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Information on EC 3.5.99.8 - 5-nitroanthranilic acid aminohydrolase
for references in articles please use BRENDA:EC3.5.99.8
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EC Tree 3 Hydrolases
3.5 Acting on carbon-nitrogen bonds, other than peptide bonds
3.5.99 In other compounds
3.5.99.8 5-nitroanthranilic acid aminohydrolase
IUBMB Comments
The enzyme catalyses the initial step in biodegradation of 5-nitroanthranilic acid by Bradyrhizobium sp. strain JS329.
The expected taxonomic range for this enzyme is: unclassified Bradyrhizobium
showSynonyms
5naa-a, 5naa deaminase, 5naa-aminohydrolase, more
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SYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
5NAA deaminase
NAAA
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
5-nitroanthranilate + H2O = 5-nitrosalicylate + NH3
-
-
-
-
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PATHWAY SOURCE
PATHWAYS
MetaCyc
5-nitroanthranilate degradation
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SYSTEMATIC NAME
IUBMB Comments
5-nitroanthranilate aminohydrolase
The enzyme catalyses the initial step in biodegradation of 5-nitroanthranilic acid by Bradyrhizobium sp. strain JS329.
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SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
5-nitroanthranilate + H2O
5-nitrosalicylate + NH3
Substrates: initial step in biodegradation of 5-nitroanthranilic acid as sole carbon, nitrogen and energy source
Products: -
Products: -
?
5-nitroanthranilate + H2O
5-nitrosalicylate + NH3
Substrates: -
Products: -
Products: -
?
5-nitroanthranilate + H2O
5-nitrosalicylate + NH3
Substrates: -
Products: -
Products: -
?
additional information
?
-
Substrates: mechanism of release of the nitro group, overview. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation
Products: -
Products: -
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additional information
?
-
-
Substrates: mechanism of release of the nitro group, overview. The nitro group is spontaneously eliminated as nitrite concomitant with the formation of a lactone from the ring fission product of 5NSA dioxygenation
Products: -
Products: -
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additional information
?
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Substrates: enzyme 5NAA-A is specific for 5-nitroanthranilate (5NAA) and cannot hydrolyze other tested derivatives, i.e. 4-nitroaniline, 4-amino-3-nitrobenzoic acid, and 2-bromo-5-nitrobenzoic acid, which are likewise poor inhibitors. The enzyme employs a nucleophilic aromatic substitution mechanism
Products: -
Products: -
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
LITERATURE
COMMENTARY
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
5-nitroanthranilate + H2O
5-nitrosalicylate + NH3
Substrates: initial step in biodegradation of 5-nitroanthranilic acid as sole carbon, nitrogen and energy source
Products: -
Products: -
?
5-nitroanthranilate + H2O
5-nitrosalicylate + NH3
Substrates: -
Products: -
Products: -
?
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COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
no added cofactors are required for the transformation
-
additional information
-
no added cofactors are required for the transformation
-
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
Mn2+
additional information
additional information
NaaA contains the sequence HEAWH (amino acid residues 108 to 112), which is similar to the zinc binding motif HEXXH
additional information
-
NaaA contains the sequence HEAWH (amino acid residues 108 to 112), which is similar to the zinc binding motif HEXXH
additional information
the enzyme is a metalloprotease family member, it can use several divalent transition metals for catalysis. The metal in 5NAA-A is labile but readily loaded in the presence of substrate. Adjacent to the 5NAA binding site is a triad of residues (His86-Glu196-Asn124) in a location equivalent to that of the corresponding M20 metalloprotease His2-Glu2-Asp metal binding motif. 5NAA-A requires metal ions for hydrolysis, and several transition metal ions are suitable. Fastest rates are observed in the presence of Mn2+, Fe2+ facilitates turnover but data cannot be fit to the Michaelis-Menten equation, like Cu2+, metal binding analysis, overview
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INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
the activity of NaaA after inhibition is restored by the addition of divalent metal ions: e.g. Co2+ to 100%, Mn2+ to 85%, Zn2+ to 81%, Fe2+ to 67%, or Ni2+ to 39%, all at 0.667 mM
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additional information
-
the activity of NaaA after inhibition is restored by the addition of divalent metal ions: e.g. Co2+ to 100%, Mn2+ to 85%, Zn2+ to 81%, Fe2+ to 67%, or Ni2+ to 39%, all at 0.667 mM
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additional information
4-nitroaniline, 4-amino-3-nitrobenzoic acid, and 2-bromo-5-nitrobenzoic acid are poor inhibitors
-
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
Michaelis-Menten kinetics
-
0.133
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Zn2+
0.14
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Co2+
0.354
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Mn2+
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.256
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Co2+
0.302
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Zn2+
0.605
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Mn2+
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kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
1.71
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Mn2+
1.83
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Co2+
2.27
5-nitroanthranilate
pH and temperature not specified in the publication, enzyme in presence of Zn2+
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pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
Highest Expressing Human Cell Lines
Cell Line LinksPlease wait a moment until the data is sorted. This message will disappear when the data is sorted.
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
metabolism
additional information
metabolism
the enzyme catalyzes the initial step in 5-nitroanthranilic acid degradation, a hydrolytic deamination to form 5-nitrosalicylic acid, followed by ring fission catalyzed by 5NSA dioxygenase, biodegradation mechanism and pathway overview
metabolism
Bradyrhizobium species strain JS329 metabolizes 5-nitroanthranilic acid (5NAA), a metabolite secreted by Streptomyces scabies, the bacterium responsible for potato scab, an agricultural disease of global economic importance. The first biodegradation enzyme is 5NAA-aminohydrolase (5NAA-A), a metalloprotease family member that converts 5NAA to 5-nitrosalicylic acid
additional information
NaaA is a hydrolytic metalloenzyme with a narrow substrate spectrum
additional information
-
NaaA is a hydrolytic metalloenzyme with a narrow substrate spectrum
additional information
the enzyme employs a nucleophilic aromatic substitution mechanism. 5-Nitroanthranilate aminohydrolase performs a unique deamination reaction whereby the amino group, which is para to the nitro substituent, is hydrolyzed to form 5-nitrosalicylic acid, and the nitro group remains intact. 5NAA binds primarily within one catalytic lobe but is stabilized by a key residue, Tyr223 from an adjacent monomer, which provides parallel-displaced Pi-Pi stacking stabilization. Within the catalytic lobe, 5NAA is held in place by Arg373 and Arg289, which stabilize the carboxyl and nitro groups, respectively, and the side chain of Glu158, which is within hydrogen bonding distance of the amino substituent. Asp160 and the main chain of Trp372 also stabilize the aforementioned adjacent Tyr223. Two loops disordered in the apo structure are located in this region of the catalytic lobe. Substrate binding organizes these loops, primarily by interactions between Tyr288 and Arg98 and between Asp95 and Lys286
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UNIPROT
ENTRY NAME
ORGANISM
NO. OF AA
NO. OF TRANSM. HELICES
MOLECULAR WEIGHT[Da]
SOURCE
SEQUENCE
LOCALIZATION PREDICTION
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable).
The reliability class of the localization prediction ranges from 1 (very reliable) to 5 (not reliable). NAAA_BRASZ
425
0
46349
Swiss-Prot
other Location (Reliability: 2)
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PDB
SCOP
CATH
UNIPROT
ORGANISM
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SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
selenomethionine-labeled apo 5NAA-A, substrate-bound 5NAA-A, product- and Mn2+-bound 5NAA-A, and the Meisenheimer complex intermediate with Zn2+ bound to the 5NAA-AR289A mutant, sitting drop method, room temperature, X-ray diffraction structure determination and analysis. 9.4 mg/ml SeMet apo 5NAA-A protein in 50 mM bicine, pH 7.0, and 150 mM NaCl crystallizes from 1.07 M NaH2PO4, and 0.83 M K2HPO4. 7 mg/ml wild-type 5NAA-A protein in 50 mM bicine, pH 7.0, 150 mM NaCl, and 5NAA (0.22 mM) crystallizes from 1.27 M NaH2PO4, and 0.63 M K2HPO4. 5NAA-A-Mn2+ complex from 5.8 mg/ml protein in 50 mM bicine, pH 7.0, 150 mM NaCl, 16 mM MnCl2, and 0.16 mM 5NSA crystallizes from 0.1 M sodium cacodylate pH 6.5, and 0.75 M trisodium citrate. 5.8 mg/ml mutant 5NAA-AR289A protein in 50 mM bicine, pH 7.0, 150 mM NaCl, 12 mM Zn(OAc)2, and 0.22 mM 5NAA crystallizes from 0.1 M sodium cacodylate, pH 6.5, and 0.65 M trisodium citrate
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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
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PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain Rosetta 2/pJS803 by nickel affinity chromatography
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta 2(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration
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CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
gene naaA, DNA and amino acid sequence determination and analysis, expression of His-tagged enzyme in Escherichia coli strain Rosetta 2/pJS803 and in strain EPI300(pJS800)
gene naaA, sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2(DE3)
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REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Qu, Y.; Spain, J.C.
Biodegradation of 5-nitroanthranilic acid by Bradyrhizobium sp. strain JS329
Appl. Environ. Microbiol.
76
1417-1422
2010
Bradyrhizobium sp. (D3WZ85), Bradyrhizobium sp.
brenda
Qu, Y.; Spain, J.C.
Molecular and biochemical characterization of the 5-nitroanthranilic acid degradation pathway in Bradyrhizobium sp. strain JS329
J. Bacteriol.
193
3057-3063
2011
Bradyrhizobium sp. (D3WZ85), Bradyrhizobium sp.
brenda
Kalyoncu, S.; Heaner, D.P.; Kurt, Z.; Bethel, C.M.; Ukachukwu, C.U.; Chakravarthy, S.; Spain, J.C.; Lieberman, R.L.
Enzymatic hydrolysis by transition-metal-dependent nucleophilic aromatic substitution
Nat. Chem. Biol.
12
1031-1036
2016
Bradyrhizobium sp. JS329 (D3WZ85)
brenda
EXTERNAL LINKS (specific for EC-Number 3.5.99.8)
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