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Literature summary for 2.4.1.87 extracted from

  • Tumbale, P.; Jamaluddin, H.; Thiyagarajan, N.; Acharya, K.R.; Brew, K.
    Screening a limited structure-based library identifies UDP-GalNAc-specific mutants of alpha-1,3-galactosyltransferase (2008), Glycobiology, 18, 1036-1043.
    View publication on PubMed

Application

Application Comment Organism
synthesis production of novel complex glycans with role in biological recognition processes with engineered enzyme with modified substrate specificity Bos taurus

Cloned(Commentary)

Cloned (Comment) Organism
PCR megaprimer method, expressed in Escherichia coli BL21(DE3) Bos taurus

Crystallization (Commentary)

Crystallization (Comment) Organism
AGGL-mutant in complex with UDP-N-acetylgalactosamine with 20 mM MES-NaOH buffer, pH 6, 10% glycerol, 10 mM MnCl2, 10 mM UDP-N-acetylgalactosamine and reservoir solution (10-15% PEG 6000, 0.1 M Tris-HCl, pH 8, 15-25% MPD), and RAAI-mutant with 0.1 M Tris-HCl, pH 8, 10-50% PEG 4000, 0.2 M sodium acetate, 10 mM MnCl2, 10 mM UDP-galactose with the same reservoir solution, vapor diffusion hanging drop method Bos taurus

Protein Variants

Protein Variants Comment Organism
H280A/A281G/A282G/L283I AGGI-mutant, instable, leucin 283 is important for enzyme stability Bos taurus
H280A/A281G/A282G/L283L AGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability Bos taurus
H280A/A281G/A282G/L283V AGGV-mutant, instable, leucin 283 is important for enzyme stability Bos taurus
H280G/A281G/A282G/L283I GGGI-mutant, instable, leucin 283 is important for enzyme stability Bos taurus
H280G/A281G/A282G/L283L GGGL-mutant, higher N-acetylgalactosamine transferase activity than galactosyltransferase activity, leucin 283 is important for enzyme stability Bos taurus
H280G/A281G/A282G/L283V GGGV-mutant, instable, leucin 283 is important for enzyme stability Bos taurus
H280R/A281A/A282A/L283I RAAI-mutant, no catalytic activity, crystallisation Bos taurus
additional information mutants with modified donor substrate specificity Bos taurus

General Stability

General Stability Organism
mutants are not stable under assay conditions (10 mM Tris-HCl, pH 7.4, 37°C), but for 20 min with 10 mM MES, pH 6.0, at 30°C Bos taurus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.04
-
UDP-galactose AGGL mutant, lactose, pH 6, 30°C Bos taurus
0.12
-
UDP-galactose wild-type, N-acetyllactosamine, pH 6, 30°C Bos taurus
0.15
-
UDP-galactose AGGL mutant, N-acetyllactosamine, pH 6, 30°C Bos taurus
0.16
-
UDP-galactose wild-type, lactose, pH 6, 30°C Bos taurus
0.25
-
UDP-N-acetylgalactosamine AGGL mutant, lactose, pH 6, 30°C Bos taurus
0.6
-
N-acetyllactosamine wild-type, UDP-galactose, pH 6, 30°C Bos taurus
0.64
-
UDP-N-acetylgalactosamine AGGL mutant, N-acetyllactosamine, pH 6, 30°C Bos taurus
13
-
N-acetyllactosamine AGGL mutant, UDP-galactose, pH 6, 30°C Bos taurus
15
-
N-acetyllactosamine AGGL mutant, UDP-N-acetylgalactosamine, pH 6, 30°C Bos taurus
30
-
lactose wild-type, UDP-galactose, pH 6, 30°C Bos taurus
98
-
lactose AGGL mutant, UDP-N-acetylgalactosamine, pH 6, 30°C Bos taurus
100
-
lactose AGGL mutant, UDP-galactose, pH 6, 30°C 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-alpha-D-galactose + N-acetyllactosamine Bos taurus
-
UDP + ?
-
?

Organism

Organism UniProt Comment Textmining
Bos taurus P14769
-
-

Purification (Commentary)

Purification (Comment) Organism
centrifugation of cell culture, pellet lysed with EasyLyse, loaded onto Ni-chelate column, washing and elution with 20 mM Tris-HCl, pH 7.9, containing NaCl and imidazole Bos taurus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
UDP-alpha-D-galactose + N-acetyllactosamine
-
Bos taurus UDP + ?
-
?
UDP-galactose + lactose
-
Bos taurus UDP + ?
-
?
UDP-galactose + N-acetyllactosamine
-
Bos taurus UDP + ?
-
?
UDP-N-acetylgalactosamine + lactose no reaction of wild-type, only of AGGL mutant Bos taurus UDP + ?
-
?
UDP-N-acetylgalactosamine + N-acetyllactosamine no reaction of wild-type, only of AGGL mutant Bos taurus UDP + ?
-
?

Synonyms

Synonyms Comment Organism
alpha-1,3-galactosyltransferase
-
Bos taurus
alpha3GT
-
Bos taurus
UDP-galactose beta-galactosyl alpha-1,3-galactosyltransferase
-
Bos taurus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.0067
-
UDP-galactose AGGL mutant, N-acetyllactosamine, pH 6, 30°C Bos taurus
0.0099
-
UDP-galactose AGGL mutant, lactose, pH 6, 30°C Bos taurus
0.045
-
UDP-N-acetylgalactosamine AGGL mutant, N-acetyllactosamine, pH 6, 30°C Bos taurus
0.095
-
UDP-N-acetylgalactosamine AGGL mutant, lactose, pH 6, 30°C Bos taurus
1.91
-
UDP-galactose wild-type, N-acetyllactosamine, pH 6, 30°C Bos taurus
4.8
-
UDP-galactose wild-type, lactose, pH 6, 30°C Bos taurus