Cloned (Comment) | Organism |
---|---|
expression of wild-type and mutant enzymes in Escherichia coli strain BL21 | Rhodococcus sp. N-771 |
Crystallization (Comment) | Organism |
---|---|
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 | Comment | Organism |
---|---|---|
S195A | catalytically inactive mutant | Rhodococcus sp. N-771 |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
1.03 | - |
Benzamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
19 | - |
acetamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
24.1 | - |
Acrylamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
34.2 | - |
Propionamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Rhodococcus sp. N-771 | Q7DKE4 | - |
- |
Purification (Comment) | Organism |
---|---|
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 | Comment | Organism | Reaction ID |
---|---|---|---|
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 |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
acetamide + H2O | - |
Rhodococcus sp. N-771 | acetate + NH3 | - |
? | |
acrylamide + H2O | low activity | Rhodococcus sp. N-771 | acrylate + NH3 | - |
? | |
benzamide + H2O | preferred substrate | Rhodococcus sp. N-771 | benzoate + NH3 | - |
? | |
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 | ? | - |
? | |
propionamide + H2O | - |
Rhodococcus sp. N-771 | propionate + NH3 | - |
? |
Subunits | Comment | Organism |
---|---|---|
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 | Comment | Organism |
---|---|---|
More | RhAmidase belongs to amidase signature, AS, family, a group of amidase families | Rhodococcus sp. N-771 |
RhAmidase | - |
Rhodococcus sp. N-771 |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
50 | 55 | assay at | Rhodococcus sp. N-771 |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | 70 | - |
Rhodococcus sp. N-771 |
Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.1 | - |
Acrylamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
20.2 | - |
acetamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
155.3 | - |
Propionamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
157.7 | - |
Benzamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
5.9 | - |
- |
Rhodococcus sp. N-771 |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5 | 8.8 | activity range, substrate acrylamide | Rhodococcus sp. N-771 |
General Information | Comment | Organism |
---|---|---|
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 Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
0.087 | - |
Acrylamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
1.14 | - |
acetamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
4.54 | - |
Propionamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 | |
153.5 | - |
Benzamide | pH 5.9, 55°C, recombinant wild-type enzyme | Rhodococcus sp. N-771 |