Identification of a hypothetical protein from podospora anserina as a nitroalkane oxidase

Article abstract of DOI:10.1021/bi100610e

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k_cat/K_M-pH profile with nitroethane shows a pK _a of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k_cat/K_M value for nitroethane over 2 orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 A, confirming its identification as an NAO.

Full text of DOI:10.1021/bi100610e

Biochemistry 2010, 49, 5035–5041 5035
DOI: 10.1021/bi100610e
Identification of a Hypothetical Protein from Podospora anserina as a
Nitroalkane Oxidase†,‡
§
§
Jose R. Tormos, Alexander B. Taylor, S. Colette Daubner, P. John Hart,§,^ and Paul F. Fitzpatrick*,§,#
ꢀ
§Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78229, Department of Biology,
St. Mary’s University, San Antonio, Texas 78228, ^Department of Veterans Affairs, Audie Murphy Division, Geriatric Research,
Education, and Clinical Center, South Texas Veterans Health Care System, San Antonio, Texas 78229, and #Department of
Chemistry, University of Texas, San Antonio, San Antonio, Texas 78249
Received April 21, 2010; Revised Manuscript Received May 17, 2010
ABSTRACT: The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of
primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a
member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum
have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for
enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal
enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and
characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the
F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in
PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The kcat/KM-pH profile with
nitroethane shows a pKa of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to
asparagine decreases the kcat/KM value for nitroethane over 2 orders of magnitude. The R406K and S373A
mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has
˚
been determined at a resolution of 2.0 A, confirming its identification as an NAO.
The flavoenzyme nitroalkane oxidase (NAO)1 from the soil
Scheme 1
fungus Fusarium oxysporum catalyzes the oxidation of nitroalk-
anes to the corresponding aldehydes or ketones with the release of
nitrite and the consumption of molecular oxygen to yield hydro-
the best substrates (12). With the slow substrate nitroethane (13),
formation of the substrate anion is rate-limiting for the reductive
half-reaction (12).
gen peroxide (Scheme 1) (1, 2). NAO is unusual, since it catalyzes
substrate oxidation by removing a substrate proton to form a
carbanion intermediate (3) (Scheme 2), whereas flavoproteins that
oxidize carbon-nitrogen and carbon-oxygen bonds typically
catalyze cleavage of the substrate carbon-hydrogen bond by
removal of a hydride rather than a proton (4-6). The NAO
reaction is initiated by abstraction of the R-proton from the
neutral form of the nitroalkane by Asp402, the active site base (7),
creating a nucleophilic nitroalkane anion (Scheme 2). The anion
then attacks the N5 position of FAD to form an adduct; this
eliminates nitrite to generate a cationic, electrophilic flavin imine
that can be attacked by hydroxide (8-11). Release of the aldehyde
or ketone product forms reduced FAD (7). In the more typical
oxidative half-reaction, the reduced FAD is oxidized by molecular
oxygen to form hydrogen peroxide (2). Release of products from
the oxidized enzyme limits turnover with primary nitroalkanes,
The proton transfer reaction between nitroethane and Asp402
catalyzed by F. oxysporum NAO provides an excellent opportu-
nity for a study of the basis for enzymatic catalysis because the
enzymatic process can be modeled by the reaction of nitroethane
and acetate in water (14). Nitroalkanes represent a prototypical
system for the study of carbon acidity, in that they exhibit
surprisingly slow deprotonation rates compared with their high
acidities, the so-called nitroalkane anomaly (15). NAO from
F. oxysporum catalyzes the ionization of nitroethane 109-fold
more effectively than acetate. Computations suggest that there
is a slight enhancement of quantum mechanical tunneling of
the proton in the active site compared to the nonenzymatic
reaction (16).
NAO is a structural member of the flavoenzyme acyl-CoA
dehydrogenase (ACAD) superfamily (10, 17, 18), even though
the sequences of the ACAD family members are only 13-23%
identical to that of NAO (1, 17). Both cofactors and the active
site bases occupy similar locations in NAO and ACAD, but the
substrates access the active site from opposite sides of the two
proteins (11, 19). Structural and mutational analyses have
identified key active site residues in F. oxysporum NAO. The
nearest residues to Asp402 are Arg409 and Ser276; together, the
three residues make up a catalytic triad. Mutation of Asp402
to alanine decreases the rate constant for abstraction of the
†This work was supported in part by NIH Grant GM 058698 to
P.F.F. and Robert A. Welch Foundation Grant AQ-1399 to P.J.H.
‡The atomic coordinates for PODANSg2158 have been deposited
with the Protein Data Bank with the file name 3MKH.
*To whom correspondence should be addressed at the Department
of Biochemistry, University of Texas Health Science Center. Phone:
(210) 567-8264. Fax: (210) 567-8778. E-mail: Fitzpatrick@biochem.
uthscsa.edu.
1Abbreviations: NAO, nitroalkane oxidase; Ni-NTA, nickel nitrilo-
triacetic acid; IPTG, isopropyl thio-2-D-galactopyranoside; DTT,
dithiothreitol; EDTA, ethylenediaminetetraacetic acid; Hepes, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid; ACAD, acyl-CoA de-
hydrogenase.
r
2010 American Chemical Society
Published on Web 05/18/2010
pubs.acs.org/Biochemistry

5036 Biochemistry, Vol. 49, No. 24, 2010
Tormos et al.
Scheme 2
(IPTG) was purchased from Research Products International
Corp. (Prospect, IL). Kanamycin monosulfate was purchased
from Fisher Bioreagents (Fair Lawn, NJ). Lysozyme was ob-
tained from Roche Molecular Biochemicals (Indianapolis, IN);
ethylenediaminetetraacetic acid (EDTA) was obtained from
Acros Organics (Morris Plains, NJ); potassium phosphate and
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) were
obtained from Fisher Scientific (Fair Lawn, NJ). The nickel
nitrilotriacetic acid (Ni-NTA) column was obtained from Invitro-
gen (Carlsbad, CA). Ampicillin was from USB Corp. (Cleveland,
OH). The pET21b(þ) vector was from Novagen (Madison, WI).
The pJ201:20714 plasmid containing an artificial gene coding
for PODANSg2158 (protein XP 001905136.1; GeneID 6189407)
from P. anserina 980 with codons optimized for expression in
Escherichia coli was obtained from DNA 2.0 (Menlo Park, CA).
Oligonucleotides were synthesized by the Nucleic Acid Core
Facility at the University of Texas Health Science Center at San
Antonio. Restriction endonucleases and Taq polymerase were
purchased from New England Biolabs (Ipswich, MA). T4 ligase
and dNTP’s were obtained from Promega (Madison, WI). Pfu
DNA polymerase was obtained from Stratagene (La Jolla, CA).
Plasmids were purified using mini- and midi-kits from Qiagen.
E. coli strain BL21(DE3) from Novagen was used for protein
expression, and strain XL10-Gold (Stratagene) was used during
DNA cloning protocols. DNA sequencing was carried out with
the Applied Biosystems Big Dye Terminator V3.1 kit and
analyzed by the Nucleic Acid Core Facility at the University of
Texas Health Science Center at San Antonio. The D399N, R40K,
and S273A mutations were generated with the QuikChange site-
directed mutagenesis kit (Stratagene).
ExpressionandPurificationofPODANSg2158. The DNA
coding for PODANSg2158 was subcloned from pJ201:20714 into
pET21b(þ) using the NcoI and EcoRI sites at the 50 and 30 ends,
respectively, to obtain pPODNE. A single colony of E. coli strain
BL21(DE3) transformed with pPODNE was used to inoculate
25 mL of LB containing 100 μg/mL ampicillin at 37 ꢀC. The
starter culture was grown for 10-12 h; 10 mL was used to
inoculate 1 L of LB containing 100 μg/mL ampicillin at 37 ꢀC.
When the A600 value of the culture reached 0.6, IPTG was added
to a final concentration of 0.25 mM, and the temperature of the
culture was lowered to 18 ꢀC. After 14-20 h, cells were harvested
by centrifugation at 5000g and 4 ꢀC for 30 min. The cells were
suspended in 10 volumes of 50 mM Hepes, pH 7.0, 1.0 mM
EDTA, 0.1 mM FAD, 0.1 mM DTT, 0.5 mM phenylmethane-
sufonyl fluoride, and 25 μg/mL lysozyme. The suspension was
sonicated for 7 min on ice and centrifuged at 12000g for 20 min.
The extract was loaded onto a Ni-NTA column that had been
previously equilibrated with 50 mM Hepes buffer, pH 8.0. The
column was eluted with a linear gradient from 0 to 2 M imidazole
(200 mL total) in the same buffer. Fractions with the highest
purity as judged by UV-visible absorbance and SDS-PAGE
were pooled and concentrated using an Amicon Ultra (Millipore)
centrifugal filter device. The mutant enzymes were expressed
and purified following the same protocol. Purified enzymes were
stored with 15% glycerol at -80 ꢀC.
nitroalkane proton by at least 1000-fold (3). The distance
between Asp402 and Arg409 is appropriate for an electrostatic
interaction, suggesting that Arg409 is involved in correctly
positioning the active site base for catalysis. Mutation of
Arg409 to lysine decreases the rate constant for proton abstrac-
tion by 100-fold and alters the position of the active site
base (20). The side chain hydroxyl of Ser276 forms hydrogen
bonds with the carboxylate oxygens of Asp402 (20). Mutation
of Ser276 to alanine decreases the rate of the proton abstraction
by 100-fold without perturbing the active site structure (11).
When the sequence of F. oxysporum nitroalkane oxidase was
first determined, its use as a query in a PSI-BLAST (21) search of
the available sequence databases identified multiple sequences of
acyl-CoA dehydrogenases, establishing the enzyme as related to
that family of flavoproteins (17). However, all of the sequences
identified in such a search contained glutamate as the active site.
Since ACADs utilize a glutamate as the active site base (22),
whereas NAO utilizes aspartate (7), all of these enzymes were
likely to be true ACADs. The lack of other NAOs rules out the
use of sequence analyses to identify residues important for
catalysis and to provide insight into the structural basis for the
divergent reactions of ACAD and NAO. We report here the
identification of several probable NAOs from other fungi and
provide kinetic and structural data firmly establishing the
hypothetical protein coded by gene PODANSg2158 of Podos-
pora anserina as an NAO.
MATERIALS AND METHODS
Extinction Coefficient of PODANSg2158. An equal
volume of 7 M urea was added to the enzyme in 50 mM Hepes,
pH 8.0 (8). Any precipitated protein was removed by centrifuga-
tion for 10 min at 14000g, and the visible absorbance spectrum of
the supernatant was recorded. The change in absorbance between
the enzyme-bound FAD and the free FAD after denaturation was
used to calculate the extinction coefficient of the purified enzyme.
Materials. FAD, 1-nitroethane, 1-[1,1-2H2]nitroethane
(99% D), 1-nitropropane, 1-nitrobutane, 1-nitropentane, 1-nitro-
hexane, dimethyl sulfoxide, sodium pyrophosphate, and phenyl-
methanesulfonyl fluoride were obtained from Sigma-Aldrich
(Milwaukee, WI). Dithiothreitol (DTT) was purchased from
Inalco Spa (Milan, Italy). Isopropyl thio-2-D-galactopyranoside

Article
Biochemistry, Vol. 49, No. 24, 2010 5037
F
IGURE 1: Alignment of active site residues of F. oxysporum NAO with likely orthologues: PODANSg2158, P. anserina DSM 980 hypothetical
protein; TSTA 069890, Talaromyces stipitatus putative acyl-CoA dehydrogenase; PMAA 028790, Penicillium marneffei electron transport
oxidoreductase; FG02379.1, G. zeaePH-1 hypotheticalprotein;SNOG 15929, Phaeosphaeria nodorum SN15 hypotheticalprotein; CHGG01268,
Chaetomium globosum CBS 148.51 hypothetical protein; BC1G 11641, Botryotinia fuckeliana B05.10 hypothetical protein; SS1G 09730,
Sclerotinia sclerotiorum 1980 hypothetical protein; NFIA 030710, Neosartorya fischeri putative acyl-CoA dehydrogenase; AN9162.2, Aspergillus
nidulans hypothetical protein; NAO, F. oxysporum nitroalkane oxidase. Conserved residues are in bold, and the active site catalytic triad Ser276,
Asp402, and Arg409 in NAO is in bold and underlined.
Assays. Nitroalkane oxidase activity was routinely measured
in 0.5 mM FAD and 50 mM Hepes, pH 8.0, by monitoring the
rate of oxygen consumption with a computer-interfaced YSI
Model 5300A biological oxygen electrode (YSI Incorporated,
Yellow Springs, OH) at 30 ꢀC, as described previously (23). When
the pH was varied, 50 mM sodium pyrophosphate was used over
the pH ranges 5.4-7.0 and 8.0-11.0, and 50 mM potassium
phosphate was used between pH 7.0 and pH 8.0. To vary the
concentration of oxygen, oxygen and argon were combined in
different ratios with a MaxBlend low-flow air/oxygen blender
(Maxtec Inc., Salt Lake City, UT), and the assay buffer was
F
IGURE 2: UV-visible absorbance spectrum of PODANSg2158.
Conditions: 50 mM Hepes at pH 8.0 and 25 ꢀC.
equilibrated with the gas mixture. Substrate solutions of nitroalk-
anes were prepared as stocks in dimethyl sulfoxide to prevent the
formation of the anionic substrate. The concentration of active
enzyme was determined using an extinction coefficient at 446 nm
F. oxysporum NAO coordinates in Protein Data Bank entry
2C0U (10) as the search model. Coordinates were refined against
the data using PHENIX (26), including simulated annealing, and
alternated with manual rebuilding using COOT (27). The co-
ordinates have been deposited in the Protein Data Bank with
accession code 3MKH.
of 11.9 mM-1 cm-1
.
Data Analysis. Steady-state kinetic data were analyzed using
the programs KaleidaGraph (Synergy Software, Reading, PA)
and IgorPro (WaveMetrics, Inc., Lake Oswego, OR). Steady-
state kinetic parameters were determined by fitting the data to the
Michaelis-Menten equation. The pH dependence of the kcat/KM
value for nitroethane was determined by fitting initial rate data to
eq 1, which applies for pH profiles that show a decrease with unit
slope at low pH. Here, Y is the kcat/KNE value at a given pH, c is
the kcat/KNE value in the pH-independent range, and K1 is the
dissociation constant for the ionization of a group that must be
unprotonated for binding or catalysis.
RESULTS
Identification of NAO Orthologues. To identify ortholo-
gues of F. oxysporum NAO, PSI-BLAST searches of the available
sequence databases were carried out. Since the use of aspartate
versus glutamate as the active site base readily distinguishes NAO
from ACAD, all positive hits containing a glutamate in the
position corresponding to Asp402 were discarded. This screen
eliminated all plant, bacterial, and vertebrate sequences. How-
ever, several fungal proteins fit this initial criterion for NAO
orthologues (Figure 1). Their sequence identities to F. oxysporum
NAO range from 43% to 86%. All contain an arginine corres-
ponding to Arg409 of the F. oxysporum enzyme. All but one
contain a serine corresponding to Ser276; the lone exception from
Gibberella zeae contains a threonine in that position. We selected
the hypothetical protein from the fungus P. anserina 980 coded by
gene PODANSg2158 for further study since it has the lowest
sequence identity to the F. oxysporum enzyme at 43%.
Characterization of PODANSg2158 Expressed in E. coli.
The DNA encoding PODANSg2158 was subcloned directly from
pJ201:20714 into pET21b(þ) using the NcoRI and EcoRI sites at
the 50 and 30 ends, respectively, for expression of the His-tagged
enzyme. Approximately 40 mg of pure NAO could be obtained
from 2 L of culture. The visible absorbance spectrum of the
purified enzyme (Figure 2) establishes that the enzyme is a
flavoprotein. Based on the small change in absorbance of the
ꢀ
ꢁ
c
log Y ¼ log
ð1Þ
1 þ ðH=K1Þ
Crystallization, Structure Determination, and Refinement.
Preliminary crystals of PODANSg2158 were grown in the
UTHSCSA X-ray Crystallography Core Laboratory from crys-
tallization screen kits (Qiagen Inc., Valencia, CA) with a Phoenix
crystallization robot (Art Robbins Instruments, Sunnyvale, CA).
Optimized crystals were grown within 1 week by the vapor
diffusion method with the protein solution mixed in a 1:1 ratio
with a buffer containing 2.5 M magnesium sulfate, 0.1 M 2-(N-
morpholino)ethanesulfonic acid buffer, pH 6, and 18% glycerol.
The crystals were flash-cooled using liquid nitrogen prior to data
collection. Data were collected at the Advanced Photon Source
beamline 24-ID-C (Argonne National Laboratory, Argonne, IL)
equipped with an ADSC Quantum 315 CCD detector. Data were
processed using HKL-2000 (24). Phases were generated by the
molecular replacement method in PHASER (25) using the

5038 Biochemistry, Vol. 49, No. 24, 2010
Tormos et al.
Scheme 3
flavin when the protein is denatured, the extinction coefficient of
the enzyme at 446 nm is 11.9 mM-1 cm-1. The crystal structure of
the enzyme shows FAD bound to the active site (see below),
establishing this as the form of the cofactor. The purified enzyme
is active with nitroethane as the substrate, with a specific activity
of 7.5 μmol min-1 mg-1. This is comparable to the value of
10.6 μmol min-1 mg-1 for F. oxysporum NAO (17). Thus, this
protein can be classified as a nitroalkane oxidase.
Steady-State Kinetic Parameters for PODANSg2158.
Previous steady-state kinetic analyses of F. oxysporum NAO have
established the steady-state kinetic mechanism as a modified ping-
pong mechanism typical of flavoprotein oxidases (Scheme 3)
(2, 7, 28). Equation 2 gives the appropriate rate equation for such
a mechanism. The appropriateness of eq 2 rather than the more
complex eq 3 for a sequential mechanism can readily be deter-
mined using the fixed ratio method (29). In this approach initial
rates are measured at different concentrations of the two sub-
strates, keeping the ratio of their concentrations constant. Cur-
vature in a double reciprocal plot of the resulting data suggests a
sequential mechanism, since there will be a term containing the
square of the concentration of one substrate in the rate equation
(eq 4, a = [O2]/[NE]). On the other hand, a linear plot will arise if
there is no term in the rate equation containing the concentration
of both substrates (eq 5). For PODANSg2158 a double reciprocal
plot of initial rate vs nitroethane concentration is linear (Figure 3),
F
centration of nitroethane at a fixed ratio of nitroethane to oxygen of 2
at pH 8.0 and 30 ꢀC.
IGURE 3: Double reciprocal plot of the initial velocity vs the con-
monotonic increase in the kcat/KM value with substrate chain
length that F. oxysporum NAO does.2
pH Dependence of kcat/KNE. The effect of pH on the kcat/KM
value with nitroethane as substrate for PODANSg2158 was
determined in order to obtain information about amino acid
residues important for binding and catalysis in that enzyme.
Figure 5 shows that the kcat/KNE value is constant at high pH and
decreases at low pH, resulting in a pKa of 5.9 ( 0.1. In
comparison the pKa for F. oxysporum NAO is 6.9 ( 0.1 (12).
Mutagenesis of Active Site Residues. To probe the role of
the catalytic triad in PODANSg2158 (Asp399, Arg406, and
Ser273), these residues were replaced with asparagine, lysine,
and alanine, respectively. The steady-state kinetic parameters of
the mutant enzymes with nitroethane are listed in Table 1. The
largest decrease was observed for the mutation of Asp399, as
there is a 400-fold decrease in kcat and ∼200-fold decrease in the
kcat/KM for nitroethane. The effect on the kcat value is a more
valid measure of the effect on the activity, in that the very low
activity of this mutant enzyme makes accurate measurement of a
kcat/KNE value difficult. Mutagenesis of Arg406 to lysine results in
establishing eq 2 as appropriate and yielding a kcat value of 15 s-1
.
For such a mechanism, the kcat/KM values of the nitroalkane and
oxygen are independent of the concentration of the other
substrate. The kcat/KM values for oxygen and nitroethane were
determined in separate analyses by varying each at a fixed
concentration of the other. These values are given in Table 1.
The KO value for PODANSg2158 is higher than that for the
2
F. oxysporum NAO (2) but within the range found for a number
of other flavoprotein oxidases (30-34).
only a 1.7-fold decrease in kcat and a 24-fold decrease in kcat/KNE
.
Mutation of the Ser273 to alanine results in only a decrease of
∼3-fold for both the kcat and kcat/KNE values.
E
1
KO
KNE
2
¼
þ
þ
ð2Þ
ð3Þ
ð4Þ
v0
kcat kcat½O2ꢀ kcat½NEꢀ
Crystal Structure of PODANSg2158. In order to provide
a more complete identification of the P. anserina enzyme as an
NAO, we determined the three-dimensional structure of the wild-
type enzyme by X-ray crystallography. The data collection and
refinement are reported in Table 2. The crystallization conditions
were different from those used for F. oxysporum NAO, yielding
E
1
KO KNE KNEKO
2
2
¼
¼
þ
þ
þ
v0
kcat kcat½O2ꢀ kcat½NEꢀ kcat½NEꢀ½O2ꢀ
E
1
K
KNE KNE
K
O2
O2
þ
þ
þ
2
˚
crystals that diffracted to 2.0 A resolution. The protein crystal-
v0
kcat kcata½NEꢀ kcat½NEꢀ
kcata½NEꢀ
KNE
lized in space group P43, with four subunits in the asymmetric
unit, consistent with the tetrameric structure of F. oxysporum
NAO (10). The structure was determined by molecular replace-
ment using the structure of that enzyme as the search model (10).
FAD could be clearly identified in each of the subunits. Figure 6
shows an overlay of the electron density map with the FAD and
active site residues. This figure also shows the chain of ordered
water molecules that extends from the FAD 20-hydroxyl to
the protein surface through a different tunnel than the substrate
binding site. A similar water chain is seen the structure of
F. oxysporum enzyme, where it has been proposed to be involved
in accepting the proton from the water molecule that forms the
hydroxide required in the mechanism in Scheme 2 (11). Figure 7
depicts an overlay of the carbon backbones for one subunit from
E
1
KO
2
¼
þ
þ
ð5Þ
v0
kcat kcata½NEꢀ kcat½NEꢀ
F. oxysporum NAO prefers linear nitroalkanes as substrates,
with primary nitroalkanes of four or more carbons having the
highest activity. To probe the substrate specificity of the P. anserina
enzyme, the kcat/KM values for a number of unbranched primary
nitroalkanes were determined. As shown in Figure 4 the latter
enzyme also prefers longer nitroalkanes, with 1-nitrohexane hav-
ing the highest activity. However, this enzyme does not show the
2Longer chain nitroalkanes are poorly soluble in aqueous solutions
and therefore were not characterized as substrates.

Article
Biochemistry, Vol. 49, No. 24, 2010 5039
Table 1: Steady-State Kinetic Parameters for Wild-Type and Mutant PODANSg2158 with Nitroethane as Substratea
kinetic parameter
cat (s-1
wild type
D399N
R406K
S273A
k
)
15 ( 1 (9.3 ( 0.7)b
13 ( 4
0.74 ( 0.20
0.39 ( 0.08
38 ( 5
0.037 ( 0.009b
10 ( 8b
0.004 ( 0.002
9.0 ( 0.5b
25 ( 4b
0.37 ( 0.04
5.0 ( 0.3b
24 ( 4b
0.21 ( 0.02
KNE (mM)
kcat/KNE (mM-1 s-1 b
)
KO (mM)c
2
kcat/KO (mM-1 s-1 c
)
2
aConditions: 50 mM Hepes, pH 8.0, 30 ꢀC. bDetermined by varying the concentration of nitroethane at 247 μM oxygen. cDetermined by varying the
concentration of oxygen at 50 mM nitroethane.
F
IGURE 4: Nitroalkane substrate specificities for F. oxysporum NAO
and PODANSg2158. Conditions for PODANSg2158: 50mMHepes,
pH 8.0 at 30 ꢀC (empty bars). Data for F. oxysporum NAO from
Gadda et al. (12) (filled bars).
F
IGURE 6: The PODANSg2158 active site superimposed on σA-
weighted electron density with coefficients 2mFo - DFc contoured at
1.2σ. The FAD isoalloxazine ring, the catalytic triad consisting of
residues S273, D399, and R406, and a chain of hydrogen-bonded water
molecules running from the active site to the bulk solvent are shown.
FIGURE 5: kcat/KM-pH profile for PODANSg2158 with nitroethane
as substrate. The line is from a fit of the data to eq 3.
Table 2: Data Collection and Refinement Statistics
data collection
space group
P43
cell dimensions
˚
a, b, c (A)
137.5, 137.5, 131.3
90, 90, 90
0.97949
R, β, γ (deg)
wavelength
F
IGURE 7: Superposition of subunit A of the F. oxysporum NAO
structure [PDB code 2C12 (10), pink] and subunit A of the PO-
DANSg2158 structure (cyan). The respective flavins are shown in
stick representation with the same color scheme.
˚
resolution (A)
50-2.0
0.089 (0.589)
a
Rsym
Ι/σΙ
17.7 (3.3)
completeness (%)
redundancy
refinement
99.7 (100)
6.2 (6.1)
each enzyme. The two proteins clearly have the same overall fold;
this is confirmed by the rmsd of 0.925 over 376 residues. An
overlay of the catalytic triad from P. anserina and F. oxysporum
NAOs, as well as the bound FAD, is shown in Figure 8. The
positions of the FAD and the active site residues are conserved in
the two enzymes.
˚
resolution (A)
44.9-2.0
152266
no. of reflections
R
work/Rfree
0.176/0.209
no. of atoms
protein
12660
224
ligand
DISCUSSION
solvent
rms deviationsb
1431
The results described here identify PODANSg2158 as an
NAO. The enzyme is a flavoprotein, and the steady-state kinetic
parameters in Table 1 establish the enzyme has nitroalkane
oxidase activity. The identities between the P. anserina and
F. oxysporum NAO are spread through the sequence; more
˚
bond lengths (A)
bond angles (deg)
0.011
1.217
aValues in parentheses are for the highest resolution shell. bRms deviations
are from idealized values for protein.

5040 Biochemistry, Vol. 49, No. 24, 2010
Tormos et al.
F
IGURE 8: Overlay of the active sites from PODANSg2158 (carbon
atoms colored in pink) and F. oxysporum NAO (carbon atoms
colored in cyan). The active site of PODANSg2158 was overlaid
with that of F. oxysporum enzyme (PDB code 2C12) (10) by super-
imposing the four carbons on the central ring in the FADs.
importantly, the active site aspartate (Asp399/402), arginine
(Arg406/409), and serine (Ser273/276) are conserved. The very
similar three-dimensional structures confirm the assignment.
These results suggest that the remaining proteins with sequences
in Figure 1 can also be described as nitroalkane oxidases. NAO
was previously proposed to be a member of the ACAD super-
family distinct from acyl-CoA dehydrogenases and acyl-CoA
oxidases (1, 17), and SCOP (35) classifies NAO as a member of
the medium-chain acyl-CoA dehydrogenase-like family, despite
the low (∼25%) sequence identity to other members of the
family. The distinct activities of NAO and acyl-CoA dehydro-
genases and the very different active site residues suggest that
NAO and related enzymes are better classified as a separate
family within the superfamily. F. oxysporum NAO has been a
model system for understanding enzyme-catalyzed carbanion
formation (16, 36). The availability of NAOs from additional
sources expands the utility of the enzyme as a model system.
While PODANSg2158 is clearly an NAO, this enzyme differs
from F. oxysporum NAO in a number of properties. The latter
enzyme prefers linear primary nitroalkanes as substrates (13).
There is an increase in the kcat/KM with each additional methy-
lene group of the substrate up to 1-nitrobutane due to an increase
in the forward commitment with increasing chain length and a
decrease of 16-fold in the Kd with each additional methylene
group (12). PODANSg2158 is similarly more active on longer
nitroalkanes, but the increase in the kcat/KM values suggests a
preference for longer chains than is the case for the F. oxysporum
enzyme. Figure 9 (top) shows the active site of NAO with
1-nitrooctane bound. The substrate binds near the flavin at the
end of a tunnel that extends to the protein surface (11). The
nitroalkane binding pocket fits 1-nitrooctane well, with a con-
striction in the tunnel just distal of the terminal carbon of the
substrate. PODANSg2158 has a similar tunnel, as shown in
Figure 9 (bottom). Comparison of the nitroalkane binding
cavities and the tunnels in the two enzymes shows that the tunnel
in PODANSg2158 is much more open. This structural difference
provides a reasonable explanation for the differences in substrate
specificities of the two enzymes.
F
IGURE 9: Active site entrances in F. oxysporum nitroalkane oxidase
[PDB code 2C12 (10), top] and P. anserina PODANSg2158 (bottom).
The placement of the 1-nitrooctane (yellow in both panels) is based
on the superposition of the backbone atoms of these structures with
the backbone atoms of PDB code 2D9E (11).
NAO and a glutamate in ACADs. Asp402 is the active site base
in F. oxysporum NAO, with a pKa of 6.9 in the free enzyme (3).
Mutagenesis of this residue to alanine or asparagine decreases
the rate constant for proton abstraction by 3 orders of magni-
tude. The structural and kinetic data presented here establish
that Asp399 is the active site base in PODANSg2158. The
decrease in the kcat of the D399N enzyme of 400-fold provides a
lower limit for the effect; with the F. oxysporum enzyme,
product release is rate-limiting for turnover, so that decreases
in the rate constant for CH bond cleavage are not fully reflected
in the kcat value (7). The pKa of 5.9 in the kcat/KNE profile
for PODANSg2158 can reasonably be assigned to Asp399. It
cannot be established from the present data whether the
differences in the pKa values of the bases in the two enzymes
truly differ by one pK unit, since the pKa value for
PODANSg2158 could be perturbed by a significant commitment
to catalysis with nitroethane as substrate. In the case of the
F. oxysporum enzyme, the pKa of 6.9 seen with nitroethane shifts
to lower values with longer chain nitroalkanes due to increasing
commitment with increased chain length (12).
A key distinction between an NAO and the acyl-CoA
dehydrogenases and oxidases that are also in the ACAD
superfamily is the use of an aspartate as the active site base in

Article
While the active site triad of an aspartate interacting with
an arginine and a serine is maintained in PODANSg2158,
the effects of mutating the arginine and serine are less in
PODANSg2158 than in F. oxysporum NAO. In the latter
Biochemistry, Vol. 49, No. 24, 2010 5041
and catalysis by nitroalkane oxidase. Arch. Biochem. Biophys. 382,
138–144.
13. Gadda, G., and Fitzpatrick, P. F. (1999) Substrate specificity of a
nitroalkane oxidizing enzyme. Arch. Biochem. Biophys. 363, 309–313.
14. Valley, M. P., and Fitzpatrick, P. F. (2004) Comparison of enzymatic
and non-enzymatic nitroethane anion formation: thermodynamics
and contribution of tunneling. J. Am. Chem. Soc. 126, 6244–6245.
15. Bernasconi, C. F. (1992) The principle of non-perfect synchroniza-
tion. Adv. Phys. Org. Chem. 27, 119–238.
16. Major, D. T., Heroux, A., Orville, A. M., Valley, M. P., Fitzpatrick,
P. F., and Gao, J. (2009) Differential quantum mechanical tunneling
in the uncatalyzed and in the nitroalkane oxidase catalyzed proton
abstraction of nitroethane. Proc. Natl. Acad. Sci. U.S.A. 106, 20734–
20739.
17. Daubner, S. C., Gadda, G., Valley, M. P., and Fitzpatrick, P. F.
(2002) Cloning of nitroalkane oxidase from Fusarium oxysporum
identifies a new member of the acyl-CoA dehydrogenase superfamily.
Proc. Natl. Acad. Sci. U.S.A. 99, 2702–2707.
18. Kim, J. J., and Miura, R. (2004) Acyl-CoA dehydrogenases and acyl-
CoA oxidases. Structural basis for mechanistic similarities and differ-
ences. Eur. J. Biochem. 271, 483–493.
enzyme, the R409K and S276A mutations decrease the kcat
/
KNE value by 64-fold and 21-fold, respectively (11, 20), compared
to decreases of 24-fold and 3-fold observed here. A significant
forward commitment to catalysis could decrease the effect of
mutating an active site residue, in addition to perturbing the pKa
value seen in the kcat/KNE-pH profile, since CH bond cleavage
would no longer be fully rate-limiting for nitroethane oxidation.
In summary, the data presented here establish that PODANS-
g2158, designated as a hypothetical protein in the sequence
database, is better classified as a nitroalkane oxidase. They also
suggest that the presence of the active site aspartate, arginine, and
serine discriminates between NAOs and true ACADs.
19. Thorpe, C., and Kim, J. P. (1995) Structure and mechanism of action
of the acyl-CoA dehydrogenases. FASEB J. 9, 718–725.
20. Fitzpatrick, P. F., Valley, M. P., Bozinovski, D. M., Shaw, P. G.,
ACKNOWLEDGMENT
This work is based upon research conducted at the North-
eastern Collaborative Access Team beamlines of the Advanced
Photon Source, supported by award RR-15301 from the
National Center for Research Resources at the National
Institutes of Health. Use of the Advanced Photon Source is
supported by the U.S. Department of Energy, Office of Basic
Energy Sciences, under Contract No. W-31-109-ENG-38. We
thank Dr. Jonathan P. Schuermann at the Advanced Photon
Source for collecting the X-ray diffraction data. Support for
the X-ray Crystallography Core Laboratory by the UTHSC-
SA Executive Research Committee and the San Antonio
Cancer Institute is also gratefully acknowledged.
ꢀ
Heroux, A., and Orville, A. M. (2007) Mechanistic and structural
analyses of the roles of Arg409 and Asp402 in the reaction of the
flavoprotein nitroalkane oxidase. Biochemistry 46, 13800–13808.
€
21. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z.,
Miller, W., and Lipman, D. J. (1997) Gapped BLAST and PSI-
BLAST: a new generation of protein database search programs.
Nucleic Acids Res. 25, 3389–3402.
22. Ghisla, S., and Thorpe, C. (2004) Acyl-CoA dehydrogenases. A
mechanistic overview. Eur. J. Biochem. 271, 494–508.
23. Gadda, G., and Fitzpatrick, P. F. (1998) Biochemical and physical
characterization of the active FAD-containing form of nitroalkane
oxidase from Fusarium oxysporum. Biochemistry 37, 6154–6164.
24. Otwinowski, Z., and Minor, W. (1997) Processing of X-ray diffraction
data collected in oscillation mode. Methods Enzymol. 276, 307–326.
25. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D.,
Storoni, L. C., and Read, R. J. (2007) Phaser crystallographic soft-
ware. J. Appl. Crystallogr. 40, 658–674.
REFERENCES
ꢀ
26. Adams, P. D., Afonine, P. V., Bunkoczi, G., Chen, V. B., Davis, I. W.,
1. Fitzpatrick, P. F., Orville, A. M., Nagpal, A., and Valley, M. P. (2005)
Nitroalkane oxidase, a carbanion-forming flavoprotein homologous
to acyl-CoA dehydrogenase. Arch. Biochem. Biophys. 433, 157–165.
2. Heasley, C. J., and Fitzpatrick, P. F. (1996) Kinetic mechanism and
substrate specificity of nitroalkane oxidase. Biochem. Biophys. Res.
Commun. 225, 6–10.
Echols, N., Headd, J. J., Hung, L.-W., Kapral, G. J., Grosse-
Kunstleve, R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read,
R. J., Richardson, D. C., Richardson, J. S., Terwilliger, T. C., and
Zwart, P. H. (2010) PHENIX: a comprehensive Python-based system
for macromolecular structure solution. Acta Crystallogr. D 66,
213–221.
3. Valley, M. P., and Fitzpatrick, P. F. (2003) Inactivation of nitroalkane
oxidase upon mutation of the active site base and rescue with a
deprotonated substrate. J. Am. Chem. Soc. 23, 8738–8739.
4. Fitzpatrick, P. F. (2004) Carbanion versus hydride transfer mechan-
isms in flavoprotein-catalyzed dehydrogenations. Bioorg. Chem. 32,
125–139.
5. Fitzpatrick, P. F. (2010) Oxidation of amines by flavoproteins. Arch.
Biochem. Biophys. 493, 13–25.
6. Gadda, G. (2008) Hydride transfer made easy in the reaction of
alcohol oxidation catalyzed by flavin-dependent oxidases. Biochem-
istry 47, 13745–13753.
7. Valley, M. P., and Fitzpatrick, P. F. (2003) Reductive half-reaction of
nitroalkane oxidase: effect of mutation of the active site aspartate to
glutamate. Biochemistry 42, 5850–8566.
8. Gadda, G., Edmondson, R. D., Russel, D. H., and Fitzpatrick, P. F.
(1997) Identification of the naturally occurring flavin of nitroalkane
oxidase from Fusarium oxysporum as a 5-nitrobutyl-FAD and con-
version of the enzyme to the active FAD-containing form. J. Biol.
Chem. 272, 5563–5570.
9. Valley, M. P., Tichy, S. E., and Fitzpatrick, P. F. (2005) Establishing
the kinetic competency of the cationic imine intermediate in nitroalk-
ane oxidase. J. Am. Chem. Soc. 127, 2062–2066.
10. Nagpal, A., Valley, M. P., Fitzpatrick, P. F., and Orville, A. M. (2006)
Crystal structures of nitroalkane oxidase: insights into the reaction
mechanism from a covalent complex of the flavoenzyme trapped
during turnover. Biochemistry 45, 1138–1150.
27. Emsley, P., and Cowtan, K. (2004) Coot: model-building tools
for molecular graphics. Acta Crystallogr., Sect D: Biol. Crystallogr.
60, 2126–2132.
28. Gadda, G., and Fitzpatrick, P. F. (2000) Mechanism of nitroalkane
oxidase: 2. pH and kinetic isotope effects. Biochemistry 39, 1406–1410.
29. Rudolph, F. B., and Fromm, H. J. (1979) Plotting methods for
analyzing enzyme rate data. Methods Enzymol. 63, 138–159.
30. Dixon, M., and Kleppe, K. (1965) D-Amino acid oxidase. II. Speci-
ficity, competitive inhibition and reaction sequence. Biochim. Biophys.
Acta 96, 368–382.
31. Newton-Vinson, P., Hubalek, F., and Edmondson, D. E. (2000) High-
level expression of human liver monoamine oxidase B in Pichia
pastoris. Protein Expression Purif. 20, 334–345.
32. Zhao, G., Song, H., Chen, Z., Mathews, S., and Jorns, M. S. (2002)
Monomeric sarcosine oxidase: role of histidine 269 in catalysis.
Biochemistry 41, 9751–9764.
33. Gaweska, H., Henderson Pozzi, M., Schmidt, D. M. Z., McCafferty,
D. G., and Fitzpatrick, P. F. (2009) Use of pH and kinetic isotope
effects to establish chemistry as rate-limiting in oxidation of a peptide
substrate by LSD1. Biochemistry 48, 5440–5445.
34. Henderson Pozzi, M., and Fitzpatrick, P. F. (2010) A lysine conserved
in the monoamine oxidase family is involved in oxidation of the
reduced flavin in mouse polyamine oxidase. Arch. Biochem. Biophys.
(in press).
35. Murzin, A. G., Brenner, S. E., Hubbard, T., and Chothia, C. (1995)
SCOP: a structural classification of proteins database for the investi-
gation of sequences and structures. J. Mol. Biol. 247, 536–540.
36. Major, D. T., York, D. M., and Gao, J. (2005) Solvent polarization
and kinetic isotope effects in nitroethane deprotonation and implica-
tions to the nitroalkane oxidase reaction. J. Am. Chem. Soc. 127,
16374–16375.
ꢀ
11. Heroux, A., Bozinovski, D. M., Valley, M. P., Fitzpatrick, P. F., and
Orville, A. M. (2009) Crystal structures of intermediates in the
nitroalkane oxidase reaction. Biochemistry 48, 3407–3416.
12. Gadda, G., Choe, D. Y., and Fitzpatrick, P. F. (2000) Use of pH and
kinetic isotope effects to dissect the effects of substrate size on binding