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Literature summary for 3.5.2.B2 extracted from

  • Gao, S.; Lu, Y.; Li, Y.; Huang, R.; Zheng, G.
    Enhancement in the catalytic activity of Sulfolobus solfataricus P2 (+)-?-lactamase by semi-rational design with the aid of a newly established high-throughput screening method (2019), Appl. Microbiol. Biotechnol., 103, 251-263 .
    View publication on PubMed
Search for more literature for 3.5.2.B2

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Rosetta (DE3) Saccharolobus solfataricus

Protein Variants

Protein Variants Comment Organism
I336R/Y388H enhanced enzyme activity Saccharolobus solfataricus
V203N/I336R enhanced enzyme activity Saccharolobus solfataricus
V203N/I336R/Y388H enhanced enzyme activity, 21fold higher enzyme efficiency (kcat/KM) compared to the wildtype enzyme. Biotransformation reaches more than 49% conversion and more than 99% enantiomeric excess at 80 °C after 2 h (with the same condition, the wild-type enzyme reached 12.3% conversion). Optimal temperature is 10°C lower than the optimum of the wild-type enzyme Saccharolobus solfataricus
V203N/Y388H enhanced enzyme activity Saccharolobus solfataricus

Inhibitors

Inhibitors Comment Organism Structure
Co2+ inhibition on the enzyme activity of both the wild-type and mutant proteins Saccharolobus solfataricus
Cu2+ inhibition on the enzyme activity of both the wild-type and mutant proteins Saccharolobus solfataricus
Zn2+ inhibition on the enzyme activity of both the wild-type and mutant proteins Saccharolobus solfataricus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
17.5
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H/I336R Saccharolobus solfataricus
22
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H Saccharolobus solfataricus
26.5
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/I336R Saccharolobus solfataricus
55.9
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme I336R Saccharolobus solfataricus
60.8
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme Y388H/I336R Saccharolobus solfataricus
61.3
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, wild-type enzyme Saccharolobus solfataricus

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ slightly enhances activity of mutant enzyme V203N/I336R Saccharolobus solfataricus
Mn2+ slightly enhances activity of mutant enzyme V203N/I336R Saccharolobus solfataricus
Sn2+ slightly enhances activity of mutant enzyme V203N/I336R Saccharolobus solfataricus

Organism

Organism UniProt Comment Textmining
Saccharolobus solfataricus P95896
-
-
Saccharolobus solfataricus ATCC 35092 P95896
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Saccharolobus solfataricus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
(+)-2-azabicyclo[2.2.1]hept-5-en-3-one + H2O
-
 Saccharolobus solfataricus + (+)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?
(+)-2-azabicyclo[2.2.1]hept-5-en-3-one + H2O
-
 Saccharolobus solfataricus ATCC 35092 + (+)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?
2-azabicyclo[2.2.1]hept-5-en-3-one + H2O
-
 Saccharolobus solfataricus (-)-2-azabicyclo[2.2.1]hept-5-en-3-one + (+)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?
2-azabicyclo[2.2.1]hept-5-en-3-one + H2O
-
 Saccharolobus solfataricus ATCC 35092 (-)-2-azabicyclo[2.2.1]hept-5-en-3-one + (+)-4-amino-cyclopent-2-enecarboxylic acid
-
 ?

Synonyms

Synonyms Comment Organism
Sslact
-
 Saccharolobus solfataricus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Saccharolobus solfataricus
80
-
 mutant enzyme V203N/Y388H/I336R Saccharolobus solfataricus
90
-
 wild-type enzyme and mutant enzymes I336R, I336H, V203N/I336R and Y388H/I366R Saccharolobus solfataricus

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
60 100 60°C: about 45% of maximal activity, 100°C: about 55% of maximal activity, mutant enzyme I336R Saccharolobus solfataricus
60 100 60°C: about 50% of maximal activity, 90°C: about 75% of maximal activity, 100°C: about 30% of maximal activity, mutant enzyme V203N/I336R/Y388H Saccharolobus solfataricus
80 100 80°C: about 80% of maximal activity, 100°C: about 75% of maximal activity Saccharolobus solfataricus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.41
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, wild-type enzyme Saccharolobus solfataricus
1.54
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H Saccharolobus solfataricus
4.76
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme I336R Saccharolobus solfataricus
7.69
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme Y388H/I336R Saccharolobus solfataricus
8.06
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/I336R Saccharolobus solfataricus
9.04
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H/I336R Saccharolobus solfataricus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.8
-
 assay at Saccharolobus solfataricus
8
-
 wild-type enzyme Saccharolobus solfataricus
9
-
 mutant enzyme V203N/I336R/Y388H Saccharolobus solfataricus

pH Range

pH Minimum pH Maximum Comment Organism
6 10 pH 6.0: about 70% of maximal activity, pH 10.0: about 45% of maximal activity, wild-type enzyme Saccharolobus solfataricus
6 10 pH 6.0: about 90% of maximal activity, pH 10.0: about 80% of maximal activity, mutant enzyme V203N/I336R/Y388H Saccharolobus solfataricus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.023
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, wild-type enzyme Saccharolobus solfataricus
0.07
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H Saccharolobus solfataricus
0.085
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme I336R Saccharolobus solfataricus
0.126
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme Y388H/I336R Saccharolobus solfataricus
0.304
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/I336R Saccharolobus solfataricus
0.517
-
 (+)-2-azabicyclo[2.2.1]hept-5-en-3-one 37°C, pH 7.8, mutant enzyme V203N/Y388H/I336R Saccharolobus solfataricus