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Literature summary for 2.4.1.133 extracted from

  • Tsutsui, Y.; Ramakrishnan, B.; Qasba, P.K.
    Crystal structures of beta-1,4-galactosyltransferase 7 enzyme reveal conformational changes and substrate binding (2013), J. Biol. Chem., 288, 31963-31970.
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene B4GALT7, recombinant expression of N-terminally His6-tagged enzyme Homo sapiens
gene B4GALT7, recombinant expression of N-terminally His6-tagged enzyme Drosophila melanogaster

Crystallization (Commentary)

Crystallization (Comment) Organism
hanging drop vapor diffusion method, using 100 mM imidazole (pH 6.5) and 750 mM sodium acetate or 100 mM HEPES buffer (pH 8.0), 100mM serinol, 1.0 M NaCl, 5% (w/v) PEG6000, and 10% 2,4-methanepentadiol Drosophila melanogaster
purified recombinant detagged enzyme in complex xylobiose, hanging drop vapor diffusion method, mixing of protein solution containing 10 mg/ml protein, 10 mM MnCl2, 10 mM UDP, and 10 mM xylobiose, with a reservoir solution containing 100mM HEPES, pH 8.0, 100mM serinol, 1.0 M NaCl, 5% PEG 6000, and 10% 2-methyl-2,4-pentanediol, crystals of enzyme mutants D318N and D211N protein in complex with UDP-Gal, by hanging drop vapor diffusion method, using a protein solution containing 10 mg/ml protein, 10 mM MnCl2, and 10 mM UDP-Gal, with the precipitating solution containing 100 mM HEPES, pH 8.0, 1.0 M NaCl, 5% PEG 6000, and 10% 2-methyl-2,4-pentanediol, X-ray diffraction structure determination and analysis Drosophila melanogaster
purified recombinant detagged wild-type and truncated enzymes, hanging drop vapor diffusion method, mixing of 15 mg/ml protein solution with reservoir solution containing 100 mM imidazole, pH 6.5, 5 mM MnCl2, and 750 mM sodium acetate, 18°C, X-ray diffraction structure determination and analysis, molecular replacement using the Drosophila melanogaster beta4GalT7 crystal structure, PDB ID 3LW6, as a search model. For crystal complexes, tetragonal crystals are soaked in the same pH 6.5 reservoir solution containing 15% 2-methyl-2,4-pentanediol at 18 °C for 20 h followed by a brief soak in 100 mM Tris, pH 8.0, 750 mM sodium acetate, 15% 2-methyl-2,4-pentanediol, 5 mM MnCl2, and 5 mM UDP-Gal at room temperature. The monoclinic crystals are obtained within 3 days using 100 mM Tris, pH 8.5, and 8% PEG 8000 as a reservoir solution by mixing it with 20 mg/ml beta4GalT7DELTA81 in 10 mM MES (pH 6.5), 150 mM NaCl, 5 mM MnCl2, and 5 mM UDP-galactose at 18 °C Homo sapiens

Protein Variants

Protein Variants Comment Organism
D211N site-directed mutagenesis, inactive mutant Homo sapiens
D211N mutant with negligible catalytic activity Drosophila melanogaster
D211N site-directed mutagenesis, the catalytic base Asp211 is mutated to Asn residue, inactive mutant, substrate-bound crystal structure analysis Drosophila melanogaster
D318N mutant with negligible catalytic activity Drosophila melanogaster
additional information construction of truncated mutant beta4GalT7DELTA81 Homo sapiens

Metals/Ions

Metals/Ions Comment Organism Structure
Mn2+ required Drosophila melanogaster
Mn2+ dependent on Drosophila melanogaster
Mn2+ dependent on, the manganese binding site is located at the N-terminal hinge region of the long loop, the conformational changes influence the manganese ion coordination, involving the Asp163-X-Asp165 and the His257-X-His259 motifs Homo sapiens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
UDP-alpha-D-galactose + O-beta-D-xylosyl-[protein] Homo sapiens
-
UDP + 4-beta-D-galactosyl-O-beta-D-xylosyl-[protein]
-
?
UDP-alpha-D-galactose + O-beta-D-xylosyl-[protein] Drosophila melanogaster
-
UDP + 4-beta-D-galactosyl-O-beta-D-xylosyl-[protein]
-
?

Organism

Organism UniProt Comment Textmining
Drosophila melanogaster Q9VBZ9
-
-
Drosophila melanogaster Q9VBZ9 gene B4GALT7
-
Homo sapiens Q9UBV7 gene B4GALT7
-

Purification (Commentary)

Purification (Comment) Organism
recombinant N-terminally His6-tagged enzyme by affinity chromatography, and cleavage of the tag by TEV protease Homo sapiens
recombinant N-terminally His6-tagged enzyme by affinity chromatography, and cleavage of the tag by TEV protease Drosophila melanogaster

Reaction

Reaction Comment Organism Reaction ID
UDP-alpha-D-galactose + [protein]-3-O-(beta-D-xylosyl)-L-serine = UDP + [protein]-3-O-(beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L-serine catalytic mechanism, oveview Homo sapiens
UDP-alpha-D-galactose + [protein]-3-O-(beta-D-xylosyl)-L-serine = UDP + [protein]-3-O-(beta-D-galactosyl-(1->4)-beta-D-xylosyl)-L-serine catalytic mechanism, oveview Drosophila melanogaster

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information donor and acceptor substrate binding, structure analysis, overview Drosophila melanogaster ?
-
?
additional information donor and acceptor substrate binding, strutcure analysis, overview Homo sapiens ?
-
?
UDP-alpha-D-galactose + O-beta-D-xylosyl-[protein]
-
Homo sapiens UDP + 4-beta-D-galactosyl-O-beta-D-xylosyl-[protein]
-
?
UDP-alpha-D-galactose + O-beta-D-xylosyl-[protein]
-
Drosophila melanogaster UDP + 4-beta-D-galactosyl-O-beta-D-xylosyl-[protein]
-
?
UDP-alpha-D-galactose + xylobiose
-
Drosophila melanogaster UDP + ?
-
?

Synonyms

Synonyms Comment Organism
beta-1,4-galactosyltransferase 7
-
Homo sapiens
beta-1,4-galactosyltransferase 7
-
Drosophila melanogaster
beta1,4-galactosyltransferase 7
-
Drosophila melanogaster
beta4GalT7
-
Homo sapiens
beta4GalT7
-
Drosophila melanogaster

General Information

General Information Comment Organism
additional information the enzyme undergoes conformational changes upon substrate binding, human enzyme in its open and closed conformations, overview Homo sapiens
physiological function the enzyme is involved in proteoglycan synthesis Homo sapiens
physiological function the enzyme is involved in proteoglycan synthesis Drosophila melanogaster