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Literature summary for 2.7.7.23 extracted from

  • Honda, Y.; Zang, Q.; Shimizu, Y.; Dadashipour, M.; Zhang, Z.; Kawarabayasi, Y.
    Increasing the thermostable sugar-1-phosphate nucleotidylyltransferase activities of the archaeal ST0452 protein through site saturation mutagenesis of the 97th amino acid position (2017), Appl. Environ. Microbiol., 83, e02291-16 .
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.7.7.23

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Escherichia coli
expression in Escherichia coli Sulfurisphaera tokodaii
gene ST0452, recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21-CodonPlus(DE3)-RIPL, subcloning in Escherichia coli strain JM109 Sulfurisphaera tokodaii

Protein Variants

Protein Variants Comment Organism
G9A site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
G9A/K147A activity is 71.8fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
G9A/K147A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
G9A/T80A activity is 35fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
G9A/T80A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
G9A/Y97A inactive mutant enzyme Sulfurisphaera tokodaii
G9A/Y97A site-directed mutagenesis, inactive mutant Sulfurisphaera tokodaii
G9A/Y97F activity is 14.5fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
G9A/Y97F site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
K147A site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
additional information ST0452 proteins exhibiting a further increase in activity are created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses show that the increased activities of the mutant proteins are principally due to increased apparent kcat values. Nine double-mutant ST0452 proteins are generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all show less than 15% of the wild-type N-acetyl-D-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibits the highest activity of the single-mutant proteins. Analysis of mutant ST0452 proteins generated using a site saturation mutagenesis strategy at the 97th position, overview Sulfurisphaera tokodaii
T80A site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
T80A/K147A activity is 11.1fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
T80A/K147A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
T80A/Y97A activity is 31.4fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
T80A/Y97A site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
T80A/Y97F activity is 25.1fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
T80A/Y97F site-directed mutagenesis, the mutant shows highly reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y103A activity is 25% higher than that of the wild-type enzyme Escherichia coli
Y103N activity is 30% higher than that of the wild-type enzyme Escherichia coli
Y103N activity is 30% lower than that of the wild-type enzyme Escherichia coli
Y97A activity is 1.83fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97A site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97A/K147A activity is 11.9fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97A/K147A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y97C activity is 1.4fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97C site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y97D activity is 1.3fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97D site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97E activity is 2.1fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97E site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y97F activity is 1.2fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97F site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97F/K147A activity is 5.4fold lower than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97F/K147A site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y97G activity is 1.2fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97G site-directed mutagenesis, the mutant exhibits slightly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97H activity is 2.9fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97H site-directed mutagenesis, the mutant exhibits highly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97I activity is 1.2fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97I site-directed mutagenesis, the mutant exhibits slightly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97K activity is 1.35fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97K site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97L activity is 1.5fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97L site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97M activity is 1.3fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97M site-directed mutagenesis, the mutant exhibits slightly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97N activity is 4.5fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97N site-directed mutagenesis, the mutant exhibits highly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97P activity is 1.5fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97P site-directed mutagenesis, the mutant shows reduced activity compared to wild-type enzyme Sulfurisphaera tokodaii
Y97Q activity is 2.3fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97Q site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97R inactive mutant enzyme Sulfurisphaera tokodaii
Y97R site-directed mutagenesis, inactive mutant Sulfurisphaera tokodaii
Y97S activity is 1.97fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97S site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97T activity is 2.1fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97T site-directed mutagenesis, the mutant exhibits increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97V activity is 3.6fold higher than that of the wild-type enzyme Sulfurisphaera tokodaii
Y97V site-directed mutagenesis, the mutant exhibits highly increased GlcNAc-1-P UTase activity compared to wild-type Sulfurisphaera tokodaii
Y97W inactive mutant enzyme Sulfurisphaera tokodaii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information kinetics of wild-type and mutant enzymes for Glc-1-P UTase activities, overview Sulfurisphaera tokodaii
0.068
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type enzyme, pH 7.5, 80°C Sulfurisphaera tokodaii
0.068
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type ST0452, pH 7.5, 80°C Sulfurisphaera tokodaii
0.097
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
0.097
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
0.147
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
0.147
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
0.861
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
0.861
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii

Metals/Ions

Metals/Ions Comment Organism Structure
Mg2+ required Sulfurisphaera tokodaii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate Sulfurisphaera tokodaii
-
 diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate Sulfurisphaera tokodaii DSM 16993
-
 diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?

Organism

Organism UniProt Comment Textmining
Escherichia coli P0ACC7
-
-
Escherichia coli K12 P0ACC7
-
-
Sulfurisphaera tokodaii Q975F9
-
-
Sulfurisphaera tokodaii Q975F9 multifunctional enzyme
-
Sulfurisphaera tokodaii DSM 16993 Q975F9
-
-

Purification (Commentary)

Purification (Comment) Organism
-
 Escherichia coli
-
 Sulfurisphaera tokodaii
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21-CodonPlus(DE3)-RIPL by nickel affinity chromatography and ultrafiltration Sulfurisphaera tokodaii

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 wild-type and mutant enzymes' Glc-1-P UTase activities, overview Sulfurisphaera tokodaii
0.21
-
 mutant enzyme G9A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.21
-
 mutant G9A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.43
-
 mutant enzyme G9A/T80A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.43
-
 mutant G9A/T80A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.48
-
 mutant enzyme T80A/Y97A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.48
-
 mutant T80A/Y97A, pH 7.5, 80°C Sulfurisphaera tokodaii
0.6
-
 mutant enzyme T80A/Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
0.6
-
 mutant T80A/Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
1.04
-
 mutant enzyme G9A/Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
1.04
-
 mutant G9A/Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
1.27
-
 mutant enzyme Y97A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
1.27
-
 mutant Y97A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
1.36
-
 mutant enzyme T80A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
1.36
-
 mutant T80A/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
2.78
-
 mutant enzyme Y97F/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
2.78
-
 mutant Y97F/K147A, pH 7.5, 80°C Sulfurisphaera tokodaii
7.04
-
 mutant enzyme Y97E, pH 7.5, 80°C Sulfurisphaera tokodaii
7.04
-
 mutant Y97E, pH 7.5, 80°C Sulfurisphaera tokodaii
10.3
-
 mutant enzyme Y97C, pH 7.5, 80°C Sulfurisphaera tokodaii
10.3
-
 mutant Y97C, pH 7.5, 80°C Sulfurisphaera tokodaii
10.31
-
 mutant enzyme Y97P, pH 7.5, 80°C Sulfurisphaera tokodaii
10.31
-
 mutant Y97P, pH 7.5, 80°C Sulfurisphaera tokodaii
15.08
-
 wild-type enzyme, pH 7.5, 80°C Sulfurisphaera tokodaii
18.12
-
 mutant enzyme Y97G, pH 7.5, 80°C Sulfurisphaera tokodaii
18.12
-
 mutant Y97G, pH 7.5, 80°C Sulfurisphaera tokodaii
18.4
-
 mutant enzyme Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
18.4
-
 mutant Y97F, pH 7.5, 80°C Sulfurisphaera tokodaii
18.68
-
 mutant enzyme Y97I, pH 7.5, 80°C Sulfurisphaera tokodaii
18.68
-
 mutant Y97I, pH 7.5, 80°C Sulfurisphaera tokodaii
19.51
-
 mutant enzyme Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
19.51
-
 mutant Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
20.37
-
 mutant enzyme Y97K, pH 7.5, 80°C Sulfurisphaera tokodaii
20.37
-
 mutant Y97K, pH 7.5, 80°C Sulfurisphaera tokodaii
21.06
-
 mutant enzyme Y97D, pH 7.5, 80°C Sulfurisphaera tokodaii
21.06
-
 mutant Y97D, pH 7.5, 80°C Sulfurisphaera tokodaii
22.03
-
 mutant enzyme Y97L, pH 7.5, 80°C Sulfurisphaera tokodaii
22.03
-
 mutant Y97L, pH 7.5, 80°C Sulfurisphaera tokodaii
27.52
-
 mutant enzyme Y97A, pH 7.5, 80°C Sulfurisphaera tokodaii
27.52
-
 mutant Y97A, pH 7.5, 80°C Sulfurisphaera tokodaii
29.77
-
 mutant enzyme Y97R, pH 7.5, 80°C Sulfurisphaera tokodaii
29.77
-
 mutant Y97S, pH 7.5, 80°C Sulfurisphaera tokodaii
31.27
-
 mutant enzyme Y97S, pH 7.5, 80°C Sulfurisphaera tokodaii
31.27
-
 mutant Y97T, pH 7.5, 80°C Sulfurisphaera tokodaii
34.6
-
 mutant enzyme Y97Q, pH 7.5, 80°C Sulfurisphaera tokodaii
34.6
-
 mutant Y97Q, pH 7.5, 80°C Sulfurisphaera tokodaii
43.7
-
 mutant enzyme Y97H, pH 7.5, 80°C Sulfurisphaera tokodaii
43.7
-
 mutant Y97H, pH 7.5, 80°C Sulfurisphaera tokodaii
52.56
-
 mutant enzyme Y103V, 37°C, pH 7.5 Escherichia coli
53.79
-
 mutant enzyme Y97T, pH 7.5, 80°C Sulfurisphaera tokodaii
53.79
-
 mutant enzyme Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
53.79
-
 mutant Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
67.11
-
 mutant enzyme Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
67.11
-
 mutant Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
75.63
-
 wild-type enzyme, 37°C, pH 7.5 Escherichia coli
93.97
-
 mutant enzyme Y103A, 37°C, pH 7.5 Escherichia coli
99.8
-
 mutant enzyme Y103N, 37°C, pH 7.5 Escherichia coli

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 Escherichia coli diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 Sulfurisphaera tokodaii diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 Sulfurisphaera tokodaii DSM 16993 diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?
UTP + N-acetyl-alpha-D-glucosamine 1-phosphate
-
 Escherichia coli K12 diphosphate + UDP-N-acetyl-alpha-D-glucosamine
-
 ?

Synonyms

Synonyms Comment Organism
amino-sugar-1-phosphate acetyltransferase
-
 Sulfurisphaera tokodaii
GlcNAc-1-P UTase
-
 Escherichia coli
GlcNAc-1-P UTase
-
 Sulfurisphaera tokodaii
GlmU
-
 Escherichia coli
ST0452
-
 Sulfurisphaera tokodaii
ST0452 protein
-
 Sulfurisphaera tokodaii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
80
-
 assay at Sulfurisphaera tokodaii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
9.97
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type enzyme, pH 7.5, 80°C Sulfurisphaera tokodaii
9.97
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type ST0452, pH 7.5, 80°C Sulfurisphaera tokodaii
10.9
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
10.9
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
48.95
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
48.95
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
49.75
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
49.75
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
 assay at Sulfurisphaera tokodaii

Cofactor

Cofactor Comment Organism Structure
UTP
-
 Sulfurisphaera tokodaii

General Information

General Information Comment Organism
evolution comparison of the activities of the ST0452 protein to those of similar enzymes from bacteria show that both the apparent Km and kcat values of the ST0452 GlcNAc-1-P UTase activity are smaller than those of Escherichia coli GlmU (EcGlmU) enzymes indicating that the archaeal ST0452 protein can accept a low concentration of substrate but that its turnover rate is lower than that of the EcGlmU enzyme Sulfurisphaera tokodaii
additional information Tyr at the 97th position of the ST0452 protein plays an important role in catalysis. Reaction scheme of the sugar-1-P UTase, overview Sulfurisphaera tokodaii
physiological function the ST0452 protein, a thermostable protein isolated from the thermophilic archaeon Sulfolobus tokodaii, is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-PNTase) and amino-sugar-1-phosphate acetyltransferase activities. ST0452 protein exhibits increased activity following single amino acid substitutions of Ala Sulfurisphaera tokodaii

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
56.9
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
56.9
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97V, pH 7.5, 80°C Sulfurisphaera tokodaii
112.4
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
112.4
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97M, pH 7.5, 80°C Sulfurisphaera tokodaii
146.6
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type enzyme, pH 7.5, 80°C Sulfurisphaera tokodaii
146.6
-
 N-acetyl-alpha-D-glucosamine 1-phosphate wild-type ST0452, pH 7.5, 80°C Sulfurisphaera tokodaii
338.4
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant enzyme Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii
338.4
-
 N-acetyl-alpha-D-glucosamine 1-phosphate mutant Y97N, pH 7.5, 80°C Sulfurisphaera tokodaii