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Literature summary extracted from

  • Ohtaki, A.; Murata, K.; Sato, Y.; Noguchi, K.; Miyatake, H.; Dohmae, N.; Yamada, K.; Yohda, M.; Odaka, M.
    Structure and characterization of amidase from Rhodococcus sp. N-771: Insight into the molecular mechanism of substrate recognition (2010), Biochim. Biophys. Acta, 1804, 184-192.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.4 expression of wild-type and mutant enzymes in Escherichia coli strain BL21 Rhodococcus sp. N-771

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
3.5.1.4 purified recombinant wild-type RhAmidase and mutant S195A in complex with benzamide, hanging drop vapor diffusion method, 20°C, mixing of 0.002 ml of protein solution containing 14 mg/ml protein in 10 mM Tris-HCl, pH 8.0, with 0.002 ml of reservoir solution containing 15% w/v PEG 4000, 100 mM magnesium chloride, 100 mM calcium chloride, 100 mM MES, pH 6.0, X-ray diffraction structure determination and analysis at 2.17 A and 2.32 A resolution, respectively, molecular replacement method Rhodococcus sp. N-771

Protein Variants

EC Number Protein Variants Comment Organism
3.5.1.4 S195A catalytically inactive mutant Rhodococcus sp. N-771

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.4 1.03
-
 Benzamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 19
-
 acetamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 24.1
-
 Acrylamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 34.2
-
 Propionamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.4 Rhodococcus sp. N-771 Q7DKE4
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.4 recombinant wild-type and mutant enzymes in Escherichia coli strain BL21 by anion exchange chromatography, ammonium sulfate fractionation, and a second different step of anion exchange chromatography Rhodococcus sp. N-771

Reaction

EC Number Reaction Comment Organism Reaction ID
3.5.1.4 a monocarboxylic acid amide + H2O = a monocarboxylate + NH3 amino acid residues Ser171, Ser195 and Lys96 are essential for catalytic activity, molecular mechanism of substrate recognition, overview Rhodococcus sp. N-771
a

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.4 acetamide + H2O
-
 Rhodococcus sp. N-771 acetate + NH3
-
 ?
3.5.1.4 acrylamide + H2O low activity Rhodococcus sp. N-771 acrylate + NH3
-
 ?
3.5.1.4 benzamide + H2O preferred substrate Rhodococcus sp. N-771 benzoate + NH3
-
 ?
3.5.1.4 additional information the Ser-cis Ser-Lys catalytic triad is located in the large domain. But the substrate-binding pocket is relatively narrow, due to the presence of the helix alpha13 in the small domain. The hydrophobic residues from the small domain are involved in recognizing the substrate. The small domain likely participates in substrate recognition and is related to the difference of substrate specificities among the AS family amidases Rhodococcus sp. N-771 ?
-
 ?
3.5.1.4 propionamide + H2O
-
 Rhodococcus sp. N-771 propionate + NH3
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.4 More RhAmidase contains three domains: an N-terminal alpha-helical domain, a small domain, and a large domain. The N-terminal alpha-helical domain is not found in other AS family enzymes. The N-terminal alpha-helical domain is involved in the formation of the dimer structure and, together with the small domain, forms a narrow substrate-binding tunnel. Structure analysis and modelling of the dimeric enzyme, overview Rhodococcus sp. N-771

Synonyms

EC Number Synonyms Comment Organism
3.5.1.4 More RhAmidase belongs to amidase signature, AS, family, a group of amidase families Rhodococcus sp. N-771
3.5.1.4 RhAmidase
-
 Rhodococcus sp. N-771

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.4 50 55 assay at Rhodococcus sp. N-771

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
3.5.1.4 20 70
-
 Rhodococcus sp. N-771

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3.5.1.4 2.1
-
 Acrylamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 20.2
-
 acetamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 155.3
-
 Propionamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 157.7
-
 Benzamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.4 5.9
-
-
 Rhodococcus sp. N-771

pH Range

EC Number pH Minimum pH Maximum Comment Organism
3.5.1.4 5 8.8 activity range, substrate acrylamide Rhodococcus sp. N-771

General Information

EC Number General Information Comment Organism
3.5.1.4 metabolism RhAmidase belongs to amidase signature family, a group of amidase families, and is responsible for the degradation of amides produced from nitriles by nitrile hydratase Rhodococcus sp. N-771

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
3.5.1.4 0.087
-
 Acrylamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 1.14
-
 acetamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 4.54
-
 Propionamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771
3.5.1.4 153.5
-
 Benzamide pH 5.9, 55°C, recombinant wild-type enzyme Rhodococcus sp. N-771