An unusually cold active nitroreductase for prodrug activations

Article abstract of DOI:10.1016/j.bmc.2012.04.004

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.

Full text of DOI:10.1016/j.bmc.2012.04.004

Bioorganic & Medicinal Chemistry 20 (2012) 3540–3550
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
An unusually cold active nitroreductase for prodrug activations
⇑
Ayhan Çelik , Gülden Yetisß
Gebze Institute of Technology, Department of Chemistry, 41400 Gebze-Kocaeli, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 November 2011
Revised 3 April 2012
Accepted 4 April 2012
Available online 11 April 2012
A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical
nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus
strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product
revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofura-
zone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with
retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could
be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered
for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was pro-
duced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical,
spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB rep-
resents a novel nitro reductase with an unusual cold active property, which has not been described pre-
viously for prodrug activating enzymes of nitroreductases.
Keywords:
Nitroreductase
Cold active enzyme
Prodrug activation
CB1954
Staphylococcus saprophyticus
Nitrofurazone
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(renamed as Aliivibrio fischeri) (FRase1),22 Rhodobacter capsulatus
(NprA, NprB),1,23 Bacillus subtilis (NfrA1, YwrO)24,25 and more re-
Activation of prodrugs by bacterial nitroreductases (NTRs) has
attracted considerable interest in recent years because of having
differential effects between tumour and normal cells in cancer pro-
drug therapy.1 The basis of the activation is that NTRs catalyse the
reduction of nitro groups in substrates to form the corresponding
hydroxylamine derivatives. These derivatives can be further
metabolised by cellular enzymes to form cytotoxic agents that
cross link with DNA, resulting in rapid cell deaths.2–4 Such reduc-
tion properties establish NTRs as prodrug activators. They have
been therefore utilised in cancer prodrug therapy including anti-
body-directed enzyme prodrug therapy (ADEPT),5 gene directed
enzyme prodrug therapy (GDEPT),6 clostridia-directed enzyme
prodrug therapy (CDEPT)7 and virus directed enzyme prodrug ther-
apy (VDEPT).8,9 In fact, FMN-dependent NTRs from Escherichia coli
(nfsB) with combination of prodrug (CB1954) have been exten-
sively studied for antitumourogenic properties6,10–12 and reached
the clinical trial stage in VDEPT.13
Nitroreductases are widely distributed among bacteria, but also
found in archaea and eukaryotic species1 and divided into two
groups; oxygen insensitive (type I) and oxygen sensitive (type II).
So far a number of oxygen insensitive NTRs have been identified
from various bacterial sources, examples include Escherichia coli
(NfsA, NfsB, YdjA),14–16 Salmonella typhimurium (Cnr, SnrA),17–19
Pseudomonas putida (PnrA),20 Vibrio harveyi (FRP),21 Vibrio fischeri
cently Bacillus licheniformis.26
Members of this family utilise FMN as a cofactor and are often
found to be homodimers. The structural aspects of nitroreductases
and roles of key active site amino acids in binding FMN as well as
substrates are now clearly defined, largely as results of extensive
structural studies of E. coli NTRs.27–31 This structural data com-
bined with recent mechanistic studies32 has allowed detailed
understanding and the rationalisation of catalytic and prodrug
binding properties of the enzymes. Based on such information,
many CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) like pro-
drug have been designed and synthesised.6,33,34 A subset of these
compounds have demonstrated better binding and cytotoxic prop-
erties than CB1954, offering a great deal of opportunities in direc-
ted enzyme prodrug therapy (DEPT).
As prodrug activating enzymes, NTRs from bacterial sources
have a number of advantages in DEPT strategy-the absence or
low catalytic activities of mammalian equivalents and differences
in substrate specificity prevent non specific prodrug activation at
the sites other than the tumour. NTR, however are required to meet
certain criteria to be used in DEPT strategy. Stability, the levels of
enzymatic activity when conjugated to antibody, sufficient cata-
lytic activity under physiological conditions and cofactor availabil-
ity are among the factors for successful applications of NTRs.
Therefore the search for ideal nitroreductases for therapeutic uses
necessitates identifying new enzymes with desired properties from
natural sources or by modification of existing enzymes by muta-
genesis, or even combination of both.
⇑
Corresponding author. Tel.: +90 262 605 3130; fax: +90 262 605 3101.
E-mail address: ayhancelik@gyte.edu.tr (A. Çelik).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.004

A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
3541
We now report the identification and characterisation of a novel
nitroreductase from uropathogenic staphylococcus, Staphylococcus
saprophyticus, which is frequently isolated from young female out-
patients suffering from uncomplicated urinary tract infections.35
The gene encoding the Ssap-NtrB from this bacterium was cloned
and the recombinant protein overexpressed in E. coli. This func-
tional nitroreductases was shown to be FMN containing flavoen-
zyme. At very low temperature, it catalyses reduction of various
electron acceptors including an antibiotic of nitrofurazone, dinitro-
benzamide family cancer prodrug of CB1954 and SN23862 and also
an artificial electron acceptor of K3[Fe(CN)6], in the expense of nic-
otinamide cofactors (NAD(P)H).
X-100 and incubated for 30 min at 37 °C. Purified genomic DNA
was diluted at a concentration of 7.6 ng/
ll in TE buffer (10 mM
Tris–Cl, pH 8.0, 1 mM EDTA).
A 627 bp portion of Ssap-NtrB gene was amplified from S. sap-
rophyticus genomic DNA using the following forward and reverse
primers. To facilitate cloning NdeI and XhoI restriction sites were
incorporated at 50-end of each primer and underlined respectively
(supplied by IDT, Leuven, Belgium): FP (50-CGGTGTCCATATGATA-
AATAATAATTTTGAAG-30) and RP (50-CACCTCGAGTTAAACGTATT-
TAGTCACAG-30). The PCR reaction mixture consisted of 2ꢀ Tag
Master Mix™ (25
1.6 pmol/ L), genomic DNA (2
(15 L) in a reaction volume of 50
l
L), forward and reverse primers (4
L, 7.6 ng/ L) and distiled water
L. The PCR reaction, carried
lL,
l
l
l
l
l
out in MJ Mini Thermal Cycler™ (BioRad), involved an initial dena-
turation step of 5 min at 95 °C, and then subjected to 29 cycles of
amplification (95 °C, 1 min; 52 °C, 1 min; 68 °C, 1.5 min) followed
by a final extension at 68 °C for 10 min. The resulting 627 bp PCR
product was gel purified using QIAprep Kit (Qiagen, ATQ Biotech-
nology, Ankara, TR). The PCR product and pET14b vector were
digested with NdeI/XhoI and gel purified using QIAprep Kit
(Qiagen). Ligation was achieved using T4 DNA ligases according
to the manufacturer instructions (NEB). Five microlitre of ligation
mixture was transferred into 50 lL E. coli TOP10 competent cell
lines according to the manufacturer instructions (Invitrogen).
Overnight cultures were prepared each of resulting colonies on
LB-Agar medium containing 100 lg/ml ampicillin. Restriction
analysis was performed using the same restriction enzymes to con-
firm successful ligation of the gene into the vector. This construct
contains the complete nitroreductase coding region and N-termi-
nal His-tag together with a thrombin cleavage site, and named as
pSsap-NtrB. The plasmid DNA was purified from transformants
using a Roche High Pure™ plasmid isolation kit. A sample of the
purified DNA was sequenced (RefGen, Ankara, TR) to confirm the
identity of the amplification product on both strands. DNA se-
quences were determined using T7 promoter and T7 terminator
primers. Nucleotide sequence data were assembled and analysed
using the BioEdit software packages (Ibis Therapeutics, CA).
Expression of the gene was under the control of IPTG (isopro-
2. Materials and methods
2.1. Chemicals and reagents
FMN and nitrofurazone (NFZ), Coomassie brilliant blue R-250, a
protease inhibitor mixture, PMSF (phenylmethylsulfonyl fluoride),
SDS (sodium dodecyl sulphate) were supplied by Sigma–Aldrich.
NADH and NADPH were obtained Roche Applied Science. IPTG
(isopropyl-b-D-thiogalactopyranoside) was supplied by Qiagen
(ATQ Biotechnology, TR). Restriction endonucleases, T4 DNA ligase,
2ꢀ Tag Master Mix™ were from New England BioLabs (NEB GmbH,
Germany). Nitrobenzamide prodrug of CB1954 (5-(aziridin-1-yl)-
2,4-dinitrobenzamide) and SN23862 (5-[N,N-bis(2-chloroethyl)-
amino]-2,4-dinitrobenzamide) were kind gift from Professor
Richard Knox (Morvus Technology Limited, UK). BSA was obtained
from Merck Biosciences. All other biochemical reagents were
obtained from various sources at highest purity possible. Reagents
from Merck, Fluka and Sigma–Aldrich were used as supplied
without prior purification.
2.2. Sequence analysis
Protein sequence of Ssap-NtrB was obtained from GeneBank
hosted in the National Centre for Biotechnology Information (NCBI)
(Accession no: gi73661517). Phylogenetic and molecular evolu-
tionary analyses were conducted using MEGA version 4 software.36
Neighbor-Joining (NJ) method was used for phylogenetic tree
searching and inference. Multiple sequence alignment was per-
formed using CLUSTRAL W program with the default parameters.37
pyl-b-D-thiogalactopyranoside)-inducible T7 RNA promoter. For
heterologous gene expression, the resulting construct (pSsap-NtrB)
was transformed into expression strain of E. coli BL21(DE3)
(Invitrogen).
2.5. Expression and purification of Ssap-NtrB
2.3. Bacterial strains, plasmids, culture media
For protein expression, E. coli BL21 (DE3) containing the expres-
sion construct of pSsap-NtrB was grown in LB medium containing
100 lg/ml ampicillin at 37 °C in a orbital shaker at 180 rpm. After
The source of chromosomal DNA for the cloning of Ssap-NtrB
was Staphylococcus saprophyticus (strain DSM 20229/ATCC
15305), obtained from DSMZ culture collection (DSMZ GmbH,
Braunschweig, Germany). The E. coli host used for cloning experi-
induction with 0.1 mM IPTG at an optical density (OD600) of 0.5,
growth was continued for up to 3 h at 20 °C before harvesting.
The cell pellet was obtained via centrifugation at 8000ꢀg for
10 min and stored ꢁ20 °C until use. Aliquots were withdrawn at
regular time points. Expression was assessed by comparing the
banding pattern obtained by SDS–PAGE analysis of whole cell
extracts with that of a negative control (i.e., E. coli BL21 (DE3) con-
taining pET14b plasmid only).
For the auto induction, the procedure was adapted from
Studier.39 Briefly, a single colony was used to inoculate a 10 ml of
starter culture for 1 L of auto induction medium, which was incu-
bated at 30 °C and 180 rpm for at least 16 h, until stationary phase
was reached. The cell pellet was obtained via centrifugation at
8000ꢀg for 10 minand stored ꢁ20 °C until use. Approximatelyevery
gram of wet cell paste from either induction procedures was sus-
pended in 4 ml of ice-cold buffer A (20 mM Tris–Cl, pH 7.8, 0.5 M
NaCl, 5 mM imidazole) containing 0.1 mM phenylmethylsulfonyl
ments was TOP10 [F-mcrA
D(mrr-hsdRMS-mcrBC) u80lacZDM15
D
lacX74 recA1 araD139 (araleu) 7697 galU galK rpsL (StrR) endA1
D
nupG], and the host used for protein expression studies was E. coli
BL21(DE3) [F-ompT hsdSB(rB-, mB-) gal dcm (DE3)] (both from
Invitrogen). The plasmid employed for both cloning and expression
was pET14b (Novagen). All organisms were cultivated in LB broth
or on LB-agar at 30 or 37 °C and supplemented with antibiotics if
necessary.
2.4. Cloning of Ssap-NtrB gene
S. saprophyticus was grown in LB medium overnight at 37 °C and
genomic DNA was obtained using phenol/chloroform extraction
method38 with minor modifications. Bacterial pellets were initially
re-suspended in 250
ll of a lysis solution containing 200 lg of
lysostaphin per ml, 20 mM Tris–Cl, 2 mM EDTA, and 1.2% Triton
fluoride (PMSF), and 100 ll of the protease inhibitor mixture

3542
A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
(Sigma–Aldrich). The cells were incubated with 80
l
l of lysozyme
heated to 120 °C for 20 min. The samples was then centrifuged at
13,500ꢀg before ultrafiltration using a Vivaspin™ micro centrifu-
gal filter with 10 kDa molecular cut off (Sartorius). The released fla-
vin content was identified via HPLC equipped with C18 RP column
(10 mg/ml) for 30 min in ice at an angle of 45° for mixing readily, be-
fore disruption by sonication using a Soniprep 150 sonicator (Sanyo,
Tokyo, Japan) fitted with a 3-mm diameter probe. The cell suspen-
sion was kept on ice and sonicated with a 15 s burst followed by a
45 s interval for six times. The resulting cell extract was centrifuged
at 28,000ꢀg for 30 min at 4 °C. The cell-free extract was carefully re-
moved and filtered through a 0.45-micron filter unit (Sartorius,
Goettingen, Germany). The clarified extract was then loaded onto
a 1-ml Nickel HiTrap™ column (Amersham Biosciences), equili-
brated in buffer A, at a flow rate of 1 ml/min. Column chromatogra-
phy was performed manually at 4 °C. The column was washed with
several times with buffer A (10 column volumes) to remove un-
bound material. The majority of contaminating proteins were then
removed by washing with 10 mM imidazole in buffer A (10 column
volumes) before elution of the recombinant His-tagged Ssap-NtrB
by 100 mM imidazole in buffer A. The buffer was exchanged to buf-
fer B (50 mM sodium phosphate, pH 7.5) using a PD-10 desalting col-
umn (Amersham Biosciences) at 4 °C. The sample volume was
reduced using a spin concentrator with a 10 kDa cut-off membrane
(Millipore, Germany). Glycerol was added to a final concentration of
50% (v/v) and the sample stored at ꢁ20 °C until use. The protein sam-
ple was saturated by commercially available FMN followed by gel
filtration chromatography using PD-10 desalting column, when
necessary.
(ACE3, 150 mm ꢀ 4.6 mm ꢀ 3
lm). The mobile phase was metha-
nol and 10 mM sodium phosphate buffer, pH 7.0 with linear gradi-
ent of 1% methanol for 5 min, up to 50% methanol for 10 minutes
with flow rate of 0.7 ml/min and monitored at 375 and 445 nm
with PDA detector (Agilent HPLC 1100). For the purpose of deter-
mination of flavin: protein ratio, the concentration of flavin was
determined by spectroscopic method at 445 nm using absorption
coefficients of 12,500 Mꢁ1 cmꢁ1 (for FMN).26 The protein content
was determined as described previously using both Bradford pro-
cedure40 and spectroscopic methods at 280 nm for confirmation.
The FMN concentration was then simply divided by protein con-
centration to obtain the flavin: protein ratio. The absorption coef-
ficients calculated for the protein were 13,370 Mꢁ1 cmꢁ1 in apo
form and 24,701 Mꢁ1 cmꢁ1 for halo form, in which absorption at
280 nm due to FMN was included in all calculation.
2.9. pH and temperature optimum, and thermal stability
determinations
The pH-activity profile of Ssap-NtrB was determined using NFZ.
The buffers used were 50 mM citric acid–sodium citrate (pH
5.4–6.2), 50 mM sodium phosphate (pH 6.0–7.9), 50 mM Tris–Cl
(pH 7.4–9.0) and 50 mM glisin-NaOH (pH 8.6–10.6) containing
4% DMSO. Activity values were determined in each of these buffers
2.6. SDS–PAGE analysis and protein concentration determination
Sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS–PAGE) was performed on a Mini-PROTEAN II electrophoresis
cell (Bio-Rad). Protein bands were visualised with Coomassie
brilliant blue R-250. Enzyme concentrations were determined
using the conventional dye-binding assay using Bradford proce-
dure.40 Bovine serum albumin (BSA) was used for generation of
standard curve. Spectroscopic determination of the enzyme con-
centration was also determined for confirmation using the calcu-
lated (based on protein sequence) extinction coefficient value of
13370 Mꢁ1 cmꢁ1 at 280 nm.41 Bradford procedure was primarily
used for standardisation of the enzyme assays. Since FMN is also
absorbs at 280 nm, the amount of FMN bound to the protein was
taken into account if the enzyme concentration was to be deter-
mined spectrophotometrically at 280 nm.
using 0.150 mM NFZ, 9.4 lg/ml Ssap-NtrB and 200 lM NADPH at
15 °C. Temperature profile of Ssap-NtrB was determined at a series
of temperatures ranged from 3 to 40 °C by NFZ assay. Activity val-
ues were determined using 0.150 mM NFZ, 12.5
and 200 M NADPH in 50 mM sodium phosphate buffer containing
4% DMSO at pH 7.4. In a typical experiment, the buffer solution
(800 l) containing NFZ was incubated at desired temperature un-
til the equilibrium reached. The enzyme sample (20 l) was added
lg/ml Ssap-NtrB,
l
l
l
and the mixture was further incubated for 3 min. The reaction was
then started by addition of NADPH. The NFZ reduction was fol-
lowed spectrophometrically at 440 nm for 2 min. The initial rates
of NFZ disappearance were calculated using extinction coefficient
of 880 Mꢁ1 cmꢁ127,42 and expressed as a percentage of the maxi-
mum activity obtained at 15 °C. The temperature in Uv–vis spec-
trometer was maintained using a circulating cooled water bath
and temperature in the cuvette was constantly monitored using
a digital thermometer.
2.7. Protein mass spectrometry of Ssap-NtrB
Mass spectrometry of the recombinant His6-tagged Ssap-NtrB
was performed by MALDI-TOF MS using MicroFlex instruments
In the enzyme thermal stability studies, the protein samples
(0.4 mg/ml, 400 ll) in 50 mM sodium phosphate buffer (pH 7.5)
(Bruker Daltonics, Billerica, MA). A protein sample (50
ml) was concentrated by precipitation using freshly prepared
500 l of 20% TCA (trichloroacetic acid). The resulting pellet was
washed twice with 250 l of cold acetone, and centrifuged at
10000ꢀg. The pellet was air dried, and re-dissolved in 40 l of
l
l of 9 mg/
were pre-incubated at 20, 30, 35, 40 and 50 °C for 0–240 min and
aliquots were taken at regular intervals and cooled. In all cases,
the remaining enzyme activity was assayed against 0.150 mM of
NFZ in 50 mM sodium phosphate buffer, pH 7.5 with 4% DMSO in
a similar manner described previously and expressed as a percent-
age of the maximum activity, for which the was no heat treatment.
l
l
l
formic acid/dH2O/isopropanol (1:3:2) mixture. The mixture was
mixed with 2ꢀ equivalent volume of matrix (saturated solution
of sinapinic acid in 50% isopropanol), and applied on a sample
target surface. The sample was air dried at room temperature to
crystallise. The MS was operated in positive ion mode (20 kV, 50
shots) and calibrated with the Bruker’s protein standards II kit
2.10. Enzyme assays with electron acceptors
Cofactor preference (NADPH or NADH) of Ssap-NtrB catalysed
reactions was carried out using K3[Fe(CN)6], as an artificial electron
acceptor. In a typical reaction, 1 mM (saturating concentration) of
K3[Fe(CN)6] in 50 mM Tris–Cl buffer pH 7.0 was incubated with
including cytochrome
c (12,360 Da), myoglobin (16,952 Da),
trypsinogen (23,982 Da), protein A (44,613 Da) and bovine serum
albumin (BSA, 66,527 Da).
50 lg/ml of Ssap-NtrB for 3 min at 15 °C and the reaction was ini-
tiated by addition of either NADPH or NADH at varying concentra-
tions. The reaction was monitored spectrophotometrically at
340 nm for 10 min. Steady state enzyme kinetics with purified
Ssap-NtrB as a prodrug activator enzyme was monitored using
both CB1954 and SN23862. In a typical reaction, series of CB1954
2.8. Flavin cofactor analysis
The amount of Ssap-NtrB bound FMN was determined as
described before.23 The purified enzyme (800
ll, 1.6 mg/ml) was

A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
3543
in the range of 0.05–4 mM or SN23862 (0–165
in 50 mM Tris–Cl (pH 7.0) buffer containing 4% DMSO. To this solu-
tion, Ssap-NtrB was added to give final concentrations of 12.5 g/
ml. The reaction was then started by addition of NADPH at final
concentration of 200 M and monitored at 420 nm (based on equal
absorption of both 2- and 4-hydroxylamine reduction product of
CB1954 at this wavelength; for CB1954
420 = 1200 Mꢁ1 cmꢁ1 43
340 = 6220 Mꢁ1 cmꢁ1 44
for
l
M) was prepared
3.2. Production of His6-Ssap-NtrB and SDS–PAGE analysis
l
The expression construct (pSsap-NtrB) was transformed into
E. coli BL21(DE3), and the cell extract was analysed by SDS–PAGE.
A protein of the expected size (ꢂ25 kDa) was readily detected on a
Coomassie-stained gel from an extract of E. coli BL21 (DE3) har-
bouring pSsap-NtrB plasmid (Fig. 1). The recombinant protein
was absent in the negative control of the same strain harbouring
pET14b. No detectable difference in the observed levels of heterol-
ogous expression was noticed between IPTG-induced and nonin-
duced cells, that suggest a degree of leakiness within the system.
When the cells were grown at a temperature of 37 °C, most of
the Ssap-NtrB accumulates in the insoluble fraction, whereas the
protein was significantly soluble in the cells grown at 25 °C. His6-
Ssap-NtrB was purified using metal ion affinity chromatography.
A single step purification protocol yields 40–50 mg protein from
a litre culture with over 90% purity based on SDS–PAGE analysis
(Fig. 1, lane 8).
l
e
)
and 340 nm (based on NADPH usage;
e
)
SN23862.
All kinetics measurements were performed using Uv–vis
spectrophotometer (Shimadzu UV-3600) equipped with a circu-
lating water bath (VWR model 1162A). Nonlinear regression anal-
ysis and Michaelis–Menten curve fitting was performed using
Origin v.7 software (OriginLab, Northampton, MA). One unit (U)
is defined as the amount of the enzyme that catalyses the conver-
sion of 1 lmol of substrate per minute under the experimental
conditions specified. The specific activity was expressed as
lmol -
minꢁ1 mgꢁ1active enzyme.
2.11. Metabolite analysis
3.3. Protein mass spectrometry of Ssap-NtrB
Product profile studies of CB1954 and SN23862 catalysed by
Ssap-NtrB were performed. In a typical reaction, CB1954 or
SN23862 was prepared in 50 mM Tris–Cl (pH 7.0) buffer, filtered
and degassed for 5 min, to give final concentration of 1 mM
(80 lM for SN23862). All reactions were prepared anaerobically
by repetitive bubbling with nitrogen for 10 min before and after
The calculated mass of His6-tagged Ssap-NtrB from its amino
acid sequences is 25,567 Da. The subunit molecular mass of the
monomer of enzyme (single charged) was determined by MALDI-
TOF and found to be 25,452 Da (Fig. 2). The calculated and mea-
sured masses are in agreement with each other and the difference
in masses is well within the instrument’s resolution power and
experimental errors.
addition of the enzyme. To this solution, Ssap-NtrB and NADPH
were added to give final concentrations of 50 lg/ml and 3.4 mM
respectively. The reactions were continued for at least 30 min at
18–20 °C before being stopped by addition of ice-cold methanol.
Samples were immediately transferred to ꢁ80 °C for at least
1.5 h followed by centrifugation at 12,000ꢀg for 10 min. The HPLC
analyses for determinations of reaction products of CB1954 were
performed using gradient HPLC system (Agilent HPLC1100)
equipped with RPC18 column (ACE5, ACE-HPLC, UK) and Uv–visi-
ble detector at 260 and 420 nm (390 and 450 nm for SN23862).
The mobile phase was acetonitrile–water mixture (v/v, 10% for
5 min, 10–60% for 20 min, 30% for 15 min.) with flow rate of
1 ml/min. For the quantification, the extinction coefficients were
3.4. Flavin cofactor analysis
The yellow colour of purified Ssap-NtrB from S. saprophyticus
indicates the presence of bound cofactor. The Uv–vis spectra of
the purified Ssap-NtrB protein showed the characteristic two peaks
at 368 and 456 nm (Fig. 3A) resembling an oxidised FMN cofactor,
which is consistent with bacterial nitroreductases. The bands were
quickly disappeared upon reduction of the flavoenzyme by addi-
tion of sodium dithionite in excess. The flavin content was subse-
quently analysed by RP-HPLC using commercial FMN as
standard and identified as flavin mononucleotide (FMN) (Fig. 3B
a
used as follows:
e
260 = 7880 Mꢁ1 cmꢁ1 for the 4-hydroxylamine
and
e
260 = 5420 Mꢁ1 cmꢁ1 for the 2-hydroxylamine.45
3. Results
3.1. Cloning of Ssap-NtrB gene
Using BLAST search46 with the sequence of E. coli nitroreduc-
tases (nfsB), a potential nitroreductase gene was identified from
the genome sequence of S. saprophyticus. Oligonucleotide primers
were designed from the sequence data covering the translational
start region of the nitroreductase gene. The forward primer con-
tained mismatches to generate an NdeI restriction site (under-
lined). Similarly, the reverse primers were designed to generate
an XhoI restriction site (underlined) for direct cloning into the
pET14b vector. The deduced product of S. saprophyticus NTR
(Ssap-NtrB) without N-terminus modification is a protein of
208 amino acid residues, 23.4 kDa, and pI of 5.49. Addition of
hexa histidine tag for the purpose of easy purification increased
the amino acid residues to 228, 25.6 kDa and pI of 6.7. Protein
BLAST database searches of the translated S. saprophyticus DNA
sequence showed high scoring similarities with putative nitrore-
ductases from Staphylococcus epidermidis and E. sibiricum (55%
identity, 72% similarity and 47% identity and 68% similarity,
respectively).
Figure 1. SDS–PAGE analysis of whole cell extracts of
a culture of E. coli
BL21(DE3)pSsap-NtrB following induction with IPTG. The arrow indicates the
position of the recombinant protein. Lane M molecular mass standards (molecular
masses in kDa are indicated on the left); lane 1 cell extracts of uninduced E. coli
BL21(DE3)pET14b (control); lane 2 cell extract of uninduced E. coli BL21(DE3)pS-
sap-NtrB; lane 3 cell extract of IPTG induced E. coli BL21(DE3)pET14b after 2 h; lane
4 cell extract of IPTG induced E. coli BL21(DE3)pSsap-NtrB after 2 h; lane 5 cell
extract of IPTG induced E. coli BL21(DE3)pSsap-NtrB after 5 h; lane 6 insoluble
fraction of E. coli BL21(DE3)pSsap-NtrB; lane 7 cell free extract; lane 8 purified His6-
tagged Ssap-NtrB

3544
A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
Figure 2. MALDI-TOF MS analysis of His6-Ssap-NtrB. The experimentally deter-
mined mass along with the predicted masses are highlighted by arrows for the
singly (M+) and doubly (M2+) charged monomers as well as singly charged dimer
(2M)2+ species. Mass for the singly monomer was found to be 25,452 Da.
and C). The flavin/protein ratio has been found to be 0.36 0.04
indicating that for per mol of enzyme, there are 0.36 mol of FMN
present. The ratio was raised to 1.10 0.20 by incubation of excess
FMN with the purified enzyme followed by gel filtration chroma-
tography for removing the unbound FMN. Furthermore, an addi-
tion of 1 mM commercial FMN to growth media has resulted in
about 1.2- and 2.3-fold increases in yield of purified protein and
flavin/protein ratio, respectively, when compared to the growth
media without external FMN supplements.
3.5. Cofactor preference of Ssap-NtrB
A steady state experiment was performed in order to determine
the pyridine-nucleotide preference of Ssap-NtrB. Using the
ferricyanide as an artificial electron acceptor, oxidation of pyri-
dine-nucleotide was monitored and resulting plots of kobs versus
nucleotide concentration can be seen in Figure 4. The NADPH-
dependent reduction of potassium ferricyanide gave a kcat(app) of
7.10 0.45 sꢁ1and KM(app) of 71.27 10.51
l
M whereas NADH gave
a kcat(app) of 4.58 0.60 sꢁ1 and KM(app) of 95.26 25.52
lM. These
results make it clear that Ssap-NtrB has similar preference for both
nucleotides with a small favour towards to NADPH.
3.6. pH and temperature optimum, and thermal stability
determinations
We have investigated the effect of pH and temperature on Ssap-
NtrB by monitoring the change of enzymatic activity over pH range
of 5.5–10.5 and temperature range of 3–40 °C as well as tempera-
ture stability of Ssap-NtrB. The activity-pH profile showed an opti-
mum pH at between 6.58 (ionic strength,
= 0.142 M) (Fig. 5) for the reduction of NFZ at 15 °C in 50 mM
phosphate buffer and in 50 mM Tris–Cl buffer (for pH 7.5,
= 0.045 M). Observations of similar activities at pH 7.5 in both
phosphate and Tris–Cl with different ionic strengths ( = 0.128 M
and = 0.045 M) could indicate that pH effect was not confounded
l = 0.081 M) and 8.0
(
l
Figure 3. Flavin analysis of purified Ssap-NtrB. (A) Absorption spectra of the
purified flavoprotein His6-Ssap-NtrB from S. saprophyticus is shown (solid line). Loss
of the FMN bands by reduction with an excess of sodium dithionite (dashed line)
together with commercial FMN standard (dotted line) are shown. Insert, the
characteristic part of the spectra (320–520 nm) was magnified for clarity. Protein
l
l
l
concentration was 1.6 mg/ml (63 lM) in 50 mM Tris–Cl buffer, pH 7.4. (B) HPLC
analysis of FMN extracted from His6-Ssap-NtrB. (C) HPLC analysis of FMN standard
by differences in ionic strength. Additionally, Ssap-NtrB lost its
obtained commercially.
activity considerably outside pH 6.0 and 8.5.
The effect of temperature on Ssap-NtrB was investigated by
monitoring the change of enzymatic activity over the range of
3–40 °C. The activity-temperature profile showed maximum
specific activity of 6.36 0.24 U/mg for the reduction of NFZ at
15–20 °C under the experimental conditions specified (Fig. 6A
(a)). (One unit is defined as the amount of the enzyme that
catalyses the conversion of 1
lmol of substrate per minute under
the experimental conditions specified.) Upon saturation the

A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
3545
Figure 4. Michaelis–Menten curves for NADPH and NADH-dependent reaction of
K3[Fe(CN)6] catalysed by Ssap-NtrB. Each point is the mean of triplicate assays.
Figure 6. Activity-temperature profile of Ssap-NtrB catalysed reduction of NFZ,
CB1954 and SN23862 (A) (a) NFZ reduction was measured by standard activity
assay with Ssap-NtrB in 50 mM sodium phosphate buffer at pH 7.0 in the
temperature range of 3–40 °C. (b) NFZ reduction was measured by standard
activity assay with Ssap-NtrB saturated with commercial FMN in 50 mM sodium
phosphate buffer at pH 7.0 in the temperature range of 3–40 °C. (B) NFZ, CB1954
and SN23862 reduction was measured in 50 mM sodium phosphate buffer at pH 7.0
at 3 and 40 °C. ⁄Activities are based upon monitoring of NADPH turnover and have
not been corrected for uncoupled oxidase activity and therefore they are upper
estimates.
Figure 5. Activity-pH profile of Ssap-NtrB profiles were determined using 50 mM
citrate (pH 5.4–6.2), 50 mM sodium phosphate (pH 6.0–7.9), 50 mM Tris–Cl (pH
7.4–9.0) and 50 mM glycine-NaOH (pH 8.6–10.6) buffers. The specific activity was
measured using NFZ as substrate and expressed as l
mol minꢁ1 mgꢁ1active enzyme
and the relative specific activity is expressed as the percentage of the maximum
specific activity obtained under the experimental conditions.
We investigated the kinetic stability of Ssap-NtrB by measuring
its half life at the different temperature points expanding from 20
to 50 °C for incubation time of 0–240 min (Fig. 7). The remaining
residual activity was determined at 20 °C using NFZ activity assay
and expressed as a percentage of activity obtained with no heat
treatment. Ssap-NtrB lost its activity notably at 30 °C within
60 min and almost complete thermo-inactivations were measured
within 3 min at above 50 °C. Saturating protein samples with com-
mercial FMN externally has raised enzyme activity 1.6-fold at opti-
mum temperature, but there was no significant contribution has
been observed for stability of Ssap-NtrB. The half life of the enzyme
at 30, 35, 40 and 50 °C have been calculated to be 160, 28, 7 and
<1 min, respectively.
enzyme with flavin cofactor (FMN) by incubation purified enzyme
with commercial FMN, followed by gel permeation chromatogra-
phy to remove unbound flavin, gave rise to an increase in activity
up to 10.40 0.19 U/mg at 20–25 °C (Fig. 6A(b)). Surprisingly,
unlike the other type I nitroreductases, over 95% and 80% activity
values relative to the maximum activity (at 15 °C) were measured
at 10 and 3 °C respectively for Ssap-NtrB without saturation. Upon
saturation, over 76% and 62% activity values relative to the maxi-
mum activity (at 20 °C) were measured at 10 and 3 °C, respectively.
At the other end of the lines, there were sharp decrease in activities
beyond the room temperature for both cases; only a fraction of the
maximum activities were obtained at 40 °C. In order to see the gen-
eralities of such observations, the temperature profile of substrates
of prodrugs including CB1954 and SN23862 were investigated at
both 15 and 3 °C monitoring either NFZ consumption and product
formation for CB1954 or cofactor consumption (NADPH reduction)
for NFZ and SN23862 (Fig. 6B). Activity profile of Ssap-NtrB against
prodrug of CB1954 and SN23862 was similar with NFZ, retaining
55 and 60% of the maximum activities, respectively.
3.7. Kinetic parameters and metabolite analysis
Preliminary activity experiments with fixed substrate concen-
tration showed that the purified enzyme was shown to be active
with NFZ, CB1954 and SN23862 (for the structure of substrates
and its metabolites, see Supplementary Scheme 1). Therefore, the

3546
A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
concentration of CB1954 was required to saturate the enzyme
(4 mM). Due to the limited solubility of CB1954, it is only possible
to obtain the initial part of the Michaelis–Menten curve for this
substrate, giving a good estimate of the specificity constant, kcat
/
KM, but poor estimates of apparent kcat or apparent KM individually.
The reduction of SN23862 resulted in mixture of product with
complex and unstable nature. Therefore, mass spectrometric
analyses was not completed and being under investigation
(Supplementary Fig. S3). The steady-state kinetic experiments
were repeated at a series of nine SN23862 concentrations ranging
from 0 to 160 lM. Any attempt to use higher concentration than
this range resulted in diminished activity of the enzyme by proba-
bly substrate inhibition. Therefore, Michaelis–Menten curve was
achieved using substrate concentration of up to 160
ies of eight different concentrations.
lM with a ser-
4. Discussion
Figure 7. Thermal stability of Ssap-NtrB. The enzyme was dissolved in 50 mM Tris–
Cl (pH 7.5) at a concentration of 0.4 mg/ml, and incubated for 0–240 min at the
temperatures indicated. The remaining residual activity was determined at 20 °C
using NFZ activity assay and expressed as a percentage of activity obtained without
heat pre-treatment of the enzyme. Each point is the mean of triplicate assays.
Homology based databank search identified a putative nitrore-
ductase gene in Staphylococcus saprophyticus. PCR based amplifica-
tion was performed and the resulting gene product was cloned into
an expression vector for evaluation. Use of pET14b vector gave a
construct that allowed production of fusion proteins with a polyhis-
tidine (His6) affinity tag site at their N-termini. Escherichia coli cells
(BL21(DE3)) that was transformed with the resulting recombinant
plasmid expressed the Ssap-NtrB protein upon induction with IPTG
in soluble form at the efficient level of 40–50 mg/L. The resulting
NTR was purified to homogeneity using metal-chelate affinity chro-
matography. Protein characterisation including sequencing, SDS–
PAGE and protein mass spectrometry confirmed its identity.
Bacterial oxygen insensitive nitroreductases could be classified
into two main groups that are representative of the E. coli NTRs:
group A (NfsA) and group B (NfsB). The phylogenetic analysis sug-
gested Ssap-NtrB to be relatively close to minor the E. coli nitrore-
ductase (NfsB) (Fig. 8). However, amino acids sequence comparison
analysis revealed that only 20% identity and 25% similarity with
NfsB.
reductive metabolism of NFZ, CB1954 and SN23862 by Ssap-NtrB
were investigated. HPLC analysis followed by mass spectrometric
analysis showed that the corresponding reduction products for
all three substrates were obtained.
In the case of NFZ, a single reduction product was obtained in
good yield under the experimental conditions (Supplementary
Fig. S1). The product of the enzymatic reduction of NFZ was iso-
lated using a reverse phase HPLC and analysed by ESI-MS in the
negative mode with a molecular ion [MꢁH]ꢁ at m/z = 183, indicat-
ing a molecular mass of 184, this corresponds to a loss of an oxygen
atom and the gain of two hydrogen atoms (a four-electron reduc-
tion) to form the hydroxylamine species as previously observed
with other nitroreductases.27 To determine the full kinetic param-
eters for NFZ reduction, the steady-state kinetic experiments were
repeated at a series of 13 NFZ concentrations ranging from 0 to
Similar to other type I nitroreductases, a concentrated solution
of Ssap-NtrB is yellow and resistant to decolouration when the en-
zyme solution was dialysed. This indicates that strong binding of
flavin to the enzyme. Spectroscopic analysis followed by HPLC
analysis proved that it is FMN that receives the reducing equiva-
lents from the pyridine nucleotides. Together, these results indi-
cate that Ssap-NtrB is a flavoprotein tightly associated with FMN.
Recombinant expression of His6-tagged Ssap-NtrB in E. coli yielded
high level of enzyme expression, but only about 30% of enzyme
contains FMN. The molar ratio of FMN:protein however can easily
be raised to 1.1 by addition of FMN externally.
Direct measurements of the reduction of the FMN group by pyr-
idine nucleotides showed that the enzyme displays no marked
preference for any of nucleotide used. The ratio of catalytic effi-
ciencies (kcat/KM) for NADPH over that of NADH for the reduction
of artificial electron acceptor (ferricyanide) is only 2.1-fold, show-
ing only a small favour towards to NADPH. This result indicates
that Ssap-NtrB has similar nucleotide preference with group B of
oxygen insensitive nitroreductases (type I),1 therefore it is named
as Ssap-NtrB. In E. coli, for examples, the nfsB uses both NADPH
and NADH to reduce NFZ and other nitro compounds, while nfsA
uses NADPH exclusively to catalyse similar reactions.14,47,48
The nitroreductase purified in this work showed maximal activ-
ity at between pH 6.0–8.0 regardless of the buffers used. This is
consistent with several well characterised bacterial nitroreducta-
ses, having an optimal pH around 7 (reviewed in1). The Ssap-NtrB
displayed, however distinct unusual characteristics in temperature
profiles with other nitroreductases. Among all major oxygen
300 lM. Ssap-NtrB catalysed reduction of NFZ obeyed Michaelis–
Menten kinetics (Supplementary Fig. S4A). All the kinetic data
were fitted to Michaelis–Menten equation by nonlinear regression
and calculated steady state parameters, kcat(app) and KM(app) are
given in Table 1. Steady state parameters for the reduction of
NFZ, CB1954 and SN23862 using other well known nitroreductases
were also shown in Table 1 for comparisons. HPLC analysis
indicated that Ssap-NtrB was able to reduce the prodrug of
CB1954 to mixture of the isomers 4HX [5-(aziridin-1-yl)-4-
hydroxyamino-2-nitrobenzamide] and 2HX [5-(aziridin-1-yl)-2-
hydroxyamino-4-nitrobenzamide] with around 1:1 molar ratio
(Supplementary Fig. S2). Under the experimental conditions,
almost all the substrate was converted to the metabolites within
30 min in the expense of NAD(P)H. Further studies by MS(ESPꢁ)
showed that the first isomer with retention time of 4.95 min could
be detected in the negative mode with a molecular ion [MꢁH]ꢁ at
m/z = 237, indicating a molecular mass of 238 and confirming that
it was indeed the 4HX (based on the information available in the
literature). Similarly, the second isomer with the retention time
of 12.86 min had the same characteristics as the first one, [MꢁH]ꢁ
at m/z = 237, indicating a molecular mass of 238 confirming that it
was the 2HX. Finally, very small amounts of metabolites (with
retention times of 19.2 and 24.5 min) were also observed.
Considering the retention times, we assumed that they were the
further reduction products of CB1954; the corresponding amines
(Supplementary Fig. S2). To examine the substrate specificity of
the enzymes, similar steady-state kinetic experiments were done
with prodrug of CB1954 and SN23862 as the substrates. High

A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
3547
Table 1
Kinetic parameters for the reduction of NFZ, CB1954 and SN23862 by purified Ssap-NtrB. Kinetic parameters for the reduction of NFZ, CB1954 and SN23862 using the major
nitroreductases have also provided for comparisons
kcat (sꢁ1
a
)
kcat/KM (sꢁ1 Mꢁ1
)
a
Enzyme
Substrates
Cofactor
KM
(l
M)
Ssap-NtrB (This work)
Ssap-NtrB (This work)
Ssap-NtrB (This work)
NFZ
NADPH
NADPH
NADPH
NADPH
NADH
NADPH
NADPH
NADH
NADPH
NADH
NADPH
NAD(P)H
49.8 3.9
2.20 0.05
2.26 0.07
0.77 0.07
10.5 0.1
10.7 0.2
16 0.48
26 2.6
44180
2120
9350
61764
66875
73000
7300
5600
110
CB1954
SN23862
NFZ
1065.2 53.1
82.4 16.9
170 13
NfsBb
NfsBb
NfsAc
NfsBc
NfsBc
AzoRc
AzoRc
ywrOd
ywrOd
NFZ
160
6
CB1954
CB1954
CB1954
CB1954
CB1954
CB1954
SN23862
220 18
3600 660
11000 2600
1400 160
6600 1000
618 169
Inactive
62 11
0.15 0.0077
0.15 0.015
8.2 1.1
23
13268
—
ND
a
b
c
These are apparent KM and kcat values at 200
Data was taken from.27
lM NADPH (for Ssap-NtrB), 250 lM NAD(P)H (for NfsA, NfsB and AzoR) and 500 lM NADPH (for ywrO).
Data was taken from.68
d
Data was taken from.24
Figure 8. Pylogenetic tree based on deduced amino acids sequences of the type I (oxygen insensitive) nitroreductases superfamily including new member, (Ç) SsapNTR (Ssap-
NtrB) from S. saprophyticus. There are two main groups in the type I nitroreductases; group A and B, which includes subgroups of B1 and B2. Members of the group A enzymes
includes the major E. coli nitroreductases of nfsA (s),61 E. cloacae NitA,62 E. cloacae SnrA,17 V. harveyi FRP,21 P. putida PrnA,20 Mycobacterium smegmatis NfnB,63 and P. putida
PnbA.20 The group B1 includes the minor E. coli nitroreductases of nfsB (d)48, Klebsiella pneumoniae NTR,64 E. cloacae NR (nfnB/nfs1),62 S. typhimurium Cnr,17 V. fischeri FRaseI,22
a hypothetical nitroreductases from Exiguobacterium sibiricum NTR,65 a hypothetical nitroreductases from S. epidermidis NTR66 and S. saprophyticus SsapNTR (Ssap-NtrB). The
group B2 includes E. coli nitroreductases of ydjA(}).30 and Staphylococcus aureus NtrA.67 Ungrouped nitroreductases from Bacillus amyloliquefaciens YwrO24 is also included.
insensitive nitroreductases characterised so far, Ssap-NtrB is
probably the first example that shows uncharacteristic tempera-
ture-activity profile; showing optimum activity at 15–20 °C and
retaining up to 85% of its optimum activity at 3 °C for the reduction
of NFZ and other substrates (3 °C was the minimum temperature at
which experiments were conducted properly. Below this
temperature, the activity measurements were hampered due to
excess condensation of water on the cell surface in the Uv–vis
spectrophometer). Saturation the enzyme with external FMN,
however, resulted in an increase in activity at about 1.6-fold, giving
specific activity of 10.40 0.19 U/mg at 20–25 °C due to probably
generating more active enzymes upon incorporation of flavin

3548
A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
(FMN). The saturated enzyme has again retained over 62% of its
optimum activity at 3 °C for the reduction of NFZ. It was noticed
that the temperature optimum was shifted slightly from 15–20
to 20–25 °C, for that we have no logical reasoning at present or
the shift may be not significant at all. Both E. coli nitroreductases
(NfsA and NfsB), for example, has optimum activity at 40 °C and
they lose their activity dramatically below 15 °C.49,50 Similar
activity-temperature profiles to E. coli nitroreductases have been
observed with other nitroreductases; examples include B. licheni-
formis (30 °C),26 Enterobacter cloacae NR (45 °C),51 Klebsiella sp. C1
Ntr (30–40 °C),52 V. fischeri FraseI (26 °C),22 R. capsulatus NprA
and NprB (30 °C)23 and Clostridium acetobutilicum NitA and NitB
(30 °C)53 (the temperature optimum were given in parentheses).
Although Ssap-NtrB shows some similar biochemical properties
with type I (group B) nitroreductases, it differs dramatically in this
aspect; being a cold active nitroreductase. The rapid temperature
dependent inactivation of the enzyme above the optimum,
strengths further the cold active nature of Ssap-NtrB. The existing
data concerning cold-adopted enzymes indicates that a high spe-
cific activity is almost always coupled with a low thermostabil-
ity.54 Saturating protein samples with commercial FMN added
externally has raised enzyme activity noticeably at optimum tem-
perature, but we have not seen any significant improvement in sta-
bility upon saturating the enzyme with externally added FMN. No
clear evidence was found to support the idea that FMN interacts
with both monomers and likely to contribute to thermal stability.
The finding, therefore, ruled out that low stability was due to lack
of FMN (or that the enzyme was being mainly in apo form) when
external FMN addition was omitted. The cold active nature of
Ssap-NtrB is quite surprising since the bacterium, from which the
nitroreductase cloned (S. saprophyticus) is known to belong to the
mesophilic bacteria, which grows best in moderate temperature
between 25 and 40 °C. Having active enzymes at low temperature
might be related to the bacterium ability for surviving in a cold
temperature environment. Finding cold-adopted nitroreductases
might, therefore shed a light on biological functions of nitroreduc-
tases in general and represent great potentials in many biotechno-
logical areas of interests.
relevant doses. We have evaluated Ssap-NtrB ability for activation
of CB1954 and another bioreductive prodrug called SN23862.
CB1954 was converted into two hydroxylamine metabolites with
equal amount as it was reported for E. coli NfsB catalysed reac-
tion.55 The product distribution or regioselectivity was postulated
to be dependent on solvent accessibility of the two nitro groups.32
Because, in silico studies using NfsB as model enzyme suggested
that the mechanism for reduction of CB1954 is by electron transfer
from FMN to the nitroaromatic ring, followed by protonation from
solvent. Although the formation of highly toxic metabolite (4HX) is
desirable as it is generally regarded as the principal cytotoxin to
form a bifunctional DNA cross-linking agent, the production of
both metabolites is also equally important. First of all, it has been
suggested that the mixture in CB1954 metabolites vary in cytotox-
icity toward different cell types. For instance, SKOV-3 cells are
more sensitive than HCT-116 to the 2-HX reduced metabolite.56
Secondly, the 2HX derivative of CB1954 was also shown to have
a greater bystander effect via its amine derivative in GDEPT, killing
neighbouring cells where the enzyme was not expressed.56 This is
particularly important since not all tumour cells will be infected
with the vector containing the gene of NTR. Finally, Ssap-NtrB
(similar to E. coli NfsB) can utilise both NADH and NADPH effi-
ciently as electron donors (whereas NfsA has a sole preference
for NADPH). E. coli NfsB shows small preference for former for
the reduction of both NFZ27 and CB195457 while Ssap-NtrB showed
a small favour towards to NADPH. Considering the intracellular
concentrations of reduced nicotinamide cofactors it could present
a small advantage over E. coli NfsB for EPT. (NADPH is usually pres-
ent at higher concentration than NADH in mammalian cells. Even
though the total amount of NAD+ and NADH is about 10 times
higher than that of NADP+ and NADPH, the ratio of NADH/NAD+
is much lower than the ratio of NADPH/NADP+; NADPH is being
the dominant form of reduced nicotinamide cofactor in the cells).58
Type I nitroreductases preferentially perform two-electron
chemistry with a ping-pong bi-bi kinetic mechanism.1 The steady
state kinetics of Ssap-NtrB with CB1954 are therefore, tentatively
compared with KM(app) and kcat(app) values with other nitroreducta-
ses1 and are summarised in Table 1 (Note that the concentration of
We have examined the kinetics and metabolite profiles of Ssap-
NtrB with nitrofurazone, CB1954 and SN23862. Nitroreductases
catalysed bioconversion of nitroaromatics to highly toxic corre-
sponding nitroso and hydroxyl amine metabolites have been
exploited in the use of nitrofuran derivatives as antibiotics. NFZ,
for example, is bactericidal for many Gram-positive and Gram-neg-
ative bacteria and this antibacterial is effective for control of Aero-
monas, Vibrio and related species. The study of Ssap-NtrB catalysed
NFZ reduction yielded a four-electron reduction product of the
hydroxylamine derivative ((E)-2-(5-hydroxyamino furan-2-yl-
methylene) hydrazinecarboxamide). The steady state kinetics re-
vealed comparable values with E. coli NfsB.27,43 Since type I nitro-
reductases follows ping-pong bi-bi mechanism, only specificity
constants (the ratio kcat/KM) are comparable meaningfully. The cat-
alytic efficiency of E. coli NfsB with NFZ is slightly better than Ssap-
NtrB under the experimental conditions (about 1.4-fold). This find-
ing is, however, not totally unexpected as Ssap-NtrB share low
amino acid sequence homology with E. coli NfsB (20% identity
and 24% similarity) and the flavin (FMN) content of the enzyme
in the recombinant form (His6-Ssap-NtrB), that is mandatory for
the enzyme reaction was found to be approximately 30%. An in-
crease of FMN/protein ratio would contribute positively in the
specificity constant.
NADPH were 200 and 250 lM for Ssap-NtrB and NfsB assays,
respectively). Even though the turnover number is lower than
E. coli NfsB, about 20-fold differences have been observed in KM(app)
indicating that Ssap-NtrB has higher affinity towards CB1954 than
NfsB. The flavin (FMN) content of the recombinant enzyme
(approximately 30%) again contributed significantly for obtaining
lower level of activity as it was explained for NFZ bioconversion.
However the specificity constant kcat/KM, which is the key param-
eter determining the rate of reaction at low substrate concentra-
tion, yielded similar values; only twofold lower specificity
constant obtained in comparison to that for NfsB.
The reduction of SN23862 yielded a mixture of products with
some complexity (Supplementary Fig. S3) and characterisation of
the metabolites were not completed and being under investigation.
The steady state kinetics of Ssap-NtrB with SN23862, however, sig-
nifies the extent of substrate specificity of the enzyme.
In conclusion, we have purified and characterised a nitroreduc-
tase enzyme from S. saprophyticus. Even though there are 20% iden-
tity and 25% similarity between this enzyme and the well known
nitroreductase; E. coli Ntr (NfsB), they share several biochemical
properties including reducing NFZ, CB1954 and SN23862 in the ex-
pense of either NADH or NADPH cofactors, pH activity-profiles and
molecular sizes. What they do not share is the activity-tempera-
ture profiles, in which E. coli Ntr (NfsB) has optimum activity at
30–40 °C with no reported activities below 15 °C, whereas Ssap-
NtrB retains considerable enzymatic activities at 15 and 3 °C. Such
unique property makes Ssap-NtrB as a versatile prodrug activating
enzyme. As in the enzyme prodrug therapy (EPT), use of E. coli NfsB
NAD(P)H-dependent nitroreductases are of importance due to
their potential in biomedicinal applications. In particular, several
cancer therapies, E. coli NfsB with cancer prodrug of CB1954 have
reached the clinical trial stages. On the other hand, poor catalytic
efficiency limits the activation of CB1954 by NfsB at clinically

A. Çelik, G. Yetißs / Bioorg. Med. Chem. 20 (2012) 3540–3550
3549
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in combination with prodrug of CB1954 has used to kill tumour
cells. To develop EPT further, a useful direction could be a combi-
nation of EPT with crymotheraphy59, a local treatment or refriger-
ation in which the tissues are exposed to a temperature of 4–10 °C.
In this case, cold-adopted nitroreductases such as Ssap-NtrB could
be an ideal candidate due to its significant activity at lowered tem-
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of damaging healthy tissues surrounding tumour cells. In addition,
Ssap-NtrB can easily be utilised to access sufficient amount of
temperature sensitive drug candidates and intermediates, enzy-
matically from prodrugs and other compounds for purpose of phar-
macological studies in vitro. Furthermore, cell ablation can be
utilised as a cure for certain disorders such as cancer and leukae-
mia. Cold active nature of Ssap-NtrB could possibly be used as a
tool to study conditional cell ablation60 in cold-blooded animals
(poikilotherms), which are becoming useful as experimental
subjects and are attracted the attention of scientists in a variety
of biological disciplines, including space biology and medical
research. Finally, application of organisms with degradative capa-
bilities, or their enzyme for eradicating pollutants from the envi-
ronment is of importance. Among the pollutants, nitroaromatic
compounds including TNT are widely used as recalcitrant explo-
sives and are required to be eliminated from the environment.
Our preliminary work indicated that Ssap-NtrB acts on simple
nitroaromatic tested so far (data not shown). As a result of this pre-
liminary work, it could be foreseen that Ssap-NtrB could therefore
be used in areas where high activity at low temperatures is re-
quired for bioremediation of compounds of explosive nature and
volatile toxic compounds.
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This work was funded by The Scientific and Technological Re-
search Council of Turkey (TUBITAK, Grant No. 110T754). We would
like to thank Professor Richard Knox (Morvus Technology Limited,
UK) for supplying cancer prodrugs (CB1954 and SN23862). We also
would like to thank Dr. Ali Turkan and Bunyamin Cosut for their
help with protein mass spectrometry and Aise Unlu for genomic
DNA preparation (all from Chemistry Department, GIT). In addi-
tion, Dr. Ferruh Ozcan and Dr. Nil Saydan (GIT, MBG Department)
have been acknowledged for their useful comments on the
manuscript.
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