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Literature summary for 3.5.1.46 extracted from

  • Negoro, S.; Kawashima, Y.; Shibata, N.; Kobayashi, T.; Baba, T.; Lee, Y.H.; Kamiya, K.; Shigeta, Y.; Nagai, K.; Takehara, I.; Kato, D.; Takeo, M.; Higuchi, Y.
    Mutations affecting the internal equilibrium of the reaction catalyzed by 6-aminohexanoate-dimer hydrolase (2016), FEBS Lett., 590, 3133-3143 .
    View publication on PubMed
Search for more literature for 3.5.1.46

Crystallization (Commentary)

Crystallization (Comment) Organism
purified wild-type and mutant (Hyb-24DNY-G187A112, Hyb-24DNY-S187A112, or Hyb-24DNY-A187A112) enzymes free or in complexes with 6-aminohexanoate, X-ray diffraction structure determination and analysis Paenarthrobacter ureafaciens

Protein Variants

Protein Variants Comment Organism
A61V/A124V/R187S/F264C/G291R/G338A/D370Y site-directed mutagenesis Paenarthrobacter ureafaciens
G181D/H266N construction of NylB mutant enzyme Hyb-24DN, the activity in Hyb-24 is enhanced 150fold by the two substitutions, where Asp181-COO- stabilizes the substrate binding by electrostatic interactions with Ald-NH3+, and Asn266 cooperatively improves the electrostatic environment with Asp181 Paenarthrobacter ureafaciens
additional information construction of chimeric NylB/NylB' mutants Hyb-24DN and Hyb-24DNY. A NylB/NylB' hybrid enzyme Hyb-24 (NylB' containing T3A-P4R-T5S-S8Q-D15G substitutions) has about 0.5% of the NylB level of Ald-hydrolytic activity. Surface structure of the entrance of the catalytic cleft of the Hyb-24DNY and its mutant enzymes, wild-type and mutant kinetics, detailed overview Paenarthrobacter ureafaciens
R187A site-directed mutagenesis, the substitution in the parental Hyb-24DNY decreases the specific activity (initial reaction rate) of synthesis to 37% of the level of Hyb-24DNY Paenarthrobacter ureafaciens
R187G site-directed mutagenesis, the substitution in the parental Hyb-24DNY decreases the specific activity (initial reaction rate) of synthesis to 15% of the level of Hyb-24DNY Paenarthrobacter ureafaciens
R187S site-directed mutagenesis, the substitution in the parental Hyb-24DNY decreases the specific activity (initial reaction rate) of synthesis to 24% of the level of Hyb-24DNY Paenarthrobacter ureafaciens
R187S/F264C/D370Y site-directed mutagenesis, the substitutions also enhance the Ald-hydrolytic activity 80fold over the parental Hyb-24 enzyme. The directly evolved enzyme (Hyb-S4M94) possesses almost no Ald-synthetic activity, although it possesses high levels of the hydrolytic activity even in 90% t-butyl alcohol. In this mutant enzyme, the R187S/F264C substitutions stabilizes the binding at the N-terminal region of Ald, while D370Y substitution stabilizes the binding at the C-terminal region of Ald Paenarthrobacter ureafaciens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics, kinetic study of wild-type and mutant enzymes Paenarthrobacter ureafaciens
1.9
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187A, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
2.1
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant enzyme Hyb-24DNY, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
2.2
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187S, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
2.6
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187G, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
6-aminohexanoate dimer + H2O Paenarthrobacter ureafaciens
-
 2 6-aminohexanoate
-
 ?

Organism

Organism UniProt Comment Textmining
Paenarthrobacter ureafaciens P07061 i.e. Flavobacterium sp. K172
-

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
6-aminohexanoate dimer + H2O
-
 Paenarthrobacter ureafaciens 2 6-aminohexanoate
-
 ?
additional information enzyme 6-aminohexanoate-dimer hydrolase catalyzes amide synthesis. The yield of this reverse reaction in 90% t-butyl alcohol varies drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft are used. The shift of the internal equilibrium between the enzyme-substrate complex and enzyme-product complex by the water-excluding effect alters the rate of the forward and reverse reactions. The local hydrophobic environment potentially provides a reaction center suitable for efficient amide synthesis Paenarthrobacter ureafaciens ?
-
 ?

Synonyms

Synonyms Comment Organism
6-aminohexanoate-dimer hydrolase
-
 Paenarthrobacter ureafaciens
Hyb-24
-
 Paenarthrobacter ureafaciens
NylB
-
 Paenarthrobacter ureafaciens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Paenarthrobacter ureafaciens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.29
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187A, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
0.63
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187S, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
0.91
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187G, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
1.17
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant enzyme Hyb-24DNY, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.3
-
 assay at Paenarthrobacter ureafaciens

General Information

General Information Comment Organism
evolution 6-aminohexanoate-dimer hydrolase (NylB) and NylB' carboxylesterase (88% homologous to NylB) are members of the penicillin-recognizing family of serine-reactive hydrolases. The NylB and NylB' utilize Ser112-Lys115-Tyr215 as catalytic triad, and catalyze the hydrolytic reaction of the Ahx-linear dimer (Ald) Paenarthrobacter ureafaciens
malfunction the yield of this reverse reaction in 90% t-butyl alcohol vaies drastically when enzyme mutants with substitutions of several amino acids located at the entrance of the catalytic cleft are used Paenarthrobacter ureafaciens
additional information residues Ser112, Lys115, and Tyr215 form the catalytic triad of enzyme NylB Paenarthrobacter ureafaciens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.153
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187A, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
0.286
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187S, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
0.35
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant Hyb-24DNY mutant R187G, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens
0.557
-
 6-aminohexanoate dimer pH 7.3, 30°C, recombinant enzyme Hyb-24DNY, hydrolysis in 90% t-butyl alcohol Paenarthrobacter ureafaciens