Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase-rhodanese fusion protein functions in sulfur assimilation

Article abstract of DOI:10.1074/jbc.M117.790170

Hydrogen sulfide (H₂S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H₂S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDOand rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO-rhodanese fusion (PRF) proteins in sulfur metabolism. Herein,we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 ? resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.

Full text of DOI:10.1074/jbc.M117.790170

JBC Papers in Press. Published on July 6, 2017 as Manuscript M117.790170
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.790170
Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese
fusion protein functions in sulfur assimilation
1
1, 2
1
1, 2
1
Nicole Motl , Meredith A. Skiba , Omer Kabil , Janet L. Smith and Ruma Banerjee *
1
Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor, MI 48109-
2
0
600, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109
Running Title: Characterization of a Persulfide Dioxygenase-Rhodanese Fusion Protein
Address correspondence to: Ruma Banerjee, Tel: (734) 615-5238; E-mail: rbanerje@umich.edu
*
ABSTRACT
and disposal as observed with the homologous
mammalian proteins.
2
Hydrogen sulfide (H S) is a signaling molecule
1
that is toxic at elevated concentrations. In
eukaryotes, it is cleared via a mitochondrial
sulfide oxidation pathway, which comprises
sulfide quinone oxidoreductase, persulfide
dioxygenase (PDO), rhodanese, and sulfite
Hydrogen sulfide (H
2
S) is a gas with toxicity
comparable to that of cyanide (1). It is also a
signaling molecule produced by prokaryotes and
eukaryotes and elicits
a
broad range of
physiological effects (2-4). Notably, it acts as a
cardioprotectant during myocardial ischemia
reperfusion and is implicated in regulation of the
response,
inflammation (5-8). Steady-state H
governed by the rates of its production and
clearance (9). In eukaryotes, H S is produced by
the transsulfuration pathway enzymes,
cystathionine β-synthase and cystathionine γ-
lyase, and by the cysteine catabolic pathway
2
oxidase and converts H S to thiosulfate and
sulfate. Natural fusions between the non-heme
iron containing PDO and rhodanese, a thiol
sulfurtransferase, exist in some bacteria.
However, little is known about the role of the
PDO-rhodanese fusion (PRF) proteins in sulfur
metabolism. Herein, we report the kinetic
properties and the crystal structure of a PRF
from the gram-negative endophytic bacterium,
cellular
stress
apoptosis,
and
2
S levels are
2
Burkholderia
phytofirmans.
The
crystal
enzyme,
mercaptopyruvate
sulfurtransferase
structures of wild-type PRF and
a
(10,11). Sulfide clearance occurs primarily via the
mitochondrial sulfide oxidation pathway (12,13).
Several enzymes are involved in the mitochondrial
sulfur oxidation pathway: sulfide quinone
oxidoreductase, the persulfide dioxygenase (PDO,
also known as ETHE1), rhodanese, and sulfite
oxidase. The main products of the sulfide
oxidation pathway are thiosulfate and sulfate.
sulfurtransferase-inactivated C314S mutant
with and without glutathione were determined
at 1.8, 2.4, and 2.7 Å resolution, respectively.
We found that the two active sites are distant
and do not show evidence of direct
communication. The B. phytofirmans PRF
exhibited
robust
PDO
activity
and
preferentially catalyzed sulfur transfer in the
direction of thiosulfate to sulfite and
glutathione persulfide; sulfur transfer in the
reverse direction was detectable only under
limited turnover conditions. Together with the
kinetic data, our bioinformatics analysis reveals
that B. phytofirmans PRF is poised to
metabolize thiosulfate to sulfite in a sulfur
assimilation pathway rather than in sulfide
stress response as seen for example with the
Staphylococcus aureus PRF or sulfide oxidation
PDO is a member of the 2-His-1Asp
mononuclear iron containing enzyme superfamily
and catalyzes the oxidation of glutathione
persulfide (GSSH) to sulfite (14,15). Mutations in
PDO result in ethylmalonic encephalopathy, an
autosomal recessive disorder (16). Over 20
mutations have been described in the ethe1 gene
ꢀ
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Abbreviations used. H S, hydrogen sulfide; PDO,
2
1
persulfide dioxygenase (also known as ETHE1);
GSSH, glutathione persulfide, PRF: PDO-
rhodanese fusion.ꢀ
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

(
17). Rhodanese is a thiosulfate sulfurtransferase;
from the two-domain human rhodanese (Fig. 1A).
In another subset, exemplified by CstB (23), the
PDO domain is fused to two rhodanese domains
(Fig. 1A). Of these, the first (or middle) rhodanese
domain is non catalytic while the second (or C-
terminal) domain houses the sulfurtransferase
activity and oxidizes bacillithiol persulfide and
coenzyme A persulfide to thiosulfate (23,30). CstB
is implicated in protecting against persulfide stress
in humans, it preferentially catalyzes sulfur
transfer from GSSH to sulfite producing
thiosulfate and GSH (Eq. 1) (18).
2
-
2-
GSSH + SO
3
→ GSH + S
2
O
3
[1]
Rhodanese domains are universal structural
modules that occur in one of three variations (19).
They can be found as tandem repeats as in human
rhodanese, which possesses a non-catalytic N-
terminal domain and a catalytically active C-
terminal domain (18,20). Alternatively, they can
be found as single domain in proteins such as
human TSTD1 (21) and E. coli GlpE (22), or as
natural fusions with other protein domains as in
the S. aureus CstB (23).
2
and H S toxicity (23).
The existence of PRFs in nature suggests that
their mitochondrial homologs, which exist as
stand-alone proteins might interact. Structures of
two PRFs are available from structural genomic
projects. The first is of CstB (PDB: 3R2U) in
which density for the catalytic rhodanese domain
is missing. The second structure is of the
biochemically uncharacterized Alicyclobacillus
acidocaldarius PRF (PDB: 3TP9) (referred to
hereafter as AaPRF), which contains a PDO
domain fused to two rhodanese domains.
Interestingly, these structures reveal the presence
of a cysteine-containing loop in the PDO active
site that is absent in the human PDO structure and
is not predicted in the sequence of BpPRF. This
additional loop is speculated to aid in substrate
transfer from the PDO domain to the rhodanese
domain (23). As CstB is the only PRF that has
been characterized biochemically to date (23,30),
there is a paucity of information on the roles of
PRFs in sulfur metabolism in bacteria.
2
In prokaryotes, H S can be generated via
several pathways. It is an end product of the
dissimilatory sulfate reducing pathway in which
sulfate is used as a terminal electron acceptor
2
during anaerobic respiration (24). H S is an
intermediate in the assimilatory sulfate-reducing
pathway where it is produced from sulfite by
sulfite reductase and is used as a substrate for
cysteine synthesis (25). Additionally, H
produced by orthologs of the eukaryotic H
biosynthetic enzymes cystathionine β-synthase
and cystathionine γ-lyase and mercaptopyruvate
sulfurtransferase, which have been identified in
several bacterial species and suggested to play a
role in defense against antibiotic-induced
2
S can be
2
S
oxidative stress (26).
H
2
S
metabolism by
In this study, we have characterized the PRF
protein from Burkolderia phytofirmans (referred
hereafter as BpPRF), a gram-negative endophyte
originally isolated from onion roots (31). In
BpPRF, the N-terminal nonheme Fe(II)-containing
PDO domain is followed by a rhodanese domain
containing the signature CRXGX(T/R) active site
motif (Fig. 1B). The N-terminal PDO domain of
BpPRF displays high sequence identity to human
PDO (56%) and displays conservation of active
site iron binding ligands.
We demonstrate that BpPRF is a bifunctional
enzyme that uses the rhodanese domain to
preferentially catalyze sulfur transfer from
thiosulfate to GSH to form sulfite and GSSH and
uses the PDO domain to oxidize GSSH to sulfite
(Eqs. 2,3).
prokaryotes combined with its toxicity at high
concentrations, necessitates mechanisms for
regulating its levels (27). The sulfide anion efflux
transporter described in Clostridium difficile
represents one mechanism for clearing sulfide via
export (28). Additionally, several bacterial species
possess orthologs of eukaryotic mitochondrial
sulfide oxidation pathway enzymes, and can
presumably dispose sulfide following its oxidation
(
23,29,30).
Recently, bacterial proteins that are natural
fusions between PDO and rhodanese have been
identified (23). Hereafter, we refer to these fusion
proteins as PRF (for PDO rhodanese fusion).
Bioinformatics analysis reveals that in a subset of
these PRFs, e.g. the one from Burkolderia
phytofirmans PsJN, the PDO domain is fused to a
single rhodanese domain, which is orthologous to
human thiol sulfurtransferase, TSTD1, and distinct
2-
2-
S
2
O
3
+ GSH→ SO
3
+ GSSH
[2]
[3]
2-
GSSH + O → GSH + SO
2
3
2

The crystal structures of BpPRF provide
insights into the architecture and the relative
juxtaposition of its active sites.
by three helices on each side (15,29,33). Density
for eight of the fifteen residues in the linker region
was missing, indicating that it is disordered (Fig.
2
A). The C-terminal domain displays
a
Results
characteristic rhodanese fold with a five-stranded
parallel β-sheet core framed by two and three α-
helices on either side of the domain (34).
Additionally, a β-hairpin extension is seen on the
N-terminal side of the rhodanese domain (Fig.
Purification and properties of BpPRF-
Recombinant wild-type and variants of BpPRF
purified using a one-step protocol, were obtained
in >95% purity as judged by SDS PAGE analysis
2
A). The two active sites are located at different
(
(
not shown). The yield for the full-length proteins
wild-type and C314S), was ~120 mg protein per
ends of the protein and are related by a rotation
angle of ~90° (Fig. S1A).
liter of culture. The rhodanese domain was
obtained with a yield of 22 mg of protein per liter
of culture. BpPRF eluted from a size exclusion
column with a molecular mass corresponding to
The PDO domain active site contains a
mononuclear non-heme iron coordinated by His58,
His114 and Asp133 comprising a 2His:1Asp facial
triad. The remaining coordination sites are
occupied by water molecules giving an octrahedral
geometry around ferrous iron (Fig. 2B). Cys314,
located in the rhodanese active site, was captured
in its persulfidated, Cys-SSH form (Fig. 2C). The
PDO active site is located at the bottom of a large
pocket framed on one side by a positively charged
ridge comprising residues Arg193-Lys216 and by
Tyr176 on the other (Fig. S1B). The rhodanese
active site is located at the bottom of a shallow
pocket that is lined by the positively charged
residues, Arg315 and Arg319 on one side, and
hydrophobic residues Ala316, Gly317 and
Gly318, on the other (Figs. S1C).
C314S BpPRF crystallized in a different space
group (Table 1) but the two domains were in the
same relative orientation as in wild-type BpPRF.
Crystals of C314S BpPRF that had been soaked
with GSH, displayed additional density in two of
the three PDO domains present in the asymmetric
unit. GSH coordinates to iron in the PDO active
site and displaces one of the iron-coordinated
waters. It is within hydrogen bonding distance of
Tyr176, Arg193, Arg142 and Lys216 (Fig. 3A).
Notably, in the C314S BpPRF structure, a chloride
ion (from the crystallization solution) sits in place
of the Cys314 persulfide seen in the wild-type
BpPRF structure. When the single-atom peak in
the electron density map was modeled as a
chloride ion, the Fo-Fc had not residual positive or
negative density, whereas modeling as a sfully
occupied water resulted in strong positive Fo-Fc
density.
4
0 kDa, consistent with it being a monomer
(
predicted mass = 41.5 kDa). The rhodanese
domain eluted as a mixture of a monomer and a
dimer (not shown). The monomeric organization
of BpPRF is distinct from that of CstB, which is
tetrameric (23).
The thermal stability of the BpPRF variants
was assessed in a turbidometric assay. The T
value for the isolated rhodanese domain (55 ± 2
m
°
°
C) was slightly higher than for wild-type (50 ± 2
C) and C314S (51.6 ± 0.8 °C) BpPRF. This result
indicates that the stand-alone rhodanese domain is
well folded and contrasts with the instability of the
excised catalytic rhodanese domain of CstB (23).
Metal Analysis-Plasma emission spectroscopy
revealed the presence of 0.54 mol iron/mol
monomer wild-type BpPRF. The metal content
was also assessed by a colorimetric assay (32),
which yielded a similar value (0.60 mol iron/mol
monomer). The iron content of C314S BpPRF was
0
.46 mol iron/mol monomer. Attempts to fully
reconstitute the metal site with FeCl
anaerobic conditions were unsuccessful.
Structure of BpPRF-The crystal structure of
BpPRF co-crystallized with thiosulfate, was
obtained at 1.79 Å resolution by molecular
replacement using the AaPRF structure (PDB
2
under
3
TP9) as template (Table 1). The final model
contains two chains in the asymmetric unit. Each
molecule consists of an N-terminal PDO domain
(
2
(
residues 1-230), a 15-residue linker (residues
31-245), and a C-terminal rhodanese domain
residues 246-357) (Fig. 2A). The PDO domain
Structural comparisons of BpPRF and
homologous human proteins-The architecture of
the BpPRF PDO active site is very similar to that
displays a typical metallo-β-lactamase-type fold
consisting of two central β-sheet clusters enclosed
3

of human PDO (15) (Fig. 3B). The substrate-
binding pocket is framed by two α-helices on one
side and by Tyr176 on the other. Key residues that
are predicted to be involved in substrate binding in
human PDO, Tyr197, Arg214, Arg163, Leu231
and Pro234 are conserved in BpPRF (Tyr176,
Arg193, Arg142, Leu212 and Pro215) and are
found in similar orientations. Lys216, which is not
conserved in human PDO, lines the entrance to the
active site and might play a role in substrate
positioning.
GSH was slightly activating (1.3-fold increase in
cat/Km(GSSH)) as seen previously with human PDO
(14). Thiosulfate was without effect, while H
and sulfite decreased kcat/Km(GSSH) 1.6- and 2.2-
fold. Hence, sulfite build up can potentially inhibit
PDO activity.
k
2
S
Sulfurtransferase
activity
of
BpPRF-
Conversion of cyanide to thiocyanate in the
presence of thiosulfate (Eq. 4) was used to monitor
the sulfurtransferase activity of BpPRF (Figs. 6A
and 7A). From this analysis, Km(thiosulfate) of 5.4± 0.7
The BpPRF rhodanese domain and the
catalytic domain of bovine rhodanese display very
similar folds (Fig. 4A). Minor structural
differences include the N-terminal β-hairpin
extension in BpPRF and two extra loops in bovine
rhodanese. The active sites are also very similar
with the conserved residues, Cys314, Arg315 and
Gly318 (BpPRF numbering), being in similar
orientations (Fig. 4B). However, Thr252 and
Lys249 in bovine rhodanese occupy the same
positions as Arg319 and Ala316, respectively, in
BpPRF. These substitutions indicate differences in
charge distribution that might contribute to
differences in substrate specificity.
2-
-
2-
-
S
2
O
3
+ CN → SO
3
+ SCN
[4]
-
1
mM, Km(CN-
)
of 22.7 ± 0.7 mM and kcat of 5.1 s at
22 ˚C were obtained (Table 3). Under the same
conditions, the sulfurtransferase activity of the
C314S mutant, carrying an inactivating mutation
in the rhodanese domain, was not detectable (data
not shown).
Since GSSH, a product of the sulfurtransferase
reaction (Eq. 2), is a substrate for the PDO domain
(Eq. 3), this activity was monitored in a “coupled”
assay, i.e. by detecting O
and 7C). This assay yielded the following
parameters: Km(GSH) = 4.7 ± 0.7 mM, Km(thiosulfate)
3.1 ± 0.2 mM and kcat = 6.2 s (Table 3). The
same reaction was also monitored directly, i.e. by
detecting sulfite (Eq. 2) colorimetrically, and
yielded essentially the same results (not shown).
Cysteine was tested as an alternate sulfur
2
consumption (Figs. 6C
=
-
1
PDO activity of BpPRF-The rate of O
consumption during conversion of GSSH to sulfite
Eq. 3) was monitored using an O electrode (Fig.
) and yielded the following kinetic parameters for
wild-type BpPRF: Km(GSSH) = 70 ± 8 µM, and
2
(
2
5
-
1
acceptor in lieu of GSH, by monitoring H
production using the lead sulfide assay (Eqs. 5,6)
Figs. 6E, 7E).
2
S
K
(
k
m(O2) = 130 ± 30 µM and kcat = 143 s at 22 ˚C,
Table 2). C314S BpPRF exhibited a 29-fold lower
cat and an ~5-fold higher K for GSSH than wild-
(
m
type BpPRF. Addition of 2.5 mM ascorbate to the
standard assay resulted in an ~20% increase in the
PDO specific activity (not shown). Cysteine
persulfide and thiosulfate were also tested as
potential substrates for the PDO domain.
However, neither displayed detectable activity.
It is important to note that the method used to
synthesize GSSH results in its contamination with
an equal concentration of GSH and an ~3-fold
2-
2-
S
2
O
3
+ Cys-SH → SO
3
+ Cys-SSH
[5]
Cys-SSH + Cys-SH → Cys-S-S-Cys + H
2
S [6]
This analysis yielded the following values: Km(Cys)
5.2 ± 0.4 mM, Km(thiosulfate) = 5.8 ± 0.8 mM and
cat = 0.2 s . Given the relatively low cellular
concentration of cysteine versus GSH and the
considerably lower kcat/Km(Cys) value (Table 3),
cysteine is unlikely to be a physiologically
relevant sulfur acceptor for BpPRF.
=
k
-1
2
excess of H S, as described previously (14). The
potential effect of GSH and sulfide on PDO
activity was examined in addition to the effect of
sulfite (a PDO product) and thiosulfate (rhodanese
substrate/product) (Table S1). Sulfite, which
consumed O , increased the background rate and
2
was therefore used at a lower concentration (0.6
mM) than the other metabolites (5 mM each).
Next,
sulfurtransferase activity in the opposite direction
i.e. reverse of Eq. 2) by tracking thiosulfate
formation under anaerobic conditions (to inhibit
GSSH consumption by PDO). However, unlike
human rhodanese (18,20), sulfur transfer activity
we
attempted
to
monitor
(
4

from GSSH to sulfite was not detected with
BpPRF under steady-state turnover conditions.
Sulfurtransferase activity of the rhodanese
domain-The isolated rhodanese domain is active
and exhibits the following kinetic parameters in
the cyanide detoxification assay (Fig. 6B, 7B):
is likely confounded by side reactions of the
reactive products, particularly GSSH (35), as it
builds up under these conditions.
Discussion
K
m(thiosulfate) = 9.2 ± 0.8 mM and Km(CN-) = 23 ± 1
Natural fusions of PDO and rhodanese are
found in bacteria and suggest a functional
interaction between them in other organisms,
where they are expressed as stand-alone proteins
-
1
mM, kcat = 4.6 s (Table 4), which are very similar
to those for full-length BpPRF. GSSH synthesis
from thiosulfate and GSH (Eq. 2) was monitored
by following H
2
S formation (Eq. 7) (Fig. 6D,7D).
(
36). In this study, we provide the first structural
and kinetic characterization of a PRF. To our
knowledge, the only other PRF that has been
biochemically characterized is CstB from S.
aureus (23,30,37), but the available CstB structure
is missing density for the catalytic rhodanese
domain. CstB is located in the cst operon (Fig.
GSSH + GSH → GSSG + H
2
S
[7]
The following values were obtained from this
analysis: Km(GSH) = 14.9 ± 0.5 mM and a
-
1
K
m(thiosulfate) = 3.8 ± 0.5 mM and kcat = 9.0 s . As
with the full-length protein, cysteine was a poor
acceptor (6-fold lower kcat/K than GSH (Fig.
F,7F) and the reverse reaction (i.e. transfer from
m
1
C). Its operonic partners include CstA, which is a
rhodanese, and a sulfide quinone oxidoreductase,
which oxidizes to polysulfides (30).
6
GSSH to sulfite) was not detectable under steady-
state turnover conditions (Table 4). Similar values
for the kinetic parameters were obtained when the
rate of sulfite production was detected (data not
shown).
Stoichiometry of PRF reaction-Our kinetic
data predicts that BpPRF can catalyze a GSH-
dependent net oxidation of thiosulfate to sulfite
2
H S
Expression of the cst operon is regulated by the
persulfide/polysulfide-sensing repressor CstR and
is induced by exogenous addition of H S or
2
polysulfides (38). CstB catalyzes the conversion of
low molecular weight persulfides to thiosulfate via
the intermediate formation of sulfite (Eq. 8) (23).
2
-
+
2
RSSH + O
2 2 2 3
+ H O→ 2S O + 2RSH + 2H [8]
(
Eqs. 2 and 3). To test this prediction, the
stoichiometry of thiosulfate and O consumed to
2
In contrast to CstB, BpPRF preferentially
sulfite formed was determined. As noted
previously, the method used to synthesize GSSH
results in an equivalent of GSH being present,
which together with unreacted GSSG and an ~3-
catalyzes sulfur transfer in the reverse direction,
i.e. from thiosulfate to sulfite (Eq. 9), which was
confirmed under single turnover conditions (Fig.
2
-
2-
+
S
2
O
3
+ O
2
+ H
2
O→ 2SO
3
+ 2H
[9]
2
fold excess of H S, would confound the reaction
stoichiometry. To circumvent this problem, GSSH
was synthesized in situ using GSH and thiosulfate
as substrates (Eq. 2).
8
). GSSH serves as the intermediate sulfur carrier,
transferring sulfur processed in the rhodanese
active site to the PDO active site. Unlike CstB,
which functions in the sulfide stress response, the
cellular context in which BpPRF functions, is not
known (38). The gene encoding BpPRF does not
reside in an operon and the metabolic context in
which it functions is not immediately obvious. The
BpPRF-encoding gene is located downstream from
a putative peroxidase and a putative LysR-type
regulator, which have been shown to be involved
in regulating bacteria mobility, virulence,
metabolism and quorum sensing (39) (Fig. 1C). A
potential fate of sulfite, which is toxic, is its
oxidation by sulfite oxidase to sulfate, which could
be utilized in an assimilatory sulfate-reducing
Under single turnover conditions, the
stoichiometry of sulfite formed/thiosulfate
consumed/oxygen consumed was 2:1:1 (Fig. 8A)
as predicted by Eqs. 2 and 3. Also, as expected,
the reaction stoichiometry of sulfite formed/GSH
consumed/thiosulfate consumed was 1:1:1 in the
absence of oxygen (Fig. 8B). However, when the
BpPRF reaction was monitored under multiple
turnover conditions in the presence of oxygen, the
stoichiometry of sulfite formed/thiosulfate
consumed/oxygen consumed switched to 1:1:1
after three turnovers (Fig. 8C). This change in
stoichiometry indicates inefficient coupling
between the PDO and rhodanese active sites, and
5

pathway. B. phytofirmans encodes a putative
sulfite oxidase. Alternatively, BpPRF might be
involved in sulfur assimilation from thiosulfate
and sulfite could be reduced by sulfite reductase
dependence observed with full-length BpPRF (Fig.
6C, 7C). This difference in behavior could be due
to the difference in the oligomeric state of the
stand-alone rhodanese domain, which exists as a
monomer/dimer mixture in solution, in contrast to
the full-length protein, which is a monomer.
Unlike human rhodanese, sulfur transfer from
GSSH to sulfite was not detectable with either the
full-length BpPRF or the stand-alone rhodanese
domain (Tables 3,4). However, the GSSH:sulfite
sulfur transfer activity was observed for both the
full-length BpPRF and the stand-alone rhodanese
domain under limited turnover conditions, i.e.
between 2-5 turnovers (data not shown). This
observation indicates that under multiple turnover
conditions as the concentrations of products (i.e.,
GSH or thiosulfate) increase, one of them inhibits
the enzyme. In fact, we found that thiosulfate but
not GSH, inhibits the GSSH:sulfite sulfur transfer
activity of BpPRF (data not shown) explaining the
lack of activity under steady state assay
conditions. The inhibitory role of thiosulfate is
consistent with studies on TSTD1 and human
rhodanese, in which thiosulfate was reported to
exhibit substrate inhibition, although at high
concentrations, for sulfur transfer from thiosulfate
to GSH (21,42,43).
(
2
encoded by B. phytofirmans) to H S, which in
turn can be used for cysteine synthesis. In B.
cenocepacia, CysB and SsuR regulate genes
encoding enzymes involved in the aliphatic
sulphonate assimilation pathway (25,40). BLAST
analysis reveals that B. phytofirmans possesses
two enzymes that have 23 and 26% identity to the
CysB and SsuR transcriptional regulators,
respectively. Bioinformatic analysis of the
sequence 100 base pairs upstream of the BpPRF
gene queried against CysB and SsuR binding sites,
was performed using the FIMO MEME suite
database (25,40,41). Several SsuR binding sites
(
Fig. S2) scored as positive motif hits in the
BpPRF promoter region (p <0.005), suggesting a
role for BpPRF in sulfur assimilation.
The
catalytic
efficiency
of
BpPRF
6
-1 -1
(kcat/Km(GSSH) = 2 × 10 M s at 22 ˚C) is 14-fold
higher than of human PDO (1.4 × 10 M s at 22 ˚
14)) and ~10 -fold greater than CstB with its
preferred substrate, CoASSH (18 × 10 M s at 25
C) (23). Mutation of the catalytic cysteine
5
-1 -1
2
(
3
-1 -1
˚
residue, C314, in the rhodanese domain decreased
iron content 1.3-fold and PDO activity, 29-fold.
This result is surprising since it indicates
communication between the rhodanese and PDO
active sites, which are however distant and
oriented 90 ˚ away from each other in the crystal
structure (Fig. S1).
It is possible that our structure of BpPRF
represents an “open” conformation versus a
“
closed” conformation in which the two domains
are proximal. We therefore attempted to model a
putative closed conformation by superimposing
the PDO and rhodanese domains on the structure
of AaPRF. In the latter, the PDO and rhodanese
active sites face each other and are separated by a
distance of 27 Å (Fig. 9 A). AaPRF contains an
additional non-catalytic rhodanese domain and a
The
thiosulfate:cyanide
sulfurtransferase
-
1
activity of BpPRF (kcat = 5.1 s at 22 ˚C) is
comparable to that of CstB (8.5 s at 25 ˚C) (23),
and 180-730-fold lower than human rhodanese
-
1
cysteine-containing
loop
(C202,
AaPRF
(
20). The thiosulfate:GSH sulfur transfer activity
-
1
numbering) located in the PDO domain active site.
This loop, while absent in BpPRF, is also present
in CstB and postulated to facilitate substrate
transfer between the PDO and catalytic rhodanese
domains (23). To adopt a similar domain
orientation, the linker region of BpPRF, which is
maximally ~36 Å long, would have to span 47 Å
requiring unfolding of a secondary structure
element. Furthermore, the calculated electrostatic
surface potential reveals that positively charged
residues line the entrance to the PDO and
rhodanese active sites, suggesting that a direct
interaction between them would be unfavorable
of BpPRF (kcat = 6.2 s at 37 ˚C) is 9-fold higher
-
1
than for human rhodanese (0.67 s at 37 ˚C),
which preferentially catalyzes the reaction in the
reverse GSSH:sulfite sulfur transfer direction (kcat
-
1
=
389 s at 25 ˚C). The isolated rhodanese domain
was well-folded as evidenced by its high T (55 ±
°C) like that of the full-length protein (50 ± 2
C) and its comparable kcat values in the
m
2
°
thiosulfate:cyanide and thiosulfate:GSH sulfur
transfer reactions (Table 4). The isolated
rhodanese domain exhibits
dependence on GSH and thiosulfate concentration
Fig 6D, 7D), in contrast to the hyperbolic
a
sigmoidal
(
6

(
Fig. 9B). The structural independence of the
Expression constructs for BpPRFs-The cDNA
encoding BpPRF was amplified from genomic
DNA isolated from Burkholderia phytofirmans
PsjN (purchased from DSMZ, Braunschweig,
Germany), using the following primers containing
NdeI and HindIII restriction sites (bold font):
forward 5′-TTAATTCATATG TTGATCTTCCG
GCAGCTATTC-3′ and reverse 5′-TTAATT
AAGCTTCTACACGCTGTTTTCAACGACGG-
3′. The resulting PCR fragment was cloned into a
pET28b vector. The C-terminal truncated BpPRF
(ΔC-BpPRF) construct missing the last 4 residues
active sites is also consistent with the high
catalytic activity of the isolated rhodanese domain.
This result is in contrast to CstB in which the
rhodanese domain once isolated, does not retain
activity (23). Fusion of the PDO and rhodanese
domains in BpPRF would serve to increase their
effective local concentration and promote transfer
of substrates between them.
An overlay of five structures, i.e. two per
asymmetric unit of wild-type BpPRF and three per
asymmetric unit of C314S BpPRF, was used to
assess significant conformational differences, if
any, between them (Fig. S3). Since the electron
density for eight residues in the linker region was
missing in these structures, the overlay was used
to assess whether the flexible linker might allow a
closer approach of the rhodanese and PDO
(
ΔC 354-357), was generated for crystallography
only, using the full-length BpPRF expression
construct as a template and the following primers
containing NdeI and HindIII restriction sites (bold
font):
forward
5′-TTAATTCATATGTTGA
TCTTCCGGCAGCTATTC-3′ and reverse 5′-TTA
ATTAAGCTTCTATTCAACGACGCGGCC-3′.
The resulting PCR fragment was cloned into a
pET28b vector. The, C314S mutant, was
generated using the QuickChange kit (Stratagene)
and the wild-type BpPRF expression construct as a
template. The isolated rhodanese domain was
subcloned from the wild-type PRF expression
construct using the following primers containing
NdeI and HindIII restriction sites (bold font):
forward 5′-TTAATTCATATGATGACCGAGCC
CGATTTGGCG-3′ and reverse 5′-TTAATTA
AGCTTCTACTGCGTGGGGACGCTCGC-3′.
The resulting PCR fragment was cloned into a
pET28b vector.
domains, permitting
a
closed conformation.
However, only slight conformational differences
were observed in the relative orientation of the two
domains (Fig. S3).
In summary, the relative orientation of the two
domains in BpPRF and their separation by a short
linker, suggests that the two active sites do not
interact. There is also no support for an alternative
model in which the protein dimerizes in a head-to-
tail manner bringing the two active sites in
promixity. In fact, the similar electrostatic
potential surfaces of the two domains also suggest
that the active sites are unlikely to interact
directly. In light of the structural data, it is
intriguing that the kinetic data provides some
evidence for allosteric communication between the
domains. For instance, mutation of the catalytic
cysteine in the rhodanese domain adversely
impacts the catalytic properties and to a lesser
extent, the iron content of the PDO domain.
Similarly, isolation of the rhodanese domain,
improves its kcat. Our kinetic data are consistent
with a role for BpPRF in sulfur assimilation rather
than in a sulfide stress response pathway.
Expression and purification of BpPRF-The
recombinant BpPRF protein was expressed in
BL21 E.coli. A 200 mL culture in Luria Bertani
o
medium was grown overnight at 37 C and used to
inoculate a 4 × 1 L culture in the same medium.
Cultures were grown at 28 °C and expression was
induced with isopropyl-β-D-thiogalactopyrano-
side (100 µM) when the optical density at 600 nm
reached 0.5. Cultures were supplemented with
ferrous ammonium sulfate at the time of induction
to a final concentration of 250 µM and growth was
continued for an additional 14 h at 28 °C. Cells
were harvested by centrifugation at 2683 × g for
Experimental Procedures
Materials Sodium sulfide, sodium sulfite,
sodium thiosulfate, GSH, oxidized glutathione, L-
cysteine, para-rosaniline hydrochloride and 2,4,6-
tripyridyl-S-triazine were purchased from Sigma.
FluoroPure grade monobromobimane was
purchased from Life Technologies.
2
0 min at 4 °C.
BpPRF was purified as follows. The cell pellet
from a 4-liter culture was resuspended in 500 mL
of 50 mM Tris buffer, pH 8, containing 0.5 M
NaCl (Buffer A), 1 tablet of protease inhibitor
7

cocktail (Roche) and 100 mg of lysozyme
Sigma). DNase (50 mg) and MgCl (10 mM final
Molecular
mass
determination-Purified
(
2
BpPRF (5 mL of 3.7 mg/mL) was loaded onto a
HiLoad™ 16/60 Superdex G-200 column
equilibrated with Buffer A at 4 °C and calibrated
with protein standards (Bio-Rad). The protein was
eluted at a flow rate of 0.5 mL min and was
monitored by absorbance at 280 nm.
Thermal denaturation assay-The thermal
stability of BpPRF and of the C314S rhodanese
domain mutant was evaluated by monitoring the
increase in absorbance at 600 nm with increasing
temperature. For this, enzyme (100 µg) in Buffer
A (final volume 200 µL) was placed in a cuvette
housed in a Cary 100 Bio spectrophotometer
equipped with a heating block connected to a
water bath. The temperature was increased from
25-70 °C in 5 °C increments.
concentration) were added to the cell suspension
and stirred at 4 °C for 60 min followed by
sonication on ice with the following pulse
sequence: 30 sec burst, 1 min rest for a total burst
time of 5 min at a power output setting of 6. The
sonicate was centrifuged at 8217 × g for 15 min at
-
1
4
°C. The resulting supernatant was diluted 2-fold
with Buffer A and loaded onto a 20 mL Ni-NTA
column equilibrated with the same buffer. The
column was washed with 500 mL of Buffer A
containing 30 mM imidazole buffer. PRF was
eluted from the column with a linear gradient
ranging from 30-500 mM imidazole in Buffer A.
Fractions containing the PRF protein were pooled,
concentrated, and dialyzed overnight against 4 L
of Buffer A and stored at -80 °C. Protein
concentration was determined using the Bradford
reagent (BioRad) with BSA as a standard. The
ΔCBpPRF, the C314S mutant and the isolated
rhodanese domain were purified using the same
protocol.
Metal analysis-Plasma emission spectroscopy
was used to analyze the total metal content of
wild-type BpPRF at the Chemical Analysis
Laboratory (University of Georgia, Athens).
Twenty metal ions are detected by this method.
The iron content of the enzyme was also measured
using a colorimetric assay described previously
Preparation of GSSH-GSSH was prepared
anaerobically as described previously (14) by
reacting oxidized glutathione (GSSG) with Na2S
in a Coy anaerobic chamber (atmosphere of 95:5
N
2
:H
GSSG + Na
Briefly, solid Na
2
) (Eq. 10).
2
S → GSSH + GSH
[10]
2
S was added in 4-fold excess, to
an anaerobic solution of 50 mM GSSG in 350 mM
sodium phosphate, pH 7.4 (final volume 5 mL).
The reaction vial was immediately sealed to
prevent loss of Na
5-30 min. The concentration of GSSH was
2
S and incubated at 37 °C for
2
(
32). This method allows for the direct
measured using the cold cyanolysis method as
described previously (14). Substrate was either
used immediately or stored at -20 °C until use.
Substrate concentration was measured both before
freezing and after thawing prior to use in the
enzyme assay. Cysteine persulfide was prepared
using the same procedure.
measurement of total iron content, the Fe(II)
content and consequently, estimation of Fe(III)
content of the enzyme. Briefly, BpPRF (10-50
µM) was denatured with 0.5 N HCl and 5% (w/v)
trichloroacetic acid (final volume 1 mL), mixed
for 30 s and subsequently centrifuged for 10 min
at 16,000 × g in a microcentrifuge. To determine
the total iron content, the supernatant (700 µL)
was mixed with 300 µL of a 1:2:1 mixture of 4
mM 2,4,6-tripyridyl-S-triazine, 50 % ammonium
acetate, and 10 % hydroxylamine chloride, and
incubated at room temperature for 5 min.
Hydroxylamine is present to reduce residual
Fe(III) to Fe(II). Absorbance of the resulting
Fe(II)-2,4,6-tripyridyl-S-triazine complex was
measured at 569 nm. The total Fe concentration
was calculated using an extinction coefficient of
Oxygen consumption assay-The PDO activity
of BpPRF was measured by monitoring O
2
consumption at room temperature (22 ˚C). The
reaction mixture consisted of 100 mM sodium
phosphate, pH 7.4, and 0.5-2 µg of enzyme (final
volume 1.6 mL) mixed in a Gilson-type chamber
containing a Clark oxygen electrode and a
magnetic stir bar. The reaction was initiated by
addition of GSSH (0.02-3 mM). The effects of
2
GSH, Na S, sulfite and thiosulfate on the PDO
-
1
-1
activity were assessed at 5 mM concentrations of
each except for sulfite, which was added to a final
concentration of 0.6 mM. Cysteine persulfide was
2
2,600 M cm for the Fe(II)-2,4,6-tripyridyl-S-
triazine complex (32).
8

tested as an alternative substrate at a final
concentration of 1 mM. The dependence of the
Thiosulfate:GSH sulfurtransferase activity
monitored by sulfite formation-The
reaction velocity on
O
2
concentration was
thiosulfate:GSH sulfurtransferase activity of
BpPRF and of the isolated rhodanese domain was
monitored by detecting sulfite formation using a
modified version of the colorimetric assay
described previously (45). The reaction mixture
contained 45 mM thiosulfate, 45 mM GSH, 0.7-3
µg enzyme and 200 mM sodium phosphate buffer,
pH 7.4 (final volume of 500 µL). The reaction was
initiated by addition of enzyme. After 5 min of
incubation at 22 ˚C, the reaction was stopped by
determined as follows. The reaction mixture
containing enzyme and the GSSH substrate in 100
mM phosphate, pH 7.4, was prepared
anaerobically in an inflatable glove bag (Cole-
Palmer) filled with N
reaction was started by injecting a known
concentration of dissolved O (generated using a
00% oxygen tank). The final O concentration
varied from 1.6-250 µM. The O concentration in
oxygenated buffer was independently determined
using the O probe.
2
(>99% purity). The the
2
1
2
2
2
the addition of 500 µl of 0.23 M HgCl . After
2
centrifugation at 16,000 × g, for 1 min, the
supernatant (125 µL) was mixed with 1 mL of a p-
rosaniline solution containing 2:1 0.04% p-
rosaniline prepared in 0.72 M HCl (w/v):0.2%
formaldehyde. The absorbance of the resulting p-
rosaniline sulfonic acid complex was measured at
Thiosulfate:cyanide sulfurtransferase assay-
The cyanide detoxification activity of BpPRF was
measured in a colorimetric assay as described
previously (44). The reaction mixture contained 60
mM thiosulfate, 60 mM potassium cyanide and 5-
1
0 µg protein in 200 mM sodium phosphate
5
70 nm. The concentration of sulfite formed was
buffer, pH 7.4 (330 µl final volume). The reaction
was initiated by addition of enzyme. After
incubation for 20 min at 22 ˚C, the reaction was
stopped by addition of 100 µL of 15 % (w/v)
formaldehyde. Thiocyanate formation was
measured by addition of 500 µL of a ferric nitrate
solution containing 165 mM ferric nitrate
nonahydrate and 13.3% (v/v) nitric acid. The
absorbance of the resulting ferric thiocyanate
complex was measured at 460 nm. The
concentration of thiocyanate formed was
determined using standard curve. The
a
concentrations of thiosulfate and GSH were varied
from 0.3-50 mM to determine the dependence of
enzyme activity on substrate concentration.
Thiosulfate:thiol sulfurtransferase activity
monitored by H
activities of BpPRF and of the isolated rhodanese
domain was measured by detecting H S formation
using the lead acetate assay described previously
2
S formation-The sulfurtransfer
2
(
(
18). The reaction mixture contained thiosulfate
0.5-25 mM) and either GSH (2-40 mM) or
determined using
a
standard curve. The
cysteine (1-50 mM) in 200 mM sodium phosphate
buffer, pH 7.4 (final volume of 1 mL) and was
pre-incubated at 37 °C for 10 min. The reaction
was initiated by addition of 0.7-15 µg enzyme.
Formation of lead sulfide was measured at 390 nm
concentrations of cyanide and thiosulfate were
varied from 0.5-60 mM to determine the
dependence of enzyme activity on substrate
concentration.
Thiosulfate:GSH sulfurtransferase activity
monitored in a coupled assay-The GSSH formed
via the sulfur transfer activity of BpPRF was
and the concentration of H
2
S formed was
calculated using an extinction coefficient of 5,500
-
1
-1
M cm for lead sulfide (18).
2
measured by coupling to O consumption during
GSSH:sulfite sulfurtransferase actvity-The
reactions were performed in a Coy anaerobic
chamber to prevent oxidation of GSSH by the
PDO domain. The standard reaction mixture
contained 1 mM GSSH and 1 mM sulfite, 0.5-50
µg enzyme, and 20 mM sodium phosphate buffer,
pH 7.4 (200 µL final volume). The derivatization
reaction was initiated by the addition of enzyme.
After 5 min of incubation, monobromobimane was
added to a final concentration of 3 mM and
incubated at room temperature for 15 min. The
GSSH utilization by the PDO activity. For this, the
reaction mixture contained 30 mM thiosulfate, 30
mM GSH, 25-30 µg enzyme and 200 mM sodium
phosphate buffer, pH 7.4 (final volume 1.6 mL)
mixed in a Gilson-type chamber containing a
Clark oxygen electrode at 22 °C. GSH and
thiosulfate concentrations were varied from 0.3-30
mM to determine the dependence of enzyme
activity on substrate concentration.
9

derivatization reaction was terminated by addition
of 100 µL of 0.2 M sodium citrate, pH 2.0.
Production of thiosulfate from GSSH and sulfite
was monitored by HPLC. For this, a 60 mM stock
solution of monobromobimane was prepared in
DMSO and protected from light during
preparation and handling. Bimane adducts of
quenched with 100 µL of 0.2 M sodium citrate, pH
2.0. The derivatized samples were processed and
separated by HPLC as described above to
determine the concentrations of sulfite formed and
thiosulfate consumed.
Reaction stoichiometry under single and
multiple
turnover
conditions-The
reaction
GSSH, Na
2
S, GSH, sulfite, and thiosulfate, were
stoichiometry of wild-type and C314S BpPRF
under single turnover conditions was monitored as
described above with the following variation. The
reaction mixture contained 250 µM thiosulfate,
250 µM GSH, and varying concentrations of
enzyme (250, 125, 83, 62.5, 50 and 42 µM). The
reaction was initiated by addition of GSH. After
incubation for 30 min at 22 °C, an aliquot of the
reaction mix (50 µL) was mixed with 20 µL of 60
mM monobromobimane and processed as
described above. For analysis of the reaction
stoichiometry under single turnover conditions in
prepared in 20 mM sodium phosphate, pH 7.4.
Each standard (final concentration 250 µM) was
incubated with a final concentration of 3 mM
monobromobimane (200 µL final volume) for 10
min at 22 ˚C.
The derivatized samples were centrifuged at
1
0,000 × g for 10 min at 4 °C, and the
supernatants were separated on a 4.6 × 150 cm
C18 reverse phase HPLC column (3 µM packing,
SunFire) using an Agilent 1100 series HPLC
system equipped with a multi-signal fluorescence
detector. The column was equilibrated with the
following solution: 80 % solvent A (897.5 mL
water, 100 mL methanol, and 2.5 mL acetic acid)
and 20 % solvent B (97.5 mL water, 900 ml
methanol, and 2.5 ml acetic acid). Samples (50
µL) were injected onto the column and resolved
using the following gradient of solvent B: 20 %
from 0-5 min, 20-45 % from 5-10 min, 45 %
isocratic from 10-15 min, 45-50 % from 15-25
min, 50 % isocratic from 25-28 min, 50-100 %
from 28-30 min, 100 % isocratic from 30-39 min,
the absence of O
derivatization reactions were performed in a Coy
anaerobic chamber (atmosphere of 95:5 N :H ).
Protein crystallization-The C-terminal
2
, the enzymatic assay and
2
2
truncated BpPRF (ΔCBpPRF) and C314S BpPRF
were crystallized by sitting drop vapor diffusion
method from a 0.75 µL:0.75 µL mixture of protein
stock and well solution. ΔCBpPRF (5 mg/mL in
5
0 mM Tris, pH 8, 0.25 M NaCl, 10 % glycerol, 5
mM thiosulfate) crystallized in 3 days at 20 °C
using a well solution of 0.2 M zinc acetate, 0.1 M
MES, pH 6, 10 % PEG-8000. A single
rhombohedral rod-shaped crystal was harvested
directly from the drop and flash-cooled in liquid
nitrogen. C314S BpPRF (5 mg/mL in 50 mM Tris,
pH 8.0, 0.25 M NaCl, 10% glycerol) crystallized
in 2 days using a well solution of 0.12 M NaCl,
1
4
00-20 % from 39-42 min, 20 % isocratic from
2-48 min. The flow rate was 0.75 min/mL.
Fluorescence of the bimane adducts was detected
using 340 nm excitation and 450 nm emission.
The concentration of thiosulfate was determined
using the peak areas and a standard curve.
2
Stoichiometry of O consumption and sulfite
0
.1 M Imidazole, pH 8.0, 25 % PEG-8000. To
formation-The stoichiometry of coupling between
the PDO and rhodanese active sites was monitored
using wild-type and C314S BpPRF as follows. The
reaction mixture contained 1 mM GSSH, 75 nM
enzyme, and 200 mM sodium phosphate buffer,
pH 7.4 (1.6 mL final volume) in a Gilson-type
chamber containing a Clark oxygen electrode. The
obtain structural data for C314S BpPRF in
complex with GSH, 5 mM GSH was added to the
drop overnight. The hexagonal rod-shaped crystals
were cryo-protected by passing harvested crystals
through 2 µL of reservoir solution with an
additional 15% glycerol followed by flash cooling
in liquid nitrogen.
Data collection and crystal structure
determination-Diffraction data were collected at
the Advanced Photon Source (APS, Argonne
National Laboratory) on the GM/CA beamline
reaction was initiated by addition of GSSH and O
2
consumption was recorded. After 5 min at 22 °C,
an aliquot of the reaction mix (50 µL) was added
to 60 mM monobromobimane (20 µL) to a final
concentration of 17 mM. After incubation for 15
min at room temperature, the reaction was
2
3ID-D or 23ID-B (Table 1). For ΔCBpPRF, data
were collected on a single crystal to 1.79 Å
1
0

resolution. For the apo- and GSH-soaked C314S
BpPRF crystals, data were collected to 2.65 and
residues excepting eight residues in the linker
region between the PDO and rhodanese domains
and the N-terminal histidine tag. The C314S
structure was solved via molecular replacement
using Phaser within the Phenix software suite with
the complete ΔCBpPRF model as a search model.
Molecular replacement successfully placed three
PDO domains and two rhodanese domains. The
third rhodanese domain is poorly ordered and was
built manually. Density is poor for this domain
compared to the rest of the structure. The GSH-
bound structure was solved via rigid body
refinement in Phenix Refine. Electrostatic surface
potentials were calculated using the APBS
program in PYMOL (51-53). Figures were
prepared using PYMOL.
2
.35 Å, respectively. For the C314S BpPRF
crystals, data quality was limited by high
mosaicity along the c axis. All data were
processed using XDS (46). The truncated
ΔCBpPRF structure was solved by molecular
replacement using BALBES (47) with the AaPRF
(
PDB ID: 3TP9), which shares 28% sequence
identity with BpPRF, as a search model. Phenix
AUTOBUILD was used to build ~70 % of the
model. To complete the model, consecutive rounds
of model building and refinement were performed
using Coot (48) and Phenix Refine (49). The
structures were validated using MolProbity (50).
Electron density is complete for all BpPRF
Acknowledgements
This work was supported in part by the National Institutes of Health (GM112455 to RB) and an NIGMS
Training Grant support (T32GM008353 to NM).
Author Disclosure Statement
No competing financial interest exists
Author contributions
N.M. designed, performed and analyzed the experiments and wrote the manuscript. N.M. and M.S. solved
the crystal structures. M.S. and J.S. assisted with the crystallography experiments, data collection and
analysis. O.K. helped design and perform the O consumption experiments. R.B. helped conceive the
2
experiments, analyzed the data and co-wrote the manuscript. All authors approved the final version of the
manuscript.
1
1

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1
4

TABLE 1. X-ray data collection and refinement statistics
ꢀ
Data Collection
PDB ID
Space group
Wild-type PRF
5VE3
C314S PRF-Apo C314S PRF-GSH
5VE4 5VE5
P6 22 P6 22
P2
1
2
1
2
1
5
5
Cell dimensions
a,b,c (Å)
63.7, 108.3, 119.6 84.5, 84.5, 549.4
83.5, 83.5, 547.6
90, 90, 120
α, β, γ (°)
90, 90, 90
90, 90, 120
APS 23ID-B
1.033
X-ray Source
Wavelength (Å)
APS 23ID-D
1.033
APS 23ID-B
1.033
1
d
min (Å)
1.79 (1.86-1.79)
0.119 (1.47)
0.045 (5.3 Å)
14.6 (1.2)
100 (96.0)
12.4 (9.0)
966,980
2.65 (2.75-2.65)
2.35 (2.43-2.35)
R-merge
Inner-shell R-merge
Avg I/σ(I)
0.126 (1.35)
0.047 (7.8 Å)
11.6 (1.7)
0.126 (1.56)
0.050 (7.0 Å)
11.0 (1.6)
2
Completeness (%)
Multiplicity
100 (100)
8.6 (8.5)
99.0 (99.0)
8.7 (9.2)
Total observations
303,925
422,379
(
67,309)
(28,966)
(43,869)
CC1/2
0.999 (0.537)
0.997 (0.898)
0.998 (0.904)
CC*
1.0 (0.836)
0.999 (0.973)
0.999 (0.975)
Refinement
Data range (Å)
Reflections used in refinement
41.26-1.79
77,982 (7,448)
16.1/19.6
6051
43.94-2.65
35,305 (3,392)
23.2/26.7
8265
41.75-2.35
48,680 (4,773)
19.7/25.2
8471
R
work/Rfree (%)
Number of non-hydrogen atoms
protein
5538
8153
8154
ligands
2
24
74
water
511
88
189
Amino acid residues
Deviation from ideality
bond lengths (Å)
bond angles (°)
Average B-factor
macromolecules
ligands
698
1047
1048
0.013
1.20
38.1
37.6
29.6
44.1
0.003
0.70
81.9
82.4
59.0
45.6
0.007
0.97
76.6
77.0
83.1
58.4
solvent
Ramachandran plot
favored (%)
98.0
2.0
0
96.0
4.0
0
97.5
2.3
allowed (%)
outliers (%)
0.2
1
2
Values in parentheses pertain to the outermost shell of data.
min inner shell
d
1
5

a
TABLE 2. Comparison of the PDO activities of wild-type and C314S BpPRF.
Enzyme
Iron
V
max
K
m
(GSSH)
K
m
(O
2
)
k
cat
k
cat/K
m
kcat/K
m
Content
(GSSH)
2
(O )
-
1
-1
-1 -1
-1 -1
mol Fe/mol µmol min
mM
mM
s
mM s
mM s
-
1
mg
Wild type 0.60 ± 0.03
207 ± 6
0.070 ± 0.008 0.13 ± 0.03 143
0.37 ± 0.04 ---
2043
14
1100
---
C314S
0.46 ± 0.02
7.3 ± 0.1
5
a
The kinetic parameters were determined by monitoring O
2
consumption in the presence of GSSH at 22
˚
C and pH 7.4, as described under Experimental Procedures.
1
6

a
TABLE 3. Kinetic parameters for the sulfurtransferase activity of BpPRF sulfurtransferase
Donor Acceptor
V
max
K
m
K
m
k
cat
k
cat/K
m
kcat/K
m
Donor
Acceptor
Donor
Acceptor
-
1
-1
-1
-1 -1
-1 -1
µmol min mg
7.4 ± 0.3
nd
mM
mM
s
mM s
mM s
2
-
-
1
. S
2
O
3
CN
5.4 ± 0.7 22.7 ± 0.7 5.1
0.9
nd
0.2
nd
2
-
2
3
4
. GSSH
SO
3
nd
nd
nd
6.2
0.2
2
2
-
-
. S
. S
2
2
O
O
3
3
GSH
Cys
8.9 ± 0.2
0.32 ± 0.01
3.1 ± 0.2 4.7 ± 0.4
5.8 ± 0.8 5.2 ± 0.4
2.0
1.3
0.04
0.04
a
The kinetic parameters for the sulfur transfer reactions 1-4 were determined at pH 7.4 as follows.
Reaction 1 monitored thiocyanate formation (22 ˚C), Reaction 2, monitored thiosulfate formation (22 ˚C),
Reaction 3, monitored sulfite formation (at 22 ˚C), or GSSH formation (via O consumption in the
coupled PDO reaction at 22 ˚C), or H S formation (37 ˚C), and Reaction 4, monitored H S formation (37
C), as described in the Experimental Procedures.
2
2
2
˚
1
7

a
TABLE 4. Kinetic parameters for the isolated rhodanese domain of BpPRF.
Donor
Acceptor
V
max
K
m
K
m
k
cat
k
cat/K
m
kcat/K
m
Donor
mM
Acceptor
mM
Donor
Acceptor
-
1
-1
-1
-1 -1
mM s
-1 -1
mM s
µmol min mg
s
2
-
-
1
. S
2
O
3
CN
18 ± 1
9.2 ± 0.8
23 ± 1
4.6
0.5
nd
0.2
nd
2
-
2
3
4
. GSSH
SO
3
nd
nd
nd
nd
2
2
-
-
. S
. S
2
2
O
O
3
3
GSH
Cys
35 ± 2
3.1 ± 0.1
3.8 ± 0.5
6.1 ± 0.4
14.9 ± 0.5 9.0
5.4 ± 0.3 0.8
2.3
0.1
0.6
0.1
a
The kinetic parameters of the sulfur transfer reactions catalyzed by the isolated rhodanese domain were
determined as summarized in Table 3 legend and described in detail under Experimental Procedures.
1
8

Figure 1. Organization of BpPRF and limited sequence comparison. A, Domain organization of PRFs.
PRFs form S. aureus (SaPRF) and A. acidocaldarius (AaPRF) have an N-terminal PDO domain, a non-
catalytic rhodanese (RHOD) domain and a catalytic rhodanese domain. BpPRF displays an N-terminal
PDO domain and C-terminal catalytic rhodanese (Rhod) domain. B, Sequence alignment of rhodanese
active site sequences from human (hTST) and bovine (RhoBov) rhodanese, human mercaptopyruvate
sulfurtransferase (MST), E. coli single domain rhodanese (GlpE), human mitochondrial single-domain
sulfurtransferase (TSTD1), S. aureus PRF (SaPRF or CstB), B. phytofirmans PRF (BpPRF), A.
acidocaldarius PRF (AaPRF). The conserved active site cysteine is highlighted in yellow. C, Comparison
of the genomic contexts of CstB (top) and BpPRF (bottom). The operon encoding CstB (pink) includes a
multi-domain rhodanese (CstA, red) and an SQR homolog (light green). The adjacent locus includes the
polysulfide sensing repressor CsTR (yellow) and TauE (purple),
a
putative sulfite
permease/transmembrane sulfur compound exporter. The loci adjacent to BpPRF (pink) include a putative
peroxidase (cyan) and pyridoxal 4-dehydrogenase (blue), a putative transmembrane (TMS)-type
transporter (dark green) and a putative LysR-type regulator (orange).
1
9

Figure 2. Crystal structure of BpPRF. The X-ray crystal structure of ΔCBpPRF was solved at 1.79 Å
resolution by molecular replacement using the AaPRF structure (PDB ID: 3TP9) as a template. A, The
structure of the BpPRF monomer consists of an N-terminal PDO domain (blue), a fifteen residue linker
region (orange) and a C-terminal rhodanese domain (red). The linker region lacked density for eight
residues (dashed lines) suggesting a flexible, disordered state. The iron (orange sphere) ligands in the
PDO domain and the active site cysteine, Cys314, in the rhodanese domain are shown in stick
representation. The rhodanese domain β-hairpin extension is highlighted by the red arrow. B, close-up of
the PDO active site with representative electron density (3.0σ Fo-Fc omit density; grey mesh) for side
chains of His58, His114, Asp133 and three water molecules (shown as red spheres) coordinated to the
iron center (orange sphere). C, Close-up of the rhodanese domain active site with representative electron
density (3.0σ Fo-Fc omit density; grey mesh) for the Cys314 side chain displaying the additional density
for the persulfide modification.
2
0

Figure 3. Close up of the PDO active site of BpPRF with GSH. A, Stereo view of the C314S BpPRF
PDO domain with GSH bound. The surrounding residues that may participate in substrate binding and/or
stabilization are shown with electron density (3.0σ Fo-Fc omit density; grey mesh) for GSH. Tyr176,
Arg142, and Lys216 are hydrogen bonded (black dashes) to GSH. Two waters (red spheres) are
coordinated to the iron center (orange sphere) in addition to the sulfur atom of GSH. B, Stereo veiw of
C314S BpPRF PDO domain (blue) in complex with GSH overlaid on human PDO (15) (PDB ID: 4CHL)
(
grey). GSH (shown in green) is displyed in stick representation. Conserved residues predicted to be
involved in substrate binding, stabilization and/or positioning (15) are Ty176, Arg193, Leu212, Arg142
and Pro215 (BpPRF numbering). Lys216 is not conserved in human PDO.
2
1

Figure 4. Comparison of the BpPRF rhodanese domain with rhodanese. A, Structural overlay of the
BpPRF rhodanese domain (red) with the catalytic domain of bovine rhodanese (PDB 1RHD, grey (34)).
The active site cysteine residues, C247 (bovine) and C314 (BpPRF) are shown in stick representation
Structural differences are shown with arrows: a β-hairpin extension seen only in BpPRF (red) and two
loop extensions seen only in bovide rhodanese (black). B, structural overlay of the rhodanese active site
loops from BpPRF (red) and bovine rhodanese (grey).
2
2

Figure 5. Kinetics of PDO activity of BpPRF. Dependence of PDO activity on GSSH concentration for
wild-type BpPRF (solid circles) and C314S BpPRF (open circles). Oxygen consumption by BpPRF in 100
mM sodium phosphate buffer, pH 7.4, was monitored at 22 ˚C in the presence of varying concentration of
GSSH. The data are representative of three independent experiments.
2
3

Figure 6. Kinetics of BpPRF-catalyzed sulfur transfer reactions. The sulfurtransferase activity
associated with the rhodanese domain in wild-type BpPRF (A, C, E) or with the isolated rhodanese
domain (Rhod domain) (B, D, F) was determined in the presence of a constant thiosulfate concentration
(
60 mM) and varying concentrations of cyanide (A and B), GSH, (C and D) or cysteine (E and F). The
data are representative of 3-4 independent experiments. The data were fitted with the Michaelis Menten,
sigmoidal or Hill equation as described under Experimental Procedures.
2
4

Figure 7. Kinetics of BpPRF-catalyzed sulfur transfer reactions. Dependence of sulfurtransferase
activty on varying thiosulfate concentrations in the presence of 60 mM cyanide (A and B), 30 mM GSH
(
(
C and D), and 50 mM cysteine (E and F) for the wild-type BpPRF and the isolated rhodanese domain
Rhod Domain). The reactions were performed as described under Experimental Procedures. The data are
representative of 3-4 independent experiments. Data were fitted with either Michaelis Menten or Hill
equation.
2
5

2
6

Figure 8. Product analysis and reaction stoichiometry of the BpPRF-The reaction stoichiometry of
BpPRF under single turnover conditions was analyzed in the presence (A) and absence (B) of oxygen. The
reactions were performed in the presence of 250 µM GSH, 250 µM thiosulfate and 250 µM enzyme. C,
The stoichiometry of BpPRF reactions under multiple turnover conditions was analyzed in the presence of
2
50 µM GSH, 250 µM thiosulfate and decreasing enzyme concentrations as described under
Experimental Procedures. Data are representative of 4-5 independent experiments.
2
7

Figure 9. Modeling of potential BpPRF domain interaction interface. A, overlay of BpPRF PDO
domain (blue) and the BpPRF rhodanese domain (red) with the PDO domain and catalyic rhodanese
domain of AaPRF (PDB: 3TP9) (shown in grey). The active site loop of AaPRF (yellos) with C202 faces
the iron center (orange sphere) in the PDO domain. B, Electrostatic surface potential of the BpPRF
domains in the same orientation as in A but rotated by 35˚. The arrows highlight locations of the PDO and
rhodanese (Rhod) active sites. Positive and negative electrical potential are shown in blue and red,
respectively and represent a range of -5 to +5 kT/e.
2
8

Structural and biochemical analyses indicate that a bacterial persulfide
dioxygenase-rhodanese fusion protein functions in sulfur assimilation
Nicole Motl, Meredith A Skiba, Omer Kabil, Janet L Smith and Ruma Banerjee
J. Biol. Chem. published online July 6, 2017
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