Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase

Article abstract of DOI:10.1016/j.bioorg.2009.07.005

A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contri

Full text of DOI:10.1016/j.bioorg.2009.07.005

Bioorganic Chemistry 37 (2009) 167–172
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Kinetic evidence for an anion binding pocket in the active site
of nitronate monooxygenase q
Kevin Francis a, Giovanni Gadda a,b,c,
*
a Department of Chemistry, Georgia State University, Atlanta, GA 30302-4098, United States
b Department of Biology, Georgia State University, Atlanta, GA 30302-4098, United States
c The Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30302-4098, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 June 2009
Available online 25 July 2009
A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula
mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase,
to investigate the structural features that contribute to the binding of the anionic nitronate substrates to
the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as
competitive inhibitors of the enzymes was observed in a plot of pKis versus the ionic volume of the anion
with slopes of 0.041 0.001 mM/Å3 and 0.027 0.001 mM/Å3 for the H. mrakii and N. crassa enzymes,
respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition con-
stants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate
monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent Kd
values of ꢀ5 mM. These results are consistent with the presence of an anion binding pocket in the active
site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a min-
imal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme.
Ó 2009 Elsevier Inc. All rights reserved.
Keywords:
2-Nitropropane dioxygenase
Nitronate monooxygenase
Anion inhibition
Alkyl nitronates
Competitive inhibition
Hansenula mrakii
Neurospora crassa
Substrate specificity
1. Introduction
of NMO that distinguishes it from the well characterized nitroal-
kane oxidase [9], which catalyzes a similar oxidation reaction
Nitronate monooxygenase (E.C. 1.13.11.32; NMO), formerly
known as 2-nitropropane dioxygenase [1] is a flavin mononucleo-
tide-dependent (FMN) enzyme that catalyzes the oxidative denitri-
fication of alkyl nitronates to their corresponding aldehyde and
keto compounds and nitrite [2,3]. The most extensively character-
ized NMOs studied to date are those from Neurospora crassa [2,4–6]
and Hansenula mrakii [3,7], although the X-ray crystallographic
structure of the enzyme from Pseudomonas aeruginosa has also
been reported [8]. Detailed mechanistic studies have been carried
out only for N. crassa NMO where it was shown that a transient an-
ionic flavosemiquinone is formed during oxidative catalytic turn-
over of the enzyme through a single electron transfer reaction
between an enzyme-bound nitronate and the flavin cofactor
(Scheme 1) [2,4]. The formation of an anionic flavosemiquinone
intermediate during oxidative catalysis is a characteristic feature
through a different mechanism that involves the formation of a
covalent flavin N(5)-adduct [10–13].
Recombinant NMO from H. mrakii was recently cloned and ex-
pressed in Escherichia coli cells and the resulting purified enzyme
was characterized in its biochemical and kinetic properties [3].
The enzyme is similar to that from N. crassa in that it contains a
single non-covalently bound FMN per monomer of enzyme and is
devoid of metal cofactors [2,3]. Moreover, an anionic flavosemiqui-
none was observed upon anaerobic mixing of H. mrakii NMO with
alkyl nitronates with chain lengths ranging from two to six carbon
atoms [3]. Neither hydrogen peroxide nor superoxide is released
during turnover of the enzyme with primary alkyl nitronates as
evident from the absence of superoxide dismutase or catalase
effects on the rates of oxygen consumption in activity assays of
H. mrakii NMO [3], a result that was also obtained with the N. cras-
sa enzyme [2].
Both H. mrakii and N. crassa NMOs are able to effectively oxidize
a number of alkyl nitronates into their corresponding carbonyl
compounds and nitrite. Such an enzymatic oxidation is of consid-
erable interest given that many alkyl nitronates are known to be
toxic or mutagenic [14–17]. Ingestion of propyl-2-nitronate, for
example, has been demonstrated to result in the formation of
Abbreviations: NMO, nitronate monooxygenase; FMN, flavin mononucleotide.
q
This work was supported in part by Grant PRF #43763-AC4 from the American
Chemical Society (to G.G.) a Molecular Basis of Disease Fellowship and a Ambrose H.
Pendergrast Fellowship from Georgia State University (K.F.).
* Corresponding author. Address: Georgia State University, Chemistry Depart-
ment, P.O. Box 4098, Atlanta, GA 30302-4098, United States. Tel.: +1 404 413 5537;
fax: +1 404 413 5551.
8-aminodeoxyguanosine and 8-oxodeoxyguanosine through
a
E-mail address: ggadda@gsu.edu (G. Gadda).
0045-2068/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.07.005

168
K. Francis, G. Gadda / Bioorganic Chemistry 37 (2009) 167–172
2.3. Pre-steady state kinetics
R
N
R
N
H C
N
O
O
H C
N
O
O
3
3
The pre-steady state kinetic parameters of H. mrakii NMO were
determined in 50 mM potassium phosphate at pH 7.4 and 30 °C
using a TgK Scientific SF-61 stopped-flow spectrophotometer.
Rates of flavin reduction were measured by monitoring the in-
crease in absorbance at 372 nm that results from anaerobic mixing
of the enzyme with substrate as previously described for N. crassa
NMO [4]. Nitronate solutions (100 mM) were prepared in water by
incubating the nitroalkane in a 1.2 M excess of potassium hydrox-
ide for at least 24 h and were diluted in water prior to use. The final
NH
NH
H C
3
N
H C
3
N
H C
C
N
H
H C
C
N
H
3
3
O
O
O
O
Scheme 1. The one-electron oxidation of ethylnitronate catalyzed by nitronate
monooxygenase.
enzyme concentration in each assay was between 9 and 20 lM,
whereas the substrate concentrations used ranged from 0.1 to
50 mM, thereby ensuring that the enzymatic reaction follows
pseudo first-order kinetics.
phenol sulfotransferase-mediated metabolic pathway in both hu-
man and rat cell lines [18,19]. Despite their toxicity, alkyl nitro-
nates are widely used in chemical industry because they provide
a quick and efficient route for the synthesis of a wide range of com-
mercially useful compounds [20–22]. An investigation of the sub-
strate specificity of NMO can therefore provide the basis for use
of the enzyme in bioremediation applications to detoxify waste
generated from industrial uses of alkyl nitronates.
In the current study, the contributions of both the nitro and
hydrocarbon moieties of the alkyl nitronate substrate of NMO for
binding and specificity were investigated through inhibition and
rapid kinetics studies. The results are consistent with the presence
of an anion binding pocket in both the H. mrakii and N. crassa en-
zymes, which interacts with the nitro group of the substrate. These
interactions are key determinants for binding and recognition of
the substrate by the enzyme, rather than the hydrophobic interac-
tions that could occur between the enzyme and the alkyl moiety of
the substrate, as in the case of nitroalkane oxidase [23,24].
2.4. UV–visible absorbance spectra of NMO in the presence of sodium
nitrite
Changes in the UV–visible absorbance spectrum of NMO upon
addition of sodium nitrite were monitored using an Agilent
Technologies diode-array spectrophotometer Model HP 8453,
thermostated at 15 °C. Spectra of the H. mrakii and N. crassa en-
zymes (at concentrations of ꢀ80
lM) were recorded before and
after addition of 1 mM sodium nitrite. Difference spectra were
then constructed by subtracting the final absorbance spectrum
of the enzyme in the presence of sodium nitrite from that of
the free enzyme.
2.5. Data analysis
Steady state kinetic data were fit with either Enzfitter (Biosoft,
Cambridge, UK) or KaleidaGraph software (Synergy Software,
Reading, PA). Stopped-flow traces monitoring the reductive half
reaction of H. mrakii NMO were fit with Eq. (1), which describes
a single exponential process where kobs is the observed first-order
rate for the increase in absorbance at 372 nm, At is the absor-
bance at time t, and A is the final absorbance. Pre-steady state
kinetic parameters were determined using Eq. (2), where kobs is
the observed rate of flavin reduction, kred is the limiting rate con-
stant for flavin reduction at saturating substrate concentrations,
and Kd is the apparent dissociation constant of the substrate (S).
Inhibition data were fit with Eq. (3), which describes a competi-
tive inhibition pattern where Kis is the dissociation constant for
the inhibitor (I).
2. Materials and methods
2.1. Materials
NMOs from H. mrakii and N. crassa were obtained through the
expression and purification protocols described previously [3,5].
Nitroethane, 1-nitropropane and 1-nitrobutane were from Sig-
ma–Aldrich (St. Louis, MO). All other reagents were of the highest
purity commercially available.
2.2. Steady state kinetics
obst
Atotal ¼ Ateꢁk þ A
ð1Þ
ð2Þ
Enzymatic activity was measured in 50 mM potassium phos-
phate at pH 7.4 and 30 °C with the method of initial rates [25] by
monitoring the rate of oxygen consumption with a computer inter-
faced Oxy-32 oxygen monitoring system (Hansatech Instrument
Ltd.). Enzyme concentrations were expressed per bound FMN con-
tent using experimentally determined values of 13,100 Mꢁ1 cmꢁ1
kred
S
kobs
¼
Kd þ S
vo
e
kcat
S
I
¼
ð3Þ
Km½1 þ ð Þꢂ þ S
Kis
(
(
e
e
446 nm) for the H. mrakii enzyme [3] and of 11,850 Mꢁ1 cmꢁ1
444 nm) for the N. crassa enzyme [2]. The final concentration of en-
3. Results
zyme used in each assay was between 25 and 65 nM, whereas sub-
strate concentrations ranged from 0.5 to 20 mM. The nitronate
form of the substrate was prepared in 100% ethanol by incubating
the corresponding nitroalkane with 1.2 M excess of potassium
hydroxide for at least 24 h at room temperature. Since the sec-
ond-order rate constant for the protonation of ethylnitronate is
15 Mꢁ1 sꢁ1 [26], enzymatic activity assays were initiated by the
addition of substrate to the reaction mixture to ensure that a neg-
ligible amount of the neutral form of the nitronate is formed during
the time required to determine initial rates of reaction (typically
ꢀ30 s).
3.1. Nitrite inhibition of NMO with respect to ethylnitronate as
substrate
In order to establish if the nitro group of the akyl nitronate sub-
strates of H. mrakii and N. crassa NMO contributes to binding and
specificity, nitrite was used as a mimic of the substrate to establish
whether it inhibits the enzymes in 50 mM potassium phosphate
pH 7.4 and 30 °C. As shown in Fig. 1A for the case of the H. mrakii
enzyme, sodium nitrite behaved as a competitive inhibitor with re-
spect to ethylnitronate as substrate for both enzymes as indicated

K. Francis, G. Gadda / Bioorganic Chemistry 37 (2009) 167–172
169
Table 1
Inorganic anion inhibition of NMO with respect to ethylnitronate as substratea.
Inhibitor
Ionic volume (Å3)b
H. mrakii, Kis
N. crassa, Kis
(mM)c
(mM)d
Sodium nitrite
55
25
47
56
64
72
1.7 0.1
10
125
30
20
12
1
Potassium fluoride
Potassium chloride
Potassium bromide
Sodium nitrate
77
2
7
11.3 0.3
4.0 0.3
1.9 0.1
2
1
1
Potassium iodide
0.95 0.06
6.0 0.4
a
Enzymatic activity was measured at varying concentrations of ethylnitronate
and several fixed concentrations of inhibitor in 50 mM potassium phosphate pH 7.4
at 30 °C. Data were fit with Eq. (3).
b
From [27].
c
The steady state kinetic parameters had the following average values:
kcat = 180 15 sꢁ1; Km = 3.6 0.2 mM and kcat/Km = 48,500 6500 Mꢁ1 sꢁ1
.
d
The steady state kinetic parameters had the following average values:
kcat = 68 1 sꢁ1; Km = 1.6 0.1 mM and kcat/Km = 42,700 3300 Mꢁ1 sꢁ1
.
ing from ꢀ80 mM with potassium fluoride to ꢀ1 mM with potas-
sium iodide. Similar results were obtained for the N. crassa NMO,
with inhibition constants ranging from ꢀ125 mM for potassium
fluoride inhibition to ꢀ5 mM for potassium iodide inhibition. Nei-
ther enzyme was inhibited by potassium phosphate at concentra-
tions as high as 100 mM, suggesting that either dianions or
anions with an ionic volume of 90 Å3 [27] are unable to bind at
the active site of both the NMOs tested.
Fig. 1. Inhibition of H. mrakii NMO by sodium nitrite with respect to ethylnitronate
as substrate. Panel A: Inhibition of H. mrakii NMO. Enzymatic activity was measured
at varying concentrations of ethylnitronate in the presence of 0 mM (ꢃ); 5 mM (o);
10 mM (j); 20 mM (h) and 50 mM (N) sodium nitrite in 50 mM potassium
phosphate at pH 7.4 and 30 °C. The lines are from fits of the data to Eq. (3). Panel B:
UV–visible absorbance spectral changes induced upon addition of 1 mM sodium
nitrite to H. mrakii (top) of N. crassa (bottom) NMO in 50 mM potassium phosphate
pH 7.4 at 15 °C. Difference spectra were constructed by subtracting the final
absorbance spectrum of the enzyme in the presence of nitrite from that of the free
enzyme.
A plot of the pKis values as a function of the ionic volume of the
inorganic anions tested as inhibitors in the range from 25 to 64 Å3
(Fig. 2) yielded a straight line with both enzymes, with the excep-
tion of sodium nitrite. The experimentally determined inhibition
constants for sodium nitrite (Table 1) were between two- to
three-times lower than the values that can be predicted from the
size of the inorganic anion by lines of Fig. 2 of 4.7 mM for the
H. mrakii and 19 mM for the N. crassa enzymes. Inhibition of the
H. mrakii enzyme was more sensitive to the volume of the inor-
ganic anion as indicated by the slope of 0.041 0.001 mM/Å3 in
the plot of pKis versus ionic volume of the anion used as a compet-
itive inhibitor for the enzyme, as compared to 0.027 0.001 mM/
Å3 for the N. crassa NMO (Fig. 2). The y-intercepts determined from
the linear fitting of the data as shown in Fig. 2 were similar to one
another irrespective of the enzyme used, with a value of ꢀ
ꢁ2.8 mM. No correlation between the electron affinities of the
inorganic anions tested and the inhibition constants of NMO was
found, as illustrated in Fig. 2. The counterions of the inhibitors
tested had no effect on the inhibition of either enzyme as indicated
by the inhibition constants of sodium nitrate, which conformed to
the linear relationship between the ionic volumes of the anions
tested using potassium salts with the inhibition constants of the
NMO.
by the pattern of lines that intersect on the y-axis in double reci-
procal plot of the initial rate of oxygen consumption versus ethyl-
nitronate concentration at different fixed concentrations of
inhibitor1
. The corresponding inhibition constants (Kis) were
1.7 0.1 mM for the H. mrakii enzyme and 9.8 0.6 mM for the
N. crassa enzyme. The effect of the nitrite on the UV–visible absor-
bance spectra of the FMN cofactor of the H. mrakii and N. crassa
NMOs was also determined. As shown in Fig. 1B, binding of sodium
nitrite to both enzymes induced spectral changes in the FMN cofac-
tor bound at the active sites of the enzymes as seen from the
increase in the absorbance intensities at 333, 414, 436 and 465 nm
along with the concomitant decrease in absorbance intensities at
381 and 485 nm. All taken together, the inhibition and spectroscopic
studies are consistent with binding of nitrite occurring at the active
site of the enzyme.
3.3. Contributions of the alkyl chain length to substrate binding in
NMO
3.2. Inorganic anion inhibition of NMO with respect to ethylnitronate
as substrate
In order to establish whether the hydrocarbon chain of the alkyl
nitronate substrate is a determinant for binding to the active site of
the enzymes, the dissociation constants for a number of nitronate
substrates and aliphatic aldehydes inhibitors with hydrocarbon
chains of various lengths were determined in 50 mM potassium
phosphate at pH 7.4 and 30 °C. As shown in Fig. 3 for the case of
ethylnitronate as substrate, stopped-flow measurements demon-
strated that the reductive half reaction of the H. mrakii NMO in-
volves the formation of an anionic flavosemiquinone as evident
from the peaks centered at ꢀ372 and 490 nm in the UV–visible
absorbance spectrum obtained after anaerobic mixing of the en-
zyme with the substrate. The observed rates of flavin reduction in-
A series of monovalent, inorganic anions were tested as inhibi-
tors for NMO in 50 mM potassium phosphate pH 7.4 and 30 °C to
determine if the enzyme contains an anion binding site for recog-
nition of the nitro group of the substrate. As shown in Table 1, inor-
ganic anions ranging in size from 25 to 64 Å3 were competitive
inhibitors for the H. mrakii enzyme, with inhibition constants rang-
1
All of the inhibition data reported in this study were also fit with equations
describing noncompetitive and uncompetitive inhibition patterns. Since the data
were all best fit with a model describing competitive inhibition, only these fits are
shown.

170
K. Francis, G. Gadda / Bioorganic Chemistry 37 (2009) 167–172
Table 2
Reductive half reaction of H. mrakii NMO at pH 7.4 at 30 °Ca.
Substrate
kred (sꢁ1
)
Kd (mM)
Ethylnitronate
Propyl-1-nitronate
Butyl-1-nitronate
210
200
350
5
5
5
5.9 0.3
5.3 0.3
5.7 0.2
a
H. mrakii NMO was anaerobically mixed with substrate in 50 mM potassium
phosphate in a stopped-flow spectrophotometer and the absorbance changes at
372 nm were monitored over time.
Each aldehyde tested was a competitive inhibitor with respect
to ethylnitronate for the H. mrakii enzyme with inhibition con-
stants of ꢀ60 mM, which further suggests that the alkyl chain
length of the substrate contributes only minimally with respect
to the nitro moiety (Kis ꢀ 2 mM) for binding of the substrate to
the enzyme (Table 3). Similar, results were obtained with the
N. crassa enzyme with propanal, butanal, pentanal or hexanal, in
that the inhibition constants for aldehyde inhibition were large
(i.e., P70 mM), though accurate determinations could not be at-
tained due to limited solubility of the inhibitors in aqueous
solution.
4. Discussion
Binding of alkyl nitronates to NMO is primarily mediated
through interactions of an anion binding pocket within the active
site of the enzyme and the nitro moiety of the substrate. Evidence
supporting this conclusion comes from inhibition studies of the
H. mrakii and N. crassa enzymes with a series of monovalent, inor-
ganic anions of varying ionic volumes. Binding of the anions likely
occurs at the active site of the enzymes as evident from the spec-
troscopic changes of the FMN cofactor induced upon incubation
of the enzyme with nitrite, which resemble those induced by bind-
Fig. 2. Anion inhibition of NMO with respect to ethylnitronate as substrate. The
inhibition constants (Kis) for a series of monovalent anions were determined by
measuring enzymatic activity of H. mrakii (ꢃ) or N. crassa (s) NMO at varying
concentrations of ethylnitronate in the presence of several fixed concentrations of
inhibitor. All assays were carried out in 50 mM potassium phosphate at pH 7.4 and
30 °C. Top panel: plots of pKis versus ionic volume. Values for the ionic volumes of
the anions used were taken from [27]. Bottom panel: plots of pKis versus electron
affinity. Values for the electron affinity were taken from [31].
ing of anthranilate to the active site of L-amino acid oxidase [28],
and the competitive inhibition pattern observed with respect to
ethylnitronate as substrate. The linear correlation between the io-
nic volumes of the inorganic anions used as inhibitors and the inhi-
bition constants that describe their binding to the enzymes
suggests the presence of a discrete binding pocket with a well-de-
fined size, where the interactions at the anion binding site of the
two enzymes between the ligand and the enzyme are maximized
as the volume of the inorganic anion increases. The lack of inhibi-
tion of either enzyme by phosphate suggests that the anion bind-
ing pocket can accommodate either monovalent ligands, but not
divalent ones, or only those with an ionic volume smaller than
90 Å3 [27] which, by assuming a spherical geometry of the inor-
ganic anion, corresponds to a diameter of the monovalent anion
that is smaller than 5.6 Å. In agreement with the estimate of the
binding pocket deriving from our kinetic analysis, the available
three dimensional structure of NMO from P. aeruginosa clearly
indicates that an anion binding site is present at the active site of
Fig. 3. Pre-steady state kinetics of H. mrakii NMO with ethylnitronate as substrate
in 50 mM potassium phosphate pH 7.4 and 30 °C. Flavin absorbance at 372 nm was
monitored over time after anaerobic mixing of the enzyme with ethylnitronate to
give final concentrations of 13 lM enzyme and 0.5 mM ethylnitronate. The UV–
visible absorbance spectra before (black) and ꢀ30 s after mixing (red) are shown.
Inset: Rates of flavin reduction determined from the fits of the stopped-flow traces
at 372 nm to Eq. (1) were plotted as function of ethylnitronate concentration (0.5 to
50 mM) and were fit with Eq. (2). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this paper.)
Table 3
Aldehyde inhibition of H. mrakii NMO with respect to ethylnitronate as substratea.
Inhibitor
Kis (mM)
Propanal
Butanal
Pentanal
Hexanal
58
60
58
60
3
1
3
2
creased hyperbolically with the concentration of nitronate and
showed similar apparent Kd values of ꢀ5 mM with ethylnitronate,
propyl-1-nitronate and butyl-1-nitronate, suggesting that the
length of the alkyl chain of the substrate is not a determinant factor
for binding of the substrate to the enzyme. The kinetic parameters
for the reductive half reaction of H. mrakii NMO are summarized in
Table 2.
a
Enzymatic activity was measured at varying concentrations of ethylnitronate
and several fixed concentrations of inhibitor in 50 mM potassium phosphate pH 7.4
at 30 °C. Data were fit with Eq. (3). The steady state kinetic parameters had the
following average values: kcat = 185
3
sꢁ1
;
Km = 3.3 0.2 mM; kcat/
Km = 55,000 2200 Mꢁ1 sꢁ1
.

K. Francis, G. Gadda / Bioorganic Chemistry 37 (2009) 167–172
171
the bacterial enzyme in proximity of the nitro moiety of the sub-
strate [8], as illustrated in Fig. 4. In that enzyme, the anion binding
pocket is comprised mainly of the side chain of His152, the peptidyl
nitrogen of Gly151 and the side chain of Ser288. Although the crystal
structures of the enzymes studied in this report are not yet avail-
able, the alignment of the amino acid sequences of the three en-
zymes show that these residues are conserved in all three NMOs
[1]. In the H. mrakii enzyme, the amino acid residues corresponding
of the sizes and geometries of the anion binding pockets at the ac-
tive sites of the enzymes from H. mrakii and N. crassa will have to
await the elucidation of the X-ray crystallographic structures of the
two enzymes, which is currently ongoing in collaboration with
Weber’s group at Georgia State University.
Substrate recognition by NMO is predominantly determined by
the interactions occurring at the active site binding pocket of the
enzyme with the nitro group of the alkyl nitronate substrate with
minimal, if any, hydrophobic interactions of the enzyme with the
alkyl chain of the substrate. Evidence supporting this conclusion
comes from the comparison of the Kis values for aldehyde inhibi-
tion with respect to nitrite inhibition, which shows that the former
are at least 10-times smaller than the latter with both the enzymes
tested. Lack of interaction of the alkyl chain of the ligand with the
enzyme is independently supported by the similar values for the
dissociation constants for substrate binding (Kd) determined for
the H. mrakii enzyme with alkyl nitronates of varying chain lengths
of between two and four carbon atoms. Indeed, one would expect a
progressive decrease in the Kd values for the substrate with
increasing lengths of the alkyl chain of the substrate if hydrophobic
interactions played a significant role for substrate binding. The re-
sults suggesting that NMO does not discriminate its ligands by
exploiting the organic moiety of the substrate are consistent with
previous studies of the enzyme from N. crassa that established that
m-nitrobenzoate effectively binds to the enzyme (i.e., Kis value of
9.1 mM at pH 7.4), despite its large size and aromatic character
[4]. Further in agreement with minimal contribution of the alkyl
chain of the substrate to binding are previous studies of the H. mra-
kii and N. crassa enzymes showing that the kcat/Km values for nitr-
onates ranging from two to six carbon atoms are independent of
the alkyl chain length of the substrate [2,3]. As illustrated in
Fig. 4, the three dimensional structure of the bacterial enzyme
from P. aeruginosa with 2-nitropropane bound at the active site
shows the presence of a wide cavity in the active site of the en-
zyme, which is large enough to accommodate substrates of various
lengths or different structures [8].
to the anion binding pocket of the bacterial enzyme are His197
,
Gly196 and Ser351. The equivalent residues in the N. crassa enzyme
are His196, Gly195 and Ser342 (or Thr344).
A comparison of the slopes in the plots of pKis versus ionic vol-
umes for the inorganic anions acting as inhibitors of the H. mrakii
and N. crassa NMOs is consistent with the anion binding pocket of
the H. mrakii enzyme being slightly smaller than that of the N. cras-
sa enzyme. In this respect, the slopes of the lines that fit the data in
the plots of Fig. 2 depend on either the values of the inhibition con-
stants that describe the binding of the inorganic anions to the en-
zyme, the ionic volumes of the inorganic anions, or both. In
principle, the binding of the inorganic anions to the binding pock-
ets in the two enzymes can be affected by a number of factors
including geometric, steric, or electrostatic effects. However, the
observation that the fits of the data in Fig. 2 yields values for the
y-intercepts for the two enzymes that are similar to one another
is consistent with the slope effect that is experimentally observed
being due primarily to the volume of the anion binding pockets of
each enzyme rather than other factors. The accurate determination
5. Conclusions
In conclusion, the results presented herein demonstrate that the
active site of yeast NMO contains an anion binding pocket, which
participates in the binding of the nitro group of the alkyl nitronate
substrates. Thus, the nitro group of the substrate is the key deter-
minant for binding of the substrate at the active site of the enzyme
as opposed to the hydrocarbon chain of the nitronate molecule act-
ing as substrate, which plays a minimal role, if any, in binding by
the enzyme. These results contrast those previously reported for
nitroalkane oxidase, whose ability to bind substrates at the active
site increases with increasing lengths of the alkyl chain of the sub-
strate and reaches a maximum value with substrates containing
four or more carbon atoms [24]. A study of the pH and kinetic iso-
tope effects on nitroalkane oxidase revealed that each methylene
group of the substrate provides approximately 2.6 kcal/mol of
binding energy [23], which was recently explained through struc-
tural studies of the enzyme that demonstrated a hydrophobic
channel leading to the active site of the enzyme [12,29,30].
Although structural studies have yet to be reported for the H. mra-
kii and N. crassa enzymes, the X-ray crystallographic structure of P.
aeruginosa NMO shows a solvent accessible active site that lacks a
hydrophobic channel like that seen in nitroalkane oxidase [8]. The
elucidation of binding pockets for anionic ligands in the active site
of enzymes through inhibition studies with inorganic anions of
various ionic volumes demonstrates a kinetic method that should
be generally applicable to any enzyme whose crystallographic
structure is not yet available.
Fig. 4. Anion binding site in P. aeruginosa NMO. An electrostatic potential map was
generated for the X-ray crystallographic structure of P. aeruginosa NMO in complex
with 2-nitropropane (PDB ID: 2GJN) using an Adaptive Poisson–Boltzmann Solver
[32] and visualized using Pymol. Panel A: Electrostatic potential surface of the active
site of P. aeruginosa NMO. Panel B: Active site amino acid residues that comprise the
anion binding site in P. aeruginosa NMO.

172
K. Francis, G. Gadda / Bioorganic Chemistry 37 (2009) 167–172
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