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Literature summary for 3.5.1.100 extracted from

  • Komeda, H.; Harada, H.; Washika, S.; Sakamoto, T.; Ueda, M.; Asano, Y.
    A novel R-stereoselective amidase from Pseudomonas sp. MCI3434 acting on piperazine-2-tert-butylcarboxamide (2004), Eur. J. Biochem., 271, 1580-1590.
    View publication on PubMed
Search for more literature for 3.5.1.100

Application

Application Comment Organism
synthesis R-amidase is the first enzyme useful for the enzymatic optical resolution of racemic piperazine-2-tert-butylcarboxamide carried out under mild conditions. Enantiomerically pure piperazine-2-carboxylic acid and its tert-butylcarboxamide derivative are important chiral building blocks for some pharmacologically active compounds such as N-methyl-D-aspartate antagonist for glutamate receptor, cardioprotective nucleoside transport blocker, and HIV protease inhibitor Pseudomonas sp.

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Pseudomonas sp.

Inhibitors

Inhibitors Comment Organism Structure
AgNO3 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
CdCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
CoCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
CuCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
CuSO4 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
Fe(NH4)2(SO4)2 1 mM, incubation at 30°C for 10 min, 67% inhibition Pseudomonas sp.
FeCl3 1 mM, incubation at 30°C for 10 min, 78% inhibition Pseudomonas sp.
HgCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
MnCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
MnSO4 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
additional information chelating reagents, e.g. o-phenanthroline, 8-hydroxyquinoline, ethylenediaminetetraacetic acid and 2,2'-dipyridyl have no significant effect on the enzyme. Carbonyl reagents such as hydroxylamine, phenylhydrazine, hydrazine, DL-penicillamine and D-cycloserine are not inhibitory toward the enzyme Pseudomonas sp.
N-ethylmaleimide 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
NiCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
p-chloromercuribenzoate 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
PbCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
ZnCl2 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.
ZnSO4 1 mM, incubation at 30°C for 10 min, complete inhibition Pseudomonas sp.

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
29500
-
 1 * 29500, SDS-PAGE Pseudomonas sp.
30128
-
 1 * 30128, calculated from sequence Pseudomonas sp.
36000
-
 gel filtration Pseudomonas sp.

Organism

Organism UniProt Comment Textmining
Pseudomonas sp. Q75SP7
-
-
Pseudomonas sp. MCI3434 Q75SP7
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Pseudomonas sp.

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.0242
-
 R-amidase from Pseudomonas sp. MCI3434 Pseudomonas sp.
4.59
-
 RamA from Escherichia coli JM109 harboring pRTB1EX Pseudomonas sp.

Storage Stability

Storage Stability Organism
-20°C, stable for more than 2 months in the buffer containing 50% glycerol Pseudomonas sp.

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(R)-piperazine-2-carboxamide + H2O hydrolysis with strict R-stereoselectivity Pseudomonas sp. (R)-piperazine-2-carboxylic acid + NH3
-
 ?
(R)-piperazine-2-carboxamide + H2O hydrolysis with strict R-stereoselectivity Pseudomonas sp. MCI3434 (R)-piperazine-2-carboxylic acid + NH3
-
 ?
(R)-piperazine-2-tert-butylcarboxamide + H2O hydrolysis with strict R-stereoselectivity, 9% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. (R)-piperazine-2-carboxylate + tert-butylamine
-
 ?
(R)-piperazine-2-tert-butylcarboxamide + H2O hydrolysis with strict R-stereoselectivity, 9% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. MCI3434 (R)-piperazine-2-carboxylate + tert-butylamine
-
 ?
(R)-piperidine-3-carboxamide + H2O 68.9% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. (R)-piperidine-3-carboxylic acid + NH3
-
 ?
(R)-piperidine-3-carboxamide + H2O 68.9% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. MCI3434 (R)-piperidine-3-carboxylic acid + NH3
-
 ?
beta-alaninamide + H2O 108% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. beta-alanine + NH3
-
 ?
beta-alaninamide + H2O 108% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. MCI3434 beta-alanine + NH3
-
 ?
D-glutamine amide + H2O no formation of glutamine, 27% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. D-glutamic acid + NH3
-
 ?
L-glutamine amide + H2O no formation of glutamine, 0.35% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. L-glutamic acid + NH3
-
 ?
additional information RamA has hydrolyzing activity toward the carboxamide compounds, in which an amino or imino group is connected to beta- or gamma-carbon, such as beta-alanine amide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutamine amide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme does not act on the other amide substrates for the aliphatic amidase: D-alanine amide, D-valine amide, D-leucine amide, D-isoleucine amide, D-proline amide, D-phenylalanine amide, D-tryptophan amide, D-methionine amide, D-serine amide, D-threoninamide, D-tyrosine amide, D-aspartic acid amide, D-glutamic acid amide, D-lysine amide, D-arginine amide, D-histidine amide, L-alanine amide, L-valine amide, L-leucine amide, L-isoleucine amide, L-proline amide, L-phenylalanine amide, L-tryptophanamide, L-methioninamide, L-serine amide, L-threonine amide, L-tyrosine amide, L-asparagine amide, L-aspartic acid amide, L-glutamic acid amide, L-lysine amide, L-arginine amide, L-histidine amide, glycine amide and (R,S)-piperidine-2-carboxamide. Carboxamides of the side chains in D-asparagine, D-glutamine, L-asparagine and L-glutamine are not hydrolyzed by the enzyme. The enzyme does not show peptidase activity toward beta-alanyl-L-alanine, beta-alanylglycine, beta-alanyl-L-histidine, glycylglycine, glycylglycylglycine, L-alanylglycine, D-alanylglycine, D-alanylglycylglycine, DL-alanyl-DL-asparagine, DL-alanyl-DL-isoleucine, DL-alanyl-DL-leucine, DL-alanyl-DL-methionine, DL-alanyl-DL-phenylalanine, DL-alanyl-DL-serine, DL-alanyl-DL-valine and L-aspartyl-D-alanine. The enzyme could not hydrolyze the following aliphatic amides, aromatic amides and nitriles: acetamide, propionamide, n-butyramide, isobutyramide, n-valeramide, n-capronamide, crotonamide, methacrylamide, cyclohexanecarboxamide, benzamide, o-aminobenzamide, m-aminobenzamide, p-aminobenzamide, p-toluamide, p-chlorobenzamide, p-nitrobenzamide, 2-picolinamide, nicotinamide, pyridine-4-carboxamide, pyrazinamide,2-thiophenecarboxamide, phenylacetamide, indole-3-acetamide, acetonitrile, propionitrile, 3-hydroxypropionitrile, n-capronitrile, methacrylonitrile, crotononitrile, glutaronitrile, 2,4-dicyanobut-1-ene, beta-phenylpropionitrile, cinnamonitrile, 2-cyanopiperidine, 2-cyanopiperazine, phenylacetonitrile, 4-methoxyphenylacetonitrile, alpha-methylbenzyl cyanide, 2-pyridineacetonitrile, 3-pyridineacetonitrile, thiophene-2-acetonitrile, beta-indoleacetonitrile, diphenylacetonitrile, 4-chlorobenzyl cyanide, benzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, p-tolunitrile, anisonitrile, 2-cyanophenol, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, pyrazinecarbonitrile, 3-cyanoindole, a-naphthonitrile, 2-thiophenecarbonitrile, terephthalonitrile and isophthalonitrile Pseudomonas sp. ?
-
 ?
additional information RamA has hydrolyzing activity toward the carboxamide compounds, in which an amino or imino group is connected to beta- or gamma-carbon, such as beta-alanine amide, (R)-piperazine-2-carboxamide (R)-piperidine-3-carboxamide, D-glutamine amide and (R)-piperazine-2-tert-butylcarboxamide. The enzyme does not act on the other amide substrates for the aliphatic amidase: D-alanine amide, D-valine amide, D-leucine amide, D-isoleucine amide, D-proline amide, D-phenylalanine amide, D-tryptophan amide, D-methionine amide, D-serine amide, D-threoninamide, D-tyrosine amide, D-aspartic acid amide, D-glutamic acid amide, D-lysine amide, D-arginine amide, D-histidine amide, L-alanine amide, L-valine amide, L-leucine amide, L-isoleucine amide, L-proline amide, L-phenylalanine amide, L-tryptophanamide, L-methioninamide, L-serine amide, L-threonine amide, L-tyrosine amide, L-asparagine amide, L-aspartic acid amide, L-glutamic acid amide, L-lysine amide, L-arginine amide, L-histidine amide, glycine amide and (R,S)-piperidine-2-carboxamide. Carboxamides of the side chains in D-asparagine, D-glutamine, L-asparagine and L-glutamine are not hydrolyzed by the enzyme. The enzyme does not show peptidase activity toward beta-alanyl-L-alanine, beta-alanylglycine, beta-alanyl-L-histidine, glycylglycine, glycylglycylglycine, L-alanylglycine, D-alanylglycine, D-alanylglycylglycine, DL-alanyl-DL-asparagine, DL-alanyl-DL-isoleucine, DL-alanyl-DL-leucine, DL-alanyl-DL-methionine, DL-alanyl-DL-phenylalanine, DL-alanyl-DL-serine, DL-alanyl-DL-valine and L-aspartyl-D-alanine. The enzyme could not hydrolyze the following aliphatic amides, aromatic amides and nitriles: acetamide, propionamide, n-butyramide, isobutyramide, n-valeramide, n-capronamide, crotonamide, methacrylamide, cyclohexanecarboxamide, benzamide, o-aminobenzamide, m-aminobenzamide, p-aminobenzamide, p-toluamide, p-chlorobenzamide, p-nitrobenzamide, 2-picolinamide, nicotinamide, pyridine-4-carboxamide, pyrazinamide,2-thiophenecarboxamide, phenylacetamide, indole-3-acetamide, acetonitrile, propionitrile, 3-hydroxypropionitrile, n-capronitrile, methacrylonitrile, crotononitrile, glutaronitrile, 2,4-dicyanobut-1-ene, beta-phenylpropionitrile, cinnamonitrile, 2-cyanopiperidine, 2-cyanopiperazine, phenylacetonitrile, 4-methoxyphenylacetonitrile, alpha-methylbenzyl cyanide, 2-pyridineacetonitrile, 3-pyridineacetonitrile, thiophene-2-acetonitrile, beta-indoleacetonitrile, diphenylacetonitrile, 4-chlorobenzyl cyanide, benzonitrile, 4-chlorobenzonitrile, 4-nitrobenzonitrile, p-tolunitrile, anisonitrile, 2-cyanophenol, 2-cyanopyridine, 3-cyanopyridine, 4-cyanopyridine, pyrazinecarbonitrile, 3-cyanoindole, a-naphthonitrile, 2-thiophenecarbonitrile, terephthalonitrile and isophthalonitrile Pseudomonas sp. MCI3434 ?
-
 ?
piperidine-4-carboxamide + H2O 0.23% of the activity with (R)-piperazine-2-carboxamide Pseudomonas sp. piperidine-4-carboxylic acid + NH3
-
 ?

Subunits

Subunits Comment Organism
monomer 1 * 29500, SDS-PAGE Pseudomonas sp.
monomer 1 * 30128, calculated from sequence Pseudomonas sp.

Synonyms

Synonyms Comment Organism
R-amidase
-
 Pseudomonas sp.
R-stereoselective amidase
-
 Pseudomonas sp.
RamA
-
 Pseudomonas sp.

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
45
-
-
 Pseudomonas sp.

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
35
-
 10 min, stable Pseudomonas sp.
40
-
 10 min, stable Pseudomonas sp.
45
-
 10 min, 13% loss of activity Pseudomonas sp.
50
-
 10 min, 97% loss of activity Pseudomonas sp.
55
-
 10 min, complete loss of activity Pseudomonas sp.

pH Stability

pH Stability pH Stability Maximum Comment Organism
6 9 30°C, 10 min, most stable in pH-range 6.0-9.0 Pseudomonas sp.