Literature summary for 3.5.99.8 extracted from

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 (2016), Nat. Chem. Biol., 12, 1031-1036 .

Cloned(Commentary)

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 (Commentary)

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

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

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

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 [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
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

Metals/Ions	Comment	Organism
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/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
5-nitroanthranilate + H2O	Bradyrhizobium sp. JS329	-	5-nitrosalicylate + NH3	-	?

Organism

Organism	UniProt	Comment	Textmining
Bradyrhizobium sp. JS329	D3WZ85	-	-

Purification (Commentary)

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 and Products (Substrate)

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

Subunits	Comment	Organism
octamer	-	Bradyrhizobium sp. JS329

Synonyms

Synonyms	Comment	Organism
5NAA deaminase	-	Bradyrhizobium sp. JS329
5NAA-A	-	Bradyrhizobium sp. JS329
5NAA-aminohydrolase	-	Bradyrhizobium sp. JS329
NAAA	-	Bradyrhizobium sp. JS329

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
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

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 [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
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