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Literature summary for 3.5.1.13 extracted from

  • Shen, W.; Chen, H.; Jia, K.; Ni, J.; Yan, X.; Li, S.
    Cloning and characterization of a novel amidase from Paracoccus sp. M-1, showing aryl acylamidase and acyl transferase activities (2012), Appl. Microbiol. Biotechnol., 94, 1007-1018.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
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 Paracoccus sp. M1-1
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

Protein Variants

Protein Variants Comment Organism
K84A no activity, part of the catalytic triad Paracoccus sp. M1-1
K84A site-directed mutagenesis, the mutant shows no detectable hydrolysis activity Paracoccus sp. M1-1
S159A no activity, part of the catalytic triad Paracoccus sp. M1-1
S159A site-directed mutagenesis, the mutant shows no detectable hydrolysis activity Paracoccus sp. M1-1
S183A no activity, part of the catalytic triad Paracoccus sp. M1-1
S183A site-directed mutagenesis, the mutant shows no detectable hydrolysis activity Paracoccus sp. M1-1

Inhibitors

Inhibitors Comment Organism Structure
1,10-phenanthroline 20-30% inhibition at 1 mM Paracoccus sp. M1-1
2-mercaptoethanol 52% inhibition at 1 mM Paracoccus sp. M1-1
4-chloromercuribenzoate
-
Paracoccus sp. M1-1
4-chloromercurybenzoate
-
Paracoccus sp. M1-1
Co2+ 1 mM: 8.5% activity; strong inhibition Paracoccus sp. M1-1
Cr2+
-
Paracoccus sp. M1-1
Cu2+ 1 mM: 8.9% activity; strong inhibition Paracoccus sp. M1-1
EDTA 20-30% inhibition at 1 mM Paracoccus sp. M1-1
Fe2+
-
Paracoccus sp. M1-1
Hg2+ 1 mM: 0% activity; complete inhibition Paracoccus sp. M1-1
iodoacetamide
-
Paracoccus sp. M1-1
Ni2+
-
Paracoccus sp. M1-1
PMSF 10 mM: 9.8% activity; strong inhibition Paracoccus sp. M1-1
SDS
-
Paracoccus sp. M1-1
Triton X-100
-
Paracoccus sp. M1-1
Tween-80
-
Paracoccus sp. M1-1
Zn2+
-
Paracoccus sp. M1-1

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.158
-
N-(3,4-dichlorophenyl)propanamide pH 7.0, 35°C Paracoccus sp. M1-1
0.158
-
3',4'-dichloropropionanilide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1
0.158
-
N-(3,4-dichlorophenyl) propanamide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ activates at 1 mM Paracoccus sp. M1-1
Mg2+ activates 1.1fold at 1 mM Paracoccus sp. M1-1
Mg2+ activates 1.3fold at 1 mM Paracoccus sp. M1-1
Mn2+ activates 1.1fold at 1 mM Paracoccus sp. M1-1
Mn2+ activates 1.3fold at 1 mM Paracoccus sp. M1-1

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
52000
-
x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation Paracoccus sp. M1-1
52000
-
x * 52000, about, sequence calculation Paracoccus sp. M1-1
52390
-
x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation Paracoccus sp. M1-1

Organism

Organism UniProt Comment Textmining
Paracoccus sp. M1-1 F6N111
-
-
Paracoccus sp. M1-1 F6N111 gene pamH
-

Purification (Commentary)

Purification (Comment) Organism
ammonium sulfate fractionation, ion exchange chromatography (DEAE-Sephadex, DEAE-Cellulose, gel filtration) Paracoccus sp. M1-1
recombinant His6-tagged PamH 5.8fold from Escherichia coli strain BL21 (DE3) by ammonium sulfate fractionation, anion exchange chromatography, and gel filtration Paracoccus sp. M1-1
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]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
39.8
-
N-(3,4-dichlorophenyl)propanamide, pH 7.0, 35°C Paracoccus sp. M1-1
39.8
-
purified recombinant His6-tagged PamH, pH 7.0, 35°C Paracoccus sp. M1-1

Storage Stability

Storage Stability Organism
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)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3',4'-dichloropropionanilide + H2O i.e. propanil Paracoccus sp. M1-1 3,4-dichloroaniline + propionate
-
?
benzamide + H2O
-
Paracoccus sp. M1-1 benzoate + NH3
-
?
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 Paracoccus sp. M1-1 ?
-
?
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. M1-1 ?
-
?
N-(3,4-dichlorophenyl) propanamide + H2O i.e. propanil Paracoccus sp. M1-1 3,4-dichloroaniline + propanoic acid
-
?
N-(3,4-dichlorophenyl)propanamide + H2O no activity with butachlor, acetochlor, 4-nitroacetanilide, p-chloracetanilide Paracoccus sp. M1-1 3,4-dichloraniline + propanoate
-
?
propanamide + H2O
-
Paracoccus sp. M1-1 propanoic acid + NH3
-
?

Subunits

Subunits Comment Organism
? x * 52000, recombinant enzyme, SDS-PAGE, x * 52390, sequence calculation Paracoccus sp. M1-1
? x * 52000, about, sequence calculation Paracoccus sp. M1-1

Synonyms

Synonyms Comment Organism
aryl acylamidase
-
Paracoccus sp. M1-1
aryl acylamidase broad substrate spectrum Paracoccus sp. M1-1
M-1 amidase
-
Paracoccus sp. M1-1
PamH
-
Paracoccus sp. M1-1

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
-
Paracoccus sp. M1-1
45
-
recombinant enzyme Paracoccus sp. M1-1

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
15 70 activity range, profile overview Paracoccus sp. M1-1

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
40
-
fairly stable up to 40°C, completely inactivated during 30 min at 60°C Paracoccus sp. M1-1
40
-
purified recombinant enzyme, fairly stable up to, over 90% activity remaining after 1 h Paracoccus sp. M1-1
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
50
-
25% residual activity after 1 h at 50°C, completely inactivated during 30 min at 60°C Paracoccus sp. M1-1
50
-
purified recombinant enzyme, 25% activity remaining after 1 h Paracoccus sp. M1-1
60
-
purified recombinant enzyme, 30 min, complete inactivation Paracoccus sp. M1-1

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.8
-
3',4'-dichloropropionanilide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1
2.8
-
N-(3,4-dichlorophenyl) propanamide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1
2.8
-
N-(3,4-dichlorophenyl)propanamide pH 7.0, 35°C Paracoccus sp. M1-1

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
8
-
-
Paracoccus sp. M1-1
8
-
recombinant enzyme Paracoccus sp. M1-1

pH Range

pH Minimum pH Maximum Comment Organism
5.5 10 activity range, profile overview Paracoccus sp. M1-1
6 9 activity range Paracoccus sp. M1-1
6.5 9 highly active Paracoccus sp. M1-1

pH Stability

pH Stability pH Stability Maximum Comment Organism
4
-
purified recombinant enzyme, 30% activity remaining after 1 h Paracoccus sp. M1-1
4.5
-
purified recombinant enzyme, 60% activity remaining after 1 h Paracoccus sp. M1-1
4.5 10 purified recombinant enzyme, over 50% activity within this range at 35°C Paracoccus sp. M1-1
5 10 more than 70% activity after preincubation for 1 h Paracoccus sp. M1-1
5 10 purified recombinant enzyme, over 70% activity remaining after 1 h Paracoccus sp. M1-1

pI Value

Organism Comment pI Value Maximum pI Value
Paracoccus sp. M1-1 isoelectric focusing
-
5.13
Paracoccus sp. M1-1 isoelectric focusing, recombinant enzyme
-
5.13

General Information

General Information Comment Organism
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 Paracoccus sp. M1-1
evolution the enzyme belongs to the amidase signature enzyme family Paracoccus sp. M1-1
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]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
17.72
-
3',4'-dichloropropionanilide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1
17.72
-
N-(3,4-dichlorophenyl) propanamide pH 7.0, 35°C, recombinant enzyme Paracoccus sp. M1-1
18
-
N-(3,4-dichlorophenyl)propanamide pH 7.0, 35°C Paracoccus sp. M1-1