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Literature summary for 2.4.1.87 extracted from

  • Pasek, M.; Ramakrishnan, B.; Boeggeman, E.; Manzoni, M.; Waybright, T.; Qasba, P.
    Bioconjugation and detection of lactosamine moiety using αl, 3-galactosyltransferase mutants that transfer C2-modified galactose with a chemical handle (2009), Bioconjug. Chem., 20, 608-618.
    View publication on PubMedView publication on EuropePMC
Search for more literature for 2.4.1.87

Application

Application Comment Organism
diagnostics identification of N-acetyllactosamine moiety of glycoproteins and glycolipids by chemiluminescence with mutated enzymes Bos taurus

Cloned(Commentary)

Cloned (Comment) Organism
PCR-amplification, expression in Escherichia coli BL21(DE3) LysS Bos taurus

Protein Variants

Protein Variants Comment Organism
H280A/A281A/A282A mutant AAA, 20 or 200 mM lactose as acceptor, 14 and 41% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
H280A/A281G/A282G mutant AGG, 20 or 200 mM lactose as acceptor, 0.4 and 1% galactosyltransferase activity (UDP-galactose as donor), 0.3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest N-acetylgalactosamnine transferase activity and ability to transfer C2-modified sugars Bos taurus
H280G/A281A/A282A protein unstable, precipitating during purification Bos taurus
H280G/A281A/A282G mutant GAG, 20 or 200 mM lactose as acceptor, 5 and 27% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
H280L/A281G/A282G mutant LGG, 20 or 200 mM lactose as acceptor, 2 and 1% galactosyltransferase activity (UDP-galactose as donor), 3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
H280S/A281G/A282G mutant SGG, 20 or 200 mM lactose as acceptor, 5 and 5% galactosyltransferase activity (UDP-galactose as donor), 2 and 11% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0, among the mutants with highest GalNac transferase activity and ability to transfer C2-modified sugars Bos taurus
H280T/A281G/A282G mutant TGG, 20 or 200 mM lactose as acceptor, 3 and 15% galactosyltransferase activity (UDP-galactose as donor), 1 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
H280V/A281G/A282G mutant VGG, 20 or 200 mM lactose as acceptor, 4 and 16% galactosyltransferase activity (UDP-galactose as donor), 0.5 and 3% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information mutated enzymes transfer N-acetylgalactosamine or C2-modified galactose from their UDP derivatives, mutations on the sugar-donor-binding residues (histidine 280, alanine 281, alanine 282) to establish changed specificity, 5-19% of original galactose transferase activity remains, , acceptor affinity is affected by mutation of histidine 280, additional mutation of S347A in all mutants does not affect the enzyme activity and kinetic parameter by itself but resembles the human blood group A and B glycosyltransferases more closely Bos taurus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.004
-
 UDP-galactose mutant AGG kia, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.014
-
 UDP-galactose mutant SGG kia, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.042
-
 UDP-galactose mutant AGG ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.068
-
 UDP-N-acetylgalactosamine mutant AGG kia, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.07
-
 UDP-galactose mutant SGG ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.101
-
 UDP-N-acetylgalactosamine mutant SGG kia, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.13
-
 UDP-galactose wild-type kia, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.5
-
 UDP-N-acetylgalactosamine mutant AGG ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.74
-
 UDP-N-acetylgalactosamine mutant SGG ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.74
-
 UDP-galactose wild-type kib, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.79
-
 UDP-galactose wild-type ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
4.5
-
 lactose wild-type kb, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
25.7
-
 UDP-galactose mutant AGG kib, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
36
-
 UDP-galactose mutant SGG kib, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
36.1
-
 UDP-N-acetylgalactosamine mutant AGG kib, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
45.8
-
 UDP-N-acetylgalactosamine mutant SGG kib, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
180
-
 lactose mutant SGG kb, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
258
-
 lactose mutant AGG kb, UDP-N-acetylgalactosamine as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
270
-
 UDP-galactose mutant AGG kb, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
330
-
 lactose mutant SGG kb, UDP-N-acetylgalactosamine as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus

Metals/Ions

Metals/Ions Comment Organism Structure
MnCl2
-
 Bos taurus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
UDP-galactose + lactose Bos taurus wild-type enzyme, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 UDP + ?
-
 ?

Organism

Organism UniProt Comment Textmining
Bos taurus P14769
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 mutant AAA, 20 or 200 mM lactose as acceptor, 14 and 41% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant AGG, 20 or 200 mM lactose as acceptor, 0.4 and 1% galactosyltransferase activity (UDP-galactose as donor), 0.3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant GAG, 20 or 200 mM lactose as acceptor, 5 and 27% galactosyltransferase activity (UDP-galactose as donor), 4 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant LGG, 20 or 200 mM lactose as acceptor, 2 and 1% galactosyltransferase activity (UDP-galactose as donor), 3 and 2% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant SGG, 20 or 200 mM lactose as acceptor, 5 and 5% galactosyltransferase activity (UDP-galactose as donor), 2 and 11% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant TGG, 20 or 200 mM lactose as acceptor, 3 and 15% galactosyltransferase activity (UDP-galactose as donor), 1 and 6% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
additional information
-
 mutant VGG, 20 or 200 mM lactose as acceptor, 4 and 16% galactosyltransferase activity (UDP-galactose as donor), 0.5 and 3% N-acetylgalactosamine transferase activity (UDP-N-acetylgalactosamine as donor), 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
1.1
-
 wild-type, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
UDP-2-acetonyl-2-deoxy-galactose + asialofetuin mutant enzymes SGG and AGG, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?
UDP-2-acetonyl-2-deoxy-galactose + N-acetyllactosaminyl-chitotriose mutant enzymes SGG, AGG, and TGG, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?
UDP-galactose + lactose wild-type enzyme, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?
UDP-galactose + lactose wild-type enzyme, and mutant enzymes AAA, GAG, AGG, SGG, TGG, LGG, VGG, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?
UDP-N-acetyl-azido-galactosamine + N-acetyllactosaminyl-chitotriose mutant enzymes SGG, AGG, and TGG, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?
UDP-N-acetylgalactosamine + lactose mutant enzymes AAA, GAG, AGG, SGG, TGG, LGG, VGG, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus UDP + ?
-
 ?

Synonyms

Synonyms Comment Organism
alpha1,3-galactosyltransferase
-
 Bos taurus
alpha3Gal-T
-
 Bos taurus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.02
-
 UDP-galactose mutant AGG, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.1
-
 UDP-galactose mutant SGG, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.3
-
 UDP-N-acetylgalactosamine mutant AGG, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.4
-
 UDP-N-acetylgalactosamine mutant SGG, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
3.4
-
 UDP-galactose wild-type, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.000074
-
 lactose mutant AGG kcat/kb, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.00055
-
 lactose mutant SGG kcat/kb, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.0007
-
 lactose mutant SGG kcat/kb, UDP-N-acetylgalactosamine as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.0011
-
 lactose mutant AGG kcat/kb, UDP-N-acetylgalactosamine as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.5
-
 UDP-galactose mutant AGG kcat/ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.5
-
 UDP-N-acetylgalactosamine mutant SGG kcat/ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.6
-
 UDP-N-acetylgalactosamine mutant AGG kcat/ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
0.75
-
 lactose wild-type kcat/kb, UDP-galactose as donor, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
1.4
-
 UDP-galactose mutant SGG kcat/ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus
4.3
-
 UDP-galactose wild-type kcat/ka, lactose as acceptor, 37°C, MnCl2, 25 mM Tris-HCl, pH 7.0 Bos taurus