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Literature summary extracted from

  • Komeda, H.; Harada, H.; Washika, S.; Sakamoto, T.; Ueda, M.; Asano, Y.
    S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase from Pseudomonas azotoformans IAM 1603 is a novel L-amino acid amidase (2004), Eur. J. Biochem., 271, 1465-1475.
    View publication on PubMed

Application

EC Number Application Comment Organism
3.5.1.101 synthesis Escherichia coli cells overexpressing the laaA gene have been demonstrated to be applicable to the S-stereoselective hydrolysis of (R,S)-piperazine-2-tert-butylcarboxamide to produce (S)-piperazine-2-carboxylic acid with high optical purity. 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 azotoformans

Cloned(Commentary)

EC Number Cloned (Comment) Organism
3.5.1.101 expression in Escherichia coli Pseudomonas azotoformans

Inhibitors

EC Number Inhibitors Comment Organism Structure
3.5.1.101 Ag+ 1 mM, complete inhibition Pseudomonas azotoformans
3.5.1.101 Cd2+ 1 mM, complete inhibition Pseudomonas azotoformans
3.5.1.101 CoCl2 1 mM, 52% inhibition Pseudomonas azotoformans
3.5.1.101 Hg2+ 1 mM, complete inhibition Pseudomonas azotoformans
3.5.1.101 iodoacetate 1 mM, 40% inhibition Pseudomonas azotoformans
3.5.1.101 NEM 1 mM, 24% inhibition Pseudomonas azotoformans
3.5.1.101 NiCl2 1 mM, 70% inhibition Pseudomonas azotoformans
3.5.1.101 p-chloromercuribenzoate 1 mM, 67% inhibition Pseudomonas azotoformans
3.5.1.101 PbCl2 1 mM, 73% inhibition Pseudomonas azotoformans
3.5.1.101 phenylhydrazine 1 mM, complete inhibition Pseudomonas azotoformans
3.5.1.101 Zn2+ 1 mM, complete inhibition Pseudomonas azotoformans

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
3.5.1.101 0.58
-
 L-proline-p-nitroanilide 30°C, pH 7.0 Pseudomonas azotoformans

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
3.5.1.101 32000
-
 gel filtration Pseudomonas azotoformans
3.5.1.101 34000
-
 1 * 34000, SDS-PAGE Pseudomonas azotoformans

Organism

EC Number Organism UniProt Comment Textmining
3.5.1.101 Pseudomonas azotoformans Q76KX0 IAM 1603
-
3.5.1.101 Pseudomonas azotoformans IAM 1603 Q76KX0 IAM 1603
-

Purification (Commentary)

EC Number Purification (Comment) Organism
3.5.1.101
-
 Pseudomonas azotoformans

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
3.5.1.101 0.000894
-
 S-stereoselective amidase from Pseudomonas azotoformans IAM 1603 Pseudomonas azotoformans
3.5.1.101 192
-
 LaaA from Escharichia coli JM109 harboring pSTB20 Pseudomonas azotoformans

Storage Stability

EC Number Storage Stability Organism
3.5.1.101 -20°C, stored without loss of activity for more than six months in the buffer containing 50% glycerol Pseudomonas azotoformans

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
3.5.1.101 (R,S)-piperazine-2-tert-butylcarboxamide + H2O % of the activity with L-proline amide Pseudomonas azotoformans (S)-piperazine-2-tert-butylcarboxylic acid + NH3
-
 ?
3.5.1.101 (R,S)-piperazine-2-tert-butylcarboxamide + H2O % of the activity with L-proline amide Pseudomonas azotoformans IAM 1603 (S)-piperazine-2-tert-butylcarboxylic acid + NH3
-
 ?
3.5.1.101 (S)-piperazine-2-carboxamide + H2O 3.7% of the activity with L-proline amide Pseudomonas azotoformans (S)-piperazine-2-carboxylic acid + NH3
-
 ?
3.5.1.101 (S)-piperazine-2-carboxamide + H2O 3.7% of the activity with L-proline amide Pseudomonas azotoformans IAM 1603 (S)-piperazine-2-carboxylic acid + NH3
-
 ?
3.5.1.101 (S)-piperazine-2-tert-butylcarboxamide + H2O 0.2% of the activity with L-proline amide Pseudomonas azotoformans (S)-piperazine-2-carboxylic acid + tert-butylamine
-
 ?
3.5.1.101 (S)-piperidine-2-carboxamide + H2O 32% of the activity with L-proline amide Pseudomonas azotoformans (S)-piperidine-2-carboxylic acid + NH3
-
 ?
3.5.1.101 L-alanine amide + H2O 10.6% of the activity with L-proline amide Pseudomonas azotoformans L-alanine + NH3
-
 ?
3.5.1.101 L-isoleucine amide + H2O 0.17% of the activity with L-proline amide Pseudomonas azotoformans L-isoleucine + NH3
-
 ?
3.5.1.101 L-leucine amide + H2O 0.46% of the activity with L-proline amide Pseudomonas azotoformans L-leucine + NH3
-
 ?
3.5.1.101 L-leucine amide + H2O 0.46% of the activity with L-proline amide Pseudomonas azotoformans IAM 1603 L-leucine + NH3
-
 ?
3.5.1.101 L-methionine amide + H2O 4.2% of the activity with L-proline amide Pseudomonas azotoformans L-methionine + NH3
-
 ?
3.5.1.101 L-phenylalanine amide + H2O 0.97% of the activity with L-proline amide Pseudomonas azotoformans L-phenylalanine + NH3
-
 ?
3.5.1.101 L-phenylalanine amide + H2O 0.97% of the activity with L-proline amide Pseudomonas azotoformans IAM 1603 L-phenylalanine + NH3
-
 ?
3.5.1.101 L-proline amide + H2O
-
 Pseudomonas azotoformans L-proline + NH3
-
 ?
3.5.1.101 L-proline-p-nitroanilide + H2O 40.9% of the activity with L-proline amide Pseudomonas azotoformans ? + NH3
-
 ?
3.5.1.101 L-serine amide + H2O 0.43% of the activity with L-proline amide Pseudomonas azotoformans L-serine + NH3
-
 ?
3.5.1.101 L-threonine amide + H2O 0.12% of the activity with L-proline amide Pseudomonas azotoformans L-threonine + NH3
-
 ?
3.5.1.101 L-tryptophan amide + H2O 0.2% of the activity with L-proline amide Pseudomonas azotoformans L-tryptophan + NH3
-
 ?
3.5.1.101 L-tyrosine amide + H2O 0.086% of the activity with L-proline amide Pseudomonas azotoformans L-tyrosine + NH3
-
 ?
3.5.1.101 additional information the following compounds are not substrates for the amidase: L-arginine amide, L-asparagine amide, L-isoasparagine, L-glutaminamide, L-isoglutamine, glycine amide, L-histidine amide, L-lysine amide, L-valine amide, D-proline amide, L-alanyl-L-alanine, L-alanylglycine, glycylglycine, L-prolyl-L-alanine and L-prolylglycine. LaaA can not act on the peptide substrates such as L-prolyl-L-alanine, L-prolylglycine, L-alanyl-L-alanine, L-alanylglycine and glycylglycine Pseudomonas azotoformans ?
-
 ?
3.5.1.101 additional information the following compounds are not substrates for the amidase: L-arginine amide, L-asparagine amide, L-isoasparagine, L-glutaminamide, L-isoglutamine, glycine amide, L-histidine amide, L-lysine amide, L-valine amide, D-proline amide, L-alanyl-L-alanine, L-alanylglycine, glycylglycine, L-prolyl-L-alanine and L-prolylglycine. LaaA can not act on the peptide substrates such as L-prolyl-L-alanine, L-prolylglycine, L-alanyl-L-alanine, L-alanylglycine and glycylglycine Pseudomonas azotoformans IAM 1603 ?
-
 ?

Subunits

EC Number Subunits Comment Organism
3.5.1.101 monomer 1 * 34000, SDS-PAGE Pseudomonas azotoformans

Synonyms

EC Number Synonyms Comment Organism
3.5.1.101 L-amino acid amidase
-
 Pseudomonas azotoformans
3.5.1.101 LaaA
-
 Pseudomonas azotoformans
3.5.1.101 S-stereoselective piperazine-2-tert-butylcarboxamide hydrolase
-
 Pseudomonas azotoformans

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
3.5.1.101 45
-
-
 Pseudomonas azotoformans

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
3.5.1.101 45
-
 stable up to Pseudomonas azotoformans

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
3.5.1.101 9
-
-
 Pseudomonas azotoformans