APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2010, p. 3590–3598
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 76, No. 11
Growth of Bacteria on 3-Nitropropionic Acid as a Sole Source of
Carbon, Nitrogen, and Energy?
Shirley F. Nishino, Kwanghee A. Shin, Rayford B. Payne,† and Jim C. Spain*
School of Civil and Environmental Engineering, Georgia Institute of Technology, 311 Ferst Dr., Atlanta, Georgia 30332-0512
Received 1 February 2010/Accepted 2 April 2010
3-Nitropropionic acid (3NPA) is a widespread nitroaliphatic toxin found in a variety of legumes and fungi.
Several enzymes have been reported that can transform the compound, but none led to the mineralization of
3NPA. We report here the isolation of bacteria that grow on 3NPA and its anion, propionate-3-nitronate (P3N),
as the sole source of carbon, nitrogen, and energy. Experiments with resting cells, cell extracts, and purified
enzymes indicate that the pathway involves conversion of 3NPA to P3N, which upon denitration yields malonic
semialdehyde, nitrate, nitrite, and traces of H2O2. Malonic semialdehyde is decarboxylated to acetyl coenzyme
A. The gene that encodes the enzyme responsible for the denitration of P3N was cloned and expressed, and the
enzyme was purified. Stoichiometry of the reaction indicates that the enzyme is a monooxygenase. The gene
sequence is related to a large group of genes annotated as 2-nitropropane dioxygenases, but the P3N mono-
oxygenase and closely related enzymes form a cluster within COG2070 that differs from previously character-
ized 2-nitropropane dioxygenases by their substrate specificities and reaction products. The results suggest
that the P3N monooxygenases enable bacteria to exploit 3NPA in natural habitats as a growth substrate.
Large-scale release of synthetic nitroaromatic compounds to
the biosphere followed the invention of nitrobenzene around
1830. In less than 200 years, microorganisms adapted to the
presence of nitroaromatic compounds in the environment by
developing catalytic pathways to exploit them as growth sub-
strates. Such rapid development suggests that the pathways did
not develop de novo but evolved from preexisting degradation
pathways such as might be found in microorganisms that de-
grade naturally occurring compounds.
3-Nitropropionic acid (3NPA) is a widespread naturally oc-
curring nitroaliphatic compound. It is a principal toxic compo-
nent of Astragalus locoweeds and has been found in hundreds
of species of legumes (20, 39) and a variety of fungi (6). The
compound causes irreversible inhibition of succinate dehydro-
genase, which makes it deadly to eukaryotes (1). Plants that
make 3NPA also contain an enzyme, 3NPA oxidase (NPAO)
(19, 20), which converts the compound to malonic semialde-
hyde (MSA) to protect the plant against the toxic effects of the
compound (20). Given its widespread occurrence, we hypoth-
esized that there must be bacteria in soil that degrade 3NPA
and play a major role in determining the flux of the compound.
Although bacteria that degrade 3NPA have previously been
sought, the focus has been on organisms that ingest 3NPA-
containing plant matter. Rumen microorganisms reduce 3NPA
to ?-alanine (4), and in the grasshopper gut, 3NPA is bound to
glycine to form inert conjugates which are then eliminated
(24). The plant enzyme NPAO converts 3NPA and O2to MSA,
nitrate, nitrite, and hydrogen peroxide (19). It is similar to
propionate-3-nitronate (P3N) oxidase (P3NO; EC 220.127.116.11)
from Penicillium atrovenetum that converts the P3N form of the
compound to MSA (36). The enzymes mentioned above are
“orphan enzymes” (28), which means that the gene(s) has not
been identified. None of the previously studied microorgan-
isms can use 3NPA as a growth substrate, and the physiological
roles of the enzymes have not been established.
MSA appears to be a central intermediate in the eukaryotic
transformation of 3NPA and its analogs. However, the trans-
formations involve distinctly different types of reactions and
metabolites released. P3N and 3NPA release nitrate and ni-
trite in a 2:1 ratio when attacked by the fungal or plant oxi-
dases. When 3NPA is reduced to ?-alanine by rumen micro-
organisms, ?-alanine is further metabolized (4), possibly by
deamination to MSA (18).
We report here the isolation from soil of aerobic bacteria
that grow on 3NPA as the sole source of carbon, nitrogen, and
energy. The genes that encode the initial enzymes of the deg-
radation pathway were cloned, and recombinant proteins were
purified and partially characterized to allow determination of
the initial steps in the catabolic pathway.
(Preliminary reports of this work have been presented pre-
viously at the 106th General Meeting of the American Society
for Microbiology [32a] and the 108th General Meeting of the
American Society for Microbiology [32b].)
MATERIALS AND METHODS
Isolation and growth of bacteria. Bacteria were isolated by selective enrich-
ment in nitrogen-free minimal medium (BLK) (5) containing 3NPA (100 ?M to
1 mM). After several transfers, the cultures were spread onto agar plates
containing BLK with 3NPA (500 ?M) and incubated at 30°C until colonies
appeared. Cultures that grew on 3NPA plates were selected for identification
and further study. 16S rRNA gene analysis was performed by MIDI Labs
Large cultures were grown in 1 liter of BLK containing twice the normal
concentration of MgSO4plus 3NPA (1 mM). When the 3NPA disappeared from
the medium, it was added twice more to a final concentration of 1 mM. Some
cultures were grown on 3NPA (500 ?M) as the nitrogen source with succinate (7
* Corresponding author. Mailing address: School of Civil and Envi-
ronmental Engineering, Georgia Institute of Technology, 311 Ferst
Dr., Atlanta, GA 30332-0512. Phone: (404) 894-0628. Fax: (404) 894-
8266. E-mail: firstname.lastname@example.org.
† Present address: Center of Marine Biotechnology, University of
Maryland Biotechnology Institute, 701 East Pratt Street, Baltimore,
?Published ahead of print on 9 April 2010.
mM) or acetate (10 mM) as the carbon source. Cultures were harvested by
centrifugation, washed twice with phosphate buffer (20 mM; pH 7.0), and stored
on ice until used in assays. Cells were broken by three passages through a French
pressure cell followed by centrifugation at either 26,000 ? g or 100,000 ? g. Cell
extracts were stored on ice until used.
Enzyme assays. 3NPA denitrase was assayed by measurement of oxygen up-
take in the presence of 3NPA or P3N. Reaction mixtures consisted of cell
extracts or purified protein (1 to 500 ?g protein) in phosphate (20 mM; pH 7.8)
or Tris buffer (100 mM; pH 7.8). The reaction was started by addition of
substrate. MSA formed from P3N was quantified spectrophotometrically by
following the NADH-dependent disappearance of MSA catalyzed by ?-hydroxy-
butyrate dehydrogenase (BDH) (36). Malonyl coenzyme A (malonyl-CoA) re-
ductase was measured in the forward direction by following the disappearance of
NADPH at 365 nm (22) and in the reverse direction by following the reduction
of NADP (22). MSA decarboxylase was assayed by following the disappearance
of MSA in the absence of cofactors (17). MSA oxidative decarboxylase was
measured by following the CoA-dependent reduction of NADP to NADPH in
the presence of MSA (29). Acetaldehyde dehydrogenase was measured by fol-
lowing the conversion of NAD to NADH in the presence of acetaldehyde (17).
Library construction. DNA (approximately 5 mg) was extracted from cultures
of Pseudomonas sp. strain JS189 with the Promega SV genomic DNA purifica-
tion system (Madison, WI) according to the manufacturer’s instructions. The
resulting DNA was sheared by vortexing. DNA fragments approximately 30 kb in
size were gel purified, ligated into the fosmid vector pCC1Fos, packaged into
phage, and transfected into Escherichia coli strain EPI300-T1 according to the
manufacturer’s directions (CopyControl fosmid library production kit; Epicentre
Biotechnologies, Madison, WI) to create a library of several thousand recombi-
nant E. coli clones.
Screening for P3N monooxygenase activity (pnoA). Clones were transferred to
96-well plates containing tryptic soy broth supplemented with NH4Cl (1 mM),
chloramphenicol (20 ?g/ml), and 1? concentrated fosmid induction solution
(Epicentre Biotechnologies, Madison, WI) and grown for 16 h at 30°C. After
growth, the library was replica plated onto BLK supplemented with NH4Cl (1
mM), chloramphenicol (20 ?g/ml), 1? concentrated fosmid induction solution,
P3N (200 ?M), and sodium succinate (200 ?M) and then incubated for 12 to 16 h
at 30°C. Clones were screened for nitrite release from P3N.
The location of pnoA on the fosmid was determined by transposon mutagen-
esis followed by a second round of screening for loss-of-function mutants. The
fosmid bearing pnoA was purified using the FosmidMax DNA purification kit
(Epicentre Biotechnologies, Madison, WI) and then randomly mutated in vitro
with the EZ-Tn5 ?KAN-2? insertion kit (Epicentre Biotechnologies, Madison,
WI). The fosmid::Ez-Tn5 ?KAN-2? fusion was reintroduced into E. coli strain
EPI300-T1 by electroporation. Mutants in which the transposon had inserted
into the fosmid were selected by growth on LB media supplemented with chlor-
amphenicol (20 ?g/ml) and kanamycin (25 ?g/ml). Kanamycin-resistant trans-
poson mutants were screened for the ability to release nitrite from P3N. Loss-
of-function mutants were sequenced using the KANREV primer (41) to
determine the insertion site of the transposon. The acquired sequence was used
to manually design additional primers for subsequent rounds of primer walking.
Approximately 10 kb of sequence was obtained and assembled into a 2-kb contig
with CAP3 (21). Open reading frames were determined by ORFFinder (http:
Cloning and expression of closely related genes. The Basic Local Alignment
Search Tool (BLAST) (3) was used to perform a homology search of the amino
acid sequence of PnoA against the protein database in GenBank. Several of the
most similar genes (Table 1) were amplified by PCR. The reaction mixture (20
?l) contained genomic DNA (68 to 115 ng), primers (0.25 ?M each), de-
oxynucleoside triphosphates (dNTPs; 0.4 mM each), buffer (1?), and Promega
GoTaq hot-start polymerase (2 U). Amplifications (30 cycles) were carried out as
follows: 95°C for 1 min, 54°C for 30 s, and 72°C for 1 min, after initial denatur-
ation at 95°C for 10 min. The PCR products were cut with BamHI and XhoI and
cloned into the pET-21a vector (Novagen, Gibbstown, NJ). The resulting con-
structs were transformed into E. coli Top10 (Invitrogen, Carlsbad, CA) for
plasmid propagation and maintenance. The constructs were transformed into E.
coli BL21 Star (DE3) (Invitrogen) for expression. Cells were grown in 30 ml of
LB medium containing Overnight Express autoinduction system I (Novagen) at
37°C. When the optical density at 600 nm (OD600) reached 0.8 to 0.9, the
temperature was reduced to 25°C, and the cultures were further incubated for
10 h. Cells were harvested by centrifugation, washed twice with phosphate buffer
(20 mM; pH 7.5) and stored on ice until used. Trees and alignments were
constructed with Geneious Pro 4.7.4.
Protein purification. His tag fusions were constructed using the pBAD102-D/
TOPO expression system (Invitrogen). Genes of interest were cloned in frame
TABLE 1. Putative P3N monooxygenases
E. coli strain
Size of protein
Pseudomonas sp. JS189
Burkholderia phytofirmans PsJN
Burkholderia phytofirmans PsJN
Pseudomonas aeruginosa PAO1
aRestriction sites are underlined.
VOL. 76, 2010 BIODEGRADATION OF 3-NITROPROPIONIC ACID3591
with the N-terminal His patch thioredoxin tag and the C-terminal His tag. The
host was E. coli Rosetta 2 (Novagen).
Single colonies of the clone containing pnoA were grown overnight with shak-
ing at 30°C in LB (5 ml) supplemented with ampicillin (125 ?g/ml) and chlor-
amphenicol (20 ?g/ml) (LBAC). The culture was diluted into fresh LBAC (1
liter) in a 1-liter bioreactor (Applikon, Netherlands) and grown overnight at
room temperature to an OD600of 0.6. Dissolved oxygen was maintained at 50%
at a stirring rate of 500 rpm, and pH was controlled at 7.0. Arabinose (0.2% final
concentration) was added to induce expression, and incubation was continued
until the culture reached stationary phase (10 h). The culture was harvested by
centrifugation and washed twice with phosphate buffer (20 mM; pH 7.4). The
pellet was stored overnight at ?80°C. The pellet was suspended in binding buffer
(20 mM phosphate, 20 mM imidazole [pH 7.4]), and the cells were broken with
a French press. The extract was clarified by centrifugation at 100,000 ? g for 45
min at 4°C. A 5-ml HiTrap chelating HP column (GE Healthcare) was charged
with Ni2?, and the protein was purified according to the manufacturer’s protocol
(14). The fractions were screened for nitrite release from P3N. Benchmark
His-tagged protein standards used to estimate the protein size were from In-
Analytical methods. High-performance liquid chromatography (HPLC) was
performed on an Agilent 1100 system equipped with a diode array detector and
a Merck Chromolith column (100 mm by 4.6 mm). The mobile phase consisted
of 95% trifluoroacetic acid (13 mM) in water (part A) and 5% trifluoroacetic acid
(6.5 mM) in acetonitrile (part B) delivered at a flow rate 1 ml min?1. The
autosampler and column heaters were maintained at 4 and 45°C, respectively.
Protein was measured using a Pierce bicinchoninic acid kit or a Nanodrop 1000
spectrophotometer by reading A280. Oxygen uptake was measured using a Clark-
type electrode and a YSI model 3600 oxygen meter. Nitrite (40), nitrate (32), and
ammonia (34) were measured colorimetrically.
Auxanography. Cells grown on1⁄4-strength tryptic soy agar plates were sus-
pended in phosphate buffer (20 mM; pH 7.2) to an OD600of ?0.2. The suspen-
sion (100 ?l) was spread onto agar plates so that the resulting growth would form
a lawn. Blank Sensi-Discs (BBL) loaded with 3 mg of P3N (five applications of
10 ?l of 500 mM P3N, then dried) were placed onto the spread plates, along with
untreated control discs. The plates were incubated at 30°C for up to 10 days (33).
Chemicals. MSA was prepared the day of use from ethyl-3,3-diethoxypropano-
ate by the method of Yamada and Jakoby (42). Nitronate forms of 3NPA and
other nitroalkanes were made immediately before use by addition of two equiv-
alents of KOH (37). Ethyl-3,3-diethoxypropanoate was from Acros Organics.
3NPA, BDH (EC 18.104.22.168), 2-nitroethanol, nitropentane, nitrocyclohexane,
?-alanine, and malonyl-CoA were from Sigma-Aldrich. Nitroethane, nitrometh-
ane, 1-nitropropane, and 2-nitropropane were from ChemService.
Bacterial strains. Pseudomonas aeruginosa PAO1 was a gift from Ronald
Olsen. Angela Sessitsch and Jerzy Nowak generously provided Burkholderia
Nucleotide sequence accession numbers. The sequences for msaD and pnoA
have been deposited in GenBank under accession number GU014557. The 16S
rRNA gene sequences for JS189 and JS190 have been deposited under accession
numbers GU354208 and GU354209, respectively.
Isolation and growth. Bacteria were isolated from soil and
water samples collected from several locations around Atlanta,
GA. Isolates that grew and accumulated nitrite and/or nitrate
in the culture medium with 3NPA as the sole source of carbon,
nitrogen, and energy were presumed to be 3NPA-degrading
bacteria. A total of 48 isolates were represented by six different
colony morphologies. Two isolates from garden soil were se-
lected for further study. Analysis of partial 16S rRNA gene
sequences identified the two strains as Cupriavidus sp., desig-
nated JS190, and Pseudomonas sp., designated JS189. When
strain JS190 was grown on 3NPA as the sole source of carbon,
nitrogen, and energy, 70% of the initial nitrogen accumulated
as nitrate, and 2% accumulated as nitrite (Fig. 1). Ammonia
was not detected. Results were similar for JS189. Cultures
grew on 3NPA at concentrations up to 2 mM, the highest
concentration tested. The molar growth yield of JS190 was
24.5 g (dry weight) per mol of 3NPA.
Oxygen uptake. Like many other nitroaliphatic compounds,
3NPA exists in solution in two ionic forms (23), the neutral
(acid) form and an anionic nitronate form. At neutral pH, only
1% of the compound is in the nitronate form (2); therefore, in
cultures, the predominant form of the compound is likely to be
the acid form. Resting cells of 3NPA-grown JS189 and JS190
were 2 to 10 times more active with the nitronate form than
with the acid form (Table 2), as measured by stimulation of
oxygen uptake. Rates of oxygen uptake with 3NPA were sim-
ilar when cells were grown on acetate or succinate (data not
shown) with 3NPA as the nitrogen source. Succinate (data not
shown)- and acetate (Table 2)-grown cultures exhibited con-
stitutive activity against P3N but not against 3NPA. No activity
was detected with any other substrate tested. Cell extracts were
specific for the nitronate (Table 3) and required no additional
cofactors for oxidation of P3N. Dialysis of cell extracts did not
affect the denitration reaction. The stoichiometry of the reac-
tion was 0.87 ? 0.14 mol of O2consumed per mol of P3N.
P3N-oxidizing enzyme. Fosmid libraries constructed from
JS189 and JS190 were screened for the ability to release nitrite
from 3NPA. One clone from Pseudomonas sp. strain JS189 was
selected for further study. Nitrite release was localized to a
10-kb BamHI-HindIII fragment of a pUC19 subclone. Several
colonies that had lost the ability to release nitrite from P3N
after transposon mutagenesis were identified. The gene dis-
rupted by Tn5 was designated pnoA.
The purified overexpressed protein encoded by pnoA cata-
FIG. 1. Growth of Cupriavidus sp. JS190 on 3NPA. Closed symbols,
experimental cultures (in triplicate); open symbols, autoclaved control
culture. Error bars represent 1 SD.
TABLE 2. Oxygen uptake by resting cells
Oxygen uptake (nmol min?1mg?1protein) by:
Pseudomonas sp. JS189
after growth on:
Cupriavidus sp. JS190
after growth on:
3NPA Acetate3NPA Acetate
71 ? 7.4b
222 ? 94
99 ? 12
176 ? 80
15 ? 12
248 ? 108 194 ? 93
aTest substrates provided at 100 ?M.
bNo activity detected with neutral or nitronate forms of 1-nitropropane, 2-ni-
tropropane, nitroethane, nitromethane, 1-nitropentane, 2-nitroethanol, and ni-
trocyclohexane or with ?-alanine.
cND, not detected.
3592 NISHINO ET AL.APPL. ENVIRON. MICROBIOL.
lyzed the denitration of P3N with a stoichiometry of 0.95 ?
0.05 mol of O2consumed per mol of P3N. The substrate range
was identical to that of extracts from wild-type cells. The the-
oretical molecular size of the His-tagged protein was predicted
to be 52.5 kDa (Fig. 2). The absorbance spectrum showed
maxima at 275 and 446 nm with a shoulder at 368 nm, consis-
tent with that of a flavoprotein (38). The purified protein was
stabilized by the addition of bovine serum albumin (BSA; 0.1
to 1.0 mg/ml) to enzyme assays. Rates of reactions in Tris
buffer were 5 to 53% of the rates of reactions in phosphate
buffer, except when BSA was present in the reaction mixture
(data not shown). Activity of the purified protein in phosphate
buffer was two to four times that of extracts from wild-type
cells (Table 3) over a range from 5 ?M to 10 mM. The Kmfor
P3N was estimated to be 168 ?M. When the pnoA gene was
amplified by PCR and recloned with the His tag but without
the His patch thioredoxin tag, the specific activity improved
6-fold, and the Kmwas estimated to be 30 ?M. Cell extracts
from JS189 and JS190 were active over similar concentration
ranges and showed the same sensitivity to Tris buffer. Both the
purified protein and the wild-type cell extracts gradually lost
the ability to catalyze further denitration after multiple (three
to four) additions of P3N. The agreement between wild type
and the purified protein in all aspects examined indicates that
pnoA is responsible for the denitration of the P3N by wild-type
The products of the denitration catalyzed by purified PnoA
are consistent with those of P3NO from Penicillium atrovene-
tum (36) and those of NPAO from Hippocrepis comosa (19),
where the enzymes oxidize P3N and 3NPA to MSA with the
loss of the nitro group (Table 4). BDH catalyzes the hydroge-
nation of MSA to 3-hydroxypropanoate (36). Both authentic
MSA and the product of PnoA-catalyzed denitration of P3N
resulted in the oxidation of NADH to NAD?in assays with
BDH; no activity was detected when any of the reaction com-
ponents was omitted. Addition of catalase to reaction mixtures
after P3N was completely consumed released an additional
2 to 4% of the original O2consumed. The stoichiometry of
the reaction catalyzed by PnoA with P3N was 178 ? 0.4 P3N ?
215 ? 10 O23 145 ? 70 NO3
?? 45 ? 5 NO2
?? 140 ? 31
FIG. 2. Absorbance spectrum of purified PnoA and estimation of
protein size. MW Stds, molecular size standards.
TABLE 3. Oxygen uptake catalyzed by P3N monooxygenase
Oxygen uptake (?mol min?1mg?1protein) by:
90.3 ? 12.5f, 553.2 ? 12.4g
22.8 ? 2.9
36.0 ? 1.9
23.2 ? 0.6
28.9 ? 0.4
68.3 ? 1.6
aNo activity detected with neutral or nitronate forms of 1-nitropropane, 2-nitropropane, nitroethane, nitromethane, 1-nitropentane, or 2-nitroethanol for PnoA, JS189, or JS190. No activity detected with neutral or
nitronate form of 2-nitropropane for JS589 to JS591.
bSubstrates provided at 500 ?M.
cSubstrates provided at 200 ?M.
dPlasmid-free host strain.
eND, no activity detected.
fPurified protein with His tag and His patch thioredoxin tag.
gPurified protein with His tag only.
VOL. 76, 2010 BIODEGRADATION OF 3-NITROPROPIONIC ACID3593
MSA ? 16 ? 7 H2O2. Based upon the similarities of the reaction
stoichiometries and the ability of BDH to catalyze NADH
oxidation in mixtures with authentic MSA and the product of
the PnoA-catalyzed reaction, we concluded that the product
MSA transformation. MSA was transformed by extracts of
cells grown on 3NPA but not by extracts of cells grown on
acetate (Table 5). Assays for various enzymes with the ability
to transform MSA (Fig. 3) showed that MSA oxidative decar-
boxylase is highly upregulated in 3NPA-grown cells but that
MSA decarboxylase, acetaldehyde dehydrogenase, and malo-
nyl-CoA reductase are not.
MSA decarboxylase was examined closely because a gene,
msaD, similar to the genes that encode MSA decarboxylase
(35) was found upstream of pnoA on a 0.5-kb fragment (Fig.
4A). The gene was cloned with a His tag and a His patch
thioredoxin tag. The purified protein rapidly converted MSA
to acetaldehyde with no additional cofactors. However, cell
extracts from JS189 and JS190 grown on 3NPA had only trace
levels of such activity. In addition, acetaldehyde decarboxylase
activity was not detected in any cell extracts. The results indi-
cate that MSA oxidative decarboxylase, but not the MSA de-
carboxylase, is responsible for the metabolism of MSA during
3NPA degradation. The location of the gene that encodes
MSA oxidative decarboxylase is currently under investigation.
Phylogenetic relationships. The proteins most similar to
PnoA all belong to COG2070, the 2-nitropropane dioxygen-
ase-like proteins. We cloned and expressed several genes with
the highest identity to pnoA (Table 1). Induced cells and cell
extracts catalyzed oxygen uptake and release of nitrite from
P3N, but not 3NPA, 2-nitropropane, or propyl-2-nitronate
(Table 3). The wild-type cells all released nitrite from P3N.
JS189, JS190, Burkholderia phytofirmans PsJN, P. aeruginosa
PAO1, Cupriavidus necator JMP134, Pseudomonas putida F1,
P. putida KT2440, and E. coli BL21 were tested by auxanog-
raphy for the ability to grow on 3NPA. The proteobacteria
were chosen because a BLAST search included their putative
2-nitropropane dioxygenases in the 50 closest matches to
PnoA. E. coli BL21(DE3) was included as a control strain
lacking a member of COG2070 and because it was the cloning
host for the pnoA homologs. All strains except E. coli used
3NPA as the sole nitrogen source. JS189, JS190, and PsJN used
3NPA as the sole source of nitrogen and carbon. When succi-
nate and ammonia were present, there were no detectable
effects on the lawns of bacteria, except for strain PsJN, with
which there was inhibition close to the source of 3NPA and a
heavier growth ring slightly farther away. PsJN also was slightly
inhibited by 3NPA in a lawn spread on1⁄4-strength tryptic soy
agar. The results indicate clearly that the genes annotated as
2-nitropropane dioxygenases in the tested strains actually en-
code P3N monooxygenases.
A variety of enzymes that transform 3NPA and P3N (Table
4) have been purified from eukaryotes. All are flavoproteins
with either FMN or FAD as a tightly bound flavin cofactor.
The subunit sizes fall within a narrow range, but the number of
subunits of the functional enzyme varies. Nitronate monooxy-
genase (NMO; EC 22.214.171.124, formerly called 2-nitropropane
TABLE 4. 3NPA-transforming enzymes
Subunit size (kDa)
(no. of subunits)
Pseudomonas sp. JS189,
Cupriavidus sp. JS190
Williopsis saturnus var. mrakii,
11, 15, 27, 31
Nitroalkane oxidase (NAO)
aReaction products as a proportion of the initial substrate where the initial substrate was 1.0.
bA/K, aldehyde or ketone.
3594 NISHINO ET AL.APPL. ENVIRON. MICROBIOL.
dioxygenase, EC 126.96.36.199) and nitroalkane oxidase (NAO;
EC 188.8.131.52) have broad substrate ranges but greatly prefer
nitroalkanes to nitropropionic acid (15, 25–27). P3NO and
NPAO have much narrower substrate ranges and are much
more active with nitropropionic acid than with nitroalkanes
(19, 36). All the enzymes that preferentially attack 3NPA re-
lease twice as much nitrate as nitrite from 3NPA, whereas
reactions catalyzed by NMO and NAO release no nitrate. The
striking similarity of the substrates and products of the PnoA-
catalyzed reaction to those of the P3NO-catalyzed reaction
from Penicillium atrovenetum (36) suggests that PnoA is a
P3NO. However, the lack of stoichiometric release of H2O2
indicates that the enzyme is a monooxygenase (13, 30), and we
have tentatively designated it P3N monooxygenase. Full char-
acterization of P3N monooxygenase is currently under way to
rigorously establish the reaction mechanism and thus the
proper EC designation.
Several lines of evidence indicate that the P3N monooxy-
genase does not catalyze the first step in the pathway. The fact
that acetate-grown cells transform P3N but not 3NPA whereas
3NPA-grown cells transform both forms suggests that the P3N
monooxygenase is constitutive. Neither cell extracts nor puri-
fied PnoA transformed 3NPA. The results suggest that the
initial step in 3NPA degradation is transformation of 3NPA to
the nitronate by an unidentified enzyme that is upregulated by
growth on 3NPA. It is possible that the failure of acetate-
grown cells to transform 3NPA indicates a transport problem
rather than lack of induction of an enzyme. Even if that were
so, the specificity of cell extracts and purified PnoA for P3N
still would suggest a requirement for an enzyme in the 3NPA
degradation pathway to catalyze the initial conversion to the
nitronate. NMO (Table 4) from Neurospora crassa (10) is the
only enzyme that readily transforms both nitronate and neutral
forms of nitroalkanes, although nitronates are the preferred
substrates. When the neutral nitroalkane is the substrate, the
initial step after enzyme-substrate complex formation is re-
moval of a proton to convert the substrate to a nitronate (11).
Thus, N. crassa NMO has the function of both tautomerase
The lack of ammonia accumulation during growth of bacte-
ria on 3NPA indicates that 3NPA is not reduced to ?-alanine
as in cattle and sheep rumen (4). Purified PnoA catalyzed
transformation of P3N with the release of nitrate and nitrite in
a 2:1 ratio, whereas growing cells released only 2% of the
nitrogen as nitrite. The observations suggest that JS189 and
JS190 incorporate nitrite as the nitrogen source, which ac-
counts for the missing nitrogen in culture fluids.
MSA is an important intermediate in multiple anabolic and
catabolic pathways, and a number of enzymes that transform
MSA have been described (Fig. 3). The lack of MSA decar-
boxylase activity was due to a lack of expression of msaD,
which is reminiscent of the myo-inositol degradation pathway
in Lactobacillus casei (43). The myo-inositol operon carries
genes for MSA oxidative decarboxylase (iolA) and MSA de-
carboxylase (iolK), but only iolA is expressed.
Based on the above results, we propose that the pathway for
degradation of 3NPA (Fig. 4B) is initiated by an inducible, but
as yet unidentified, enzyme that converts 3NPA to P3N. The
key step in the pathway is the denitration of P3N by the action
of a constitutive P3N monooxygenase, encoded by the pnoA
gene. An inducible MSA oxidative decarboxylase then converts
MSA to acetyl-CoA, which enters central metabolic pathways.
Additional sequencing will be required to locate the genes that
encode the MSA oxidative decarboxylase and the hypothesized
initial enzyme. The facile isolation of bacteria that grow on
3NPA suggests a highly evolved and widespread degradation
pathway that may have evolved to exploit plant or fungal pro-
duction of 3NPA.
The function of all other enzymes that transform 3NPA and
FIG. 3. Potential transformations of MSA.
TABLE 5. MSA enzyme assays
Sp act (?mol min?1mg?1protein) of:
Pseudomonas sp. JS189 after
Cupriavidus sp. JS190 after
MSA oxidative decarboxylase
Malonyl-CoA reductase (forward reaction)
Malonyl-CoA reductase (reverse reaction)b
0.24 ? 0.02
0.04 ? 0.01
1.21 ? 0.41
aND, not detected.
VOL. 76, 2010 BIODEGRADATION OF 3-NITROPROPIONIC ACID3595
nitroalkanes has been ascribed to detoxification or protection,
but their physiological roles have not been established. P3NO
and 3NPAO have been found only in organisms that also
produce 3NPA. The presence of 3NPA in plants has been
attributed to antiherbivory strategies, while the presence of
3NPA in fungi remains to be explained. In contrast to the other
enzymes, PnoA in Cupriavidus sp. JS190 and Pseudomonas sp.
JS189 clearly serves as a means to exploit 3NPA as a growth
substrate. The P3N monooxygenase described here is the only
member of the group whose physiological role has been estab-
lished and the first P3N monooxygenase for which a gene
sequence has been reported.
Gene sequences that encode dioxygenases related to 2-ni-
tropropane dioxygenase constitute COG2070, which comprises
53 proteins distributed among 30 genomes (http://www.ncbi
.nlm.nih.gov/COG/grace/wiew.cgi?COG2070), with over 2,100
nucleotide sequences in GenBank (Fig. 5). Many organisms
contain multiple genes annotated as encoding 2-nitropropane
dioxygenase. Despite the apparent widespread distribution of
the COG, prior to this study, only proteins from Williopsis
saturnus var. mrakii (formerly Hansenula mrakii) (27, 31), Neu-
rospora crassa (11, 15), Pseudomonas aeruginosa (16), and
Streptomyces achromogenes (9, 44) had been purified and char-
acterized to various degrees. Although called 2-nitropropane
dioxygenase, no evidence established 2-nitropropane as the
physiological substrate of any of the enzymes included in
COG2070. The recent reclassification of 2-nitropropane dioxy-
genase as nitronate monooxygenase (13) was based on the
recharacterization of purified proteins from the ascomycetes.
The P3N monooxygenase is biochemically distinct from the
well-characterized nitronate monooxygenases and falls within
COG2070, but with only ?20 to 25% amino acid identity to the
biochemically characterized enzymes. Many of the current an-
notations are wrong (12), and the situation with 2-nitropro-
pane dioxygenase seems to be another example of annotation
based only on modest sequence similarity without functional
information. Our preliminary investigation of putative ni-
tronate monooxygenases from B. phytofirmans PsJN and P.
aeruginosa PAO1 confirms that the three closely related genes
encode P3N monooxygenase rather than 2-nitropropane dioxy-
genase, based on the specificity of the enzymes for P3N and the
lack of activity for 2-nitropropane. Ha et al. crystallized a
2-nitropropane dioxygenase from P. aeruginosa PAO1 and an-
alyzed and aligned the amino acid sequence with several
closely related enzymes (16) but did not determine the physi-
ological substrate of the enzyme. Six motifs were described,
and 10 highly conserved residues that interact with FMN were
identified. When the P3N monooxygenases described here
were added to the alignment, only three of the highly con-
served residues interacting with FMN along with the His152
identified as the catalytic base were conserved across both
2-nitropropane dioxygenase and P3N monooxygenase (Fig. 6).
Motifs II and IV, the latter being described as the most highly
conserved motif by Ha et al., are disrupted in the P3N mono-
oxygenases. Taken together, the evidence suggests that P3N
monooxygenases form a separate cluster within COG2070 and
that many of the proteins annotated as “2-nitropropane dioxy-
genase” are in fact P3N monooxygenases (Fig. 5). The argu-
ment is supported by the fact that 3NPA is much more likely to
FIG. 4. (A) Context of the sequenced genes. (B) Proposed pathway for degradation of 3NPA.
FIG. 5. Distribution of selected members of COG2070 based on
amino acid sequences of 2-nitropropane dioxygenases and P3N mono-
oxygenase. Boldface lettering denotes biochemically characterized en-
zymes. Asterisks denote P3N monooxygenases identified in the present
3596 NISHINO ET AL.APPL. ENVIRON. MICROBIOL.
be widespread in natural ecosystems than are 2-nitropropane
and other nitroalkanes.
The facile isolation of bacteria that grow on 3NPA suggests
a highly evolved and widespread degradation pathway that may
have evolved to exploit plant or fungal production of 3NPA.
The ability of the pnoA-containing strains to grow on 3NPA as
a nitrogen source but not always as a carbon source suggests
the lack of a complete degradation pathway or an inability to
regulate such a pathway in some strains. It is possible that, in
some bacteria, the presence of P3N monooxygenase might be
a detoxification mechanism similar to the proposed function in
fungi and plants. It is also a plausible mechanism of scavenging
nitrogen. There may be a continuum of enzymes with functions
from protection to growth represented in the diversity of genes
in COG2070. The 3NPA-degrading bacteria are the only or-
ganisms reported to grow well on 3NPA as a sole source of
carbon, nitrogen, and energy. An Alcaligenes sp. was reported
to produce nitrate and nitrite from 3NPA; however, after 28
days, only a fraction of the initial 3NPA was consumed, and
minimal growth was observed (7). The failure of E. coli to
either grow on or be inhibited by 3NPA suggests alternate
means of detoxification of 3NPA or insensitivity to its effects.
We are currently more fully characterizing the biochemistry of
the P3N monooxygenase and ecological roles of bacteria that
This work was supported by the Defense Threat Reduction Agency
and Army Research Office grant W911NF-07-1-0077.
We thank Giovanni Gadda and Kevin Francis for helpful discussions
and for reviewing the manuscript.
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