Cloned (Comment) | Organism |
---|---|
gene naaA, sequence comparisons and phylogenetic analysis, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain Rosetta 2(DE3) | Bradyrhizobium sp. JS329 |
Crystallization (Comment) | Organism |
---|---|
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 | Bradyrhizobium sp. JS329 |
Protein Variants | Comment | Organism |
---|---|---|
D88A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
D88N | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
E158A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
E196A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
H86A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
N124A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
R289A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
R373A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
Y223A | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
Y223F | site-directed mutagenesis, inactive mutant | Bradyrhizobium sp. JS329 |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
additional information | 4-nitroaniline, 4-amino-3-nitrobenzoic acid, and 2-bromo-5-nitrobenzoic acid are poor inhibitors | Bradyrhizobium sp. JS329 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | Michaelis-Menten kinetics | Bradyrhizobium sp. JS329 | |
0.133 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Zn2+ | Bradyrhizobium sp. JS329 | |
0.14 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Co2+ | Bradyrhizobium sp. JS329 | |
0.354 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Mn2+ | Bradyrhizobium sp. JS329 |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Co2+ | activates | Bradyrhizobium sp. JS329 | |
Mn2+ | activates | Bradyrhizobium sp. JS329 | |
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 | Bradyrhizobium sp. JS329 | |
Zn2+ | activates | Bradyrhizobium sp. JS329 |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
5-nitroanthranilate + H2O | Bradyrhizobium sp. JS329 | - |
5-nitrosalicylate + NH3 | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Bradyrhizobium sp. JS329 | D3WZ85 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain Rosetta 2(DE3) by nickel affinity chromatography, ultrafiltration, and gel filtration | Bradyrhizobium sp. JS329 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
5-nitroanthranilate + H2O | - |
Bradyrhizobium sp. JS329 | 5-nitrosalicylate + NH3 | - |
? | |
additional information | 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 | Bradyrhizobium sp. JS329 | ? | - |
- |
Subunits | Comment | Organism |
---|---|---|
octamer | - |
Bradyrhizobium sp. JS329 |
Synonyms | Comment | Organism |
---|---|---|
5NAA deaminase | - |
Bradyrhizobium sp. JS329 |
5NAA-A | - |
Bradyrhizobium sp. JS329 |
5NAA-aminohydrolase | - |
Bradyrhizobium sp. JS329 |
NAAA | - |
Bradyrhizobium sp. JS329 |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.256 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Co2+ | Bradyrhizobium sp. JS329 | |
0.302 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Zn2+ | Bradyrhizobium sp. JS329 | |
0.605 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Mn2+ | Bradyrhizobium sp. JS329 |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme is a metalloprotease family member | Bradyrhizobium sp. JS329 |
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 | Bradyrhizobium sp. JS329 |
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 | Bradyrhizobium sp. JS329 |
kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.71 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Mn2+ | Bradyrhizobium sp. JS329 | |
1.83 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Co2+ | Bradyrhizobium sp. JS329 | |
2.27 | - |
5-nitroanthranilate | pH and temperature not specified in the publication, enzyme in presence of Zn2+ | Bradyrhizobium sp. JS329 |