Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member

Article abstract of DOI:10.1016/j.bbapap.2014.03.016

An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe-4S]^{2 +/1 +} centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.

Full text of DOI:10.1016/j.bbapap.2014.03.016

Biochimica et Biophysica Acta 1844 (2014) 1238–1247
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbapap
Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM
family member
a
b
a
b
Susana A.L. Lobo , Andrew D. Lawrence , Célia V. Romão , Martin J. Warren ,
a,
a,
Miguel Teixeira ⁎, Lígia M. Saraiva ⁎
a
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal
Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
b
a r t i c l e i n f o
a b s t r a c t
Article history:
An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus
and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen
III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into
haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we re-
port the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the
pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM)
and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5′-
deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains
Received 28 February 2014
Received in revised form 28 March 2014
Accepted 31 March 2014
Available online 5 April 2014
Keywords:
Desulfovibrio
Alternative haem biosynthesis
Iron-coproporphyrin III
S‐adenosylmethionine
iron-sulfur protein
2
+/1+
two [4Fe–4S]
centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III
induces conformational modifications in both centres. Amino acid sequence comparisons indicated that
D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich se-
quences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of
D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-
coproporphyrin III is discussed.
©
2014 Elsevier B.V. All rights reserved.
1
. Introduction
oxidase (HemF or HemN). Subsequently the macrocyle is oxidised by
the removal of six hydrogens to give protoporphyrin IX in a reaction
catalysed by protoporphyrinogen oxidase (HemG or HemY) and finally
iron is inserted into the core of the substrate template by ferrochelatase
(HemH) to give haem b [1,2]. However, in sulfate reducing bacteria of
the Desulfovibrio genus and in methanogenic Archaea, haem b is pro-
duced along a distinct branch of the modified tetrapyrrole pathway
where sirohaem acts as an intermediate precursor (Fig. 1) [3–5].
Along this route uroporphyrinogen III is transformed into sirohaem
by methylation at C2 and C7, oxidation of the macrocycle and
ferrochelation. Sirohaem is then converted into haem b by the enzymes
of the alternative haem biosynthetic pathway (Ahb). Initially, sirohaem
is acted upon by the AhbA and AhbB proteins that promote the decar-
boxylation of the acetic acid chains attached to C12 and C18 yielding
12,18-didecarboxysirohaem (Fig. 1). This decarboxylated form of
sirohaem is modified by AhbC, which catalyses the loss of the acetic
acid side chains at C2 and C7 in an S-adenosylmethionine (SAM)-
dependent reaction, to form iron-coproporphyrin III (Fe-Cp III)
All modified tetrapyrroles, which include some of the major cofac-
1
tors of life such as haem, sirohaem, haem d , chlorophylls and cobala-
mins (vitamin B12) are synthesised along a branched biosynthetic
pathway. Recently, the discovery of a new branch of the tetrapyrrole
pathway revealed that haem b can be made by one of two distinct
routes. In the classic haem b synthesis pathway, found in eukaryotes
and many bacteria, four enzymatic steps are required to transform
uroporphyrinogen III, the primogenitor of all modified tetrapyrroles,
into haem b. The first step of the classic pathway is catalysed by
uroporphyrinogen decarboxylase (HemE), which mediates the sequen-
tial decarboxylation of the four acetic acid side chains of the substrate to
give coproporphyrinogen III. Next, the two propionic acid side chains at-
tached to C3 and C8 undergo oxidative decarboxylations to generate
protoporphyrinogen IX in a reaction catalysed by coproporphyrinogen
(
Fig. 1). The final step is also a SAM-dependent reaction promoted by
Abbreviations: SAM, S‐adenosylmethionine; HPLC, high-performance liquid chroma-
tography; MS, mass spectrometry; Ahb, alternative haem biosynthetic proteins; Fe-Cp
III, iron-coproporphyrin III
AhbD that converts the two propionate side chains attached to C3 and
C8 of Fe-Cp III into vinyl groups forming haem b (Fig. 1) [3]; therefore,
the conversion of sirohaem into haem b via the alternative haem path-
way involves several reactions that are linked to radical SAM chemistry.
⁎
Corresponding authors.
E-mail addresses: miguel@itqb.unl.pt (M. Teixeira), lst@itqb.unl.pt (L.M. Saraiva).
http://dx.doi.org/10.1016/j.bbapap.2014.03.016
570-9639/© 2014 Elsevier B.V. All rights reserved.
1

S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
1239
Fig. 1. Alternative haem b biosynthesis pathway in which sirohaem is converted to haem b via the AhbA, AhbB, AhbC and AhbD enzymes.
Radical SAM enzymes are a vast family of proteins with a wide diver-
sity of functions participating, for example, in the biosynthesis of cofac-
predicted the tridimensional structure of AhbD and propose the puta-
tive pockets for the binding of the iron–sulfur centres and the SAM
and iron-coproporphyrin III substrates.
tors, vitamins, DNA precursors, and antibiotics. One of the features of
these enzymes is the presence of, at least, one [4Fe–4S]2
+/1+
cluster co-
ordinated by only three cysteine residues arranged in a conserved
Cx Cx C motif located at the N-terminal region of the protein [6–10].
The catalytically active species of the cluster is the reduced [4Fe–4S]
3
2
2. Materials and methods
1
+
form that promotes the reductive cleavage of SAM to yield methionine
and a highly reactive 5′-deoxyadenosyl (dAdo) radical. Due to the oxy-
gen lability of this iron–sulfur cluster these reactions usually occur
under anaerobic conditions. This large family of enzymes also includes
the oxygen-independent coproporphyrinogen oxidase, HemN, from the
canonical haem b pathway [11,12]. Radical SAM enzymes may have ad-
2.1. Cloning and expression of recombinant D. vulgaris AhbD
The gene encoding the D. vulgaris Hildenborough AhbD (gene locus
DVU0855) was amplified in a PCR reaction using genomic DNA and two
different pairs of oligonucleotides to produce a His-tagged and a non-
labelled protein. The DNA fragment amplified with the pair 5′-CAC
ATA TGG GAG CGC ATC CCA CTG CCC-3′ and 5′-CAC TCG AGG CGG
ditional iron–sulfur centres, dubbed “auxiliary clusters” [13]. For exam-
ple, the biotin synthase, BioB, contains an extra [2Fe–2S]2
+/1+
cluster
GAG GGC GTC ATA C-3′ was cloned into NdeI/XhoI pET23b vector
+
[
14], whereas the methylthiotransferases RimO and MiaB bind an
(Novagen), which allowed production of a C-terminal 6x-HisTag fused
protein. The DNA fragment amplified with primers 5′-CGG GCG GCC
ATA TGG GAG CG-3′ and 5′-CAT GTC GGT GGA GAA TTC CCC TGC-3′
2
+/1+
auxiliary [4Fe–4S]
cluster coordinated by three invariant cysteines,
which is proposed to be involved in activation of sulphide or
+
methylsulfide [15,16]. In the molybdenum cofactor biosynthetic
was cloned into NdeI/EcoRI pET28a vector (Novagen), excised with
enzyme MoaA, the additional [4Fe–4S]2
+/1+
centre is also coordinated
the same restriction enzymes and cloned into pET23b to generate a
+
by three cysteines [17] while in the anaerobic sulfatase maturating
protein with no fusion tags, designated AhbD. All recombinant plasmids
were sequenced to confirm the absence of errors in the gene sequences
and then transformed in E. coli BL21 Star(DE3)pLysS cells (Invitrogen).
For protein expression, E. coli cells carrying the recombinant plasmids
were grown in 1 l flasks fully filled with LB medium, to which the appro-
priate antibiotics were added. Cells growing at 37 °C that reached an
OD600 of approximately 0.5 were supplemented with 400 μM isopropyl
β-D-1-thiogalactopyranoside (IPTG) and 30 μM Fe (in the form of
enzyme AnSME from Clostridium perfringens the two auxiliary
2
+/1+
[4Fe–4S]
centres are coordinated by four cysteine residues [18].
In this work, the AhbD enzyme involved in the last step of the
alternative haem b biosynthetic pathway in Desulfovibrio vulgaris
Hildenborough was biochemically and spectroscopically characterised
and shown to be a member of the radical SAM superfamily of proteins
2
+/1+
having two [4Fe–4S]
centres. Based on modelling studies, we

1240
S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
2
FeSO4.7H O), and after an overnight growth, at 20 °C, were harvested by
(2.1 × 150 mm; Advanced Chromatography Technologies Ltd) attached
centrifugation.
to an Agilent 1100 series HPLC equipped with a diode array detector and
coupled to a micrOTOF-Q II (Bruker) mass spectrometer. The tetrapyr-
role derivatives were detected at 390 nm and separation was achieved
by applying a binary gradient at a flow rate of 0.2 ml min with 0.1%
trifluoroacetic acid (TFA) as solvent A and acetonitrile as solvent B.
The column was first equilibrated with 20% solvent B and after sample
injection the concentration of solvent B was increased to 100%. The
total duration of each run was 50 min [3].
For the analysis of SAM derived products, reaction mixtures contain-
ing ~40 μM D. vulgaris AhbD, 1 mM sodium dithionite and 15 μM Fe-Cp
III in a final 0.5 ml volume of buffer A were anaerobically prepared, and
the reaction was initiated by adding 1 mM SAM. After overnight incuba-
tion, 100 μl aliquots were taken and immediately quenched by addition
2
.2. Cell free lysates preparation
−
1
D. vulgaris cells were grown anaerobically in lactate/sulfate medium
[19], at 37 °C, in 1 l closed flasks until an OD600 correspondent to the sta-
tionary phase (OD600 ~1.2), at which point cells were collected by cen-
trifugation and kept at −80 °C.
In an anaerobic chamber (Belle Technology, b2 ppm O
2
), D. vulgaris
and the E. coli BL21 Star(DE3)pLysS cells overexpressing D. vulgaris
AhbD were resuspended in degassed 50 mM Tris–HCl pH 8 (buffer A),
lysed by sonication and centrifuged for 20 min at ~30,000 ×g for remov-
al of unbroken cells. The resulting cell free lysates of D. vulgaris and of
E. coli overexpressing AhbD were used in the activity assays as described
below.
2 4
of an equal volume of 100 mM H SO . The mixtures were then centri-
fuged for 20 min at 16,000 ×g, and 50 μl of the supernatants containing
the SAM related products were analysed by HPLC–MS. As control, a mix
of SAM, 5′-deoxyadenosine (dAdo), 5′-methylthioadenosine (MTA) and
S-adenosyl-L-homocysteine (SAH) was also prepared and the products
were separated and analysed by mass spectrometry as previously de-
scribed [3], at a flow rate of 0.2 ml/min and with a gradient of acetoni-
trile in 0.1% TFA as follows: 0–1 min isocratic 0% B (acetonitrile), 1–
15 min ramp to 10% B, 15–20 min ramp to 30% B, 20–25 min ramp to
50% B, 25–30 min ramp to 100% B, 30–35 min isocratic 100% B, 35–
40 min ramp to 0% B and 40–50 min to 0%. The elution profile was mon-
itored by UV–visible spectroscopy at 260 nm.
2
.3. Protein purification
For protein isolation, E. coli BL21 Star(DE3)pLysS cells overexpress-
ing D. vulgaris AhbD were resuspended in buffer A and disrupted by
passing three times through a French Press, at 900 Psi under aerobic
conditions, after which the suspensions were centrifuged for 30 min
at ~30,000 ×g. The resulting supernatant was submitted to an argon at-
mosphere and further manipulated in a Coy model A-2463 anaerobic
chamber containing a gas mixture of 95% argon plus 5% hydrogen.
The supernatant was applied to a 3 ml Ni2 saturated Chelating
Sepharose resin (GE, Healthcare), previously equilibrated in buffer A
with 500 mM NaCl and 10 mM imidazole (binding buffer). A step gradi-
ent of imidazole in the same buffer was applied and the protein was
eluted at 250 mM imidazole, concentrated in an Amicon Stirred Ultrafil-
tration Cell using a 10 kDa membrane (Millipore), and submitted to
buffer exchange by means of a PD10 column (GE Healthcare) that
used buffer A as running buffer. The purity of the protein was evaluated
by SDS-PAGE and the protein concentration was determined by the
Pierce Bicinchoninc acid Protein Assay kit (Thermo Scientific) using
Sigma protein standards. UV–visible spectra were recorded inside the
anaerobic chamber in a Shimadzu UV-1800 spectrophotometer.
+
2.5. Binding assay
The binding of Fe-Cp III to AhbD was analysed by monitoring the
changes of the UV–visible spectrum of the reduced Fe-Cp III (15 μM)
upon successive additions of reduced AhbD protein (2 to 36 μM). The re-
duced form of Fe-Cp III was obtained by previous incubation with
2 molar equivalents of sodium dithionite in buffer A, while reduction
of AhbD was achieved by addition of 8 molar equivalents of sodium
dithionite. The shift of the Soret band and the variation of the absor-
bance at 550 nm were monitored. The association constant (K
the maximal number of binding sites (Bmax) values were determined
by fitting the data to the equation Y = Bmax * X / (X + K ) by nonlinear
A
) and
The molecular mass and quaternary structure of the protein were
determined by passage in a Superdex 200 10/300GL column (GE,
Healthcare), equilibrated with 50 mM sodium-phosphate pH 7 buffer
containing 150 mM NaCl. The iron content of the pure protein was de-
termined by the 2,4,6-tripyridyl-1,2,3-triazine (TPTZ) method [20].
A
regression using GraphPad Prism software version 5.0 for Windows,
GraphPad Software, San Diego, California, USA (www.graphpad.com).
In this equation Y was calculated from the absorbance at 550 nm or
the shift of the Soret band maximum and X represents the concentration
of the protein. The titration data were normalised between 0 and 100 in
relation to the smallest and the largest values of each data set.
2
.4. Analysis of the reaction products by high-performance liquid
chromatography–mass spectrometry (HPLC–MS)
2.6. EPR studies
Independent assays were performed using the purified protein as
well as the cell free lysates of D. vulgaris and of E. coli BL21Star(DE3)
pLysS cells overexpressing D. vulgaris AhbD.
Four samples prepared under anaerobic conditions were analysed
by EPR spectroscopy, all containing the same concentration of AhbD
(160 μM): sample 1, constituted by the as-purified AhbD; sample 2,
consisting of purified enzyme reduced with sodium dithionite
(2 mM); sample 3, a mixture of AhbD with SAM (1 mM) and sodium
dithionite (2 mM); and sample 4, a mixture of AhbD, SAM (1 mM),
Fe-Cp III (175 μM) and sodium dithionite (2 mM). All samples were pre-
pared in buffer A with a final volume of 300 μl, loaded into EPR tubes,
sealed with caps and immediately frozen in liquid nitrogen. EPR spectra
were acquired at several temperatures and microwave powers in a
Bruker EMX spectrometer equipped with an Oxford Instruments con-
tinuous flow helium cryostat. Spectra simulation was done with the
SPINCOUNT program (http://www.chem.cmu.edu/groups/hendrich/
facilities/index.html). Several independent protein preparations from
distinct cell cultures were studied yielding consistently the same results.
The reduction potential of AhbD was determined by means of an an-
aerobic redox titration (under constant flux of argon) of the purified
AhbD, monitored by EPR. To this end, 200 μM AhbD resuspended in
Anaerobically purified D. vulgaris AhbD (15 μM) was mixed with
1
5 μM Fe-Cp III, 1 mM SAM and 0.5 mM sodium dithionite in a final vol-
ume of 1 ml and left to react for 3 h and overnight, at room temperature,
under anaerobic conditions.
Reaction mixtures of 4 ml in buffer A containing D. vulgaris cell ly-
sate, 20 μM Fe-Cp III (Frontier Scientific), 0.1 mM SAM, 10 mM sodium
dithionite and 0.5 mM NADH were prepared and anaerobically incubat-
ed overnight, at room temperature. Reaction mixtures of 4 ml in buffer A
with E. coli cell lysate, 20 μM Fe-Cp III, 0.5 mM SAM and 10 mM sodium
dithionite were also prepared and incubated overnight.
In all cases, after the completion of the reaction, the tetrapyrrole in-
termediates were purified by a haem extraction protocol [21] and
analysed by reverse phase chromatography and mass spectrometry.
To this end, the final extraction solvent, ethyl acetate, was evaporated
and the intermediates were resuspended in a mixture of 90% acetoni-
trile–10% methanol (HPLC grade) and resolved on an Ace 5 AQ column

S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
1241
buffer A was titrated by stepwise addition of buffered sodium dithionite
10, 50 and 100 mM), in the presence of a standard redox mediators
(
mixture (100 μM): 1,2 naphtoquinone (E′0 = 180 mV), 1,4-
naphtoquinone (E′0 = 60 mV), methylene blue (E′0 = 11 mV), mena-
dione (E′0 = 0 mV), indigo tetrasulfonate (E′0 = −30 mV), plumbagin
(
(
(
E′0 = −40 mV), resorufin (E′0 = −51 mV), indigo trisulfonate
E′0 = −70 mV), indigo disulfonate (E′0 = −110 mV), phenazine
E′0 = −125 mV), 2-hydroxy-1,4-naphtoquinone (E′0 = −152 mV),
anthraquinone sulfonate (E′0 = −225 mV), phenosafrine (E′0 =
−
−
255 mV), safranine (E′0 = −280 mV), neutral red (E′0 =
325 mV), benzyl viologen (E′0 = −345 mV) and methyl viologen
(
E′0 = −440 mV). A mixture of catalase (130 U/ml), glucose oxidase
(
4 U/ml) and glucose (3 mM) was also added to the sample mixture
to further ensure that anaerobic conditions were maintained. Combined
silver/silver chloride and platinum electrodes calibrated against a satu-
rated quinhydrone solution at pH 7 were used. Potentials are reported
relative to the standard hydrogen electrode. The experimental data
were fitted to a single one-electron Nernst curve (n = 1).
Fig. 3. Titration of Fe-Cp III with D. vulgaris AhbD. UV–visible spectra of reduced Fe-Cp III
(15 μM) obtained upon successive additions of AhbD (spectral shifts are indicated by
arrows) and respective titration curve (inset) considering the shift of the Soret band max-
imum (black squares) and the absorbance increase at 550 nm (open diamonds); the
values were normalised to their final stage. The solid line corresponds to the fitting of
the curves with an equation for a 1:1 binding stoichiometry and a binding constant of
3
. Results
3
.1. Biochemical and UV–visible spectroscopic analysis of D. vulgaris AhbD
The recombinant D. vulgaris AhbD was purified under anaerobic con-
8
μM (see Materials and methods).
ditions in order to protect any putative oxygen-sensitive clusters. Nev-
ertheless, the stability of the AhbD clusters was tested by exposure of
the protein to oxygen and no degradation was observed. The protein
was eluted from an analytical gel filtration chromatography as a mono-
mer with an apparent molecular mass of ~40 kDa, in agreement with its
gene-derived amino acid sequence. The as-isolated protein exhibits a
brown colour with a visible spectrum displaying two absorption bands
at 325 and 415 nm (Fig. 2). These features are similar to those of the
oxidised form of iron–sulfur cluster containing proteins. Indeed, metal
analysis also showed that AhbD contains 6 ± 2 mol of iron per mol of
protein, suggesting the presence of more than one iron–sulfur cluster.
seen in control samples that contained all components except AhbD.
We observed that the Soret band of Fe-Cp III shifted from 405 nm to
4
13 nm, concomitantly with the development of bands at 528
and 550 nm. These alterations are consistent with the binding of the
Fe-Cp III to the protein and the formation of a low-spin hexa-
coordinated Fe-Cp III. Furthermore, the trace resultant from the subtrac-
tion of the final titration spectrum from that of the iron-coproporphyrin
alone exhibits maxima at 424, 518 and 550 nm. These maxima are sim-
ilar to those previously observed for D. desulfuricans ATCC2774
bacterioferritin, which is naturally isolated with iron-coproporphyrin
as the haem cofactor and that has two haem-iron axial ligands [22,23].
We also evaluated the affinity of the Fe-Cp III substrate to D. vulgaris
AhbD by performing titration experiments of AhbD with Fe-Cp III which
were monitored by following the alteration of the Soret band and the
absorbance increase at 550 nm (see Materials and methods). The data
3
.2. D. vulgaris AhbD binds Fe-Cp III
In the alternative biosynthetic pathway operative in D. vulgaris,
AhbD converts iron-coproporphyrin III into haem b. In this work, we
have analysed the binding of the substrate by following changes in the
visible spectrum of Fe-Cp III upon addition of AhbD. The mixing of
AhbD with oxidised Fe-Cp III caused no alterations in the spectrum of
Fe-Cp III. However, addition of reduced AhbD to the sodium dithionite
reduced iron-tetrapyrrole induced shifts in all the porphyrin-
associated absorption bands (Fig. 3). Note that these shifts were not
allowed the determination of an association constant (K ), consistent
A
with a single (1:1) binding stoichiometry, of 8 ± 1 μM (Fig. 3).
3.3. D. vulgaris AhbD promotes cleavage of SAM and converts Fe-Cp III into
haem b
Since the step promoted by AhbD is proposed to be a SAM-
dependent reaction, the products of SAM cleavage were analysed by
HPLC–MS after incubation of the purified protein with SAM and sodium
dithionite. Under these conditions, the formation of 5′-deoxyadenosine
(
dAdo) product was detected (Fig. 4A). The reaction mixture containing
the same concentration of all components but done in the presence of
the Fe-Cp III substrate was also analysed. We observed that the addition
of the second substrate led to an approximately 10-fold increase of the
amount of the dAdo formed, indicating that the metalloporphyrin po-
tentiates the SAM cleavage (Fig. 4C). The data show that D. vulgaris
AhbD performs a reaction that is typical of the radical SAM enzymes,
namely the reductive cleavage of SAM into methionine and the dAdo
radical. As with other members of this family of proteins [24] the SAM
derived products were observed in the absence of the second substrate.
The products formed during the conversion of Fe-Cp III into haem b
catalysed by D. vulgaris AhbD were also determined by HPLC–MS. The
reaction was performed separately with D. vulgaris free cell lysates,
E. coli cell extracts overproducing an untagged form of D. vulgaris
AhbD and purified D. vulgaris AhbD. The reaction mixtures consisting
Fig. 2. UV–visible spectrum of the as-isolated D. vulgaris AhbD (5 μM) in 50 mM Tris–HCl
pH 8, recorded under anaerobic conditions.

1242
S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
a quasi-isotropic signal with g-values at around 2.02 (2.03, 2015, 2.005)
as determined by simulating the spectrum using the program
SPINCOUNT (Fig. 6Aa), characteristic of an oxidised [3Fe–4S]1
+/0
clus-
ter. This resonance accounts 10–15% of the spectral intensity of the
dithionite reduced sample, under the same non saturating conditions,
and most likely results from a minor oxidative degradation of one or
two of the tetranuclear centres, a common feature in low-reduction po-
tential [4Fe–4S]2
+/1+
centres. Reduction of AhbD with sodium
dithionite led to development of a more complex signal with g-values
ranging from g ~2.06 to 1.92, which are typical of reduced bi- or
tetranuclear iron–sulfur centres (Fig. 6Ab). These resonances were de-
tected without significant line broadening only up to a temperature of
~
30 K. Hence, we concluded that are not originated from binuclear cen-
tres but from tetranuclear iron–sulfur clusters which agrees with the
cysteine motifs present in the AhbD amino acid sequence (see below).
The resonances were deconvoluted by varying the temperature and mi-
crowave power. When, at 10 K, the microwave power was increased a
resonance with gmax = 2.083 developed. This is was clearly discernible
at 40 mW (Fig. 6Ba) while at lower g-values no further resonances were
detected. Also, resonances at low magnetic fields, which can originate
from cluster spin states higher than ½, were not observed even at
high microwave powers and low temperatures down to 4.6 K (data
not shown). The experimental data at 10 K could be rationalised as
resulting from two reduced tetranuclear centres, with distinctly dis-
cernible gmax values and quite different relaxation properties: a fast
relaxing species, only observable at low temperatures and high micro-
wave power, with g-values at 2.083, 1.921 and 1.84, and a slower
relaxing one with g-values at 2.057, and 1.925 (Fig. 6Ba). Within the
limitations resulting from the distinct relaxation properties, these spe-
cies are present essentially in equimolar amounts.
To further characterise D. vulgaris AhbD, the redox potential of
the iron–sulfur centres was determined by performing a redox titration
followed by EPR. The results were analysed with a one-electron Nernst
curve and allow determining a single reduction potential value of −390
±
10 mV (Fig. 7). The data also indicate that the reduction potentials of
the two centres are similar as the individual reduction potentials of the
two iron–sulfur centres could not be deconvoluted. Moreover, the lack
of a plateau observed under the tested conditions shows that the
metal centres of AhbD have very low reduction potentials, whose
upper limit is −390 mV.
We have also analysed the EPR spectra of dithionite-reduced AhbD
samples upon addition of the substrates. In the presence of SAM,
some modifications of the spectra occurred and the data could again
be interpreted as resulting from two main components, with g-values
only slightly distinct from those of the reduced sample (Fig. 6Ac and
Bb). The minor alterations observed upon addition of SAM only are
not unexpected as EPR spectroscopy, although sensitive to minute mod-
ifications of the paramagnetic centre electronic structure, does not nec-
essarily reflect specific changes of, e.g., the coordination of the iron ions
in the clusters.
Major differences were observed when the substrates SAM and
Fe-Cp III were both added: two sets of resonances are clearly affected,
but to differing extents. By varying the microwave power, two major
species were defined, one that was more similar to the previously
identified slow relaxing centre, with g-values at 2.064 and 1.92, and
a second one from a faster relaxing centre with very broad reso-
nances and g-values of 2.13, and ~1.92, underneath the gmed of the
first species (Fig. 6Ad and Bc).
Fig. 4. HPLC–MS analysis of the cleavage derived products of SAM promoted by D. vulgaris
AhbD. From top to bottom: HPLC trace of the standards 5′-deoxyadenosine (dAdo) and
S-adenosylhomocysteine (SAH); (A) AhbD incubated with SAM in the presence of sodium
dithionite; (B) AhbD and SAM done in the absence of the reducing agent (control sample);
(C) AhbD incubated with SAM and Fe-Cp III under reducing conditions; (D) AhbD mixed
with SAM and Fe-Cp III without the reducing agent (control reaction). HPLC traces were
acquired at 390 nm after an overnight incubation of the reaction mixture. In reaction mix-
tures A-D SAM was eluted as two peaks with the same m/z value of 399.
of Fe-Cp III, SAM and the purified AhbD protein or the D. vulgaris free cell
lysates overproducing AhbD were incubated overnight, and the extract-
ed samples separated and analysed. Peaks typically corresponding to
haem b (m/z 616), a mono-vinyl derivate of Fe-Cp III (m/z 662) and
the unreacted form of Fe-Cp III (m/z 708) were detected indicating
that the conversion was incomplete (Fig. 5A and D). However, when
E. coli cell free extracts overexpressing AhbD were used in the presence
of SAM, a complete conversion of Fe-Cp III into haem b was observed
after an overnight incubation (Fig. 5B), while control reactions per-
formed in the absence of SAM contained essentially only Fe-Cp III
(
Fig. 5C).
Altogether, these results show that AhbD converts Fe-Cp III into
haem b and that the reaction occurs in two consecutive steps.
Above ~40 K, the spectra display solely a signal with g-values of
1+
2
.035, 2.023, and 2.01, quite distinct from that of the [3Fe–4S] centre
detected in the oxidised protein; this species is also observed in the 10 K
spectra (Fig. 6A and B, marked by an asterisk). This species, which has a
slow relaxation rate (it is not detected at high microwave powers) and
of unknown origin, was observed in all spectra of the reduced protein
(including in the presence of the substrates), and in different prepara-
tions, albeit accounting always to less than 10% of the total spectral
2
+/1+
3
.4. D. vulgaris AhbD contains two [4Fe–4S]
centres
The nature of the iron–sulfur clusters in AhbD was studied in detail
by EPR spectroscopy. The EPR spectrum of the as-isolated AhbD exhibits

S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
1243
Fig. 5. HPLC–MS analysis of tetrapyrrole derivatives derived from reaction mixtures of cell free extracts or purified D. vulgaris AhbD. The reaction mixtures contained: (A) D. vulgaris cell
free extracts, Fe-Cp III, SAM and sodium dithionite; (B) E. coli cell free extracts overexpressing D. vulgaris AhbD, Fe-Cp III, SAM and sodium dithionite; (C) purified D. vulgaris AhbD, Fe-Cp III
and sodium dithionite; (D) purified D. vulgaris AhbD, Fe-Cp III, SAM and sodium dithionite. HPLC traces were acquired at 390 nm after an overnight incubation of the reaction mixture. Fe-
Cp III eluted from the HPLC column as two peaks (marked with an asterisk) with the same m/z value of 708.
intensity at 10 K. Since this species corresponds to a minor component,
its origin was not further investigated.
alternative haem b pathway, namely the D. vulgaris AhbC that converts
12,18-didecarboxysirohaem into Fe-Cp III (Figs. 1 and 8).
Altogether, the data show that D. vulgaris AhbD contains two [4Fe–
Fig. 8 compares the amino acid sequence of D. vulgaris AhbD with
those of enzymes involved in the synthesis of tetrapyrroles and decar-
boxylation reactions (Fig. 8). D. vulgaris AhbD shares 25% identity, 39%
similarity with Paracoccus denitrificans NirJ, which participates in
S]2
+/1+
centres that are perturbed upon addition of the substrates
4
SAM and Fe-Cp III. It should be noted that all experiments were per-
formed in the presence of excess sodium dithionite, and for this reason
reoxidation of the clusters was not observed upon addition of SAM; the
iron of Fe-Cp III will be in the 2+ state and is therefore EPR silent in the
conditions used, which is consistent with our previous observation that
the ferrous Fe-Cp III adopts a low-spin (S = 0) configuration when
bound to the protein. The largest changes at one of the clusters probably
reflect the binding of SAM to the non-cysteine coordinated iron of the
cluster in the protein N-terminal domain, as has been shown for several
other radical SAM enzymes (e.g. [15,25–28]. The major effects after ad-
dition of the Fe-Cp III substrate suggest that it induces alterations in the
conformation of the AhbD protein, and that the Fe-Cp III binding pocket
is probably close to the iron–sulfur clusters.
1
the synthesis of haem d . In relation to E. coli HemN, the oxygen-
independent coproporphyrinogen III oxidase of the classical haem b
pathway that performs the oxidative decarboxylation of the propionate
side chains of rings A and B of coproporphyrinogen III to vinyl groups
producing protoporphyrinogen IX, a low degree of similarity is seen
(10% identity, 20% similarity) [3,26,30]. The low degree of similarity
among these proteins suggests that despite the small structural differ-
ences of the several tetrapyrrole substrates their conversion requires
enzymes with highly specific structures.
A comparative analysis of the amino acid sequence of D. vulgaris
AhbD against the NCBI protein database shows that the protein belongs
to the radical SAM enzymes superfamily. The protein has a radical
SAM motif in the N-terminal region and a SPASM-like motif in the
C-terminus (Fig. 8). The SAM motif contains a cysteine rich region
3
.5. Amino acid sequence analysis of D. vulgaris AhbD
A BLAST search against Desulfovibrio spp. and Archaea genomes
3 3
(C31x C35xHC38) typical of SAM proteins, namely the Cx CxXC se-
showed that homologs of D. vulgaris AhbD are widespread in
Desulfovibrio spp., sharing 27%–89% identity and 42%–92% similarity
when considering the first 100 retrieved amino acid sequences. For ex-
ample, AhbD from D. vulgaris and D. desulfuricans ATCC 27774 have 70%
identity and 77% similarity between them. Orthologs of D. vulgaris AhbD
also occur in methanogenic Archaea (e.g., Methanosarcina barkeri AhbD,
quence, where X is an aromatic residue; these three cysteines bind
the catalytic [4Fe–4S]2
+/1+
centre, leaving the fourth iron free to in-
teract with SAM (e.g., [6,18,31]). The N-terminal region of D. vulgaris
AhbD has also a GGD triad in place of the GGE motif, which is present in
several radical SAM proteins and is involved in the binding of SAM [31].
The C-terminal region of AhbD houses a second cysteine rich region
5
0% identity, 68% similarity). In agreement with our data, recent studies
2 5 2
C326x C329x C335x C338, characteristic of a SPASM motif, whose
showed that the M. barkeri AhbD also catalyses the conversion of Fe-Cp
name is derived from the following biochemically characterised mem-
bers of this subfamily: subtilosin A maturase AlbA, pyrroloquinoline
quinone E PqqE, anaerobic Sulfatase maturating enzyme, and
mycofactocin peptide maturase [32–34]. The SPASM domain is quite
2
+/1+
III into haem and the authors proposed that it contains [4Fe–4S]
centres [29]. Interestingly, D. vulgaris AhbD shares ~26% identity and
4% similarity with the enzyme involved penultimate step of the
4

1244
S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
Fig. 6. EPR spectra of D. vulgaris AhbD: oxidised and reduced forms and upon incubation with SAM and Fe-Cp III. (A) EPR spectra of AhbD: as prepared, oxidised form (a); sodium-dithionite
reduced (b); sodium-dithionite reduced AhbD mixed with SAM (c); sodium-dithionite reduced AhbD incubated with SAM and Fe-Cp III (d). Spectra were recorded at 10 K and 2 mW.
(B) Power dependence of the EPR spectra of AhbD: sodium-dithionite reduced form (a); sodium-dithionite reduced AhbD mixed with SAM (b); sodium-dithionite reduced AhbD protein
mixed with SAM and Fe-Cp III (c). Spectra were recorded at 10 K and 2, 20, 40 and 63 mW, as indicated. Asterisk denotes the unknown species (b10%).
conserved in members of the radical SAM family and is described
4. Discussion
to bind auxiliary [4Fe–4S]2
+/1+
centres. The biochemical and
spectroscopic studies presented above indicate that in D. vulgaris
In this work, D. vulgaris AhbD is shown to be a monomeric cytoplas-
AhbD the SPASM motif is also involved in the binding of one auxiliary
mic protein and a member of the radical SAM superfamily. The
2
+/1+
2+/1+
[4Fe–4S]
cluster, most possibly coordinated by four cysteine resi-
protein harbours two [4Fe–4S]
centres, one located at the N-
dues. For most of the enzymes, the function of the additional cluster re-
mains, so far, unclear [13].
terminal domain and coordinated by three cysteines and the other at
the C-terminus putatively ligated by four cysteines. Under reductive
conditions and in the presence of SAM, AhbD promotes the sequential
decarboxylation of the two propionate side chains attached to C3 and
C8 of Fe-Cp III to form vinyl groups, i.e., generating haem b.
Using two of the more similar structural protein analogues of
D. vulgaris AhbD, namely the AnSME from C. perfringens [18] and
MoaA of S. aureus [17], we predicted a three dimensional model
for the structure of AhbD (Fig. 9). AnSME, which has two auxiliary
4Fe–4S]2
+/1+
clusters each coordinated by four cysteines located at
[
the C-terminus, shares with D. vulgaris AhbD 15% identity, 31% similar-
ity, while MoaA that has one additional [4Fe–4S]2
+/1+
coordinated by
only three cysteines shares 14% identity and 34% similarity with the
D. vulgaris enzyme (Fig. 8). The D. vulgaris AhbD structure model has
β/α motifs arranged in a TIM barrel-like motif, the common feature of
most radical SAM enzymes [6], although some of the beta strands ap-
pear as loops probably due to the low amino acid sequence similarity.
As predicted from the amino acid sequence, the N-terminal region cor-
responds to the SAM radical protein domain, which includes the three
cysteines that bind the catalytic [4Fe–4S]2
+/1+
cluster, the two glycine
Fig. 7. Redox titration of the [4Fe–4S] 2+/1+ clusters of D. vulgaris AhbD. Titration was
monitored by EPR spectroscopy, measuring the peak-to peak intensity of the g = 1.91 res-
onance, at 10 K. The solid line corresponds to a one-electron Nernst curve, with a reduction
potential of −390 mV.
residues, Gly75 and Gly76, and the aspartate residue Asp77 that inter-
acts with the SAM molecule. The C-terminal domain contains four cys-
teines (Cys326, Cys329, Cys335 and Cys356) that are predicted to be

S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
1245
Fig. 8. Amino acid sequence alignment of D. vulgaris Hildenborough AhbD (gene locus DVU0855) with D. desulfuricans ATCC 27774 AhbD (Ddes0287), Methanosarcina barkeri AhbD
MbarA1458) D. vulgaris Hildenborough AhbC (DVU0857), Methanosarcina barkeri AhbC (MbarA1793), D. desulfuricans ATCC 27774 AhbC (Ddes0289), P. denitrificans haem d biosynthetic
(
1
enzyme NirJ (Pden2494), Clostridium perfringens AnSME (CPF_0616), Staphylococcus aureus MoaA (SA2063) and Escherichia coli HemN (b2955). The two domains of D. vulgaris AhbD
namely, the AdoMet radical domain and the auxiliary iron–sulfur binding SPASM domain are indicated. The amino acid residues of the AdoMet radical motif for the binding of the con-
served iron–sulfur centre CX CX C are shaded in black and marked with stars. Residues of the GG(E/D)P motif involved in the binding of SAM are shaded in dark grey and indicated
3 2
with open circles. Cysteine residues of the SPASM motif are shaded in light grey. The alignment was constructed using the amino acid sequence of D. vulgaris AhbD to search for similar
sequences against all microbial genomes using NCBI Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/), performed in ClustalX 2.1 [37] and edited in GeneDoc [38].

1246
S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
Fig. 9. 3D model of D. vulgaris AhbD. (A) Cartoon representation of the predicted D. vulgaris AhbD structure. (B) Tridimensional structure of C. perfringens AnSME displayed in the same
orientation as in panel A. (C) Surface representation of D. vulgaris AhbD model. (D) Surface representation of D. vulgaris AhbD model rotated by 90° from the view in panel C. The figure
depicts a channel (marked with an arrow) and the predicted binding site of the SAM molecule which is located at the end of the channel. The model was generated by the I-TASSER server
[39–41] using as template the structure of C. perfringens AnSME (PDB ID: 4K36). The root-mean-square deviation between the two models is 0.987 Å. In panels A and B, the monomer is
coloured in gradient, starting from blue (N-terminal) to red (C-terminal) and the region of the conserved cysteines involved on the iron–sulfur clusters is boxed and zoomed. The amino
acid residues, the SAM molecule and the iron–sulfur clusters are represented as sticks. Pictures were built with the PyMOL program [42].
the ligands of the auxiliary [4Fe–4S]2+/1+ cluster equivalent to auxiliary
centre II in the C. perfringens AnSME (Fig. 9A). Both iron–sulfur clusters
are expected to be close to the protein surface, which may allow
exchange of electrons with yet unknown physiological partners. Inter-
estingly, in the place where C. perfringens AnSME binds the auxiliary
cluster I, the D. vulgaris enzyme has still two cysteine residues

S.A.L. Lobo et al. / Biochimica et Biophysica Acta 1844 (2014) 1238–1247
1247
[
17] P. Hanzelmann, H. Schindelin, Crystal structure of the S-adenosylmethionine-de-
pendent enzyme MoaA and its implications for molybdenum cofactor deficiency
in humans, Proc. Natl. Acad. Sci. U. S. A. 101 (2004) 12870–12875.
(
Cys288 and Cys338) (Fig. 9B), which are conserved in all AhbD homo-
logues so far sequenced. In the protein surface representation of AhbD
Fig. 9C and D) it is striking the presence of a cavity that remarkably re-
(
[18] P.J. Goldman, T.L. Grove, L.A. Sites, M.I. McLaughlin, S.J. Booker, C.L. Drennan, X-ray
structure of an AdoMet radical activase reveals an anaerobic solution for
formylglycine posttranslational modification, Proc. Natl. Acad. Sci. U. S. A. 110
sembles the haem binding cavity of hemophores [35,36], and which is
absent in the model templates. We hypothesize that it will be in this
cavity that the substrate Fe-Cp III binds pointing the two propionate
side chains to the interior of the protein and being close to the SAM
binding pocket which will enable their decarboxylation; the Fe-Cp III
is also predicted to be located near the D. vulgaris AhbD auxiliary cluster,
in a pocket resembling that containing the auxiliary cluster I binding site
in C. perfringens AnSME.
(2013) 8519–8524.
[19] J. Le Gall, W.J. Payne, L. Chen, M.Y. Liu, A.V. Xavier, Localization and specificity of cy-
tochromes and other electron transfer proteins from sulfate-reducing bacteria,
Biochimie 76 (1994) 655–665.
[
20] D.S. Fischer, D.C. Price, A simple serum iron method using the new sensitive chro-
mogen tripyridyl-S-triazine, Clin. Chem. 10 (1964) 21–31.
[21] M. Lubben, K. Morand, Novel prenylated hemes as cofactors of cytochrome oxidases.
Archaea have modified hemes A and O, J. Biol. Chem. 269 (1994) 21473–21479.
[
[
22] C.V. Romao, M. Regalla, A.V. Xavier, M. Teixeira, M.Y. Liu, J. Le Gall, A bacterioferritin
from the strict anaerobe Desulfovibrio desulfuricans ATCC 27774, Biochemistry 39
(2000) 6841–6849.
23] C.V. Romao, R. Louro, R. Timkovich, M. Lubben, M.Y. Liu, J. LeGall, A.V. Xavier, M.
Teixeira, Iron-coproporphyrin III is a natural cofactor in bacterioferritin from the an-
aerobic bacterium Desulfovibrio desulfuricans, FEBS Lett. 480 (2000) 213–216.
24] K.S. Duschene, J.B. Broderick, The antiviral protein viperin is a radical SAM enzyme,
FEBS Lett. 584 (2010) 1263–1267.
In conclusion, we show that D. vulgaris AhbD is a two [4Fe–4S]2+/1+
containing haem synthase that adds a new function to the widespread
superfamily of radical SAM enzymes.
[
[
Funding
25] S.R. Wecksler, S. Stoll, H. Tran, O.T. Magnusson, S.P. Wu, D. King, R.D. Britt, J.P.
Klinman, Pyrroloquinoline quinone biogenesis: demonstration that PqqE from
Klebsiella pneumoniae is a radical S-adenosyl-L-methionine enzyme, Biochemistry
The work was funded by PEst-OE/EQB/LA0004/2011, FCT project
PTDC/BBB-BQB/0937/2012 and the FCT student fellowship SFRH/BPD/
6
4
8 (2009) 10151–10161.
[26] A.A. Brindley, R. Zajicek, M.J. Warren, S.J. Ferguson, S.E. Rigby, NirJ, a radical SAM
family member of the d heme biogenesis cluster, FEBS Lett. 584 (2010) 2461–2466.
3944/2009.
1
[
27] J.M. Demick, W.N. Lanzilotta, Radical SAM activation of the B12-independent glycerol
References
dehydratase results in formation of 5′-deoxy-5′-(methylthio)adenosine and not
5′-deoxyadenosine, Biochemistry 50 (2011) 440–442.
[
[
[
1] H.A. Dailey, Terminal steps of haem biosynthesis, Biochem. Soc. Trans. 30 (2002)
90–595.
2] I.U. Heinemann, M. Jahn, D. Jahn, The biochemistry of heme biosynthesis, Arch.
Biochem. Biophys. 474 (2008) 238–251.
[28] J. Feng, J. Wu, N. Dai, S. Lin, H.H. Xu, Z. Deng, X. He, Discovery and characterization
of BlsE, a radical S-adenosyl-L-methionine decarboxylase involved in the blasticidin
S biosynthetic pathway, PLoS One 8 (2013) 68545.
[29] M. Kühner, K. Haufschildt, A. Neumann, S. Storbeck, J. Streif, G. Layer, The alternative
route to heme in the methanogenic archaeon Methanosarcina barkeri, Archaea,
2014, 2014.
[30] G. Layer, E. Kervio, G. Morlock, D.W. Heinz, D. Jahn, J. Retey, W.D. Schubert, Structural
and functional comparison of HemN to other radical SAM enzymes, Biol. Chem. 386
(2005) 971–980.
[31] H.J. Sofia, G. Chen, B.G. Hetzler, J.F. Reyes-Spindola, N.E. Miller, Radical SAM, a novel
protein superfamily linking unresolved steps in familiar biosynthetic pathways with
radical mechanisms: functional characterization using new analysis and informa-
tion visualization methods, Nucleic Acids Res. 29 (2001) 1097–1106.
[32] D.H. Haft, M.K. Basu, Biological systems discovery in silico: radical S-
adenosylmethionine protein families and their target peptides for posttranslational
modification, J. Bacteriol. 193 (2011) 2745–2755.
5
3] S. Bali, A. Lawrence, S.L. Lobo, S.L. M., B. Golding, D. Palmer, M. Howard, S. Ferguson,
M.J. Warren, Molecular hijacking of siroheme for the synthesis of heme and d
heme, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 18260–18265.
1
[
[
4] S. Storbeck, S. Rolfes, E. Raux-Deery, M.J. Warren, D. Jahn, G. Layer, A novel pathway
for the biosynthesis of heme in Archaea: genome-based bioinformatic predictions
and experimental evidence, Archaea 2010 (2010) 175050.
5] B. Buchenau, J. Kahnt, I.U. Heinemann, D. Jahn, R.K. Thauer, Heme biosynthesis
in Methanosarcina barkeri via a pathway involving two methylation reactions,
J. Bacteriol. 188 (2006) 8666–8668.
6] J.L. Vey, C.L. Drennan, Structural insights into radical generation by the radical SAM
superfamily, Chem. Rev. 111 (2011) 2487–2506.
7] G. Layer, D.W. Heinz, D. Jahn, W.D. Schubert, Structure and function of radical SAM
enzymes, Curr. Opin. Chem. Biol. 8 (2004) 468–476.
8] S.J. Booker, T.L. Grove, Mechanistic and functional versatility of radical SAM en-
zymes, F1000 Biol. Rep. 2 (2010) 52.
[
[
[
[
[33] D.H. Haft, Bioinformatic evidence for a widely distributed, ribosomally produced
electron carrier precursor, its maturation proteins, and its nicotinoprotein redox
partners, BMC Genomics 12 (2011) 21.
[34] A. Benjdia, S. Subramanian, J. Leprince, H. Vaudry, M.K. Johnson, O. Berteau, Anaerobic
sulfatase-maturating enzyme—a mechanistic link with glycyl radical-activating
enzymes? Febs J. 277 (2010) 1906–1920.
9] D.P. Dowling, J.L. Vey, A.K. Croft, C.L. Drennan, Structural diversity in the AdoMet
radical enzyme superfamily, Biochim. Biophys. Acta 1824 (2012) 1178–1195.
[
[
[
10] Q. Zhang, W.A. van der Donk, W. Liu, Radical-mediated enzymatic methylation: a
tale of two SAMS, Acc. Chem. Res. 45 (2012) 555–564.
[35] P. Arnoux, R. Haser, N. Izadi-Pruneyre, A. Lecroisey, M. Czjzek, Functional aspects of
the heme bound hemophore HasA by structural analysis of various crystal forms,
Proteins 41 (2000) 202–210.
[36] H. Cwerman, C. Wandersman, F. Biville, Heme and a five-amino-acid hemophore re-
gion form the bipartite stimulus triggering the has signaling cascade, J. Bacteriol. 188
(2006) 3357–3364.
[37] M.A. Larkin, G. Blackshields, N.P. Brown, R. Chenna, P.A. McGettigan, H. McWilliam,
F. Valentin, I.M. Wallace, A. Wilm, R. Lopez, J.D. Thompson, T.J. Gibson, D.G. Higgins,
Clustal W and Clustal X version 2.0, Bioinformatics 23 (2007) 2947–2948.
[38] B. Nicholas, B. Karl, P. Nicholas, B. Hugh Jr., GeneDoc: a tool for editing and annotat-
ing multiple sequence alignments, 1997.
[39] A. Roy, A. Kucukural, Y. Zhang, I-TASSER: a unified platform for automated protein
structure and function prediction, Nat. Protoc. 5 (2010) 725–738.
[40] A. Roy, J. Yang, Y. Zhang, COFACTOR: an accurate comparative algorithm for
structure-based protein function annotation, Nucleic Acids Res. 40 (2012) 471–477.
[41] Y. Zhang, I-TASSER server for protein 3D structure prediction, BMC Bioinforma. 9
(2008) 40.
11] G. Layer, K. Verfurth, E. Mahlitz, D. Jahn, Oxygen-independent coproporphyrinogen-
III oxidase HemN from Escherichia coli, J. Biol. Chem. 277 (2002) 34136–34142.
12] G. Layer, K. Grage, T. Teschner, V. Schunemann, D. Breckau, A. Masoumi, M. Jahn, P.
Heathcote, A.X. Trautwein, D. Jahn, Radical S-adenosylmethionine enzyme
coproporphyrinogen III oxidase HemN: functional features of the [4Fe–4S] cluster
and the two bound S-adenosyl-L-methionines, J. Biol. Chem. 280 (2005) 29038–29046.
13] N.D. Lanz, S.J. Booker, Identification and function of auxiliary iron–sulfur clusters in
radical SAM enzymes, Biochim. Biophys. Acta 1824 (2012) 1196–1212.
[
[
14] M.M. Cosper, G.N. Jameson, H.L. Hernandez, C. Krebs, B.H. Huynh, M.K. Johnson,
Characterization of the cofactor composition of Escherichia coli biotin synthase, Bio-
chemistry 43 (2004) 2007–2021.
[
15] K.H. Lee, L. Saleh, B.P. Anton, C.L. Madinger, J.S. Benner, D.F. Iwig, R.J. Roberts, C. Krebs,
S.J. Booker, Characterization of RimO, a new member of the methylthiotransferase
subclass of the radical SAM superfamily, Biochemistry 48 (2009) 10162–10174.
16] F. Forouhar, S. Arragain, M. Atta, S. Gambarelli, J.M. Mouesca, M. Hussain, R. Xiao, S.
Kieffer-Jaquinod, J. Seetharaman, T.B. Acton, G.T. Montelione, E. Mulliez, J.F. Hunt,
M. Fontecave, Two Fe–S clusters catalyze sulfur insertion by radical-SAM
methylthiotransferases, Nat. Chem. Biol. 9 (2012) 333–338.
[
[42] W.L. DeLano, The PyMOL Molecular Graphics System, http://www.pymol.org 2002.