Nitroreductase from Salmonella typhimurium: Characterization and catalytic activity

Article abstract of DOI:10.1039/b926274a

The biocatalytic activity of nitroreductase from Salmonella typhimurium (NRSal) was investigated for the reduction of α,β-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics. The synthesized gene was subcloned into a pET28 overexpression system in E.coli BL21 strain, and the corresponding expressed protein was purified to homogeneity with 15% protein mass yield and 41% of total activity recovery. NRSal showed broad substrate acceptance for various nitro compounds such as 1-nitrocyclohexene and aliphatic nitroalkenes (alkene reductase activity), as well as nitrobenzene (nitroreductase activity), with substrate conversion efficiency of > 95%. However, the reduction of enones was generally low, proceeding albeit with high stereoselectivity. The efficient biocatalytic reduction of substituted nitroalkenes provides a route for the preparation of the corresponding nitroalkanes. NRSal also demonstrated the first single isolated enzyme-catalyzed reduction of nitrobenzene to aniline through the formation of nitrosobenzene and phenylhydroxylamine as intermediates. However, chemical condensation of the two intermediates to produce azoxybenzene currently limits the yield of aniline.

Full text of DOI:10.1039/b926274a

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Nitroreductase from Salmonella typhimurium: characterization and catalytic
activity
Yanto Yanto,a Me´lanie Halla and Andreas S. Bommarius*a,b
Received 14th December 2009, Accepted 27th January 2010
First published as an Advance Article on the web 15th February 2010
DOI: 10.1039/b926274a
The biocatalytic activity of nitroreductase from Salmonella typhimurium (NRSal) was investigated for
the reduction of a,b-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatics. The
synthesized gene was subcloned into a pET28 overexpression system in E.coli BL21 strain, and the
corresponding expressed protein was purified to homogeneity with 15% protein mass yield and 41% of
total activity recovery. NRSal showed broad substrate acceptance for various nitro compounds such as
1-nitrocyclohexene and aliphatic nitroalkenes (alkene reductase activity), as well as nitrobenzene
(nitroreductase activity), with substrate conversion efficiency of > 95%. However, the reduction of
enones was generally low, proceeding albeit with high stereoselectivity. The efficient biocatalytic
reduction of substituted nitroalkenes provides a route for the preparation of the corresponding
nitroalkanes. NRSal also demonstrated the first single isolated enzyme-catalyzed reduction of
nitrobenzene to aniline through the formation of nitrosobenzene and phenylhydroxylamine as
intermediates. However, chemical condensation of the two intermediates to produce azoxybenzene
currently limits the yield of aniline.
In our previous work, we have characterized three un-related
enoate reductases: XenA from Pseudomonas putida, KYE1 from
Introduction
Flavoenzymes are biocatalysts that catalyze a wide range of
Kluyveromyces lactis, and Yers-ER from Yersinia bercovieri. We
biochemical reactions ranging from dehydrogenation, one- and
have shown that these three enoate reductases feature broad
two-electron transfer redox reactions, oxygen activation during
substrate specificity but different substrate preferences on a set
of various a,b-unsaturated carbonyl compounds.6 Since enoate
oxidation and hydroxylation reactions, to light emission in bi-
oluminescent bacteria.1–2 As part of flavoenzymes, the recently
reductases often possess nitroreductase activity, we initiated a new
growing “Old Yellow Enzyme” (OYE) family was reported to cat-
search for a candidate that possesses broad substrate specificity
alyze the reduction of various substrates such as a,b-unsaturated
for nitro compounds, however, applying a different strategy: the
investigation of nitroreductases for their enoate reductase activity,
withspecific emphasis onthe biocatalytic reductionofnitroalkenes
to provide an efficient route for the synthesis of chiral substituted
nitroalkanes. This strategy was based on the observation that some
nitroreductases had shown reductase activity on quinones.57,58,59
Nitroalkanes are potential intermediates in the synthesis of
amines, aldehydes, carboxylic acids or denitrated compounds.15
The traditional organic synthesis of enantiomerically pure ni-
troalkanes occurs through asymmetric conjugate addition to
nitroalkenes,17–19 reduction of nitroalkenes with transition metal
catalyst,20 hydrogenation with Jacobsen-type organocatalyst,21 or
addition of a,b-unsaturated bonds onto nitroalkanes.22 Due to
various restrictions on these organic synthesis processes (such as
growing environmental restrictions on the type of catalyst used,
narrow substrate acceptance), an efficient biocatalytic route for
chiral nitroalkanes is highly desirable. Recently, several published
works have demonstrated stereoselective reduction of nitroalkenes
using: OPR1 and OPR3 from Lycopersicon esculentum,13 OYE
from Saccharomyces carlsbergensis and OYE2-3 from Saccha-
romyces cerevisiae,12 NCR from Zymomonas mobilis,12 YqjM from
Bacillus subtilis,13 PETN reductase from Enterobacter cloacae,11
and crude enzyme preparation from Clostridium sporogenes.16
These enzymatic processes are a valuable addition to the estab-
lished synthetic toolbox and inspire the search for new candidates.
aldehydes, ketones, imides, esters, nitriles, and carboxylic acids.3–6
=
The asymmetric bioreduction of C C bonds employing OYE
family proteins can create up to two chiral centers and therefore
constitutes an interesting route towards enantiomerically pure
=
compounds. The C C bond reduction catalyzed by enoate
reductases proceeds via a ping-pong mechanism, where the flavin
is first reduced at the expense of a nicotinamide cofactor in the
reductive half-reaction, followed by the asymmetric reduction
of the substrates through trans-hydrogenation (oxidative half-
reaction).7–9 The OYE family also features nitroreductase activity
through biodegradation of explosive compounds such as PETN,
TNT, and nitroglycerin. Several members of the OYE family such
as XenA and XenB from Pseudomonas sp.,10 PETN reductase from
Enterobacter cloacae,11 OYEs from Saccharomyces sp.,12 YqjM
from Bacillus subtilis,13 and OPRs from Arabidopsis thaliana14 have
been characterized for their biocatalytic activity on both nitro-
aromatics and aliphatic nitro compounds.
aSchool of Chemical and Biomolecular Engineering, Parker H. Petit Institute
of Bioengineering and Biosciences, Georgia Institute of Technology, 315
Ferst Drive, Atlanta, GA, 30332-0363, USA. E-mail: andreas.bommarius@
chbe.gatech.edu; Fax: +1 404 894 2291; Tel: +1 404 385 1334
bSchool of Chemistry and Biochemistry, Georgia Institute of Technology,
901 Atlantic Drive, Atlanta, GA, 30332-0400, USA; Fax: +1 404 894 2866;
Tel: +1 404 894 1838
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Table 1 Purification of wild type NRSal monitored with nitrofurazone activitya
Purification step
Total protein/mgb
Total activity [U]
Specific activity [U/mg]
Yield (%)
Lysate
53.3
22.4
22.0
8.20
285
136
149
116
5.34
5.02
6.78
14.2
100
48
52
(NH4)2SO4
Dialysis
DEAE
41
a Dry cell weight 1.1 g L-1. b Nitrofurazone activity assay: 50 mM TrisHCl buffer pH 7.2, 0.05 mM Nitrofurazone, 0.1 mM NRSal, GDH recycling system:
(8 mM NADPH, 2 mM Glucose, 10 U GDH). The activity was calculated by monitoring the decrease of nitrofurazone wavelength at 375 nm with
extinction coefficient of e375nm = 15,000 (M cm)-1.30
In this context, we came across a homolog of nitroreduc-
tase from Enterobacter cloacae: nitroreductase from Salmonella
typhimurium (NRSal). NRSal was first discovered in 1989 from
Salmonella typhimurium strain TA1538NR,23 and represents a
new class of flavin-dependent nitroreductase enzymes.24–27 The
nucleotide sequence of this oxygen-insensitive FMN-containing
NRSal encodes for 217 amino acids, with a calculated molec-
ular weight of 23,955 Da. This enzyme has been shown to
reduce some nitroaromatic compounds, such as p-nitrophenol or
p-nitrobenzoic acid.28 Since then however, there has been no
published work about any further characterization of NRSal,
and especially its catalytic activity, which renders it an interesting
candidate for us to explore. In this paper, we examine the ability
of NRSal to catalyze the reduction of a,b-unsaturated carbonyl
compounds, nitroalkenes, and nitroaromatics.
tag. The addition of the polyhistidine tag was for ease of enzyme
purification.
The purification of wild-type NRSal was optimized compared
to previously published method.28 The original six steps of
NRSal purification were reduced to two steps, with first am-
monium sulfate precipitation to remove the majority of non-
proteinaceous biological material, followed by anion exchange
column chromatography with DEAE-Sepharose column to iso-
late pure NRSal with 15% protein mass recovery from the
cell lysate sample, and with total activity recovery of 41%.
The purification of wild-type NRSal was monitored with the
standard activity assay on nitrofurazone28 (Table 1) followed
by SDS-PAGE analysis (Fig. 2). The final elution showed a
single band upon SDS-PAGE analysis. The NRSal with N- or
C-terminal polyhistidine tag was purified according to the stan-
dard Ni2+-NTA bead protocol from Qiagen (Valencia, CA).
However, the activity of NRSal with polyhistidine tag was
found to be much lower compared to the wild type. Activity
check was performed by monitoring the reduction of nitro-
furazone (known substrate for NRSal28) and cyclohexenone
(common substrate for enoate reductases). NRSal with N- or
C-terminal polyhistidine tag showed specific activity of 4.40 U/mg
and 3.27 U/mg respectively for nitrofurazone reduction, which
was much lower compared to wild type specific activity of
14.2 U/mg. An analogous pattern was observed for the reduction
of cyclohexenone, where wild type NRSal showed significantly
higher specific activity (0.39 U/mg) compared to NRSal with
N-terminal (0.16 U/mg) and C-terminal (0.17 U/mg) polyhisti-
dine tag. Since the presence of the polyhistidine tag considerably
decreased enzymatic activity of NRSal, further characterization
was continued only with wild-type, untagged-NRSal.
Results & discussion
Cloning, overexpression, and purification
The gene coding for NRSal was purchased from BioBasic Inc (On-
tario, Canada) according to the published sequence (UnitProtKB
accession # P15888).23,29 Sequence analysis revealed that NRSal
from Salmonella typhimurium shares high amino acid identity and
similarity with nitroreductase from Enterobacter cloacae with 89%
identity and 93% similarity (Fig. 1). The gene product was cloned
into vector pET28a(+) (Novagen, San Diego, CA) at the following
restriction sites: NcoI and HindIII with stop codon for expression
of wild type NRSal, NcoI and HindII for expression of NRSal
with C-terminal polyhistidine tag, and NdeI and HindIII with
stop codon for expression of NRSal with N-terminal polyhistidine
Fig. 1 Amino acid alignment of nitroreductase NRSal from Salmonella
typhimurium (P15888, NR_Sal) and nitroreductase from Enterobacter
cloacae (Q01234, NR_ENT) using EMBOSS Pairwise Alignment Needle
Algorithm (http://www.ebi.ac.uk).
Fig. 2 SDS-PAGE analysis for purification of wild type NRSal: lane
1 protein ladder, lane 2 cell lysate sample, lane 3 70% (NH4)2SO4
precipitation, lane 4 DEAE-Sepharose column.
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Table 2 Half-life of NRSal measured at various temperatures using 1-
nitrocyclohexene as substrate
Enzymatic activity & stability study
We first investigated the effect of temperature and pH on NRSal
by monitoring the change of enzymatic activity over the range
of 10–60 ◦C (Fig. 3), and pH range of 4–9 (Fig. 4). The
activity-temperature profile showed maximum specific activity
of 13.6 U/mg for reduction of nitrofurazone at 45 ◦C and
pH 7.5, which was 3.2-fold higher than the activity at room
temperature (20 ◦C). Arrhenius plots yield an activation energy
T/◦C
Half-life
kd,obs
a
37
45
50
365 h
17.9 h
2.3 h
0.0019 h-1
0.0388 h-1
0.3100 h-1
a kd,obs is the observed deactivation rate constant
◦
Ea of 42.7 kJ mol-1 for the temperature range of 10–45 C (r2 =
0.963), and a deactivation energy Ed of -31.5 kJ mol-1 for the
of NRSal at all temperatures followed first order deactivation
kinetic (data not shown). Also, NRSal seemed reasonably stable
with shortest half-life of 2.3 h measured at 50 ◦C, and half-life
decreased exponentially with increasing temperatures.
◦
temperature range of 45–60 C (r2 = 0.965). The activity-pH
profile showed an optimum pH at 7.2 with highest specific activity
of 8.10 U/mg for reduction of nitrofurazone at 20 ◦C in 50 mM
TrisHCl buffer. We also found that NRSal had lower activity (but
similar activity–pH pattern with a maximum activity at pH 7.2) in
50 mM phosphate buffer compared to 50 mM TrisHCl buffer. The
activity of NRSal is therefore buffer dependent and TrisHCl is the
optimum buffer for reactions run between pH 6–7.5. Additionally,
NRSal considerably lost its activity beyond pH 8.
Substrate spectrum characterization
With finding of a broad enoate reductase activity in a ni-
troreductase as one of our goals, we investigated the catalytic
activity of purified NRSal with several common a,b-unsaturated
carbonyl substrates for enoate reductases (Table 3. Entries 1–
4). We first tested NRSal activity on cyclohex-2-enone (1) and
related substrate 3-methyl-2-cyclohexenone (2).31–33 The nitrore-
ductase enzyme displayed enoate reductase activity on both
substrates. Both compounds 1 and 2 were reduced, albeit with
maximum substrate conversion of only 12% and 5.2% respectively.
NRSal showed strong stereospecificity in the reduction of 2 and
yielded (R)-3-methyl-2-cyclohexanone with 84% e.e. Substrates
ketoisophorone (3) and citral (4) were also tested due to their large-
scale industry application to produce the corresponding levodione
and citronellal, respectively.34–37 NRSal showed modest maximum
conversion of 12% with 3 to produce (R)-levodione with 87% e.e,
and did not show any biocatalytic reduction of aldehyde 4. This
=
Fig. 3 Activity–temperature profile of NRSal. Activity was measured
with standard nitrofurazone activity assay in 50 mM phosphate buffer at
pH 7.5.
remarkable stereoselectivity for a nitroreductase in reducing C C
bonds may point at a mechanism similar to that of OYEs, as OYEs
proceed via a ping pong Bi Bi mechanism, as do nitroreductases
in the reduction of the nitro group.7–9,57,58
Despite the low conversions with a,b-unsaturated carbonyl
substrates, we hypothesized that NRSal, as a nitroreductase, would
display stronger alkene reductase activity in the presence of a nitro
group. Indeed, NRSal showed broader substrate acceptance for
nitro related compounds (Table 3. Entries 5–8). First, we observed
=
the reduction of C C bond on nitro compounds 5 to 7. Maxi-
mum conversion of 1-nitrocyclohexene (5) was 97% to produce
the reduced product 1-nitrocyclohexane. Aliphatic nitroalkenes
(6–7) were also shown to be favored substrates of NRSal with
overall conversion efficiency of > 95%. Both compounds 6 and 7
were reduced to the corresponding alkanes, in case of compound 7
to the racemic product; either the enzyme was not stereoselective
for that compound, or alternatively the potential enantiomeric
product could have racemized due to the acidity of the proton
on Ca. Finally, we observed a more typical behavior for a
nitroreductase in the reduction of aromatic nitro compound such
as nitrobenzene (8), which was reduced with conversion of > 99%
to produce nitrosobenzene, phenylhydroxylamine, but also aniline
and the chemical condensation product azoxybenzene. To our
knowledge, this is the first report of a single enzyme-catalyzed
reduction of nitrobenzene to aniline (reaction detailed in the next
section).
Fig. 4 Activity–pH profile of NRSal in three different buffers: 50 mM
TrisHCl (x), 20 mM citrate buffer (ꢀ), and 50 mM phosphate buffer (ꢀ).
Activity was measured with standard nitrofurazone activity assay at 20 ◦C.
Since thermal stability of enzymes is a key consideration for
application in larger scale processes, we investigated the kinetic
stability of NRSal by measuring its half-life at three different
temperature points of 37, 45, and 50 ◦C (Table 2). The deactivation
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Table 3 Biocatalytic conversion of various a,b-unsaturated carbonyl compounds, nitroalkenes, and nitroaromatic substrates with NRSal (* represents
a center of chirality)
Entry
Substrate
Product
Conversion (NADPH/NADH)
12%/12%
1
2
3
5.2%/4.8% (R) 84% e.e
12%/10% (R) 87% e.e
4
5
N A-1
No conversion
95%/97%
6
7
8
95%/95%
Both >99% Racemic mixture
>99%/>99% Overall nitrobenzene
conversion
Overall, NRSal accepts both cofactors NADPH/NADH
equally well, with higher substrate acceptance for compounds
containing the electron withdrawing nitro group. Importantly,
the diverse catalytic activity of NRSal was reflected in the broad
substrate spectrum and range of conversion efficiency.
reduced, resulting into the corresponding Meisenheimer complex,
followed by subsequent release of nitrite.41–42 The formation of the
Meisenheimer complex has been observed for several members
of the OYE family such as PETN reductase from Enterobacter
cloacae,43 XenB from Pseudomonas fluorescens,44 and NemA from
Escherichia coli,45 which also display nitroreductase activity on the
nitro group.
Enzymatic reduction of nitrobenzene to aniline
Published work on the reduction of nitrobenzene to aniline was
mainly accomplished with metal support catalysts (e.g Ru/SBA,
Ni, Pt/C catalysts) or whole-cell biocatalysts (e.g Rhodotorula
mucilaginosa, Arracacia xanthorrhiza, Candida guilliermondii,
Saccharomyces cerevisiae).46–50,55 The single enzyme-catalyzed re-
duction of nitrobenzene (e.g with nitrobenzene nitroreductase
from Pseudomonas pseudoalcaligenes JS45) was reported to pro-
duce phenylhydroxylamine, a highly reactive intermediate.51–52
The enzymatic reduction of nitroaromatic compounds can follow
two pathways: the reduction of the nitro group or the reduction
of the aromatic ring. The nitro group reduction is either a one-
electron or two-electron reduction mechanism.38–40 Both mech-
anisms involve the reduction of the nitro group to the amine
through highly reactive nitroso and hydroxylamino intermediates.
The aromatic ring in nitroaromatic compounds can also be
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To the best of our knowledge, the discovery of NRSal reduc-
tion of nitrobenzene represents the first single isolated enzyme-
catalyzed reduction of nitrobenzene to aniline. The mechanism
for NRSal nitrobenzene reduction was shown to follow the two-
electron reduction pathway of the nitro-group: nitrobenzene was
reduced to form nitrosobenzene first and phenylhydroxylamine
next, followed by subsequent reduction of phenylhydroxylamine
to aniline. At the same time, chemical condensation between
the nitrosobenzene and phenylhydroxylamine resulted in the
production of azoxybenzene (Fig. 5).53–54 Control experiments
were performed to demonstrate the condensation reaction by
mixing of these standards under similar reaction conditions but
without enzyme present. Real-time analysis was performed to
quantify the reduction of 1 mM nitrobenzene over time course
of 160 min: nitrobenzene was completely converted within 40 min
(with conversion of > 99%), along with the formation of 0.26 mM
nitrosobenzene, 0.09 mM phenylhydroxylamine, 0.06 mM aniline,
and 0.35 mM azoxybenzene (Fig. 6). Nitrobenzene and phenyl-
hydroxylamine were found to be chemically unstable and were
completely degraded after 6 h, with no further increase of aniline
as final product. In an effort to boost the percentage formation of
aniline in the overall reaction, one possible method was to limit the
condensation between nitrosobenzene and phenylhydroxylamine.
Experiments were performed to remove molecular oxygen from the
reaction system as initial hypothesis that O2 was essential in the
condensation reaction. However, azoxybenzene formation was still
detected at similar concentration level, implying that its formation
is a condensation reaction without oxidation.
3. Conclusion
In summary, nitroreductase NRSal from Salmonella typhimurium
is a versatile biocatalyst that displays both nitroreductase and
enoate reductase activity. The enzyme showed strong preference in
=
the reduction of C C bonds for nitro-containing substrates, with
=
less satisfactory biocatalytic activity towards the reduction of C C
bonds in enones, still with a high stereoselectivity. It is interesting
that a true nitroreductase presents strong enoate reductase activity
with substrate stereopreference, is an FMN-bound enzyme and is
still not associated with OYE family. The feasibility of biocatalytic
reduction of nitroalkenes to nitroalkanes provides an addition
as part of the synthetic toolbox to obtain various intermediates
of interest. This is also the first isolated single enzyme-catalyzed
reduction of nitrobenzene to aniline, which provides a possible
perspective for a green production process of anilines other
than traditional synthesis through chemical hydrogenation and
metal-supported catalyst.46 Although overall aniline formation
is still limited due to condensation reaction in the system, we
are currently investigating for optimized reaction conditions and
larger scale reaction system.
Experimental section
NRSal primer sequences, cloning, and expression
The DNA sequence of NRSal from Salmonella thypimurium
was synthesized by BioBasic Inc (Ontario, Canada) according
to UnitProtKB accession # P15888. The corresponding specific
5¢- and 3¢-primers were synthesized at Eurofins-mwg|operon
Biosciences (Huntville, AL). The primers contain a gene-encoding
segment (italic), a restriction site (underlined), and an overhang.
The 5¢-primers of wild type and C-terminal polyhistidine tagged
introduced a NcoI restriction site (5¢-CAT GCC ATG GCA ATG
GAT ATT GTT TCC GTA GCT CTG C-3¢), and N-terminal
tagged introduced a NdeI restriction site (5¢-GGG AAT TCC
ATA TGA TGG ATA TTG TTT CCG TAG CTC TGC-3¢) into the
PCR fragment. The 3¢-primer introduced a HindIII restriction site
in to the PCR fragment (with stop codon: 5¢-CCC AAG CTT TTA
CAC TTC AGT CAG GGT GGT TTC CA-3¢, and without stop
codon: 5¢- CCC AAG CTT CAC TTC AGT CAG GGT GGT
TTC CA-3¢).
Fig. 5 Proposed nitrobenzene reduction pathway to aniline and azoxy-
benzene by NRSal.
DNA amplification was performed with standard PCR protocol
with Pfu polumerase from New England Biolabs (Marlborough,
MA) and PCR buffer from Stratagene (La Jolla, CA). DNA was
amplified for 30 cycles in an Eppendorf Gradient Thermocycler
(Eppendorf, Hamburg, Germany) followed by gel purification
with standard Qiagen gel kit (Qiagen, Valencia, CA). PCR assay:
100 ng of DNA, 200 mM of each dNTP, 10 mM of each primers, 1
U of Pfu polymerase, and 5 mL of Pfu buffer in a final volume of
50 mL. Each DNA amplification cycle: 1 min denaturation at 95 ◦C,
1 min◦annealing step at 50, 55, and 60 ◦C, 3.5 min extension step
at 68 C. The final PCR products were digested with restriction
enzymes and ligated into pET28a (+) (Novagen, San Diego) and
transformed into competent E. coli XL1-Blue cells. The NRSal
genes were sent for DNA sequencing at Eurofins-mwg|operon
Biosciences (Huntville, AL).
The NRSal genes were transformed into E. coli BL21 for protein
expression. Starter culture consisted of 5 ml LBKan inoculated from
Fig. 6 Conversion-time plot for the reduction of nitrobenzene by NRSal
in the presence of NADH.
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frozen stock and grown overnight at 37 ◦C. The starter culture were
used for inoculation of 300 ml culture (0.1% v/v), which was gently
aerated until OD600 reached 0.5 for addition of 0.1 mM of IPTG
and left overnight for protein expression. Harvested cell pellets
were kept for -80 ◦C for protein purification.
1–8 and ee values of the corresponding products were determined
with the columns and methods described below.
2-Cyclohexen-1-one (1) and nitrobenzene (8) were analyzed
using Shimadzu SHRX5 column (15 m, 0.25 mm, 0.25 mm).
Temperature program for 1: injector and detector temperature at
220 ◦C; split ratio 25 : 1; start at 100 ◦C, hold 2 min, 10 ◦C min-1 to
160 ◦C, hold 2 min. Retention time: 2-cyclohexen-1-one 6.3 min,
cyclohexanone 4.8 min. Temperature program for 8: injector and
detector temperat◦ure at 300 ◦C; split ratio 25 : 1; start at 80 ◦C,
hold 5 min, 10 C min-1 to 290 ◦C, hold 5 min. Retention
time: nitrobenzene 5.8 min, aniline 2.9 min, nitrosobenzene
2.0 min, phenylhydroxylamine 3.9 min, azoxybenzene 14.8 min.
Compounds 3-methyl-2-cyclohexenone (2), ketoisophorone (3),
citral (4), 1-nitro-cyclohexenone (5), and trans-b-nitrostyrene (6)
were analyzed using RESTEK Rt-BDEXcst chiral column (30
m, 0.32 mm, 0.25 mm). Temperature program for 2: injector and
de◦tector temperature at 200 ◦C; split ratio 20 : 1; start at 90 ◦C,
Purification of NRSal
The cell pellets of wild type NRSal were re-suspended in 50 mM
TrisHCl buffer pH 7.5 (buffer A) and disrupted by sonication.
The supernatant was separated after centrifugation, where NRSal
was precipitated between 40–70% of saturated ammonium sulfate.
The precipitate was dissolved again in buffer A and dialyzed over
6 h. The dialyzed sample was applied to DEAE-SepharoseTM FF
(GE Healthcare, Sweden) column and washed with 25 mM step
increment of NaCl in buffer A. Purified NRSal was collected
at elution of 150 mM NaCl with 0.1 mM of free FMN added
to account for the loss of FMN during the purification (only
20% FMN occupancy measured before addition of free FMN).
Although experiments were performed to measure for the FMN
occupancy at the end of purification, free FMN in the sample
seemed to saturate the spectrophotometer measurement even
with multiple wash cycles. The NRSal with N- or C-terminal
polyhistidine tag were purified with Ni2+-NTA beads according
to the standard protocols of Qiagen (Valencia, CA). The protein
concentration was determined by the Bradford method with
Coomassie Plus Protein assay reagent and BSA assay (Pierce
Chemical) as the calibration curve. SDS-PAGE analysis was
performed with 50 mg protein sample elution into each well. The
protein samples were mixed with 2X sample buffer (125 mM
TrisHcl, pH 6.8, 4% SDS, 50% glycerol. 0.02% bromophenol blue,
and 10% 2-mercaptoethanol). The mixed samples were incubated
at 100 ◦C for 5 min and loaded onto 12% PAGETM Gold precast
gel and run in BioRad Mini Protean chamber (BioRad, Hercules
CA) at 150 V for 45 min with Tris-HEPES-SDS running buffer
(12.1 g L-1 Tris, 23.8 g L-1 HEPES, 1 g L-1 SDS). Biorad Precision
Plus protein ladder was used as standards in the gel analysis.
◦
1
C min-1 to 130 ◦C, 5 C min-1 to 180 ◦C. Retention time:
3-methyl-2-cyclohexenone 14.9 min, (R)-3-methyl-2-cyclohexanone
13.0 min, (S)-3-methyl-2-cyclohexanone 13.1 min. Temperature
program for 3: injector and det◦ector temperature at 210◦◦C; split
ratio 25 : 1; start at 100 ◦C, 3 C min-1 to 130 ◦C, 20 C min-1
to 200 ◦C, hold 3 min. Retention time: ketoisophorone 11.0 min,
(R)-levodione 6.8 min, (S)-levodione 7.1 min. Temperature pro-
gram for 4: injector and detector temperature at 220 ◦C; split
◦
rat◦io 6.4 : 1; start 80 ◦C, 2.5 C min-1 to 130 ◦C, hold 10 min,
5
C min-1 to 180 ◦C, hold 5 min. Retention time: citral
20.1 min. Temperature program for 5: injector and detector
temperature at 220 ◦C; split ratio 5 : 1; start 120 ◦C, 10 ◦C min-1 to
210 ◦C; hold 5 min. Retention time: 1-nitrocyclohexenone 6.8 min,
1-nitrocyclohexanone 5.7 min. Temperature program for 6: in-
jector and detector temperature at 220 ◦C; split ratio 25 : 1;
◦
start 120 ◦C, hold 3 min, 10 C min-1 to 210 ◦C; hold 5 min.
Retention time: trans-b-nitrostyrene 7.2 min, phenylnitroethane
3.6 min. Compound 1-phenyl-2-nitropropene (7) was analyzed
using Agilent Cyclosil-B chiral column (30 m, 0.32 mm, 0.25 mm).
Tem◦perature program for 7: injector ◦and detector tempe◦rature at
220 C; split ratio 25 : 1; start◦at 100 C, hold 10 min, 5 C min-1
to 130 ◦C, hold 15 min, 5 C min-1 to 220 ◦C, hold 2 min.
Retention time: phenyl-2-nitropropene 11.5 min, racemic phenyl-
2-nitropropane 9.6 min and 10.5 min.
R
GelCodeꢀ Blue Stain Reagent (Pierce, Rockford IL) was used
for final gel staining.
Enzyme Assay
NRSal kinetic measurement were performed by monitoring the
oxidation of NAD(P)H at 340 nm using e340nm of 6.22 mM-1cm-1.
Reaction assays were analyzed with Shimadzu GC-2010 using
RT-BDEXcsf chiral column (30 m 0.32 mm 0.25 mm, Restek)
for entry # 1–4; Cyclosil-B chiral column (30 m 0.32 mm
0.25 mm, J&W Scientific) for entry # 6–8; and SHRX5 column
(15 m 0.25 mm 0.25 mm Shimadzu) for entry # 5. All substrates
and products were purchased commercially from Sigma Aldrich
or VWR with highest purity available except products of entry
6–8, and phenyl-hydroxylamine. Prof. Jim Spain Research Group
(Georgia Institute of Technology) kindly provided synthesized
phenylhydroxylamine.56 Products of 6–7 were synthesized accord-
ing to previous published protocols.16
Acknowledgements
We thank the Eckert-Liotta Research Group (especially Ryan
Hart) at the Georgia Institute of Technology for access and help
with GC-MS and Jim Spain’s Research Group (especially Shirley
F Nishino) at the Georgia Institute of Technology for help in
phenylhydroxylamine analysis with HPLC.
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