Catalytic role of a conserved cysteine residue in the desulfonation reaction by the alkanesulfonate monooxygenase enzyme

Article abstract of DOI:10.1016/j.bbapap.2009.09.014

Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to either serine or alanine had little effect on FMNH₂ binding. The k_cat/K_m value for the C54S SsuD variant increased 3-fold, whereas the k_cat/K_m value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH₂ was mixed against C54S SsuD (k_obs, 111 s^{- 1}) in oxygenated buffer that was 10-fold faster than wild-type SsuD (k_obs, 12.9 s^{- 1}). However, there was no initial fast phase observed in similar kinetic traces obtained for C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-(hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-(hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.

Full text of DOI:10.1016/j.bbapap.2009.09.014

Biochimica et Biophysica Acta 1804 (2010) 97–105
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Catalytic role of a conserved cysteine residue in the desulfonation reaction by the
☆
alkanesulfonate monooxygenase enzyme
Russell A. Carpenter a,b, Xuanzhi Zhan a,c, Holly R. Ellis a,⁎
a
The Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama, 36849, USA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331-4501, USA
Pharmacology Department, Vanderbilt University, Nashville, TN 37232-6600, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2009
Received in revised form 8 September 2009
Accepted 11 September 2009
Available online 19 September 2009
Detailed kinetic studies were performed in order to determine the role of the single cysteine residue in the
desulfonation reaction catalyzed by SsuD. Mutation of the conserved cysteine at position 54 in SsuD to
either serine or alanine had little effect on FMNH
increased 3-fold, whereas the kcat/K value for C54A SsuD decreased 6-fold relative to wild-type SsuD. An
initial fast phase was observed in kinetic traces obtained for the oxidation of flavin at 370 nm when FMNH
2 m
binding. The kcat/K value for the C54S SsuD variant
m
2
−
1
was mixed against C54S SsuD (kobs, 111 s ) in oxygenated buffer that was 10-fold faster than wild-type
Keywords:
Alkanesulfonate monooxygenase
FMN reductase
SsuE
SsuD
−
1
SsuD (kobs, 12.9 s ). However, there was no initial fast phase observed in similar kinetic traces obtained for
C54A SsuD. This initial fast phase was previously assigned to the formation of the C4a-(hydro)peroxyflavin
in studies with wild-type SsuD. There was no evidence for the formation of the C4a-(hydro)peroxyflavin
with either SsuD variant when octanesulfonate was included in rapid reaction kinetic studies, even at low
octanesulfonate concentrations. The absence of any C4a-(hydro)peroxyflavin accumulation correlates with
the increased catalytic activity of C54S SsuD. These results suggest that the conservative serine substitution
is able to effectively take the place of cysteine in catalysis. Conversely, decreased accumulation of the C4a-
Rapid reaction kinetic
(
hydro)peroxyflavin intermediate with the C54A SsuD variant may be due to decreased activity. The data
described suggest that Cys54 in SsuD may be either directly or indirectly involved in stabilizing the C4a-
hydro)peroxyflavin intermediate formed during catalysis through hydrogen bonding interactions.
2009 Elsevier B.V. All rights reserved.
(
©
1
. Introduction
Sulfur is a critical element for the growth of bacterial organisms.
and a monooxygenase (SsuD). SsuD belongs to a family of reduced
flavin-dependent monooxygenases that use flavin as a substrate
instead of a bound prosthetic group [4]. It catalyzes the oxygenolytic
Many bacteria have inorganic sulfur requirements for the production
of sulfur-containing compounds. Inorganic sulfur is poorly repre-
sented in aerobic soil, therefore bacteria in these environments must
have an alternative process for obtaining this crucial element [1,2]. To
meet its sulfur requirements under sulfate-starvation conditions,
Escherichia coli synthesizes taurine dioxygenase and alkanesulfonate
monooxygenase proteins for the acquisition of sulfur from alternative
sources [3,4]. The expression of these proteins allows the organism to
utilize a broad range of alkanesulfonates during growth when
inorganic sulfur is limiting.
cleavage of 1-substituted alkanesulfonates in the presence of FMNH
2
and dioxygen, producing the corresponding aldehyde and sulfite. In
the proposed mechanism, the C4a-peroxyflavin (II in Scheme 1)
makes a nucleophilic attack on the sulfonate functional group to form
an initial alkanesulfonate peroxyflavin intermediate (III). A Baeyer-
Villiger rearrangement of the flavin adduct (III) followed by proton
abstraction by an active site base (IV) generates the aldehyde and
sulfite products (V). The reaction catalyzed by SsuD has been partially
evaluated through steady-state and rapid reaction kinetic analyses
[5]. The octanesulfonate substrate is unable to bind to SsuD unless
reduced flavin is present, suggesting that reduced flavin binds first to
induce a protein conformational change that allows the alkanesulfo-
nate to bind. The reactions catalyzed by many two-component flavin-
dependent monooxygenases have been shown to involve a C4a-
(hydro)peroxyflavin intermediate. The formation of the C4a-(hydro)
peroxyflavin intermediate in bacterial luciferase in the presence and
absence of the aldehyde substrate has been identified through kinetic
analyses [6–9]. This flavin intermediate was isolated at low tempera-
tures in the absence of the aldehyde substrate demonstrating the
stability of this intermediate in bacterial luciferase [10]. Rapid reaction
The two-component alkanesulfonate monooxygenase system is
1
composed of both an NAD(P)H -dependent flavin reductase (SsuE)
2
Abbreviations: FMN, flavin mononucleotide; FMNH , reduced flavin mononucleo-
tide; NAD(P)H, nicotinamide adenine dinucleotide (phosphate); SsuE, alkanesulfonate
flavin reductase; SsuD, alkanesulfonate monooxygenase
☆
This work was supported by the National Science Foundation (MCB-0545048 to
H.R.E.).
Corresponding author. Tel.: +1 334 844 6991; fax: +1 334 844 6959.
E-mail address: ellishr@auburn.edu (H.R. Ellis).
⁎
1570-9639/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2009.09.014

98
R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
Scheme 1.
kinetic evaluation of SsuD also suggests that the C4a-(hydro)
peroxyflavin intermediate is formed as proposed in the mechanism,
but this flavin intermediate is not as stable as the intermediate
generated in the bacterial luciferase reaction [5].
The SsuD enzyme exists as a homotetramer containing four active
sites, with each subunit composed of a triose phosphate isomerase
located in an insertion region containing an unstructured loop that is
thought to cover the active site once the substrates are bound.
Although direct substitution of Arg291 has not been performed,
deletion of this unstructured loop region in bacterial luciferase
resulted in a decrease in bioluminescence [23]. The catalytic role of
Cys106 in bacterial luciferase has been studied in extensive detail.
Initial studies demonstrated that chemical modification of the
reactive thiol of Cys106 resulted in the inactivation of the enzyme,
leading to the assumption that this residue was directly involved in
catalysis [24–26]. Substitution of Cys106 was shown to reduce
(
TIM)-barrel fold. The location of the SsuD active site has been
proposed to be located at the C-terminal end of the β-barrel, where
the active sites of most TIM-barrel enzymes are found [11]. The
identification of the active site is supported by earlier biochemical
studies, and the location of conserved amino acid residues in SsuD.
Although the amino acid sequence identity is low, SsuD is strikingly
similar in overall structure with bacterial luciferase and long-chain
alkane monooxygenase (LadA) [12–17]. While luciferase uses a long-
chain aliphatic aldehyde as a substrate to catalyze the production of
visible light and a corresponding carboxylic acid, LadA oxidizes long-
chain alkanes to primary alcohols. These enzymes catalyze distinctly
different biochemical reactions; however, they all are members of the
FMN-dependent monooxygenase family.
Several amino acids located in the putative active site of SsuD
(
His228, Tyr331, Arg297, and Cys54) are in a similar spatial
arrangement as proposed catalytically relevant amino acid residues
from bacterial luciferase (His44, Tyr110, Arg291, and Cys106) and
LadA (His311, Tyr63, and Cys14) (Fig. 1) [12–17]. The His44 residue in
the α-subunit of bacterial luciferase has been reported to be the
catalytic base in the bioluminescence reaction. Mutation of His44
residue to alanine showed marked differences in the intensity of light
emitted [18]. The bioluminescence activity could be reconstituted by
chemical rescue with the addition of imidazole to the reaction [19].
Substitution of the corresponding residue in LadA was shown to
inactivate the enzyme [17]. The conserved Tyr residue has been
evaluated in LadA where variants of this residue were shown to
completely abolish activity [17]. In the structure of bacterial luciferase
with bound FMN, Tyr110 is proposed to be involved in hydrogen
bonding interactions with flavin binding residue Asp113 [16,20–22]. A
conserved arginine residue in SsuD is also located in a similar position
to Arg291 in bacterial luciferase. In both enzymes, this residue is
Fig. 1. Putative active site of SsuD. The highlighted amino acids are spatially
conserved in bacterial luciferase (His44, Tyr110, Arg291, and Cys106) and LadA
(His311 and Tyr63). The active site was generated with Swiss PDBViewer (PDB
ID:1M41) and rendered with the MegaPov program (7).

R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
99
bioluminescence, and was suggested to be involved in the stabiliza-
tion of the C4a-(hydro)peroxyflavin intermediate in bacterial lucifer-
ase [27,28]. The Cys106 residue is not directly involved in catalysis,
but appears to play an indirect role in maintaining the proper active
site conformation for optimal stabilization of the C4a-(hydro)
peroxyflavin. Variants of the corresponding Cys14 residue in LadA
were unable to catalyze the oxidation of the alkane substrate [17].
Earlier studies demonstrated that cysteine-labeling of SsuD with
methylmercury leads to inactivation of the enzyme suggesting a
possible role of Cys54 in catalysis [12]; however, the role of the
spatially conserved cysteine residue in the desulfonation reaction
catalyzed by SsuD has not been evaluated.
phosphate buffer, pH 7.5, at 25 °C. Buffer exchange was performed to
remove all NaCl and glycerol using an Amicon Ultra Centrifugal Filter
(Millipore, Billerica, MA) with a MWCO of 10,000 kDa. Far-UV circular
dichroism spectra were recorded on a JASCO J-810 spectropolarimeter
(Easton, MD). Measurements were taken in 0.2 nm increments in
continuous scanning mode from 270 to 180 nm in a 0.1 cm path length
cuvette with a bandwidth of 1 nm and a scanning speed of 20 nm/min.
Each spectrum is the average of 8 accumulated scans.
2.4. pK
a
determination of Cys54
The pK
a
of Cys54 was determined by measuring the absorbance at
The three-dimensional structures of flavin-dependent enzymes
that utilize a proposed C4a-peroxyflavin in catalysis are increasingly
being determined [12–17,29,30]. A striking feature observed in these
structures is the importance of active site residues in maintaining the
proper architecture and environment for optimal catalysis. Therefore,
understanding the catalytic roles of these active site residues is highly
significant in order to fully understand the mechanistic features of
these enzymes. Although SsuD is grouped with bacterial luciferase
and LadA because of structural similarities, the mechanism of SsuD is
quite distinct from these enzymes. Due to the expression of this
protein under times of sulfur limitation, Cys54 is the only cysteine
residue located in SsuD. The inactivation of cysteine-modified SsuD
combined with the spatial similarity of this residue to the functionally
significant cysteine residues in bacterial luciferase and LadA suggests
that Cys54 may be important in catalysis. In order to determine the
function of Cys54 in the desulfonation reaction, variants of the SsuD
enzyme were constructed in which the cysteinyl residue was replaced
by either serine or alanine. The experiments described in this work
define the functional role of the conserved cysteine residue in the
catalytic mechanism of SsuD.
240 and 280 nm over a pH range of 6.2–10.2 in the appropriate buffer
(50 mM Bis–Tris, pH 6.2–7.2; 50 mM Tris–HCl, pH 7.2–9.0; 50 mM
glycine, pH 9.0–10.2, and 100 mM NaCl). The SsuD enzyme (10 μM
final concentration) was equilibrated in each buffer for 10 min prior to
measuring the absorbance values. The final protein concentration in
solution was determined using the absorption coefficient of SsuD at
280 nm (ɛ280 =4.79×104
M
−1
cm ). The amount of SsuD in
−1
solution and A240 values were used to calculate the ɛ240 at each pH
value. The ɛ240 value was plotted against the pH and the pK
determined by a fit to Eq. (1).
a
was
hꢀ
ꢁ
ꢀ
ꢁi
−
pH
−pKa
A × 10
+
B × 10
−pH
y =
ꢂ
ꢃ
ð1Þ
−pKa
1
0
+ 10
where y is the ɛ240, A is the lower plateau at high pH, and B is the
upper plateau at low pH.
2.5. Steady-state kinetic studies
For the variant and wild-type SsuD enzymes the reaction was
initiated with the addition of NADPH (500 μM) into a reaction mixture
containing SsuD (0.2 μM), SsuE (0.6 μM), FMN (2 μM), NADPH
2
. Materials and methods
(
5
500 μM), and varying concentrations of octanesulfonate (10–
00 μM) in standard buffer with 100 mM NaCl at 25°C. Because of
2
.1. Materials
decreased enzymatic activity, a final concentration of 1.0 μM was used
in assays with C54A SsuD protein. Following quenching of the
reaction, the amount of sulfite product was determined as previously
described [5].
QuikChange site-directed mutagenesis kit and super-competent
cells E. coli BL21(DE3) were from Stratagene (La Jolla, CA).
Oligonucleotide primers were from Invitrogen (Carlsbad, CA).
Potassium phosphate (monobasic and dibasic), flavin mononucleo-
tide phosphate (FMN), nicotinamide adenine dinucleotide phosphate
NADPH), Trizma base, Bis–Tris, glycine, and NaCl were purchased
from Sigma (St. Louis, MO). Glycerol was obtained from Fisher
Pittsburgh, PA). Octanesulfonate was from Fluka (Milwaukee, WI).
2.6. Substrate binding studies
(
Flavin binding to variant and wild-type SsuD enzymes was
(
monitored by spectrofluorimetric titration with either oxidized or
reduced flavin. Spectra were obtained on a Perkin Elmer LS 55
luminescence spectrometer (Palo Alto, CA) with an excitation
wavelength of 280 nm and emission wavelength of 344 nm. For the
titration of SsuD with FMN, a 1.0 mL solution of variant or wild-type
SsuD enzyme (1 μM) in standard buffer containing 100 mM NaCl was
titrated with a solution of FMN (1.5–65.0 μM for C54S and 4.4–90.8 μM
for C54A), and the fluorescence spectra recorded after each addition.
The standard phosphate buffer contained 25 mM potassium phos-
phate, pH 7.5, and 10% glycerol unless otherwise noted.
2
.2. Site-directed mutagenesis, expression, and purification
Mutagenesis of the pET21a plasmid containing the ssuD gene was
performed with the Stratagene QuikChange Site-Directed Mutagen-
esis Kit. The TGC codon for Cys54 was replaced with GCT and GCG for
Ser and Ala, respectively. The ssuD Cys54 variants were identified by
sequence analysis at Davis Sequencing (Davis, CA). Plasmids harbor-
ing the appropriate ssuD Cys54 variants were transformed into E. coli
BL21(DE3) super-competent cells for protein expression and stored at
2
Similar experiments were performed with FMNH , however care had
to be taken to keep the solution anaerobic as previously described [5].
Anaerobic solutions of the SsuD variants (0.5 μM) in a glass titration
cuvette were titrated with FMNH
2
(0.21–6.0 μM for C54S and 0.21–
9
.4 μM for C54A) in an air-tight titrating syringe. The bound FMN or
−
80 °C. The mutants generated are referred to as C54S (Cys54 to Ser)
FMNH
2
was determined by the following equation:
and C54A (Cys54 to Ala) SsuD. The expression and purification of the
variant and wild-type SsuD proteins were performed as previously
described [31].
I0 − Ic
I0 − If
½
FMNꢀbound = ½SsuDꢀ
ð2Þ
2
.3. Far-UV circular dichroism
where [SsuD] is the initial concentration of enzyme, I
fluorescence intensity of SsuD prior to titration, I
intensity of SsuD following each addition of FMN or FMNH
the final fluorescence intensity. The concentration of bound FMN or
0
is the initial
c
is the fluorescence
, and I is
The spectra of the variant and wild-type SsuD enzymes were
2
f
obtained with an enzyme concentration of 1.2 μM in 25 mM potassium

100
R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
FMNH
dissociation constant (K
2
was plotted against the total FMN or FMNH
) according to Eq. (3):
2
to obtain the
d
Bmax
ðKd + XÞ
X
Y =
ð3Þ
where Bmax is the maximum binding at equilibrium with the
d
maximum concentration of substrate, and K is the dissociation
constant for the substrate [5].
For the binding of octanesulfonate to variant or wild-type SsuD, a
1
.0 mL solution of SsuD (1 μM) with reduced flavin (2 μM) was made
anaerobic in a glass titration cuvette as described previously [5].
Aliquots of an anaerobic solution of octanesulfonate (1.3–57.5 μM for
C54S and 1.3–73.2 μM for C54A) in an air-tight titrating syringe were
2
added to the SsuD–FMNH complex. The fluorescence spectra were
recorded with an excitation wavelength at 280 nm and emission
intensity measurements at 344 nm. The concentration of bound
octanesulfonate was determined by Eq. (1), and plotted against the
concentration of free octanesulfonate to determine the dissociation
Fig. 2. Monitoring cysteine thiolate absorption at varying pH values to determine the
pK of Cys54. The SsuD enzyme (5 μM) was measured at 240 and 280 nm over a range of
pH values (6.2–10.5). The 240 values at each pH were calculated using the
concentration of SsuD in solution and the absorbance obtained at A240. The ɛ240 was
plotted against the pH and fit to Eq. (1).
a
constant (K
.7. Rapid reaction kinetic experiments
Rapid reaction kinetic analyses were carried out using an Applied
d
) according to Eq. (3).
ɛ
2
There was a clear dependence on pH with a single apparent pK
a
value
Photophysics SX.18 MV stopped-flow spectrophotometer. Anaerobi-
osis was established as previously described [5]. All experiments were
of 9.3± 0.1. The difference in the protonated and deprotonated states
−1
−1
of Cys54 was 6,000 M
cm , which agrees with previously
performed by mixing 15 μM FMNH
45 μM) in air-saturated buffer in the other drive syringe. When
included in the reaction, varied concentrations of octanesulfonate
0.05–2.0 mM) were mixed with the protein solutions in air-saturated
2
in one drive syringe against SsuD
published molar extinction coefficients for cysteine thiolates
(
− 1
− 1
(
4,000–6,000 M
cm ) [32]. The results indicate that the
protonated form of Cys54 likely predominates in SsuD at the pH
utilized in the steady-state and rapid reaction kinetic assays.
(
buffer. All experiments were carried out in single-mixing mode by
mixing equal volumes of the solutions, and monitoring the reactions
by single wavelength analyses at 370 and 450 nm. The single-
wavelength traces of each cysteine variant at 370 and 450 nm were
fitted to the following equations using the KaleidaGraph software
3
.2. Steady-state kinetic parameters for octanesulfonate
Steady-state kinetic analyses were performed with each cysteine
(
Abelbeck Software, Reading, PA):
variant to determine if the substitution of Cys54 led to an alteration in
the kinetic parameters previously determined for wild-type SsuD.
Results from circular dichroism spectroscopy experiments revealed that
there were no obvious perturbations in the overall secondary structure
between the Cys54 variants and wild-type SsuD, suggesting that
substitution of Cys54 did not cause any major consequences to the
protein structure. Because SsuD requires reduced flavin for activity, a
coupled assay that includes SsuE in the reaction must be employed. This
A = A expð−k tÞ + A expð−k tÞ + C
ð4Þ
ð5Þ
1
1
2
2
A = A expð−k tÞ + A expð−k tÞ + A expð−k tÞ + C
1
1
2
2
3
3
where k
1
, k
2
, and k
3
are the apparent rate constants for the different
, A , and A are amplitudes of
phases, A is the absorbance at time t, A
1
2
3
each phase, and C is the absorbance at the end of the reaction.
The data for the concentration dependence of octanesulfonate on
the first phase were fitted with the following equation:
2
assay included an excess of SsuE so FMNH would not be limiting in the
reaction. The steady-state kinetic parameters for the Cys54 variants and
wild-type SsuD were obtained by measuring the amount of TNB anion
formed at 412 nm from the reaction of DTNB with the sulfite product.
The steady-state kinetic parameters were determined by a fit of the
data to the hyperbolic dependence of each enzyme on octanesulfonate
concentration. The C54S SsuD enzyme showed an approximate 6-fold
kobs = klim½Sꢀ= ðKd + ½SꢀÞ
ð6Þ
where kobs is the observed rate constant, klim is the limiting rate
constant for flavin oxidation at saturating octanesulfonate concentra-
tions, K
d
is the dissociation constant for the enzyme-substrate
decrease in the K
in an overall increase in the kcat/K
(
m
value and a 2-fold decrease in the kcat value resulting
value compared to wild-type SsuD
Table 1). The less conservative C54A SsuD variant showed a 2-fold
complex, and S is the substrate concentration.
m
3
. Results
.1. pK determination of Cys54
The pK value of Cys54 was evaluated to establish the protonation
decrease in the K
compared to wild-type, leading to a decrease in the kcat/K
results suggest that serine was effectively able to substitute for cysteine
m
value and a 9-fold decrease in the kcat value
value. These
m
3
a
a
Table 1
Dissociation constants and steady-state kinetics for C54S, C54A, and wild-type SsuD.
state of the single active site cysteine by monitoring the absorbance of
the cysteine thiolate at 240 nm [32–36]. Because SsuD contains only
k
cat
K
m
,
k
cat/K
m
K
d
, FMN
K
(μM)
d
, FMNH
2
(min−1) octanesulfonate (μM
(μM)
−1
min−1
) (μM)
one cysteine residue, determining the pK
a
of Cys54 by this method is
not complicated by the contribution of additional cysteine thiolates.
Inhibition assays with iodoacetamide are less reliable due to the
utilization of DTNB in the standard steady-state kinetic SsuD assays.
The ɛ240 values representing the absorbance of the cysteine thiolate
were determined at varying pH values between 6.2 and 10.2 (Fig. 2).
WT SsuDa 51.7± 2.1 44.0± 8.3
C54S SsuD 28.1± 0.4 7.5± 0.9
5.6± 0.1 26.4± 1.7
1.2± 0.2
3.8± 0.7
0.2± 0.1
10.2± 0.4 0.32± 0.15
11.3± 0.6 0.47± 0.09
13.7± 0.4 0.53± 0.09
C54A SsuD
a
Previously reported (5).

R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
101
under steady-state conditions, while the alanine substitution led to a
decrease in the steady-state kinetic parameters for SsuD.
the values obtained for wild-type SsuD (17.5± 0.9 μM) by similar
methods [5]. These results suggest that each variant has an increased
affinity for octanesulfonate and indicate that the binding of octane-
sulfonate is affected by the substitution of Cys54 to either serine or
alanine.
3
.3. Substrate binding affinity to the Cys54 SsuD variants
Spectrofluorimetric titrations were performed to determine
whether Cys54 substitutions affect the binding of FMN or FMNH
2
.
3.4. Kinetic studies of flavin oxidation by the SsuD variants
The dissociation constants for the binding of oxidized and reduced
flavin to the SsuD variants were determined by titrating either FMN
In order to further define the function of Cys54 in the
desulfonation reaction by SsuD, rapid reaction kinetic studies
were performed with each of the cysteine variants. Stopped-flow
or FMNH
intrinsic protein fluorescence emission due to the binding of FMN or
FMNH was monitored at 344 nm for each variant. The concentra-
2
into a sample of C54S or C54A SsuD. The decrease in the
2
2
kinetic studies were initially performed by mixing FMNH against
tion of bound and free flavin was calculated using Eq. (1), and the
concentration of flavin bound to each variant was plotted against
C54S and C54A SsuD in air-saturated buffer in the absence of the
alkanesulfonate substrate, and the oxidation of the flavin monitored
at 370 and 450 nm. The Cys54 SsuD variants were maintained at a
higher concentration (45 μM) relative to FMNH (15 μM) to ensure
2
single-turnover conditions. The rate constants obtained with each
the concentration of free flavin. The dissociation constants (K
FMN and FMNH are summarized in Table 1. The average K
for FMNH binding to each SsuD variant was 0.47± 0.09 μM and
.53± 0.09 μM for C54S or C54A SsuD, respectively. The dissociation
d
) for
2
d
value
2
0
variant are summarized in Table 2. The kinetic trace was best fit to
−
1
constants for FMN binding to C54S and C54A SsuD were determined
to be 11.3± 0.6 μM and 13.7± 0.4 μM, respectively (data not
shown). These results indicate that mutation of the putative active
site cysteine residue to either serine or alanine had little effect on
a triple exponential equation with rate constants of 111± 15 s
−
1
−1
(k
1
), 24.1± 0.8 s
(k
2
), and 0.33± 0.01 s
3
(k ) for SsuD C54S (○,
Fig. 4A). An initial fast phase (k
1
) was observed in the reaction at
370 nm for the C54S SsuD variant that represents the generation of
the C4a-(hydro)peroxyflavin intermediate [5]. The rate constants
obtained for the last two phases have been assigned to the decay of
the flavin intermediate back to the oxidized form. The C54S SsuD
variant was able to generate the C4a-(hydro)peroxyflavin, but the
rate of formation was increased 10-fold relative to wild-type. The
rate constants for the subsequent breakdown of the C4a-(hydro)
FMN or FMNH
value of 0.32± 0.15 μM (FMNH
wild-type SsuD [5].
2
binding when compared to the previously reported
K
d
2
) and 10.2± 0.4 μM (FMN) for
It was previously shown that ordered substrate binding occurs in
the SsuD reaction, with reduced flavin cofactor binding first before the
octanesulfonate substrate can bind [5]. The binding of octanesulfonate
to the C54S or C54A SsuD/FMNH
2
complex was performed to
peroxyflavin were 13-, and 4-fold over wild-type for k
2 3
, and k ,
determine whether the binding of the substrate was affected by the
cysteine substitutions. Dissociation constants for the binding of
octanesulfonate to the Cys54 SsuD variants were obtained under
anaerobic conditions similar to those previously described for
respectively. The kinetic trace at 450 nm was best fit to a double
−
1
exponential equation with rate constants of 9.76± 0.19 s
(k
) (●, Fig. 4A). The C54A SsuD kinetic trace at
370 nm did not exhibit an initial fast phase and was best fit to a
double exponential equation with rate constants of 4.70± 0.10 (k
and 0.35± 0.01 (k ) at 370 nm (○, Fig. 4B). Rate constants of 1.99±
0.04 (k ) and 0.32± 0.01 (k ) for C54A SsuD were obtained from the
1
)
−
1
and 0.33± 0.01 s
(k
2
titration of each SsuD variant with FMNH
2
[5]. The concentration of
1
)
octanesulfonate substrate bound to each SsuD variant/FMNH
2
2
complex was calculated according to Eq. (1) and plotted against the
concentration of free octanesulfonate after each addition (Fig. 3,
1
2
fit of the kinetic traces at 450 nm (●, Fig. 4B). These results suggest
that the C4a-(hydro)peroxyflavin is only formed with the more
conservative cysteine to serine substitution, while substitution of Cys
to Ala led to decreased accumulation of the flavin intermediate.
titration of C54S SsuD). The K
nate to each Cys54 SsuD variant/FMNH
and 4.97± 0.58 μM for C54S and C54A SsuD, respectively. The K
values obtained for the SsuD variants show a distinct decrease from
d
value for the binding of octanesulfo-
2
complex was 2.68± 0.60 μM
d
3.5. Kinetic studies of flavin oxidation by SsuD variants in the presence of
octanesulfonate
Rapid reaction kinetic analyses were also employed to determine if
flavin oxidation by the Cys54 SsuD variants was affected by the addition
of the octanesulfonate substrate relative to wild-type SsuD. The
oxidation of flavin by the SsuD variants in the presence of octanesulfo-
nate substrate was monitored by stopped-flow analyses at 370 and
4
2
50 nm by mixing free FMNH (15 μM) with either C54S or C54A SsuD
(
45 μM) and octanesulfonate (0.05–2.0 mM) in air-saturated buffer.
Three phases were previously observed in kinetic traces for wild-type
SsuD at low octanesulfonate concentrations (≤100 μM) [5]. The initial
fast phase at 370 nm was attributed to formation of the C4a-
peroxyflavin (II in Scheme 1) or an octanesulfonate peroxyflavin
Table 2
Rate constants for the oxidation of reduced flavin by C54S, C54A, and wildtype SsuD in
the absence of octanesulfonate.
2
Fig. 3. Fluorimetric titration of the C54S SsuD/FMNH complex with octanesulfonate.
C54S SsuD (1.0 μM) was titrated with octanesulfonate (1.3–57.5 μM). Emission
intensity measurements at 344 nm were obtained with an excitation wavelength of
3
70 nm
450 nm
(s−1
1.80± 0.03 0.08± 0.01 2.37± 0.01 0.10± 0.01
(s−1)
k
2
(s−1
)
k
(s−1
)
k
1
)
2
k )
(s−1
k
1
3
280 nm. The change in the intrinsic fluorescence intensity for the SsuD/FMNH
2
WT SsuDa
C54S SsuD
12.9± 0.3
111± 15
complex following the addition of octanesulfonate was converted to concentration of
bound octanesulfonate (Eq. (2)) and plotted against the concentration of free
octanesulfonate. Inset: Change in fluorescence emission intensity at 344 nm. The
solid line represents a fit of the titration curve to Eq. (3).
24.1± 0.8
0.33± 0.01 9.76± 0.19 0.33± 0.01
1.99± 0.04 0.32± 0.01
C54A SsuD 4.70± 0.10 0.35± 0.01
a
Previously reported (5).

102
R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
units of these enzymes adopt a TIM-barrel fold, and there are several
conserved amino acid residues in SsuD with spatial counterparts in
the active site of bacterial luciferase and LadA. The spatial correlation
between conserved amino acid residues in these enzymes suggests
that the active site of SsuD is located at the C-terminal end of the TIM-
β-barrel [12]. While the function of these conserved residues in LadA
have not been fully evaluated, the catalytic role of several of these
amino acid residues has been identified for bacterial luciferase. The
amino acid Cys106 in bacterial luciferase lies along a wall of a small
cavity that projects off a larger pocket, and is proposed to be in close
contact with the pyrimidine structure of the isoalloxazine ring of the
flavin [13–15]. It was established that chemical modification of this
active site cysteine results in the complete loss of enzymatic activity
[26]. Substitution of the cysteine residue to alanine, valine, and serine
resulted in an active luciferase enzyme with decreased biolumines-
cence. Although the rate of formation of the C4a-(hydro)peroxyflavin
intermediate was not compromised in these variants, the rate of decay
for the flavin intermediate was increased relative to wild-type [28].
The luciferase enzyme from Vibrio fischeri contains a valine residue at
position 106 in the α subunit in a similar position as the cysteine in
luciferase from V. harveyi based on sequence alignments, suggesting
Fig. 4. Kinetics of flavin oxidation by C54S and C54A SsuD in the absence of
octanesulfonate substrate. Stopped-flow kinetic experiments were performed with
the C54S or C54A SsuD variant (45 μM) combined with FMNH
FMNH in a tonometer was mixed against C54S SsuD in air-saturated buffer. The kinetic
traces shown are an average of three separate experiments following flavin oxidation at
70 (○) and 450 nm (●). The solid lines are fits of the kinetic traces to Eq. (3) or (4). B:
FMNH in a tonometer was mixed against C54A SsuD in air-saturated buffer. The kinetic
traces shown are an average of three separate experiments following flavin oxidation at
70 (○) and 450 nm (●). The solid lines are the fits of the kinetic traces to Eq. (4) or (5).
2
(15 μM) at 4 °C. A:
2
3
2
3
intermediates (III or IV) [5]. At higher octanesulfonate concentrations
there were only two phases observed that correspond to flavin
oxidation, suggesting the C4a-(hydro)peroxyflavin does not accumu-
late to significant levels with wild-type SsuD. The initial fast phase at
3
70 nm was not observed for either the C54S or C54A SsuD variants
even at low octanesulfonate concentrations, and all kinetic traces were
best fit to a double exponential equation (Fig. 5A and B). With both SsuD
variants, the kobs values for the first phase at 370 nm showed a
hyperbolic dependence on substrate concentration, giving a K
d
and
−
1
limiting rate constant of 160± 20 μM and 1.58± 0.08 s
for C54S
−
1
SsuD, and 190± 40 μM and 1.55± 0.13 s for C54A SsuD (Fig. 5A and
B, insets). These results from the rapid reaction kinetic studies indicate
that there were no substantial changes in the K
d
or kobs values
, 93 μM; kobs
.4 s ). The absence of an initial fast phase suggests that the C4a-
hydro)peroxyflavin does not accumulate with the Cys54 SsuD variants
in the presence of octanesulfonate.
compared to the values obtained for wild-type SsuD (K
d
,
−
1
Fig. 5. Kinetics of flavin oxidation by C54S and C54A SsuD in the presence of
2
octanesulfonate. Stopped-flow kinetic experiments were performed by mixing FMNH
15 μM) in a tonometer against C54S or C54A SsuD (45 μM) in the presence of variable
2
(
(
amounts of octanesulfonate (0.025 (●), 0.05 (○), 0.1 (■), 0.25 (□), 0.5 (▴), and 1.0 (Δ)
mM) in oxygenated buffer. The kinetic traces shown represent an average of three
4
. Discussion
separate experiments. A: Anaerobic FMNH
concentrations of octanesulfonate substrate. Inset: Dependence on the kobs at varying
octanesulfonate concentration for the second phase at 370 nm. B: Anaerobic FMNH
mixed against C54A SsuD with varying concentrations of octanesulfonate substrate.
Inset: Dependence on the kobs at varying octanesulfonate concentrations for the second
phase at 370 nm. The solid lines are the fits of the kinetic traces to Eq. (6).
2
mixed against C54S SsuD with varying
2
Even though they share low amino acid sequence identity, the
alkanesulfonate monooxygenase from E. coli is similar in overall
structure to bacterial luciferase and LadA [12–17]. The monomeric

R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
103
that a thiol is not directly essential for bioluminescence activity
27,28]. The increase in the decay of the C4a-(hydro)peroxyflavin
Evidence exists for the formation of the C4a-(hydro)peroxyflavin
intermediate in the SsuD catalyzed reaction through rapid reaction
kinetic studies [5]. The flavin intermediate was only detectable if SsuD
and reduced flavin were not premixed in the tonometer, leading to
conversion of the complex to the proposed inactive form. As illustrated
[
intermediate suggested that the cysteine is involved in stabilizing this
intermediate. While studies suggest the interaction between the thiol
and flavin is not direct, it is essential in preserving the correct
structural conformation for C4a-(hydro)peroxyflavin stabilization. A
recent three-dimensional structure of bacterial luciferase with flavin
bound shows Cys106 projecting toward the C-4a position on the
isoalloxazine ring of the flavin moiety at a distance of 4.1 Å [16,37].
Substitution of the corresponding cysteine to alanine in LadA
eliminated the catalytic activity of the enzyme.
Due to its role in sulfur assimilation, the SsuD enzyme contains a
limited number of sulfur-containing amino acids. Therefore, the
location of the single cysteine residue within the putative active site of
SsuD suggests a possible role for Cys54 in catalysis. In addition, this
residue is highly conserved in all putative SsuD enzymes from a
diverse group of bacteria. Initial studies have shown that labeling of
the conserved Cys54 residue in the active site of SsuD led to
inactivation of the enzyme, but the role of this residue in catalysis
was not evaluated [12]. Site-directed mutagenesis was used to
substitute the Cys54 residue with serine and alanine. In steady-state
kinetic assays, the substitution of Cys54 altered the kinetic parameters
of SsuD. The SsuD C54S and C54A variants gave kcat values that were
in the initial kinetic investigations of SsuD, this species could be more
−
specifically called a C4a-peroxyflavin intermediate (FMN-OO
)
resulting in a nucleophilic attack on the sulfonate group of the
alkanesulfonate to form an alkanesulfonate peroxyflavin adduct.
Kinetic studies were performed to determine if the C4a-(hydro)
peroxyflavin intermediate was observed in stopped-flow reactions
when reduced flavin was mixed with the Cys54 SsuD variants. Three
phases were identified in the kinetic trace of C54S SsuD mixed against
−
1
2 1
FMNH at 370 nm. An initial fast phase (k =111 s ) was observed
that is attributed to the formation of the C4a-(hydro)peroxyflavin
intermediate, and is 10-fold greater than the rate constant obtained
with wild-type SsuD. As previously observed for wild-type, this fast
phase was not present in the kinetic trace obtained at 450 nm. The rate
constants obtained for the last two phases at 370 nm were
2 3
approximately 13- and 4-fold greater for k and k , respectively.
These phases have previously been assigned to the decay of the C4a-
(hydro)peroxyflavin. Therefore, although there was increased accu-
mulation of the flavin intermediate, the rate of breakdown for this
intermediate also increased. The kinetic traces obtained at 370 and
450 nm for the C54A SsuD variant were similar with no initial fast
phase observed at 370 nm, suggesting that there was no accumulation
of the proposed C4a-(hydro)peroxyflavin with this variant. It is
uncertain whether the C4a-(hydro)peroxyflavin does not form with
this C54A SsuD variant in the absence of substrate, or if the flavin
intermediate does not accumulate to detectable levels in the absence
of substrate. Stopped-flow analyses on essential thiol variants of
bacterial luciferase, where the reactive cysteinyl residue at position
106 in the α subunit was replaced with alanine, serine, and valine,
were also performed in the absence of the aldehyde substrate [28].
When mixing the luciferase variants and reduced flavin with
oxygenated buffer, a C4a-(hydro)peroxyflavin intermediate was
observed in all three mutants with rate constants equal to that of
wild-type luciferase [28].
2
- and 9-fold lower than wild-type SsuD, respectively. However, the
concentration of the C54A SsuD enzyme had to be increased 5-fold
over the concentration of C54S and wild-type SsuD in order to detect
m
any appreciable activity. The K values with octanesulfonate were
decreased 6- and 2-fold for the C54S and C54A SsuD variants,
respectively. The changes in the steady-state kinetic parameters led to
a 3-fold increase in the kcat/K value for C54S SsuD, while the kcat/K
m m
value for C54A SsuD decreased 6-fold. These results suggest that the
less conservative cysteine to alanine substitution had a greater effect
on the steady-state kinetic parameters, while the serine was able to
adequately substitute for cysteine.
Comparable steady-state kinetic parameters for the C54S SsuD
variant as wild-type indicated that Cys54 may be involved in substrate
binding through hydrogen bonding and/or maintaining the structural
integrity of the active site, as serine would also be able to provide
similar interactions. However, the K
d
values determined for reduced
Rapid reaction kinetic experiments were also performed for each
Cys54 SsuD variant in the presence of octanesulfonate. Similar kinetic
experiments with wild-type SsuD showed an initial fast phase
attributed to the formation of the C4a-(hydro)peroxyflavin only at
low octanesulfonate concentrations (≤100 μM). The reaction was
thought to occur rapidly in the presence of increasing concentrations
of the octanesulfonate substrate so that no appreciable levels of C4a-
(hydro)peroxyflavin intermediate accumulated. With both SsuD
variants there was no initial fast phase at 370 nm representing the
formation of the C4a-(hydro)peroxyflavin even at low octanesulfo-
nate concentrations, and the kinetic traces were best fit to a double
exponential equation. At concentrations of octanesulfonate greater
than 100 μM, the first phase representing flavin oxidation at 370 nm
displayed a hyperbolic dependence on octanesulfonate concentration
or oxidized flavin binding to the C54A SsuD variant were similar to the
values obtained for wild-type SsuD indicating that Cys54 was not
directly involved in flavin binding. This is in contrast to the results
from fluorescence titration experiments with bacterial luciferase,
where mutation of the cysteine at position 106 to alanine, serine, or
d 2
valine resulted in an increase in the K value for FMNH [27]. Previous
kinetic evaluation of SsuD demonstrated that two conformational
changes likely occur with the binding of reduced flavin and
octanesulfonate. Therefore, optimal contacts by the cysteine residue
may only be obtained with both substrates bound, and the affinity of
the flavin for the Cys54 SsuD variants in the absence of octanesulfo-
nate would likely be similar to wild-type SsuD.
The K
with each of the SsuD variants by titrating FMNH
SsuD with octanesulfonate. The K value for both variants led to an
d
values for octanesulfonate binding were also determined
2
-bound C54S or C54A
[5]. The kobs and K
the values obtained with wild-type SsuD. There was an alteration in
the K values obtained using this method compared to static
d
values for the Cys54 SsuD variants were similar to
d
increase in the binding affinity. It was previously demonstrated through
kinetic studies that incubation of wild-type SsuD with reduced flavin in
the absence of oxygen results in the conversion of the complex to an
inactive form that must undergo an isomerization to the active form of
the enzyme before catalysis can occur [5]. Because this conversion may
only occur in the presence of dioxygen, octanesulfonate is likely binding
to the inactive complex in the static anaerobic titration experiments.
d
fluorescent titration experiments with octanesulfonate due to
inherent differences in the two experimental procedures. In the
titration experiments the octanesulfonate substrate is likely binding
to the inactive form of the SsuD–FMNH
obtained may not be relevant to the catalytic conditions. The increase
in K values for the variants does not seriously impact catalysis, as the
value for C54S SsuD was actually 3-fold higher than wild-type.
2
complex, and the values
d
Titration of octanesulfonate into the inactive form of the FMNH
2
-bound
kcat/K
m
SsuD variants may lead to alternative interactions of the substrate and
active site amino acid residues in the absence of dioxygen leading to a
greater affinity of SsuD for octanesulfonate. However, these interactions
may not be favorable for catalysis.
Although there was no initial fast phase at 370 nm representing
formation of the C4a-(hydro)peroxyflavin with either cysteine
variant, both variants possessed activity. Therefore, the flavin
intermediate must be generated in the reaction. The kcat/K
m
value

104
R.A. Carpenter et al. / Biochimica et Biophysica Acta 1804 (2010) 97–105
increased with the C54S SsuD variant, and an increased rate of
formation and decay of the C4a-(hydro)peroxyflavin was observed in
the manuscript. We would also like to thank Dr. Evert Duin for the use
of his anaerobic tent to set up the anaerobic conditions described.
the absence of octanesulfonate. Conversely, the kcat/K
m
value
decreased with the C54A SsuD variant, and there was no initial rate
representing C4a-(hydro)peroxyflavin formation in the absence of
octanesulfonate. These results suggest that the decreased accumula-
tion of the C4a-(hydro)peroxyflavin intermediate at low octanesulfo-
nate concentrations with C54S SsuD may be due to increased
reactivity of the flavin intermediate; while the decreased accumula-
tion with C54A SsuD is likely due to destabilization of the active site
environment.
The results from rapid reaction kinetic studies with the Cys54 SsuD
variants support the importance of hydrogen bonding interactions in
the stabilization of the C4a-(hydro)peroxyflavin intermediate either
through direct interactions or by preserving the structural integrity of
the active site. The cysteine thiol and not the thiolate is likely involved
in stabilizing this intermediate, as the serine residue can compensate
for cysteine in catalysis. The less conservative C54A SsuD variant
would be unable to stabilize the flavin intermediate and would likely
lead to alterations in the local environment of the C4a-(hydro)
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