In denitri-Keywords heme d 1 biosynthesis; precorrin-2; Pseudomonas aeruginosa; SAM-dependent uroporphyrinogen III methyltransferase; uroporphyrinogen III Correspondence G.. We pro-duced
Trang 1S-adenosyl-L-methionine-dependent uroporphyrinogen III
Sonja Storbeck1, Johannes Walther1, Judith Mu¨ller1, Vina Parmar2, Hans Martin Schiebel3,
Dorit Kemken4, Thomas Du¨lcks4, Martin J Warren2and Gunhild Layer1
1 Institute of Microbiology, Technische Universita¨t Braunschweig, Germany
2 Department of Biosciences, University of Kent, Canterbury, UK
3 Institute of Organic Chemistry, Technische Universita¨t Braunschweig, Germany
4 Institute of Organic Chemistry, University of Bremen, Germany
Introduction
Some bacteria, such as Pseudomonas aeruginosa, use
denitrification as an alternative form of respiration
under conditions of low oxygen tension in the presence
of nitrogen oxides (e.g nitrate or nitrite) [1] During denitrification, the dissimilatory nitrite reductase cata-lyzes the reduction of nitrite to nitric oxide In
denitri-Keywords
heme d 1 biosynthesis; precorrin-2;
Pseudomonas aeruginosa; SAM-dependent
uroporphyrinogen III methyltransferase;
uroporphyrinogen III
Correspondence
G Layer, Institute of Microbiology,
Technische Universita¨t, Braunschweig,
Spielmannstr 7, 38106 Braunschweig,
Germany
Fax: +49 531 391 5854
Tel: +49 531 391 5813
E-mail: g.layer@tu-bs.de
Website: http://www.tu-braunschweig.de/
ifm
(Received 23 June 2009, revised 10 August
2009, accepted 14 August 2009)
doi:10.1111/j.1742-4658.2009.07306.x
Biosynthesis of heme d1, the essential prosthetic group of the dissimilatory nitrite reductase cytochrome cd1, requires the methylation of the tetrapyr-role precursor uroporphyrinogen III at positions C-2 and C-7 We pro-duced Pseudomonas aeruginosa NirE, a putative S-adenosyl-l-methionine (SAM)-dependent uroporphyrinogen III methyltransferase, as a recombi-nant protein in Escherichia coli and purified it to apparent homogeneity by metal chelate and gel filtration chromatography Analytical gel filtration of purified NirE indicated that the recombinant protein is a homodimer NirE was shown to be a SAM-dependent uroporphyrinogen III methyltransfer-ase that catalyzes the conversion of uroporphyrinogen III into precorrin-2
in vivo and in vitro A specific activity of 316.8 nmol of
precorrin-2 h)1Æmg)1 of NirE was found for the conversion of uroporphyrinogen III
to precorrin-2 At high enzyme concentrations NirE catalyzed an overme-thylation of uroporphyrinogen III, resulting in the formation of trimethylpyrrocorphin Substrate inhibition was observed at uroporphyri-nogen III concentrations above 17 lm The protein did bind SAM, although not with the same avidity as reported for other SAM-dependent uroporphyrinogen III methyltransferases involved in siroheme and cobala-min biosynthesis A P aeruginosa nirE transposon mutant was not comple-mented by native cobA encoding the SAM-dependent uroporphyrinogen III methyltransferase involved in cobalamin formation However, bacterial growth of the nirE mutant was observed when cobA was constitutively expressed by a complementing plasmid, underscoring the special require-ment of NirE for heme d1biosynthesis
Abbreviations
HR-ESI-MS, high-resolution electrospray mass spectrometry; NirS, cytochrome cd 1 nitrite reductase; SAH, S-adenosyl- L -homocysteine; SAM, S-adenosyl- L -methionine; SUMT, SAM-dependent uroporphyrinogen III methyltransferase; Trx, thioredoxin.
Trang 2fying bacteria there are two different types of
dissimi-latory nitrite reductases One is a copper-containing
enzyme (NirK) and the other is the
tetrapyrrole-containing cytochrome cd1 nitrite reductase (NirS) [2]
P aeruginosapossesses the latter enzyme [3] NirS
con-tains the tetrapyrroles heme c and heme d1as essential
prosthetic groups [4] Heme d1 is a
dioxo-isobacterio-chlorin, which is structurally related to siroheme and
cofactor F430 and is not a real heme [5] The unique
structural features of heme d1 are the oxo groups on
C-3 and C-8, the acrylate substituent on C-17 and the
combination of acetate groups and methyl groups on
C-2 and C-7, leading to partially saturated pyrrole
rings A and B (Fig 1A)
The multistep biosynthesis of heme d1 is not
under-stood All cyclic tetrapyrroles share the common
precursor uroporphyrinogen III, which is converted
into either hemes and (bacterio)chlorophylls via
proto-porphyrin IX, or into siroheme, cofactor F430 and
cobalamin via precorrin-2 [6] For the biosynthesis of heme d1, a pathway via precorrin-2 was suggested because the formation of heme d1 requires methylation
of tetrapyrrole rings A and B at positions C-2 and C-7 Indeed, it was found that the heme d1 methyl groups attached to C-2 and C-7 are derived from methionine, probably via S-adenosyl-l-methionine (SAM) [7] During the biosyntheses of siroheme and cobalamin, SAM-dependent uroporphyrinogen III methyltransferases (SUMTs) catalyze the methylation
of uroporphyrinogen III to precorrin-2 (Fig 1B) Several SUMTs from diverse organisms involved
in siroheme (CysG, SirA, UPM1) and cobalamin (CobA) biosynthesis have been purified and bio-chemically characterized [8–13] Most SUMTs are homodimeric proteins, except for the enzyme from Bacillus megaterium that was shown to be a monomer [9] Some SUMTs show inhibition by the substrate uroporphyrinogen III and by the product
S-adenosyl-A B
C
Fig 1 Structure of heme d 1 (A), SAM-dependent methylation of uroporphyrinogen III (B) and amino acid sequence alignment of NirE, CysG and CobA from Pseudomonas aeruginosa (C) (A) The unique structural features of heme d1are the methyl-group ⁄ acetate-group combina-tions and the oxo-groups on rings A and B and the acrylate side chain on ring D (B) SUMT proteins catalyze the SAM-dependent methyla-tion of uroporphyrinogen III, at posimethyla-tions C-2 and C-7, to precorrin-2 (C) Amino acid sequence alignment of P aeruginosa NirE with
P aeruginosa CysG (SUMT domain = amino acid residues 219-461) and CobA shows that NirE exhibits 30% identity and 47% homology with the two other SUMTs Identical residues are highlighted with black boxes.
Trang 3l-homocysteine (SAH) [9,12] Based on amino acid
sequence analysis, two different types of SUMT have
been found to exist Members of the first type are
usu-ally proteins of around 30 kDa and possess SUMT
activity only (SirA, CobA) By contrast, members of
the second type are usually proteins of larger size and
possess, in addition to their SUMT activity, other
cat-alytic activities such as siroheme synthase activity
(CysG) or uroporphyrinogen III synthase activity
(CobA+HemD) [14–16] Some of the SUMTs show
an overmethylation activity that catalyzes a third
methyl transfer at position C-12, which results in the
formation of trimethylpyrrocorphin [10,11,17–19]
Crystal structures are available for the monofunctional
SUMT CobA and the multifunctional SUMT CysG
[20,21]
So far, there are no reports about the enzyme which
catalyzes the methylation of tetrapyrrole C-atoms C-2
and C-7 during heme d1 biosynthesis However, in the
late 1990s a gene cluster was identified in P aeruginosa
(the so-called nir operon), of which several genes
encode proteins potentially involved in heme d1
bio-synthesis [22] Based on amino acid sequence analysis,
one of these genes, nirE, was proposed to encode a
SUMT NirE shares around 30% amino acid sequence
identity and 47% homology with the two other
SUMTs from P aeruginosa (CysG, CobA; Fig 1C)
that are involved in siroheme and cobalamin
bio-synthesis Therefore, it was proposed that the NirE
protein could be the SUMT required for heme d1
formation [22] However, so far, this has not been
demonstrated experimentally Here we report the
pro-duction, purification and characterization of
recombi-nant NirE from P aeruginosa We show that NirE is
indeed a SUMT which catalyzes the methylation of
uroporphyrinogen III to precorrin-2 in vivo and in vitro
Results and Discussion
Production of recombinant P aeruginosa NirE
The recombinant P aeruginosa NirE protein was
pro-duced either as a fusion protein carrying a C-terminal
His-tag (NirE-His) or as a fusion protein carrying both
N-terminal thioredoxin (Trx)- and S-tags and a
C-ter-minal His-tag (Trx-S-NirE-His) In both cases the
recombinant protein was purified to apparent
homoge-neity in a single chromatographic step on Ni
Sepha-roseTM6 Fast Flow (Fig 2A) Initial experiments were
performed using NirE-His; however, this protein
showed a tendency to precipitate at concentrations
above 3 mgÆmL)1 By contrast, the Trx-S-NirE-His
protein was soluble at high enzyme concentrations
A
B
C
Fig 2 Production and purification of recombinant NirE from Pseu-domonas aeruginosa and characterization of in vivo accumulated tetrapyrroles (A) SDS ⁄ PAGE analysis of the production and purifi-cation of recombinant NirE Lane 1, proteins within a cell-free extract prepared from Escherichia coli BL21(DE3) carrying pET32a-nirE-Trx after induction with isopropyl thio-b- D -galactoside (IPTG); lane 2, recombinant Trx-S-NirE-His after chromatography on Ni Sepharose TM Fast Flow; lane 3, S-NirE-His after thrombin cleavage and gel filtration chromatography; lane 4, purified recombinant NirE-His; lanes M, marker proteins with Mrvalues indicated (B) Tetra-pyrroles accumulated during NirE production in E coli were extracted from the soluble protein fraction using C 18 reversed-phase material and were characterized using UV-visible absorption spectroscopy and mass spectrometry The UV-visible absorption spectrum of the extracted tetrapyrroles is characteristic of tri-methylpyrrocorphin [10,11,17–19] (C) HR-ESI-MS results; the experimental mass and isotopic pattern of the [M-H]) ion of compound 872 are shown HR-ESI-MS revealed an exact mass of 871.2687 for the [M-H]) ion, which corresponds to the chemical formula C43H44N4O16, in accordance with the mass and chemical formula of trimethylpyrrocorphin in its trilactone form.
Trang 4After thrombin cleavage and removal of the Trx-tag
by gel filtration, the remaining S-NirE-His could be
concentrated up to 20 mgÆmL)1 Both NirE constructs
(NirE-His and S-NirE-His) exhibited a slight reddish
colour after concentration of the protein UV-visible
absorption spectroscopy indicated the presence of
trimethylpyrrocorphin (data not shown), the NirE
reaction product produced during protein production
in Escherichia coli (see below) NirE thus seems to bind
this overmethylation reaction product rather tightly
because it remained bound to the protein, at least
partially, during protein purification This tight binding
of trimethylpyrrocorphin seems to be a feature unique
to NirE because it has not been reported for other
SUMT proteins However, a physiological role of this
binding phenomenon can be excluded because
tri-methylpyrrocorphin does not represent a physiological
intermediate during heme d1biosynthesis
The native molecular mass of NirE was determined
by gel filtration chromatography of NirE-His A native
relative molecular mass of 60 000 ± 3000 was deduced
from this experiment, suggesting a dimeric structure
for the NirE protein (with a subunit molecular mass of
30 kDa) Other SUMT proteins involved in siroheme
(CysG) and cobalamin (CobA) biosynthesis were also
reported to be dimeric proteins [20,21]
NirE carries SUMT activity in vivo
During the production of both NirE-His and
Trx-S-NirE-His in E coli a red compound accumulated in
the cells This compound was extracted from the
solu-ble protein fraction using C18-reversed phase material
and analysed by UV-visible absorption spectroscopy
and mass spectrometry In both cases (NirE-His and
Trx-S-NirE-His production) the UV-visible absorption
spectrum of the extracted compound exhibited an
absorption maximum at 354 nm and was very similar
to the previously reported absorption spectrum of
trimethylpyrrocorphin, the overmethylation product of
SUMT proteins (Fig 2B) [10,11,17–19] Analysis of
the extracted compound by high-resolution
electro-spray mass spectrometry (HR-ESI-MS) in the negative
ion mode revealed an exact mass of 871.2687 for the
[M-H]) ion, which corresponds to an elemental
com-position of C43H44N4O16with a deviation of 0.8 p.p.m
This elemental composition is in accordance with the
mass and chemical formula of trimethylpyrrocorphin
in its trilactone form (Fig 2C) Isolation of lactone
derivatives of isobacteriochlorins has been reported
previously [23,24] and lactone formation probably
occurs during the tetrapyrrole extraction procedure
However, our results showed that the production of
recombinant NirE in E coli leads to the accumulation
of trimethylpyrrocorphin in vivo, an observation that has been reported for most of the known SUMT proteins [10,11,17–19]
NirE carries SUMT activity in vitro Next, we tested the NirE protein for SUMT activity
in vitro The standard NirE activity assay was per-formed as described in the Materials and methods sec-tion with enzymatically produced uroporphyrinogen III The formation of the NirE reaction product, pre-corrin-2, was followed using UV-visible absorption spectroscopy Figure 3 shows the absorption spectra obtained from enzyme reactions after overnight incu-bation The spectrum obtained from a reaction mix-ture containing the uroporphyrinogen III producing enzymes HemB, HemC and HemD did not show any characteristic features in the 300–700 nm region, as was expected for a solution containing colourless uro-porphyrinogen III under anaerobic conditions Upon the addition of recombinant purified NirE and SAM
to the reaction mixture, an absorption spectrum was observed that exhibited very broad absorption features (between 500–400 nm and 400–350 nm), in agreement with the yellow colour of the corresponding reaction mixture This spectrum is characteristic for precorrin-2 [17,25] When the precorrin-2 dehydrogenase SirC and NAD+ were also included in the above reaction
Fig 3 UV-visible absorption spectra of NirE activity assays The substrate uroporphyrinogen III (urogen III in the figure) was pro-duced from 5-aminolevulinic acid by an enzyme cocktail containing purified, recombinant Pseudomonas aeruginosa HemB and Bacil-lus megaterium HemC and HemD (dashed line) Precorrin-2 forma-tion was observed (dotted line) upon the addiforma-tion of purified NirE and SAM Precorrin-2 formed by NirE was converted into sirohydro-chlorin by SirC in the presence of NAD+(solid line).
Trang 5mixture, the solution turned red instead of yellow,
indicating the conversion of precorrin-2 (produced by
NirE) into sirohydrochlorin The UV-visible
absorp-tion spectrum obtained from such a reacabsorp-tion mixture
corresponds indeed to the typical absorption spectrum
of sirohydrochlorin (Fig 3) [25] When SAM was
omitted from the NirE activity assay no precorrin-2
formation was observed (data not shown) These
results clearly show that NirE is able to catalyze the
two SAM-dependent methylation reactions to convert
uroporphyrinogen III into precorrin-2 in vitro
Initially we performed enzyme assays with all three
NirE proteins – NirE-His, Trx-His and
S-NirE-His – and compared their catalytic activities We
observed that Trx-S-NirE-His and S-NirE-His showed
similar activities By contrast, NirE-His showed only
half of the activity of the other two proteins Therefore,
S-NirE-His was used for all subsequent enzyme assays
and experiments We observed the highest catalytic rates
with chemically produced substrate uroporphyrinogen
III at a concentration of 17 lm, a SAM concentration
of 200 lm and at NirE concentrations of 1.5 lm Under
these assay conditions a specific activity of 316.8 nmol
of precorrin-2 h)1Æmg)1of NirE was observed
Previously, it was reported that SUMT proteins
catalyze a third methylation of uroporphyrinogen III
to generate a trimethylpyrrocorphin, not only in vivo
but also in vitro at high enzyme concentrations in the
assay [17,18] As the production of recombinant NirE
in E coli leads to the accumulation of
trimethylpyrro-corphin in vivo, we tested if high concentrations of
NirE in our enzyme assay also formed this compound
in vitro We observed the formation of
trimethylpyrro-corphin in our activity assays at NirE concentrations
above 10 lm in the presence of 500 lm SAM (data not
shown) Thus, NirE is indeed a SUMT that shows the
same catalytic behaviour in vivo and in vitro as the
SUMTs for siroheme and cobalamin biosynthesis
NirE exhibits substrate inhibition by
uroporphyrinogen III and product inhibition
by SAH
SUMT proteins were reported to exhibit inhibition by
their substrate uroporphyrinogen III, as well as by the
reaction by-product SAH Therefore, we tested NirE
for such inhibition phenomena In our enzyme assay
using chemically produced uroporphyrinogen III we
observed substrate inhibition at uroporphyrinogen III
concentrations above 17 lm, as shown in Figure 4A
In order to test NirE for inhibition by SAH we added
increasing amounts of SAH to our activity assay We
observed inhibition of the NirE reaction at SAH
concentrations above 2 lm (Fig 4B) NirE thus displays the same inhibition phenomena as those previously reported for other SUMT proteins [9,12] However, the question of whether these substrate and product-inhibition characteristics are physiologically relevant, or if they only represent in vitro assay artefacts, requires further investigation
SAM binding
In previous studies, rapid SAM-binding assays were performed in order to characterize SUMT proteins [10,26,27] Therefore, we also tested the NirE protein for its ability to bind SAM After incubation of NirE with radioactively labelled SAM, the mixture was
A
B
–1 × NirE (nmol)
–1 × NirE (nmol)
Fig 4 Inhibition of NirE activity by the substrate uroporphyrinogen III and by the product SAH (A) NirE activity assays (1.5 l M NirE,
200 l M SAM) were performed with increasing amounts of chemi-cally synthesized uroporphyrinogen III Initial rates of precorrin-2 formation were plotted against the uroporphyrinogen III concentra-tion (B) Increasing amounts of S-adenosyl- L -homocysteine (SAH) were added to the NirE activity assay (1.5 l M NirE, 200 l M SAM,
17 l M uroporphyrinogen III) Initial rates of precorrin-2 formation were plotted against the SAH concentration.
Trang 6passed over a desalting column and the elution
frac-tions were analyzed for radioactivity using a liquid
scintillation counter As a control, the same experiment
was carried out with BSA In the BSA control
experi-ment all radioactivity eluted in the small-molecules
fractions By contrast, when SAM was mixed with
NirE, the label was found to co-elute with the
protein-containing fractions (data not shown) We also tested
whether SAM remained bound to NirE during
dena-turing electrophoresis, as observed for other SUMT
proteins [10,26,27] After incubation of NirE with
radioactively labelled SAM, the protein was subjected
to SDS⁄ PAGE and fluorography No radioactivity was
found to be associated with the protein after
denatur-ing electrophoresis (data not shown) These
experi-ments show that NirE binds SAM; however, the
binding seems to be weaker than for other SUMT
proteins because SAM did not remain bound to the
protein under denaturing conditions
P aeruginosa cobA is able to complement a
P aeruginosa nirE mutant
We have unambiguously shown, from the results
described in the previous section, that the NirE protein
is a SAM-dependent uroporphyrinogen III
methyl-transferase Although P aeruginosa also possesses the
genes encoding the SUMTs involved in cobalamin
(cobA) and siroheme (cysG) biosynthesis, the nirE gene
product was found to be essential for heme d1
biosyn-thesis A P aeruginosa nirE knockout mutant was
unable to synthesize heme d1 and produced only heme
d1-lacking, inactive cytochrome cd1 [22] This absolute
requirement for NirE during heme d1 biosynthesis is
surprising considering the identical catalytic abilities of
NirE and CobA CobA catalyzes the SAM-dependent
methylation of uroporphyrinogen III to form
precor-rin-2 during cobalamin biosynthesis The third SUMT
in P aeruginosa, the trifunctional siroheme synthase
CysG, probably does not release precorrin-2 during
siroheme formation and therefore cannot provide this
precursor for heme d1 biosynthesis in the absence of
NirE The observation that CobA is apparently not
able to replace NirE during heme d1 formation may
have several explanations One possibility could be
that specific protein–protein interactions between NirE
and the subsequent heme d1 biosynthesis protein are
required to allow substrate channelling of the highly
labile precorrin-2 Another explanation may be that
CobA, although probably present under anaerobic
denitrifying conditions in order to sustain cobalamin
biosynthesis for cobalamin-dependent enzymes,
such as class II ribonucleotide reductase [28], is
produced in amounts too low to sustain efficient heme
d1biosynthesis
In order to investigate these possibilities, we tested whether P aeruginosa cobA, when constitutively expressed from a plasmid, was able to complement a
P aeruginosa PAO1 nirE transposon mutant (strain PAO1 ID35553) For these experiments, wild-type
P aeruginosa PAO1 and P aeruginosa PAO1 ID35553 carrying diverse complementation plasmids were grown
as described in the Materials and methods When anaerobic growth conditions were reached (after about
4 h), strain PAO1 ID35553 as well as this strain carry-ing the basic plasmid pUCP20T showed greatly impaired growth when compared with the wild-type strain (Fig 5) By contrast, strain PAO1 ID35553, car-rying the complementation plasmid pUCP20T-nirE, grew almost as well as the wild-type strain, as expected Interestingly, strain PAO1 ID35553, carrying plasmid pUCP20T-cobA, showed growth behaviour similar to that of the wild-type strain and the same growth behaviour as the nirE-complemented strain PAO1 ID35553 (Fig 5) Therefore, cobA was able to complement the P aeruginosa nirE mutant strain when constitutively expressed from a complementation plas-mid By contrast, the concentration of native CobA produced under anaerobic denitrifying growth condi-tions was apparently not sufficient to restore efficient heme d1 biosynthesis in the nirE)background Indeed, cobA transcript levels were found to be absent in Affymetrix microarray analyses of anaerobically grown
Fig 5 Growth curves of wild-type Pseudomonas aeruginosa PAO1 and of P aeruginosa strain PAO1 ID35553 P aeruginosa was grown under anaerobic growth conditions in the presence of nitrate, as described in the Materials and methods Strain PAO1 ID35553 (D) and this strain carrying plasmid pUCP20T (.) showed impaired growth under these growth conditions compared with the wild-type strain ( ) Bacterial growth of strain PAO1 ID35553 was restored by plasmids pUCP20T-nirE (•) and pUCP20T-cobA (s).
Trang 7P aeruginosa(M Schobert, personal communication).
These results are in agreement with the fact that the
genes for nitrite reductase NirS and the proposed heme
d1 biosynthesis proteins, including NirE, are organized
in one large operon [22] The transcription of the nir
operon genes was found to be highly up-regulated
in P aeruginosa under anaerobic conditions in the
presence of nitrate [28] Under such conditions the
co-transcription of heme d1 biosynthesis genes and
the co-production of both NirE and the other heme
d1 biosynthesis proteins, in high amounts ensures the
efficient and highly concerted action of the proteins In
order to cope with the high demand for heme d1under
denitrifying conditions, such a concerted action of
heme d1biosynthesis proteins is required and therefore
native cobA, which is not co-transcribed with the nir
genes, is not able to replace nirE Thus, the NirE
protein is a SAM-dependent uroporphyrinogen III
methyltransferase which is specifically required for
heme d1 biosynthesis
Materials and methods
Chemicals
Unless stated otherwise, all chemicals, reagents and
antibi-otics were obtained from Sigma-Aldrich (Taufkirchen,
Germany) or Merck (Darmstadt, Germany) DNA
polymerase, restriction endonucleases and PCR requisites
were purchased from New England Biolabs (Frankfurt
a.M., Germany) Oligonucleotide primers were purchased
from metabion international AG (Martinsried, Germany)
Kits for PCR purification and gel extraction were
pur-chased from Qiagen GmbH (Hilden, Germany) Ni
Sepha-roseTM 6 Fast Flow was purchased from GE Healthcare
(Mu¨nchen, Germany) [Methyl-14
C]-S-adenosyl-l-methio-nine was obtained from Hartmann Analytic (Braunschweig,
Germany) Uroporphyrin III was obtained from Frontier
Scientific Europe (Carnforth, UK)
Plasmids, bacteria and growth conditions
The E coli strain DH10B was used as the host for cloning,
and E coli BL21(DE3) was used as the host for protein
production For complementation studies a P aeruginosa
PAO1 mutant was used, which carries a transposon
inser-tion in the nirE gene (strain PAO1 ID35553) [29] This
mutant was transformed with plasmids pUCP20T-nirE,
pUCP20T-cobA or pUCP20T For anaerobic growth
condi-tions [30], LB (Luria–Bertani) medium was supplemented
with 50 mm nitrate and carbenicillin at a final
concentra-tion of 250 lgÆmL)1 P aeruginosa precultures were grown
aerobically overnight and the anaerobic cultures (140-mL
bottles filled with 135 mL of LB medium) were inoculated
with appropriate volumes of these precultures to obtain
a final A of 0.05 at 578 nm Culture was carried out at
37C
The plasmids used for the production of recombinant
P aeruginosa NirE were pET32a-nirE-Trx and pET22b-nirE (see below) The plasmids used for the production of recombinant P aeruginosa HemB [31] and B megaterium HemC, HemD and SirC [32] were described previously For the production of recombinant proteins, E coli BL21(DE3) cells, carrying the respective plasmid, were grown at 37C in 500 mL of LB medium supplemented with ampicillin at a final concentration of 100 lgÆmL)1
At a A of 0.6 at 578 nm, protein production was induced
by the addition of 50 lm isopropyl thio-b-d-galactoside (IPTG) The cells were then cultured further at 17C After
18 h of culture the cells were harvested by centrifugation and stored at)20 C until required
Construction of vectors
For the construction of nirE expression vectors, the nirE gene from P aeruginosa PAO1 was PCR amplified using the primers nirE_Pa_BamHI_for (GCCGGGATCCAT GAACACTACCGTGATTC) and nirE_Pa_XhoI_rev (GACTCGAGGGCGCATGCGAC) containing BamHI and XhoI restriction sites (underlined), respectively, for cloning the nirE gene into pET32a (Novagen, Darmstadt, Germany) For cloning the nirE gene into pET22b (Nov-agen), the PCR primers NirE_NdeI (GTCATATGACA CTACCGTGATTCC) and NirE_HindIII (GTAAGCTT GCATGCGACGGCCTCG), containing NdeI and HindIII restriction sites (underlined), respectively, were used The plasmid pHAE2 [22], containing a fragment of the P aeru-ginosa PAO1 nir operon, was used as the DNA template The resulting PCR fragments were digested with BamHI and XhoI, or with NdeI and HindIII, and ligated into the appropriately digested vectors pET32a or pET22b to gener-ate pET32a-nirE-Trx and pET22b-nirE, respectively For construction of complementation vectors the nirE gene and the cobA gene, including a 50-bp upstream region bearing the ribosome-binding sites, were PCR amplified using the primers NirE_Compl_for (GAGAATTCGGA AATCGGCCTCG) and NirE_Compl_rev (CTAAGCTTT CAGGCGCATGCG) for the nirE gene and CobA_ Compl_for (GAGAATTCACTGCTGGCGGCC) and CobA_Compl_rev (CTAAGCTTTCAGGCGCTCAGGG) for the cobA gene, respectively, containing EcoRI and HindIII restriction sites (underlined) A colony of
P aeruginosa PAO1 was used as a template The resulting PCR fragments were digested with EcoRI and HindIII and ligated into the appropriately digested vector pUCP20T to generate pUCP20T-nirE and pUCP20T-cobA, respectively The vectors were transferred into strain PAO1 ID35553 by diparental mating using E coli ST18 [33] as a donor Stan-dard procedures were used for PCR amplification, agarose
Trang 8gel electrophoresis, dephosphorylation, ligation and
trans-formation of chemocompetent E coli cells [34] Restriction
enzymes were used as recommended by the manufacturer
Purification of enzymes
All protein purification steps were carried out at 4C
Harvested E coli cells, harbouring recombinantly
pro-duced NirE protein, were resuspended in buffer A [50 mm
Tris⁄ HCl (pH 7.5), 200 mm KCl, 10% (w ⁄ v) glycerol]
containing 1 mm phenylmethanesulfonyl fluoride The cells
were disrupted by a single passage through a French press
at 1000 p.s.i (68947.57 hPa) and then centrifuged for
60 min at 175 000 g The supernatant was applied to
1 mL of silica gel 100 C18-reversed phase material,
acti-vated first with methanol then equilibrated with buffer A,
to extract accumulated tetrapyrroles The flow-through,
containing the NirE protein, was applied to 1.5 mL of Ni
SepharoseTM 6 Fast Flow equilibrated with buffer A
After extensive washing with buffer A, the recombinant
NirE protein was eluted with 2.5 mL of buffer A
contain-ing 200 mm imidazole After elution of NirE, buffer
exchange was performed in an anaerobic chamber (Coy
Laboratories, Grass Lake, MI, USA) by passing the
pro-tein solution through a NAP-25 column (GE Healthcare)
that had been equilibrated with buffer A containing 5 mm
dithiothreitol When NirE was produced as a fusion
pro-tein with an N-terminal Trx-tag, the Trx-tag was cleaved
off with thrombin using the Thrombin Cleavage Capture
Kit (Novagen) according to the manufacturer’s
instruc-tions NirE was separated from the Trx-tag by gel
filtra-tion chromatography on a HiLoad 16⁄ 60 Superdex 200
column (GE Healthcare) equilibrated with buffer A
taining 5 mm dithiothreitol Protein solutions were
con-centrated by ultrafiltration (Amicon, Millipore GmbH,
Eschborn, Germany) The purified NirE protein was
stored at)20 C The N-terminal amino acid sequences of
the purified proteins were determined by Edman
degrada-tion and were found to be identical to those expected
from the cloning strategy (MNTTVIP for NirE-His and
GSGMKET for S-NirE-His) Recombinant P aeruginosa
HemB and B megaterium HemC, HemD and SirC were
purified as previously described [31,32]
Determination of protein concentration
The Bradford Reagent (Sigma-Aldrich) was used to
deter-mine protein concentrations, according to the
manufac-turer’s instructions, using BSA as a standard
Molecular mass determination
The native molecular mass was estimated from gel filtration
chromatography using a SuperdexTM 200 10⁄ 300 GL
column attached to an A¨KTATM Purifier system (GE Healthcare) The column was equilibrated with buffer A containing 5 mm dithiothreitol Protein samples of 150 lL were loaded onto the column and chromatographed at a flow rate of 0.5 mLÆmin)1 Protein elution was monitored
by determining the absorption of the eluate at 280 nm The column was calibrated using the protein standards carbonic anhydrase (29 000 Da), BSA (66 000 Da), conalbumin (77 000 Da), alcohol dehydrogenase (150 000 Da) and b-amylase (200 000 Da)
Extraction of tetrapyrrole compounds
In vivo accumulated tetrapyrrole compounds were extracted from the soluble protein fraction by passing the cell-free extracts over a 1-mL silica gel 100 C18-reversed phase col-umn, activated first with methanol then equilibrated with buffer A The silica gel was washed with water and the bound tetrapyrroles were eluted with methanol The solvent was removed by evaporation and the dried tetrapyrroles were stored at)20 C
UV-visible absorption spectroscopy
UV-visible absorption spectra of extracted tetrapyrroles were recorded using a V-550 spectrophotometer (Jasco, Gross-Umstadt, Germany)
NirE activity assay
In vitro NirE activity assays were performed under anaero-bic conditions in an anaeroanaero-bic chamber (Coy Laboratories)
in a final volume of 1 mL of thoroughly degassed buffer containing 50 mm Tris⁄ HCl (pH 8.0), 100 mm KCl, 5 mm MgCl2and 50 mm NaCl The final NirE concentration was 1.5 lm The substrate uroporphyrinogen III was generated from 1 mm 5-aminolevulinic acid using a coupled assay system including HemB (0.14 lm), HemC (0.15 lm) and HemD (0.17 lm) Alternatively, uroporphyrin III was reduced chemically and used at a final concentration of
8 lm The reaction was started by the addition of SAM to
a final concentration of 200 lm and was incubated at 37C
in the dark The reaction was monitored using a Lambda 2 spectrophotometer (PerkinElmer Instruments, U¨berlingen, Germany) In order to quantify the precorrin-2, it was converted to sirohydrochlorin (e376 = 2.4· 105
m)1Æcm)1 [35]) by the addition of SirC (1.5 lm) and 100 lm NAD+
Preparation of uroporphyrinogen III
Uroporphyrinogen III was prepared by chemical reduction
of uroporphyrin III with 3% sodium amalgam, as described previously for coproporphyrinogen III [36]
Trang 9SAM-binding assay
The SAM-binding assay was performed as described
previ-ously [26] Briefly, 100 lm purified NirE protein was
incu-bated with 0.5 lCi of [methyl-14C]-S-adenosyl-l-methionine
in a final volume of 250 lL of buffer A at 25C for 1 h
The protein solution was then passed over a NAP-5 column
(GE Healthcare) and eluted with 3 mL of buffer A
Fractions of 100 lL were collected and analysed for
radio-activity using a Liquid Scintillation Analyzer Tri-Carb 2900
TR (PerkinElmer Life Sciences) Analysis of SAM binding
by fluorography was performed as described previously
[26]
Mass spectrometry of tetrapyrroles
HR-ESI-MS data were acquired using a Bruker
microTOF-Q II equipped with an Apollo ESI ion source (Bruker
Daltonik, Bremen, Germany) Samples were dissolved in
methanol and introduced, via direct infusion, at a flow rate
of 4 lLÆmin)1
Acknowledgements
We thank Professor H Arai (University of Tokyo,
Japan) for the gift of plasmid pHAE2, and Professor
S Ha¨ußler (Helmholtz-Centre for Infection Research,
Braunschweig, Germany) for the gift of the P
aerugin-osa nirE transposon mutant (strain PAO1 ID35553)
We thank Dr Jan Willmann (Bruker Daltonik,
Bre-men, Germany) for HR-ESI-MS measurements We
also thank Prof D Jahn and Drs J Moser and M
Schobert for helpful discussions This work was
sup-ported by the Emmy-Noether-Program of the
Deut-sche Forschungsgemeinschaft and by funds from the
Fonds der Chemischen Industrie to G.L
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