BRENDA - Enzyme Database show

Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities

Shen, W.; Chen, H.; Jia, K.; Ni, J.; Yan, X.; Li, S.; Appl. Microbiol. Biotechnol. 94, 1007-1018 (2012)

Data extracted from this reference:

Cloned(Commentary)
EC Number
Commentary
Organism
3.5.1.4
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.
3.5.1.13
gene pamH, DNA and amino acid sequence determination and analysis, sequence comparison, cloning and expression of His6-tagged PamH in Escherichia coli strains DH5alpha and BL21 (DE3), respectively; 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. M1-1
Engineering
EC Number
Amino acid exchange
Commentary
Organism
3.5.1.4
K84A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.4
S159A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.4
S183A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.13
K84A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
3.5.1.13
S159A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
3.5.1.13
S183A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
Inhibitors
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
1,10-phenanthroline
20-30% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
2-mercaptoethanol
52% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
4-chloromercurybenzoate
-
Paracoccus sp.
3.5.1.4
Co2+
strong inhibition
Paracoccus sp.
3.5.1.4
Cr2+
-
Paracoccus sp.
3.5.1.4
Cu2+
strong inhibition
Paracoccus sp.
3.5.1.4
EDTA
20-30% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
Fe2+
-
Paracoccus sp.
3.5.1.4
Hg2+
complete inhibition
Paracoccus sp.
3.5.1.4
iodoacetamide
-
Paracoccus sp.
3.5.1.4
Ni2+
-
Paracoccus sp.
3.5.1.4
PMSF
strong inhibition
Paracoccus sp.
3.5.1.4
SDS
-
Paracoccus sp.
3.5.1.4
Triton X-100
-
Paracoccus sp.
3.5.1.4
Tween-80
-
Paracoccus sp.
3.5.1.4
Zn2+
-
Paracoccus sp.
3.5.1.13
1,10-phenanthroline
; 20-30% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
2-mercaptoethanol
52% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
4-chloromercuribenzoate
-
Paracoccus sp. M1-1
3.5.1.13
4-chloromercurybenzoate
-
Paracoccus sp. M1-1
3.5.1.13
Co2+
; 1 mM: 8.5% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
Cr2+
-
Paracoccus sp. M1-1
3.5.1.13
Cu2+
; 1 mM: 8.9% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
EDTA
; 20-30% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Fe2+
-
Paracoccus sp. M1-1
3.5.1.13
Hg2+
; 1 mM: 0% activity; complete inhibition
Paracoccus sp. M1-1
3.5.1.13
iodoacetamide
-
Paracoccus sp. M1-1
3.5.1.13
Ni2+
-
Paracoccus sp. M1-1
3.5.1.13
PMSF
; 10 mM: 9.8% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
SDS
-
Paracoccus sp. M1-1
3.5.1.13
Triton X-100
-
Paracoccus sp. M1-1
3.5.1.13
Tween-80
-
Paracoccus sp. M1-1
3.5.1.13
Zn2+
-
Paracoccus sp. M1-1
KM Value [mM]
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
2.8
-
Acrylamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp.
3.5.1.13
0.158
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
0.158
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1
3.5.1.13
0.158
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
Metals/Ions
EC Number
Metals/Ions
Commentary
Organism
Structure
3.5.1.4
Ca2+
activates at 1 mM
Paracoccus sp.
3.5.1.4
Mg2+
activates at 1 mM
Paracoccus sp.
3.5.1.4
Mn2+
activates at 1 mM
Paracoccus sp.
3.5.1.13
Ca2+
activates at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Mg2+
activates 1.1fold at 1 mM; activates 1.3fold at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Mn2+
activates 1.1fold at 1 mM; activates 1.3fold at 1 mM
Paracoccus sp. M1-1
Molecular Weight [Da]
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
52000
-
x * 52000, about, sequence calculation
Paracoccus sp.
3.5.1.13
52000
-
x * 52000, about, sequence calculation; x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
3.5.1.13
52390
-
x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
Organism
EC Number
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
3.5.1.4
Paracoccus sp.
G9FKH7
gene pamH
-
3.5.1.13
Paracoccus sp. M1-1
F6N111
; gene pamH
-
Purification (Commentary)
EC Number
Commentary
Organism
3.5.1.4
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.
3.5.1.13
ammonium sulfate fractionation, ion exchange chromatography (DEAE-Sephadex, DEAE-Cellulose, gel filtration); recombinant His6-tagged PamH 5.8fold from Escherichia coli strain BL21 (DE3) by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration; 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. M1-1
Specific Activity [micromol/min/mg]
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
3.5.1.4
14.33
-
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate acrylamides
Paracoccus sp.
3.5.1.4
39.82
-
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate benzamide
Paracoccus sp.
3.5.1.13
39.8
-
N-(3,4-dichlorophenyl)propanamide, pH 7.0, 35°C; purified recombinant His6-tagged PamH, pH 7.0, 35°C
Paracoccus sp. M1-1
Storage Stability
EC Number
Storage Stability
Organism
3.5.1.13
rapid loss of activity during purification and storage in Tris-HCl (10 mM, pH 8.0) buffer at 4°C or 20°C, with the addition of 10% glycerol and 50 mM NaCl prior to freezing activity is maintained for up to 3 months with only marginal losses
Paracoccus sp. M1-1
Substrates and Products (Substrate)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.1.4
acetamide + H2O
-
718672
Paracoccus sp.
acetate + NH3
-
-
-
?
3.5.1.4
acrylamide + H2O
-
718672
Paracoccus sp.
acrylate + NH3
-
-
-
?
3.5.1.4
benzamide + H2O
-
718672
Paracoccus sp.
benzoate + NH3
-
-
-
?
3.5.1.4
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
718672
Paracoccus sp.
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
low activity, reaction of EC 3.5.1.19
718672
Paracoccus sp.
nicotinate + NH3
-
-
-
?
3.5.1.4
phenylacetamide + H2O
-
718672
Paracoccus sp.
phenylacetate + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
-
718672
Paracoccus sp.
propionate + NH3
-
-
-
?
3.5.1.4
pyrazinamide + H2O
low activity
718672
Paracoccus sp.
pyrazinate + NH3
-
-
-
?
3.5.1.13
3',4'-dichloropropionanilide + H2O
i.e. propanil
718672
Paracoccus sp. M1-1
3,4-dichloroaniline + propionate
-
-
-
?
3.5.1.13
benzamide + H2O
-
718672
Paracoccus sp. M1-1
benzoate + NH3
-
-
-
?
3.5.1.13
additional information
substrate specificity, overview. PamH is highly active on aromatic and short-chain aliphatic amides, e.g. benzamide and propionamide, moderately active on amino acid amides, and possesses weak urease activity. Of the anilides examined, only propanil is a good substrate for PamH. PamH is also able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide, it shows the highest reaction rate with isobutyramide
718672
Paracoccus sp. M1-1
?
-
-
-
-
3.5.1.13
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
718672
Paracoccus sp. M1-1
?
-
-
-
-
3.5.1.13
N-(3,4-dichlorophenyl) propanamide + H2O
i.e. propanil
718672
Paracoccus sp. M1-1
3,4-dichloroaniline + propanoic acid
-
-
-
?
3.5.1.13
N-(3,4-dichlorophenyl)propanamide + H2O
no activity with butachlor, acetochlor, 4-nitroacetanilide, p-chloracetanilide
718672
Paracoccus sp. M1-1
3,4-dichloraniline + propanoate
-
-
-
?
3.5.1.13
propanamide + H2O
-
718672
Paracoccus sp. M1-1
propanoic acid + NH3
-
-
-
?
Subunits
EC Number
Subunits
Commentary
Organism
3.5.1.4
?
x * 52000, about, sequence calculation
Paracoccus sp.
3.5.1.13
?
x * 52000, about, sequence calculation; x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
Temperature Optimum [°C]
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
45
-
recombinant enzyme
Paracoccus sp.
3.5.1.13
45
-
; recombinant enzyme
Paracoccus sp. M1-1
Temperature Range [°C]
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
15
70
activity range, profile overview
Paracoccus sp.
3.5.1.13
15
70
activity range, profile overview
Paracoccus sp. M1-1
Temperature Stability [°C]
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
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.
3.5.1.13
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. M1-1
3.5.1.13
40
-
fairly stable up to 40°C, completely inactivated during 30 min at 60°C; purified recombinant enzyme, fairly stable up to, over 90% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
50
-
25% residual activity after 1 h at 50°C, completely inactivated during 30 min at 60°C; purified recombinant enzyme, 25% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
60
-
purified recombinant enzyme, 30 min, complete inactivation
Paracoccus sp. M1-1
Turnover Number [1/s]
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
3.5.1.13
2.8
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
2.8
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1
3.5.1.13
2.8
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
pH Optimum
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
8
-
recombinant enzyme
Paracoccus sp.
3.5.1.13
8
-
; recombinant enzyme
Paracoccus sp. M1-1
pH Range
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
5.5
10
activity range, profile overview
Paracoccus sp.
3.5.1.13
5.5
10
activity range, profile overview
Paracoccus sp. M1-1
3.5.1.13
6
9
activity range
Paracoccus sp. M1-1
3.5.1.13
6.5
9
highly active
Paracoccus sp. M1-1
pH Stability
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
3.5.1.4
4.5
10
purified recombinant enzyme, over 50% activity within this range at 35°C
Paracoccus sp.
3.5.1.13
4
-
purified recombinant enzyme, 30% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
4.5
10
purified recombinant enzyme, over 50% activity within this range at 35°C
Paracoccus sp. M1-1
3.5.1.13
4.5
-
purified recombinant enzyme, 60% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
5
10
more than 70% activity after preincubation for 1 h; purified recombinant enzyme, over 70% activity remaining after 1 h
Paracoccus sp. M1-1
pI Value
EC Number
Organism
Commentary
pI Value Maximum
pI Value
3.5.1.4
Paracoccus sp.
isoelectric focusing
-
5.13
3.5.1.13
Paracoccus sp. M1-1
isoelectric focusing; isoelectric focusing, recombinant enzyme
-
5.13
Cloned(Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.1.4
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.
3.5.1.13
gene pamH, DNA and amino acid sequence determination and analysis, sequence comparison, cloning and expression of His6-tagged PamH in Escherichia coli strains DH5alpha and BL21 (DE3), respectively; 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. M1-1
Engineering (protein specific)
EC Number
Amino acid exchange
Commentary
Organism
3.5.1.4
K84A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.4
S159A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.4
S183A
site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp.
3.5.1.13
K84A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
3.5.1.13
S159A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
3.5.1.13
S183A
no activity, part of the catalytic triad; site-directed mutagenesis, the mutant shows no detectable hydrolysis activity
Paracoccus sp. M1-1
Inhibitors (protein specific)
EC Number
Inhibitors
Commentary
Organism
Structure
3.5.1.4
1,10-phenanthroline
20-30% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
2-mercaptoethanol
52% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
4-chloromercurybenzoate
-
Paracoccus sp.
3.5.1.4
Co2+
strong inhibition
Paracoccus sp.
3.5.1.4
Cr2+
-
Paracoccus sp.
3.5.1.4
Cu2+
strong inhibition
Paracoccus sp.
3.5.1.4
EDTA
20-30% inhibition at 1 mM
Paracoccus sp.
3.5.1.4
Fe2+
-
Paracoccus sp.
3.5.1.4
Hg2+
complete inhibition
Paracoccus sp.
3.5.1.4
iodoacetamide
-
Paracoccus sp.
3.5.1.4
Ni2+
-
Paracoccus sp.
3.5.1.4
PMSF
strong inhibition
Paracoccus sp.
3.5.1.4
SDS
-
Paracoccus sp.
3.5.1.4
Triton X-100
-
Paracoccus sp.
3.5.1.4
Tween-80
-
Paracoccus sp.
3.5.1.4
Zn2+
-
Paracoccus sp.
3.5.1.13
1,10-phenanthroline
; 20-30% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
2-mercaptoethanol
52% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
4-chloromercuribenzoate
-
Paracoccus sp. M1-1
3.5.1.13
4-chloromercurybenzoate
-
Paracoccus sp. M1-1
3.5.1.13
Co2+
; 1 mM: 8.5% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
Cr2+
-
Paracoccus sp. M1-1
3.5.1.13
Cu2+
; 1 mM: 8.9% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
EDTA
; 20-30% inhibition at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Fe2+
-
Paracoccus sp. M1-1
3.5.1.13
Hg2+
; 1 mM: 0% activity; complete inhibition
Paracoccus sp. M1-1
3.5.1.13
iodoacetamide
-
Paracoccus sp. M1-1
3.5.1.13
Ni2+
-
Paracoccus sp. M1-1
3.5.1.13
PMSF
; 10 mM: 9.8% activity; strong inhibition
Paracoccus sp. M1-1
3.5.1.13
SDS
-
Paracoccus sp. M1-1
3.5.1.13
Triton X-100
-
Paracoccus sp. M1-1
3.5.1.13
Tween-80
-
Paracoccus sp. M1-1
3.5.1.13
Zn2+
-
Paracoccus sp. M1-1
KM Value [mM] (protein specific)
EC Number
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
3.5.1.4
2.8
-
Acrylamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp.
3.5.1.13
0.158
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
0.158
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1
3.5.1.13
0.158
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
Metals/Ions (protein specific)
EC Number
Metals/Ions
Commentary
Organism
Structure
3.5.1.4
Ca2+
activates at 1 mM
Paracoccus sp.
3.5.1.4
Mg2+
activates at 1 mM
Paracoccus sp.
3.5.1.4
Mn2+
activates at 1 mM
Paracoccus sp.
3.5.1.13
Ca2+
activates at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Mg2+
activates 1.1fold at 1 mM; activates 1.3fold at 1 mM
Paracoccus sp. M1-1
3.5.1.13
Mn2+
activates 1.1fold at 1 mM; activates 1.3fold at 1 mM
Paracoccus sp. M1-1
Molecular Weight [Da] (protein specific)
EC Number
Molecular Weight [Da]
Molecular Weight Maximum [Da]
Commentary
Organism
3.5.1.4
52000
-
x * 52000, about, sequence calculation
Paracoccus sp.
3.5.1.13
52000
-
x * 52000, about, sequence calculation; x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
3.5.1.13
52390
-
x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
Purification (Commentary) (protein specific)
EC Number
Commentary
Organism
3.5.1.4
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.
3.5.1.13
ammonium sulfate fractionation, ion exchange chromatography (DEAE-Sephadex, DEAE-Cellulose, gel filtration); recombinant His6-tagged PamH 5.8fold from Escherichia coli strain BL21 (DE3) by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration; 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. M1-1
Specific Activity [micromol/min/mg] (protein specific)
EC Number
Specific Activity Minimum [µmol/min/mg]
Specific Activity Maximum [µmol/min/mg]
Commentary
Organism
3.5.1.4
14.33
-
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate acrylamides
Paracoccus sp.
3.5.1.4
39.82
-
purified recombinant wild-type enzyme, pH 7.0, 35°C, substrate benzamide
Paracoccus sp.
3.5.1.13
39.8
-
N-(3,4-dichlorophenyl)propanamide, pH 7.0, 35°C; purified recombinant His6-tagged PamH, pH 7.0, 35°C
Paracoccus sp. M1-1
Storage Stability (protein specific)
EC Number
Storage Stability
Organism
3.5.1.13
rapid loss of activity during purification and storage in Tris-HCl (10 mM, pH 8.0) buffer at 4°C or 20°C, with the addition of 10% glycerol and 50 mM NaCl prior to freezing activity is maintained for up to 3 months with only marginal losses
Paracoccus sp. M1-1
Substrates and Products (Substrate) (protein specific)
EC Number
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
3.5.1.4
acetamide + H2O
-
718672
Paracoccus sp.
acetate + NH3
-
-
-
?
3.5.1.4
acrylamide + H2O
-
718672
Paracoccus sp.
acrylate + NH3
-
-
-
?
3.5.1.4
benzamide + H2O
-
718672
Paracoccus sp.
benzoate + NH3
-
-
-
?
3.5.1.4
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
718672
Paracoccus sp.
?
-
-
-
-
3.5.1.4
nicotinamide + H2O
low activity, reaction of EC 3.5.1.19
718672
Paracoccus sp.
nicotinate + NH3
-
-
-
?
3.5.1.4
phenylacetamide + H2O
-
718672
Paracoccus sp.
phenylacetate + NH3
-
-
-
?
3.5.1.4
propionamide + H2O
-
718672
Paracoccus sp.
propionate + NH3
-
-
-
?
3.5.1.4
pyrazinamide + H2O
low activity
718672
Paracoccus sp.
pyrazinate + NH3
-
-
-
?
3.5.1.13
3',4'-dichloropropionanilide + H2O
i.e. propanil
718672
Paracoccus sp. M1-1
3,4-dichloroaniline + propionate
-
-
-
?
3.5.1.13
benzamide + H2O
-
718672
Paracoccus sp. M1-1
benzoate + NH3
-
-
-
?
3.5.1.13
additional information
substrate specificity, overview. PamH is highly active on aromatic and short-chain aliphatic amides, e.g. benzamide and propionamide, moderately active on amino acid amides, and possesses weak urease activity. Of the anilides examined, only propanil is a good substrate for PamH. PamH is also able to catalyze the acyl transfer reaction to hydroxylamine for both amide and anilide substrates, including acetamide, propanil, and 4-nitroacetanilide, it shows the highest reaction rate with isobutyramide
718672
Paracoccus sp. M1-1
?
-
-
-
-
3.5.1.13
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
718672
Paracoccus sp. M1-1
?
-
-
-
-
3.5.1.13
N-(3,4-dichlorophenyl) propanamide + H2O
i.e. propanil
718672
Paracoccus sp. M1-1
3,4-dichloroaniline + propanoic acid
-
-
-
?
3.5.1.13
N-(3,4-dichlorophenyl)propanamide + H2O
no activity with butachlor, acetochlor, 4-nitroacetanilide, p-chloracetanilide
718672
Paracoccus sp. M1-1
3,4-dichloraniline + propanoate
-
-
-
?
3.5.1.13
propanamide + H2O
-
718672
Paracoccus sp. M1-1
propanoic acid + NH3
-
-
-
?
Subunits (protein specific)
EC Number
Subunits
Commentary
Organism
3.5.1.4
?
x * 52000, about, sequence calculation
Paracoccus sp.
3.5.1.13
?
x * 52000, about, sequence calculation; x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation
Paracoccus sp. M1-1
Temperature Optimum [°C] (protein specific)
EC Number
Temperature Optimum [°C]
Temperature Optimum Maximum [°C]
Commentary
Organism
3.5.1.4
45
-
recombinant enzyme
Paracoccus sp.
3.5.1.13
45
-
; recombinant enzyme
Paracoccus sp. M1-1
Temperature Range [°C] (protein specific)
EC Number
Temperature Minimum [°C]
Temperature Maximum [°C]
Commentary
Organism
3.5.1.4
15
70
activity range, profile overview
Paracoccus sp.
3.5.1.13
15
70
activity range, profile overview
Paracoccus sp. M1-1
Temperature Stability [°C] (protein specific)
EC Number
Temperature Stability Minimum [°C]
Temperature Stability Maximum [°C]
Commentary
Organism
3.5.1.4
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.
3.5.1.13
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. M1-1
3.5.1.13
40
-
fairly stable up to 40°C, completely inactivated during 30 min at 60°C; purified recombinant enzyme, fairly stable up to, over 90% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
50
-
25% residual activity after 1 h at 50°C, completely inactivated during 30 min at 60°C; purified recombinant enzyme, 25% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
60
-
purified recombinant enzyme, 30 min, complete inactivation
Paracoccus sp. M1-1
Turnover Number [1/s] (protein specific)
EC Number
Turnover Number Minimum [1/s]
Turnover Number Maximum [1/s]
Substrate
Commentary
Organism
Structure
3.5.1.13
2.8
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
2.8
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1
3.5.1.13
2.8
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
pH Optimum (protein specific)
EC Number
pH Optimum Minimum
pH Optimum Maximum
Commentary
Organism
3.5.1.4
8
-
recombinant enzyme
Paracoccus sp.
3.5.1.13
8
-
; recombinant enzyme
Paracoccus sp. M1-1
pH Range (protein specific)
EC Number
pH Minimum
pH Maximum
Commentary
Organism
3.5.1.4
5.5
10
activity range, profile overview
Paracoccus sp.
3.5.1.13
5.5
10
activity range, profile overview
Paracoccus sp. M1-1
3.5.1.13
6
9
activity range
Paracoccus sp. M1-1
3.5.1.13
6.5
9
highly active
Paracoccus sp. M1-1
pH Stability (protein specific)
EC Number
pH Stability
pH Stability Maximum
Commentary
Organism
3.5.1.4
4.5
10
purified recombinant enzyme, over 50% activity within this range at 35°C
Paracoccus sp.
3.5.1.13
4
-
purified recombinant enzyme, 30% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
4.5
10
purified recombinant enzyme, over 50% activity within this range at 35°C
Paracoccus sp. M1-1
3.5.1.13
4.5
-
purified recombinant enzyme, 60% activity remaining after 1 h
Paracoccus sp. M1-1
3.5.1.13
5
10
more than 70% activity after preincubation for 1 h; purified recombinant enzyme, over 70% activity remaining after 1 h
Paracoccus sp. M1-1
pI Value (protein specific)
EC Number
Organism
Commentary
pI Value Maximum
pI Value
3.5.1.4
Paracoccus sp.
isoelectric focusing
-
5.13
3.5.1.13
Paracoccus sp. M1-1
isoelectric focusing; isoelectric focusing, recombinant enzyme
-
5.13
General Information
EC Number
General Information
Commentary
Organism
3.5.1.4
evolution
the enzyme belongs to the amidase signature enzyme family
Paracoccus sp.
3.5.1.4
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.
3.5.1.13
evolution
PamH belongs to the amidase signature, AS, enzyme family. The Ser-Ser-Lys catalytic residues are highly conserved, indicating that there is an evolutionary relationship between the enzymes in the AS family; the enzyme belongs to the amidase signature enzyme family
Paracoccus sp. M1-1
3.5.1.13
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. M1-1
General Information (protein specific)
EC Number
General Information
Commentary
Organism
3.5.1.4
evolution
the enzyme belongs to the amidase signature enzyme family
Paracoccus sp.
3.5.1.4
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.
3.5.1.13
evolution
PamH belongs to the amidase signature, AS, enzyme family. The Ser-Ser-Lys catalytic residues are highly conserved, indicating that there is an evolutionary relationship between the enzymes in the AS family; the enzyme belongs to the amidase signature enzyme family
Paracoccus sp. M1-1
3.5.1.13
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. M1-1
KCat/KM [mM/s]
EC Number
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
3.5.1.13
17.72
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
17.72
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
18
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1
KCat/KM [mM/s] (protein specific)
EC Number
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
3.5.1.13
17.72
-
3',4'-dichloropropionanilide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
17.72
-
N-(3,4-dichlorophenyl) propanamide
pH 7.0, 35°C, recombinant enzyme
Paracoccus sp. M1-1
3.5.1.13
18
-
N-(3,4-dichlorophenyl)propanamide
pH 7.0, 35°C
Paracoccus sp. M1-1