Cloned (Comment) | Organism |
---|---|
gene pamh, genetic organization, DNA and amino acid sequence determination and analysis, sequence comparison, recombinant expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3). The enzyme is only successfully expressed using autoinduction media under low-temperature incubation at 25°C, otherwise, the expressed proteins forms inclusion bodies | Paracoccus sp. |
Protein Variants | Comment | Organism |
---|---|---|
K84A | site-directed mutagenesis, the mutant shows no detectable hydrolysis activity | Paracoccus sp. |
S159A | site-directed mutagenesis, the mutant shows no detectable hydrolysis activity | Paracoccus sp. |
S183A | site-directed mutagenesis, the mutant shows no detectable hydrolysis activity | Paracoccus sp. |
Inhibitors | Comment | Organism | Structure |
---|---|---|---|
1,10-phenanthroline | 20-30% inhibition at 1 mM | Paracoccus sp. | |
2-mercaptoethanol | 52% inhibition at 1 mM | Paracoccus sp. | |
4-chloromercurybenzoate | - |
Paracoccus sp. | |
Co2+ | strong inhibition | Paracoccus sp. | |
Cr2+ | - |
Paracoccus sp. | |
Cu2+ | strong inhibition | Paracoccus sp. | |
EDTA | 20-30% inhibition at 1 mM | Paracoccus sp. | |
Fe2+ | - |
Paracoccus sp. | |
Hg2+ | complete inhibition | Paracoccus sp. | |
iodoacetamide | - |
Paracoccus sp. | |
Ni2+ | - |
Paracoccus sp. | |
PMSF | strong inhibition | Paracoccus sp. | |
SDS | - |
Paracoccus sp. | |
Triton X-100 | - |
Paracoccus sp. | |
Tween-80 | - |
Paracoccus sp. | |
Zn2+ | - |
Paracoccus sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
2.8 | - |
Acrylamide | pH 7.0, 35°C, recombinant enzyme | Paracoccus sp. |
Metals/Ions | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | activates at 1 mM | Paracoccus sp. | |
Mg2+ | activates at 1 mM | Paracoccus sp. | |
Mn2+ | activates at 1 mM | Paracoccus sp. |
Molecular Weight [Da] | Molecular Weight Maximum [Da] | Comment | Organism |
---|---|---|---|
52000 | - |
x * 52000, about, sequence calculation | Paracoccus sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Paracoccus sp. | G9FKH7 | gene pamH | - |
Purification (Comment) | Organism |
---|---|
recombinant His6-tagged wild-type enzyme 5.82fold from Escherichia coli strain BL21 (DE3) by ammonium sulfate fractionation, dialysis, anion exchange chromatography, gel filtration, and ultrafiltration | Paracoccus sp. |
Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
---|---|---|---|
14.33 | - |
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate acrylamides | Paracoccus sp. |
39.82 | - |
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate benzamide | Paracoccus sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
acetamide + H2O | - |
Paracoccus sp. | acetate + NH3 | - |
? | |
acrylamide + H2O | - |
Paracoccus sp. | acrylate + NH3 | - |
? | |
benzamide + H2O | - |
Paracoccus sp. | benzoate + NH3 | - |
? | |
additional information | the PamH enzyme exhibits amidase activity, aryl acylamidase activity (EC 3.5.1.13), and acyl transferase activity. It shows excellent activity toward the majority of the aromatic and aliphatic amides, such as acetamide, propionamide, phenylacetamide, and benzamide. The aromatic amides, with substitutions of one or two carbons in the ring by a nitrogen, have a negative influence on amidase activity, leading to low specific activity values for pyrazinamide and nicotinamide. No activity is detectable on long-chain aliphatic amide hexanoamides. Amino acid amides are also hydrolyzed by the enzyme. The enzyme possesses urease activity, but N-methyl substituted is not hydrolyzed by the enzyme. The amidase shows low activity on asparagines (9%), L-glutamine (17%), and D-glutamine (13%) corresponding to benzamide (100%). The anilide substrate range of the enzyme is very narrow and cannot hydrolyze butachlor, acetochlor, 4-nitroacetanilide, p-chloroacetanilide, or other structurally analogous compounds | Paracoccus sp. | ? | - |
? | |
nicotinamide + H2O | low activity, reaction of EC 3.5.1.19 | Paracoccus sp. | nicotinate + NH3 | - |
? | |
phenylacetamide + H2O | - |
Paracoccus sp. | phenylacetate + NH3 | - |
? | |
propionamide + H2O | - |
Paracoccus sp. | propionate + NH3 | - |
? | |
pyrazinamide + H2O | low activity | Paracoccus sp. | pyrazinate + NH3 | - |
? |
Subunits | Comment | Organism |
---|---|---|
? | x * 52000, about, sequence calculation | Paracoccus sp. |
Synonyms | Comment | Organism |
---|---|---|
PamH | - |
Paracoccus sp. |
Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|
45 | - |
recombinant enzyme | Paracoccus sp. |
Temperature Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
15 | 70 | activity range, profile overview | Paracoccus sp. |
Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
---|---|---|---|
40 | 60 | the purified recombinant wild-type enzyme is fairly stable up to 40°C, has 25% residual activity at 50°C after 1 h, and is completely inactivated at 60°C after 30 min | Paracoccus sp. |
pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
8 | - |
recombinant enzyme | Paracoccus sp. |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
5.5 | 10 | activity range, profile overview | Paracoccus sp. |
pH Stability | pH Stability Maximum | Comment | Organism |
---|---|---|---|
4.5 | 10 | purified recombinant enzyme, over 50% activity within this range at 35°C | Paracoccus sp. |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Paracoccus sp. | isoelectric focusing | - |
5.13 |
General Information | Comment | Organism |
---|---|---|
evolution | the enzyme belongs to the amidase signature enzyme family | Paracoccus sp. |
additional information | the enzyme maintains a core alpha/beta/alpha structure and the G-(GAV)-S-(GS)2-GX-(GSAE)-(GSAVYCT)-X-(LIVMT)-(GSA)-X6-(GSAT)-X-(GA)-X-(DE)-X-(GA)-X-S-(LIVM)-R-X-P-(GSACTL) sequence motif | Paracoccus sp. |