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Literature summary for 3.5.1.11 extracted from

  • Torres, L.L.; Cantero, A.; del Valle, M.; Marina, A.; Lopez-Gallego, F.; Guisan, J.M.; Berenguer, J.; Hidalgo, A.
    Engineering the substrate specificity of a thermophilic penicillin acylase from Thermus thermophilus (2013), Appl. Environ. Microbiol., 79, 1555-1562.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene pac, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Thermus thermophilus

Protein Variants

Protein Variants Comment Organism
I57F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, Km for penicillin G is 4.5fold, while the catalytic efficiency remains at wild-type value Thermus thermophilus
L188F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 10fold improved compared to the wild-type enzyme Thermus thermophilus
L188R site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G Thermus thermophilus
L188R/S189F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G Thermus thermophilus
L24F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 12fold improved compared to the wild-type enzyme Thermus thermophilus
L24F/I57F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G Thermus thermophilus
L24F/I57F/L188F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G Thermus thermophilus
L24F/I57F/L188R/S189F site-directed mutagenesis, inactive mutant Thermus thermophilus
additional information substrate specificity change through engineering: generation of a set of enzyme variants containing between one and four amino acid replacements, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position alpha188, alpha189, or beta24 improves the Km for penicillin G between 9 and 12fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6fold Thermus thermophilus
S189F site-directed mutagenesis, the substrate specificity of the mutant shifts from penicillin K to penicillin G, penicillin G binding is 9fold improved compared to the wild-type enzyme Thermus thermophilus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics, overview Thermus thermophilus
0.05
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
0.1
-
penicillin K pH 5.5, 65°C, recombinant mutants L188F and S189F Thermus thermophilus
0.12
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
0.15
-
penicillin K pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
0.27
-
penicillin K pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus
0.32
-
penicillin K pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus
13.4
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
15.5
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F Thermus thermophilus
17.8
-
penicillin G pH 5.5, 65°C, recombinant mutant S189F Thermus thermophilus
36.5
-
penicillin G pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus
37.6
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
43.3
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/S189F Thermus thermophilus
44.4
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
164
-
penicillin G pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
penicillin G + H2O Thermus thermophilus
-
phenylacetate + 6-aminopenicillanate
-
?
penicillin K + H2O Thermus thermophilus
-
octanoic acid + 6-aminopenicillanate
-
?

Organism

Organism UniProt Comment Textmining
Thermus thermophilus Q72G76 gene pac
-

Purification (Commentary)

Purification (Comment) Organism
recombinant enzyme from Escherichia coli strain BL21(DE3) by heat shock treatment of 20 min at 65°C, and immobilized metal ion affinity chromatography Thermus thermophilus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information the acylase shows a marked preference for penicillin K over penicillin G, substrate specificity, overview Thermus thermophilus ?
-
?
penicillin G + H2O
-
Thermus thermophilus phenylacetate + 6-aminopenicillanate
-
?
penicillin K + H2O
-
Thermus thermophilus octanoic acid + 6-aminopenicillanate
-
?
penicillin K + H2O preferred substrate for the wild-type enzyme Thermus thermophilus octanoic acid + 6-aminopenicillanate
-
?

Synonyms

Synonyms Comment Organism
penicillin acylase
-
Thermus thermophilus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
65
-
assay at Thermus thermophilus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/S189F Thermus thermophilus
0.13
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
0.3
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
0.32
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
0.36
-
penicillin K pH 5.5, 65°C, recombinant mutant S189F Thermus thermophilus
0.43
-
penicillin K pH 5.5, 65°C, recombinant mutant L188F Thermus thermophilus
0.44
-
penicillin G pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus
0.54
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F Thermus thermophilus
0.65
-
penicillin G pH 5.5, 65°C, recombinant mutant S189F Thermus thermophilus
0.75
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
0.85
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
1.43
-
penicillin K pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
1.56
-
penicillin G pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus
4.91
-
penicillin K pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus
5.6
-
penicillin K pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
5.5
-
assay at Thermus thermophilus

General Information

General Information Comment Organism
additional information three-dimensional modeling with identification of the catalytic residues and the substrate binding pocket, overview. The substrate binding and active site orientation is predited by docking and molecular homology structure modeling Thermus thermophilus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0005
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/S189F Thermus thermophilus
0.003
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
0.008
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
0.009
-
penicillin G pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus
0.012
-
penicillin G pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus
0.034
-
penicillin G pH 5.5, 65°C, recombinant mutant L188F Thermus thermophilus
0.036
-
penicillin G pH 5.5, 65°C, recombinant mutant S189F Thermus thermophilus
0.063
-
penicillin G pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
3.6
-
penicillin K pH 5.5, 65°C, recombinant mutant S189F Thermus thermophilus
4.2
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F Thermus thermophilus
4.3
-
penicillin K pH 5.5, 65°C, recombinant mutant L188F Thermus thermophilus
9.5
-
penicillin K pH 5.5, 65°C, recombinant mutant L188F/L24F/I57F Thermus thermophilus
15
-
penicillin K pH 5.5, 65°C, recombinant mutant L24F/I57F Thermus thermophilus
16.1
-
penicillin K pH 5.5, 65°C, recombinant wild-type enzyme Thermus thermophilus
18.2
-
penicillin K pH 5.5, 65°C, recombinant mutant I57F Thermus thermophilus