteins (α4β4) were present in the number of binding sites and dissociation
complex (Table 3). For the second peak the constants for effector nucleotides. The wild
densitometric analyses indicated two NrdB type P. aeruginosa NrdA displayed the
polypeptides per each NrdA, and the observed canonical allosteric response with an initial
molecular mass indicated an αβ2 complex. activation by dATP followed by an inhibition of
Surprisingly, also the truncated protein enzyme activity at higher dATP concentrations,
NrdAΔ147 can form a tight complex to the wild whereas the truncated NrdA∆147 responded to
type NrdB protein. The complex eluted in a dATP activation but lacked the dATP inhibitory
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single peak with an estimated molecular weight response. Competition experiments with dATP
of 250 kDa corresponding to a heterodimeric in presence of dTTP convincingly showed that
structure (α2β2) of the active complex (Table 3), the remaining ATP-cone (ATP-c2) in the
which was corroborated by the densitometric truncated NrdA∆147 protein lacked nucleotide-
analysis. binding properties. ATP-c2 lacks some of the
side chains known to be involved in nucleotide
P. aeruginosa NrdA and NrdB form a tight binding in E. coli NrdA (Fig. 1b), e.g. the
complex in presence of ATP equivalent of Lys-21 (10), and also His-59
In our gel filtration experiments we recently shown to be important for allosteric
observed distinct complexes between the P. regulation (21), offering a plausible explanation
aeruginosa NrdA and NrdB components for the lack of allosteric response in ATP-c2.
indicating that the proteins formed strong Secondly, the P. aeruginosa NrdAB
complexes. We titrated the NrdB protein over holoenzyme forms at least a 7-fold stronger
the NrdA wild type or NrdAΔ147 proteins in complex than e.g. the E. coli NrdAB
the presence of ATP and estimated a KD of ca. holoenzyme complex (30) and appears to have
60 nM (based on NrdA and NrdB polypeptide a different quaternary structure. The truncated
concentrations) for the NrdA wild type and the P. aeruginosa NrdA∆147/NrdB complex also
NrdAΔ147 complexes (Table 4, and has a strong KD, whereas the additionally
truncated NrdA∆182 does not interact with
Supplement Fig. 4). The KDs were also
NrdB, suggesting that residues between
measured in the presence of dTTP as a positive
positions 148 and 182 in NrdA may influence
effector. The results showed a considerably
weaker complex with a KD of 0.41 µM the strength of the complex. The strong
interaction of P. aeruginosa NrdAB was
(polypeptide concentrations) for a wild type
reaffirmed with gel filtration experiments in
complex and a KD of 0.79 µM (polypeptide
which the active complex chromatographed
concentrations) for a NrdAΔ147 complex
with an apparent heterotetrameric structure
(Table 4).
(α4β4) in contrast to the α2β2 structure found for
DISCUSSION E. coli NrdAB (29,31,32), and the hexameric
form (α6β6) of mouse NrdAB in presence of
In this study we have characterized the ATP (33). Interestingly, the oligomeric
P. aeruginosa class Ia RNR, one of the three structure of the NrdA∆147/NrdB complex was
physiologically relevant RNRs in this heterodimeric (α2β2) suggesting that the N-
opportunistic pathogen (15,16). Three terminal ATP-cone (ATP-c1) in P. aeruginosa
unexpected features of the class Ia RNR NrdA is crucial for the wild type quaternary
8structure of the NrdAB complex as well as for of a class Ia is widespread. Figure 7 clearly
the allosteric regulation of overall enzyme shows that P. aeruginosa NrdA clusters
activity. together with NrdAs from some β- and γ-
Thirdly, the P. aeruginosa NrdB diiron- proteobacteria and the Chlamydiaceae.
tyrosyl radical center formed with about the Interestingly, all these proteins have a
same rate as the corresponding center in E. coli duplication (or triplication) of the ATP-cone
(34,35), but the P. aeruginosa radical-diiron domain. Other NrdAs in the β- and γ-
center is short lived and the radical and diiron proteobacteria are clustered in a different
sites decomposed together. This could reflect a subgroup, and all these sequences have a single
more open structure and have functional ATP-cone domain. The observed tree topology
reasons. Interestingly, the iron ions remained (a tree based on only the catalytic part of the
bound to the protein and the radical was protein has the same topology) suggests that the
efficiently reactivated during assay conditions. alteration in the N-terminal region has occurred
We also observed that the functional enzyme recently and is not a rare event. Why is this
needs continuous supply of oxygen, as expected duplication maintained and widely distributed?
if the diiron-radical center has to be reformed Our results show that only one ATP-cone
continuously for reduction of ribonucleotides. (ATP-c1) is allosterically functional, whereas
The oxygen requirement of the native class Ia the role of the other ATP-cone (ATP-c2)
RNR activity was previously observed in crude remains unclear. We suggest that part of the
extracts from P. aeruginosa (15). As also ATP-c2 domain might be important for NrdB
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shown earlier expression of class Ia RNR in P. interaction, since the deletion of only 35
aeruginosa drops 6-fold in early stationary additional residues (NrdA∆182) mimicking an
phase, when the expression of class II RNR NrdE protein knocked out enzyme activity and
increases 6-7-fold (16). The atypical continuous interaction with the NrdB protein.
oxygen-requirement of the P. aeruginosa class The widespread duplication of the
Ia RNR could imply that enzyme activity is ATP-cone in RNRs is an illustration of the
inactivated in microaerophilic conditions and plasticity of this domain. Interestingly, in
may explain why this organism also encodes a Chlamydia the catalytic domain and the
class II RNR. The presence of functional genes adjacent ATP-cone seem to have eukaryotic
for all three classes of RNRs may thus allow P. origin whereas the two proximal copies are
aeruginosa to proliferate at any given oxygen more similar to prokaryotic counterparts
availability that it encounters. (13,14). Hence it was suggested that a
Interestingly, analysis of the flanking recombination between eukaryotic and bacterial
regions of the P. aeruginosa nrdAB operon (16) genes might have resulted in a protein with
revealed that it is preceded by a transposase multiple ATP-cones. We suggest that class
gene (PA1153, 177 residues) and followed by diversification within the RNR family is an
an open reading frame of phage origin example of protein modularity. The modularity
(PA1154, 184 residues). These two proteins are concept is an attractive hypothesis that has
probably reminiscent footprints of a phage and implications for how proteins may have
support the idea that the class Ia genes might evolved in Nature. It has been suggested that
have been introduced in the P. aeruginosa construction of new types of proteins with
genome via a lateral gene transfer event (3). diverse functions could occur by use of simple
Crucial for the long-term persistence of newly building blocks or modules (36,37). In RNR the
acquired DNA is that it confers a selectable central catalytic core of the enzyme (the 10-
function. As the different RNRs have different stranded β/α-barrel structure) has been highly
oxygen dependencies it is expected that constrained during evolution, as seen in the
multiple RNRs will present selective conserved three-dimensional structure (5-8),
advantages for organisms that explore several whereas the different domains (modules)
different ecosystems, e.g. the Pseudomonas connected to the core region are more labile.
genus. Subtle shuffling and recombination involving
A comparison of the P. aeruginosa the central catalytic domain, the allosteric ATP-
NrdA protein with other bacterial class Ia cone domain(s) and docking sites for other
proteins gives surprising results because the ligands may have formed the three well-defined
presence of a duplication in the N-terminal part contemporary RNR classes. Without these extra
9domains, the enzyme core could not sustain the prerequisites of the environments. Therefore,
required fine-tuning of the dNTP pools needed contemporary RNRs are a functional and
for accurate DNA synthesis and repair. Neither structural mosaic family of proteins as obvious
could it achieve the radical-based reaction for instance with the availability of different
mechanism, which is initiated in additional RNR protein sequences (38).
domains and/or components as specified by the
ACKNOWLEDGEMENTS
We are grateful to Patrick Young and Anthony Poole for valuable discussions and critical comments
on the manuscript. This study was supported by grants from the Swedish Cancer Foundation and the
Swedish Research Council (to BMS) and from Carl Trygger Foundation (to MS). ET was supported
by a postdoctoral fellowship from the Spanish Ministerio de Educación y Ciencia.
ABBREVIATIONS
BSA, bovine serum albumin; DTT, dithiothreitol; EPR, electron paramagnetic resonance; IPTG,
isopropyl-1-thio-β−D-galactopyranoside; PCR, polymerase chain reaction; RNR, ribonucleotide
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reductase; SDS-PAGE, sodium dodecyl sulphate polyacrylamide gel electrophoresis
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11Table 1. Radical content and specific activity of P. aeruginosa NrdB as a function of added iron
during reconstitution.
Iron added per NrdBa (mol/mol) Radical content (µM) Enzyme activityd (mU)
0b 0 ≤26d
0.5 2.1 185
1 4.2 280
1.5 5.2 350
2 5.5 280
2.5 5.7 374
3 5.3 403
3 (after ≈90 min, plus desalting)cTable 3. Complexes formed with P. aeruginosa RNR wild type proteins and with NrdA∆147
NrdA wild type NrdA∆147 NrdB
Additions a a
kDa Composition kDa Composition kDa Compositiona
528 ±81 α4-6b
none 202 ±11 α2 184 ±10 β4
≈120 αb
634 ±40 α4β4
NrdB 250 ±19 α2β2 n.a.c
217 ±11 αβ2
a
closest integer(s)
b
the major component is α4-6, whereas α is a minor component
c
n.a., not applicable
Table 4. P. aeruginosa NrdAB RNR forms a strong complex in presence of ATP.a
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KD(NrdAB) (µM)b
NrdA protein
in presence of ATP in presence of dTTP
NrdA wt 0.059 ±0.010 0.41 ±0.07
NrdA∆147 0.057 ±0.015 0.79 ±0.43
a
All protein concentrations are given for monomers; KD values were calculated using the equation
published by Climent & al. (20).
b
Concentrations of NrdA proteins were 0.10 µM when ATP (5 mM) was used as allosteric effector
and 0.6 µM when dTTP (0.1 mM) was used.
13FIGURE LEGENDS
Figure 1. Schematic alignment of P. aeruginosa and E. coli NrdA proteins.
A, Wild type and mutated P. aeruginosa NrdA with the E. coli NrdA protein. Nucleotides binding to
the allosteric sites in E. coli NrdA (4) are shown. The cysteines corresponding to those in the active
site of E. coli (C225, C439 and C462) as well as the C-terminal cysteines involved in transthiolation
are shown. In addition, a Pfam domain architecture (25) is represented.
B, Sequence of the E. coli allosteric activity site (ATP-cone) and the two activity sites sequences in P.
aeruginosa (ATP-c1 and ATP-c2). Protein fragments were aligned using ClustalX as previously
described (16). Black arrows indicate E. coli residues at the activity site involved in nucleotide binding
(10). Black shadow denotes identity in all three sequences, dark grey similarity in the three sequences
and light grey identity in only two sequences.
Figure 2. SDS-PAGE analysis of final purified Nrd proteins.
The proteins were run on a 10% polyacrylamide gel and Coomassie-stained after the run. 4 µg of
protein for each sample were loaded onto the gel. Lane 1, NrdA wild type; lane 2, NrdAΔ147; lane 3,
NrdAΔ182 and lane 4, NrdB protein.
Figure 3. Tyrosyl radical decay and iron content in P. aeruginosa NrdB as a function of time.
A 20 µM NrdB monomer solution was reconstituted in the cuvette at pH 7.6 by addition of 30 µM
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Fe2+, and spectra were recorded at 90 s intervals. The top spectrum in the inset shows that a radical
concentration of 4.7 µM was reached 40 s after addition of iron. The arrows indicate iron charge
transfer bands and tyrosyl radical absorption. The lowest spectrum (filled triangles in inset) was
registered 1 hour and 54 minutes after iron addition. The radical decay (open circles) was determined
after subtraction of a spectrum at 1600 s after iron addition. Filled squares show iron bound to NrdB at
different time points during radical decay.
Figure 4. EPR spectra at 93 K.
a) Purified reconstituted NrdB from P. aeruginosa frozen 4 s after addition of 1 Fe2+/polypeptide, 37
µM radical, b) NrdB radical from P. aeruginosa in cell suspension of overproducing E. coli cells was
determined to 5 µM from subtraction of a fraction of the spectrum in a) leaving a background signal of
4 µM; 9 scans, spectrum divided by 2 for layout reasons, c) NrdB tyrosyl radical from purified E. coli
protein normalized to 37 µM radical. Recording parameters: microwave power 1 mW; modulation
amplitude 0.2 mT. The hyperfine coupling of the doublets was measured to 1.9 mT in a) and 2.1 mT in
c).
Figure 5. Stimulation of the CDP reduction by ATP and dATP.
Pure proteins, 30 nmoles NrdA () or NrdA∆147 () and 60 nmoles NrdB were assayed under
standard conditions except for the concentrations of ATP (in A) or dATP (in B) indicated on the
abscissa. In B. the maximum activity for the NrdA wild type was 130 mU and for the NrdA∆147 190
mU.
Figure 6. Effects of dATP in absence and presence of dTTP.
A, binding assays with dATP. B, competition assays with dTTP (1 mM) and increasing concentrations
of dATP. Binding and activity assays were under standard conditions using 0.6 µM of wild type NrdA
() or NrdA∆147 () protein.
Figure 7. Unrooted phylogenetic tree of representative NrdA proteins.
NrdA sequences with duplication or triplication in the N-terminal region are marked in dark grey
shadow. The β− and γ-proteobacteria without domain duplication in the NrdA protein are shown in
light grey. All sequences were from the Ribonucleotide Reductase Database (38). The alignment and
the tree were performed in the same way as previously described (16). Only bootstrap values below
950 are shown.
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Enzyme Catalysis and Regulation:
Ribonucleotide reductase modularity:
atypical duplication of the ATP-cone
domain in Pseudomonas aeruginosa
Eduard Torrents, MariAnn Westman,
Margareta Sahlin and Britt-Marie Sjöberg
J. Biol. Chem. published online July 7, 2006
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