Information on EC 1.13.12.16 - nitronate monooxygenase

-

-

?

1-hydroxybutyl-2-nitronate + O2

? + HNO2

-

anionic, expression on the basis of the reactivity of propyl-2-nitronate with 2-nitropropane dioxygenase: 31.5

-

?

1-hydroxyethyl-2-nitronate + O2

glycoaldehyde + HNO2

-

-

?

1-nitrobutane + O2

? + HNO2

-

-

?

1-nitrobutane + O2

butyraldehyde + HNO2

1-nitrobutane + O2

butyraldehyde + nitrite

-

-

?

1-nitrohexane + O2

? + HNO2

-

-

?

1-nitrohexane + O2

hexanaldehyde + HNO2

1-nitrohexane + O2

hexanaldehyde + nitrite

-

-

?

1-nitropentane + O2

? + HNO2

1-nitropentane + O2

pentanaldehyde + HNO2

1-nitropentane + O2

pentanaldehyde + nitrite

-

-

?

1-nitropropane + O2

1,1-dinitropropane + HNO2

-

639226

-

?

1-nitropropane + O2

? + HNO2

-

-

?

1-nitropropane + O2

propionaldehyde + HNO2

10 entries

1-nitropropane + O2

propionaldehyde + nitrite

-

-

?

1-nitropropane + O2 + H2O

propionaldehyde + HNO2 + H2O2

-

-

?

2 Cu((CH3)2CNO2)(PPh3)2 + O2

2 Cu(O2N)(PPh3)2 + 2 propan-2-one

-

using a copper(I) aci-2-nitropropanate complex

698266

-

?

2-hydroxybutyl-3-nitronate + O2

? + HNO2

-

anionic, expression on the basis of the reactivity of propyl-2-nitronate with 2-nitropropane dioxygenase: 26.7

-

?

2-hydroxypentyl-3-nitronate + O2

2-hydroxy-pentane-3-one + HNO2

-

-

?

2-hydroxypentyl-3-nitronate + O2

? + HNO2

-

anionic, expression on the basis of the reactivity of propyl-2-nitronate with 2-nitropropane dioxygenase: 32.3

-

?

2-nitro-1-butanol + O2

1-hydroxy-butane-2-one + HNO2

2-nitro-1-propanol + O2

1-hydroxy-propane-2-one + HNO2

2-nitro-1-propanol + O2

? + HNO2

-

-

?

2-nitro-1H-indene-1,3(2H)-dione + Cu(0) + N,N,N',N'-tetramethylethylenediamine + O2

1H-indene-1,2,3,-trione + (NO2)CuN,N,N',N'-tetramethylethylenediamine

-

with N,N-dimethylformamid (conversion: 30%) as solvent

-

?

2-nitroethanol + O2

? + HNO2

-

-

?

2-nitroethanol + O2

glycoaldehyde + HNO2

-

13% of the activity with 2-nitropropane

-

?

2-nitroethanol + O2 + H2O

? + HNO2 + H2O2

-

8.4% relative activity (1-nitropropane: 100%)

-

?

2-nitropropane + O2

?

-

-

?

2-nitropropane + O2

? + HNO2

-

-

?

2-nitropropane + O2

acetone + HNO2

14 entries

2-nitropropane + O2

acetone + nitrite

-

-

?

2-nitropropane + O2 + H2O

acetone + HNO2 + H2O2

-

96.9% relative activity (1-nitropropane: 100%)

-

?

3 propionate-3-nitronate + O2

3 malonic semialdehyde + nitrite + 2 nitrate + H2O2

3-nitro-1-butanol + O2 + H2O

? + HNO2 + H2O2

-

15.7% relative activity (1-nitropropane: 100%)

-

?

3-nitro-2-butanol + O2

3-hydroxy-butane-2-one + HNO2

5 entries

3-nitro-2-butanol + O2

? + HNO2

-

-

?

3-nitro-2-butanol + O2 + H2O

? + HNO2 + H2O2

-

6.5% relative activity (1-nitropropane: 100%)

-

?

3-nitro-2-pentanol + O2

2-hydroxy-pentane-3-one + HNO2

5 entries

3-nitro-2-pentanol + O2

? + HNO2

-

-

?

3-nitro-2-pentanol + O2 + H2O

2-hydroxy-pentane-3-one + HNO2 + H2O2

-

116% relative activity (1-nitropropane: 100%)

-

?

3-nitropropionate + O2

?

-

-

?

3-nitropropionate + O2 + H2O

? + HNO2 + H2O2

-

0.5% relative activity (1-nitropropane: 100%)

-

?

3-nitropropionic acid + O2

?

butyl-1-nitronate + O2

?

-

-

?

butyl-1-nitronate + O2

? + nitrite

butyl-1-nitronate + O2

NO2- + butanal

-

-

?

cyclohexyl nitronate + O2

? + HNO2

-

-

?

ethyl nitronate + O2

? + nitrite

ethyl nitronate + O2

acetaldehyde + HNO2

-

-

?

ethylnitronate + Cu(0) + N,N,N',N'-tetramethylethylenediamine + O2

acetone + (NO2)CuN,N,N',N'-tetramethylethylenediamine

-

with N,N-dimethylformamid (conversion: 60%) and pyridine (conversion: 90%) as solvent

-

?

ethylnitronate + O2

acetaldehyde + HNO2

ethylnitronate + O2

acetaldehyde + nitrite + other products

-

-

?

ethylnitronate + O2

NO2- + acetaldehyde

-

-

?

ethylnitronate + O2 + FMNH2

acetaldehyde + nitrite + FMN + H2O

5 entries

hexyl-1-nitronate + O2

? + nitrite

hexyl-1-nitronate + O2

NO2- + hexanal

-

-

?

nitrocyclohexane + O2

? + HNO2

nitrocyclohexane + O2

cyclohexanone + HNO2

nitrocyclohexane + O2 + H2O

cyclohexanone + HNO2 + H2O2

-

99.8% relative activity (1-nitropropane: 100%)

-

?

nitrocyclopentane + O2

? + HNO2

-

-

?

nitroethane + O2

? + nitrite

-

-

?

nitroethane + O2

acetaldehyde + HNO2

11 entries

nitroethane + O2

acetaldehyde + HNO2 + 1,1-dinitroethane

-

in contrast with the unambiguous stoichiometry of 2-nitropropane oxidation, the nitroethane oxidation is stoichiometrically complicated; 1,1-dinitroethane and nitrate are formed as minor products

639226

-

?

nitroethane + O2

acetaldehyde + nitrite

nitroethane + O2

ethanal + nitrite

nitroethane + O2

ethylnitronate

-

2-nitropropane dioxygenase utilizes a branched catalytic mechanism with nitroethane as substrate. The branch point occurs at the enzyme-ethylnitronate complex and involves either the release of the nitronate or an oxidative denitrification reaction. The partitioning of the enzyme-nitronate complex results in the formation of multiple products from independent catalytic pathways with nitroethane as substrate for the enzyme. In the nonoxidative pathway, nitroethane is deprotonated by histidine 196 to generate ethylnitronate which is subsequently released from the enzyme as a reaction product. The oxidative denitrification pathway was established in previous studies of the enzyme and involves the oxidation of ethylnitronate by the enzyme bound flavin to generate acetaldehyde and nitrite as product

-

r

nitroethane + O2 + H2O

? + HNO2 + H2O2

-

51.8% relative activity (1-nitropropane: 100%)

-

?

nitroethane + O2 + H2O

ethanal + nitrite + H2O2

nitromethane + O2

? + HNO2

-

-

?

nitromethane + O2

formaldehyde + HNO2

nitromethane + O2 + H2O

? + HNO2 + H2O2

-

7.3% relative activity (1-nitropropane: 100%)

-

?

pentane-1-nitronate + Cu(0) + N,N,N',N'-tetramethylethylenediamine + O2

pentaldehyde + (NO2)CuN,N,N',N'-tetramethylethylenediamine

-

with N,N-dimethylformamid (conversion: 28%) and pyridine (conversion: 21%) as solvent

-

?

pentyl-1-nitronate + O2

?

-

-

?

pentyl-1-nitronate + O2

? + HNO2

-

-

?

pentyl-1-nitronate + O2

? + nitrite

pentyl-1-nitronate + O2

NO2- + pentanal

-

-

?

propionate 3-nitronate + O2

?

-

-

?

propionate-3-nitronate + O2

?

propyl-1-nitronate + O2

?

propyl-1-nitronate + O2

? + nitrite

propyl-1-nitronate + O2

NO2- + propionaldehyde

-

-

?

propyl-1-nitronate + O2

propionaldehyde + HNO2

-

-

?

propyl-1-nitronate + O2 + FMNH2

? + nitrite + FMN + H2O

-

-

?

propyl-2-nitronate + Cu(0) + 1,10-phenanthroline + O2

propan-2-one + ?

-

with methanol (conversion: 42%), MeCN (conversion: 24%), and N,N-dimethylformamid (conversion: 43%)

-

?

propyl-2-nitronate + Cu(0) + 2,2'-bipyridine + O2

propan-2-one + ?

-

with methanol (conversion: 44%), MeCN (conversion: 54%), and N,N-dimethylformamid (conversion: 37%)

-

?

propyl-2-nitronate + Cu(0) + N,N,N',N'-tetramethylethylenediamine + O2

propan-2-one + (NO2)CuN,N,N',N'-tetramethylethylenediamine

-

with methanol (conversion: 70%), MeCN (conversion: 49%), N,N-dimethylformamid (conversion: 71%), and pyridine (conversion: 67%) as solvent

-

?

propyl-2-nitronate + Cu(0) + O2

propan-2-one + ?

-

without ligand and without solvent (conversion: 12%)

-

?

propyl-2-nitronate + O2

?

-

-

?

propyl-2-nitronate + O2

? + nitrite

propyl-2-nitronate + O2

acetone + HNO2

propyl-2-nitronate + O2

NO2- + acetone

-

-

?

propyl-2-nitronate + O2 + FMNH2

? + nitrite + FMN + H2O

propylnitronate + O2

? + HNO2

-

anionic, expression on the basis of the reactivity of propyl-2-nitronate with 2-nitropropane dioxygenase: 57.9

-

?

undecan-6-nitronate + Cu(0) + N,N,N',N'-tetramethylethylenediamine + O2

undecan-6-one + (NO2)CuN,N,N',N'-tetramethylethylenediamine

-

with N,N-dimethylformamid (conversion: 66%) and pyridine (conversion: 67%) as solvent

-

?

additional information

?

-

19 entries

3 propionate-3-nitronate + O2

3 malonic semialdehyde + nitrite + 2 nitrate + H2O2

ethylnitronate + O2 + FMNH2

acetaldehyde + nitrite + FMN + H2O

nitroethane + O2

acetaldehyde + nitrite

propyl-1-nitronate + O2 + FMNH2

? + nitrite + FMN + H2O

-

-

?

propyl-2-nitronate + O2 + FMNH2

? + nitrite + FMN + H2O

additional information

?

-

cytochrome c

-

cytochrome c also inhibits the reaction and half-inhibition is found in the presence of 17 microM cytochrome c. The addition of cytochrome c to the reaction mixture containing 2-nitropropane and 2-nitropropane dioxygenase causes an increase in absorbance at 550 nm indicating the reduction of cytochrome c

FAD

6 entries

FMN

12 entries

riboflavin 5'-phosphate

-

can partially replace FAD

Fe

-

enzyme contains 1.09 gatom of iron per mol of enzyme; enzyme contains 1.09 gatom of iron per mol of enzyme

639222

Fe3+

-

contains 1 g atom of non-heme iron per mol of enzyme

Iron

stoichiometry of 0.02 FMN per 1 monomer of enzyme

additional information

6 entries

2-mercaptoethanol

3-(3,4-dihydroxyphenyl)-L-Ala

-

3-nitro-1-propionate

-

; competitive inhibitor, pH 7.0, 30°C

4-hydroxy-2,2,6,6-tetramethyl-piperidinooxy radical

-

5-hydroxytryptophan

-

8-hydroxyquinoline

-

; strong inhibition

acetone

-

inhibits competitively against 2-nitropropane, no inhibitory effect against O2

alpha-Naphthol

-

catechol

-

cysteine

-

; marked decrease in enzyme activity

cytochrome c

dithiothreitol

EDTA

-

1 mM, relative activity remaining 95%

epinephrine

ethylnitronate

-

GSH

HgCl2

hydroquinone

-

iodoacetate

-

; 1 mM, complete inhibition

KCN

-

; strongly

m-Nitrobenzoate

-

N-ethylmaleimide

NaBH4

-

NADH

NADPH

-

competitive inhibitor. oxygenation is inhibited approximately 100% by 0.1 mM NADPH, respectively, although NAD+ and NADP+ are ineffective even at 1 mM. During the inhibition, NADH and NADPH are oxidized

NaHSO3

-

NEM

nitrite

-

inhibits noncompetitively against 2-nitropropane, functioned as a noncompetitive inhibitor against oxygen in the presence of excess 2-nitropropane (50 mM)

Nitro blue tetrazolium

Nitromethane

-

inhibits noncompetitively

p-chloromercuribenzoate

PCMB

phloroglucinol

-

pyrogallol

-

resorcinol

-

serotonin

-

Superoxide dismutase

-

Thiophenol

-

Tiron

additional information

6 entries

-

2,2'-dipyridyl

-

2,4,6-tripyridyl-triazine

-

-

o-phenanthroline

-

15 - 17.9

1-nitrobutane

1.4

1-nitrohexane

in 50 mM Tris-HCl, pH 8 at 30°C

7.1

1-nitropentane

in 50 mM Tris-HCl, pH 8 at 30°C

1.54 - 57

1-Nitropropane

1.61 - 33

2-Nitropropane

9 entries

0.59 - 4.2

3-Nitro-2-butanol

1.04 - 6.8

3-Nitro-2-pentanol

0.58

3-nitropropionate

-

pH 7.4, 30°C

10

butyl-1-nitronate

in 50 mM Tris-HCl, pH 8 at 30°C

3.1 - 20

ethyl nitronate

5 - 15.9

ethylnitronate

0.00133

FAD

-

1.4

hexyl-1-nitronate

in 50 mM Tris-HCl, pH 8 at 30°C

0.9

Nitrocyclohexane

-

1 - 29

nitroethane

11 entries

0.005 - 0.3

O2

9 entries

0.06

propionate 3-nitronate

-

pH 5.5, 30°C

0.11 - 0.34

propionate-3-nitronate

5.5 - 8.3

propyl-1-nitronate

1.61 - 3.1

propyl-2-nitronate

additional information

-

3.6 - 6.2

1-nitrobutane

1.3 - 1.7

1-nitrohexane

3 - 4.3

1-nitropentane

8.3 - 1300

1-Nitropropane

0.82 - 6400

2-Nitropropane

4.4

3-nitropropionate

-

pH 7.4, 30°C

42 - 56

butyl-1-nitronate

25 - 4100

ethyl nitronate

9 entries

16.2 - 350

ethylnitronate

21 - 22

hexyl-1-nitronate

3.5 - 2000

nitroethane

7 entries

35

pentyl-1-nitronate

pH 8.0, 30°C

860

propionate 3-nitronate

-

pH 5.5, 30°C

430 - 1300

propionate-3-nitronate

42 - 1300

propyl-1-nitronate

15 - 6400

propyl-2-nitronate

additional information

kcat at different conditions, overview, effects of presence of superoxide dimutase or catalase on the turnover rate of the enzyme

-

0.35

1-nitrobutane

in 50 mM Tris-HCl, pH 8 at 30°C

1.1

1-nitrohexane

in 50 mM Tris-HCl, pH 8 at 30°C

0.61

1-nitropentane

in 50 mM Tris-HCl, pH 8 at 30°C

0.83

1-Nitropropane

in 50 mM Tris-HCl, pH 8 at 30°C

7.6

3-nitropropionate

-

pH 7.4, 30°C

5.6 - 112

butyl-1-nitronate

2.5 - 300

ethyl nitronate

1.02 - 129

ethylnitronate

16 - 130

hexyl-1-nitronate

0.04 - 0.56

nitroethane

0.001 - 11000

O2

1.2 - 121

pentyl-1-nitronate

14330

propionate 3-nitronate

-

pH 5.5, 30°C

1300 - 11818.2

propionate-3-nitronate

11.4 - 186.71

propyl-1-nitronate

1.6 - 29

propyl-2-nitronate

additional information

-

investigation of the pH effects on the kcat/Km-values

672094

-

3.1

3-nitro-1-propionate

-

; competitive inhibitor, pH 7.0, 30°C

1.2 - 100

ethylnitronate

0.46

-

13

cell free extract

15.5

-

49

after DEAE-Sepharose column chromatography purification only

80

after DEAE-Sepharose column chromatography and octyl-Sepharose column chromatography purification; after DEAE-Sepharose column chromatography and octyl-Sepharose column chromatography purification; at 25°C assay temperature, purified recombinant enzyme, substrate ethyl nitronate; at 25°C assay temperature, purified recombinant enzyme, substrate ethyl nitronate

397

-

;

10000

-

5.5

-

assay at

6.5

7

7.4

-

assay at

7.4 - 8

-

assay at

7.4

-

assay at

726808, 727018

7.4 - 8

-

assay at

7.4

7.5

-

assay at

8

8.5

-

9.5

-

assay at

additional information

-

when the enzyme is dialyzed against 10 mM potassium phosphate buffer (pH 7.0) immediately after reduction by dithionite, the absorption spectrum similar to that of the native enzyme appears with concomitant restoration of approximately 80% of the activity

6 - 10

-

assays are carried out at pH 6, 8, 9.5, and 10

6 - 9

-

investigation of the pH effects on the kcat/Km-values

6 - 9.5

-

7 - 8.5

-

; when the enzyme is acidified to pH 3.0 and treated in the same way, the prosthetic groups do not dissociate from the protein and almost full activity remained

7

Cyberlindnera mrakii IF0 0895

-

assay at

30

37

40

20 - 45

20 - 50

-

IF0 0895

639223, 639224

-

Cyberlindnera saturnus mrakii

-

695891, 726808, 727018

-

-

-

-

-

-

Q9FDD4

UniProt

mycelium

evolution

-

NMO is a member of the group H flavin-dependent monooxygenases

malfunction

physiological function

additional information

-

four conserved motifs are identified in PA4202. Oxidation of propionate-3-nitronate by NMO yields malonic semialdehyde, which is an important metabolite that can be converted to acetyl-CoA, acetate, or 3-hydroxypropionate and enter various catabolic or anabolic pathways. Active site structure analysis, overview

6bka, download, 3D-view

4q4k, download, 3D-view

SCOPe (4q4k)

CATH (4q4k)

Q9HWH9

Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

5lsm, download, 3D-view

SCOPe (5lsm)

CATH (5lsm)

Q8EJJ2

Shewanella oneidensis (strain MR-1)

25000

-

1 * 25000 + 1 * 39000, SDS-PAGE

37000

-

dynamic light scattering analysis

39000

-

1 * 25000 + 1 * 39000, SDS-PAGE

39920

calculated; calculated from the amino acid composition of the enzyme

40000

42000

47000

-

4 * 47000, SDS-PAGE

60000

-

gel filtration

64000

-

equilibrium sedimentation

70000

-

gel filtration

80000

recombinant enzyme, gel filtration

185000

-

gel filtration

dimer

homodimer

2 * 40000, gel filtration, heat denaturation, and mass spectroscopic analysis

monomer

tetramer

-

4 * 47000, SDS-PAGE

additional information

crystallization of the substrate and the complex

-

698266

hanging drop vapour diffusion method using 10% (w/v) polyethylene glycol monomethyl ether 5000

-

purified recombinant His-tagged enzyme, vapor diffusion hanging drop method at room temperature, 0.002 ml of 14 mg/ml protein in 50 mM Tris-Cl, pH 8.0, 100 mM NaCl, 20 mM 2-nitropropane, with 0.002 ml of reservoir solution containing 14% w/v PEG 5000 monomethylether and 0.1 M Na-HEPES, pH 7.0, and equilibration against 1 ml of reservoir solution, 7-10 days, X-ray diffraction structure determination and analysis at 1.44 A resolution, molecular replacement

-

crystal structure of NAO from Streptomyces ansochromogenes is determined. It consists of two domains, a TIM barrel domain bound to FMN and C-terminal domain with a novel folding pattern

Q9FDD4

H199A

-

mutant shows no enzymatic function

H196N

-

site-directed mutagenesis, does to catalyze the formation of ethylnitronate from nitroethane. It is a better catalyst than the wild-type enzyme for oxidative turnover with ethylnitronate; the H196N variant form of the enzyme does to catalyze the formation of ethylnitronate from nitroethane. The H196N variant is a better catalyst than the wild-type enzyme for oxidative turnover with ethylnitronate

H152A

-

His152 likely functions as the catalytic base that initiates oxidation of neutral substrates by abstracting a proton from the alpha-carbon; site-directed mutagenesis. His152 likely functions as the catalytic base that initiates oxidation of neutral substrates by abstracting a proton from the alpha-carbon

S288A

-

mutant enzyme forms inclusion bodies when overexpressed in Escherichia coli; site-directed mutagenesis. Mutant enzyme forms inclusion bodies when overexpressed in Escherichia coli

H179D

Q9FDD4

mutant enzyme shows no activity. Crystal structure of mutant H179D is determined: The structural superimposition of wild-type Sa-NAO and mutant H179D-nitroethane shows no significant structural variation

H179K

Q9FDD4

mutant enzyme shows no activity

H179V

Q9FDD4

mutant enzyme shows no activity

additional information

-

construction of an enzyme deletion mutant by the in vivo recombination system of the yeast Saccharomyces cerevisiae strain InvSc1, overview. Quantitative RT-PCR enzyme expression analysis. The expression of ddlA is not changed in the DELTAnmoA mutant compared to the wild-type

745219

6 - 10

-

stable

639225

40

-

5 min, no loss of activity

45

-

5 min, 14% loss of activity

50

-

5 min, 21.5% loss of activity

55

-

5 min, 60.6% loss of activity

60

-

5 min, 91% loss of activity

65

-

5 min, complete inactivation

denaturation by sodium dithionite

-

gradual and irreversible loss of activity after treatment with 1% sodium lauryl sulfate or 6 M guanidine HCl, activity is reduced to 50% of its initial activity after 50 min at 37°C, complete loss of activity after 10 h

-

immediate denaturation with 8 M urea or thiourea

-

-20°C, 1 mM or 10 mM potassium phosphate buffer, pH 7.0, at least 3 months

-

-20°C, 50 mM potassium phosphate, pH 7.4, 6 months, no loss of activity

-

; to homogeneity

DEAE fast flow column chromatography and Hi-Prep 16/10 octyl fast flow column chromatography; DEAE fast flow column chromatography and Hi-Prep 16/10 octyl fast flow column chromatography; recombinant enzyme from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, DEAE and octyl resin chromatography; recombinant enzyme from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, DEAE and octyl resin chromatography

HiLoad XK 16 Superdex 200 prep-grade column gel filtration and Mono Q HR5/5 column chromatography

-

purified to homogeneity

-

recombinant His-tagged enzyme from Escherichia coli strain Rosetta(DE3)pLysS by affinity chromatography

-

to homogeneity

-

using Ni-NTA chromatography

-

expressed as a His-tagged fusion protein in Escherichia coli

-

expressed in Escherichia coli

expressed in Escherichia coli BL21(DE3) cells; gene ncd-2, expression in Escherichia coli strain BL21(DE3), subcloning in strain XL-1 Blue; gene ncd-2, expression in Escherichia coli strain BL21(DE3), subcloning in strain XL-1 Blue; expressed in Escherichia coli BL21(DE3) cells

expressed in Escherichia coli C41(DE3) cells

-

expression in Escherichia coli

-

gene PA4202 or nmoA, monocistronic, low expression level gene, has an intergenic region of 97 nucleotides with neighbouring gene PA4201 (ddlA) encoding D-alanine alanine ligase, and an intergenic region of 107 nucleotides with gene PA4203 encoding a LysR regulator, PA4203 (nmoR) represses its own transcription and the expression of PA4202 (nmoA), presence of a single NmoR binding site between nmoA and nmoR, and interaction of NmoR with the intergenic nmoA-nmoR control region. In silico analysis of the ddlA-nmoA-nmoR-ppgL locus in Pseudomonas aeruginosa genomes, transcriptome analysis

-

745219

gene pa4202, DNA and amino acid sequence determination and analysis, recombinant expression of His-tagged enzyme in Escherichia coli strain Rosetta(DE3)pLysS

-