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Literature summary extracted from

  • Chen, L.; Zhang, Y.; Feng, Y.
    Structural dissection of sterol glycosyltransferase UGT51 from Saccharomyces cerevisiae for substrate specificity (2018), J. Struct. Biol., 204, 371-379 .
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.173 gene AGT26, recombinant expression of N-terminally His6-tagged UGT51 residues 722-1198 from pET28a vector with a thrombin cleavage site in Escherichia coli srain BL21(DE3) CodonPlus Saccharomyces cerevisiae

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.173 purified recombinant UGT51 alone and in complex with UDP-glucose, hanging drop vapor diffusion method, mixing of 20 mg/ml protein in 20 mM Tris-HCl, 500 mM NaCl, and 2 mM DTT, with reservoir solution containing 0.2 M ammonium acetate, 0.1 M HEPES, pH 7.5, 25% w/v PEG 3350 for the free enzyme, or containing 0.2 M MgCl2, 0.1 M Tris, pH 8.5, and 25% w/v PEG 3350 for the enzyme complex, receiving microcrystals, optimized crystallization protocol involves dispensing 0.0015 ml of 20 mg/ml protein and 0.001 ml of reservoir solution (containing 0.2 M MgCl2, 0.1 M Tris pH 8.5 and 25% w/v PEG 3350) and 500 nl of 0.001 diluted crystal seed stock solution, rod protein crystals appear after 3 days, 14°C, crystals are soaked in cryoprotectant solution with 5 mM UDP-glucose, X-ray diffraction structure determination and analysis at 2.77 A and 1.9 A resolutions, respectively, molecular replacement using the structure of GtfD (PDB ID 1rrv) as the search model, modeling Saccharomyces cerevisiae

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.173 S801A/L802A/V804A/K812A/E816K/S849A/N892D site-directed mutagenesis, the changed unique interaction network in mutant M7_1 with an 1800fold activity improvement toward an unnatural substrate protopanaxadiol (PPD), might influence its substrate preference Saccharomyces cerevisiae

Localization

EC Number Localization Comment Organism GeneOntology No. Textmining
2.4.1.173 membrane associated Saccharomyces cerevisiae 16020
-

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.1.173 UDP-glucose + a sterol Saccharomyces cerevisiae
-
 UDP + a sterol 3-beta-D-glucoside
-
 ?
2.4.1.173 UDP-glucose + a sterol Saccharomyces cerevisiae ATCC 204508
-
 UDP + a sterol 3-beta-D-glucoside
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.173 Saccharomyces cerevisiae Q06321
-
-
2.4.1.173 Saccharomyces cerevisiae ATCC 204508 Q06321
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.173 recombinant N-terminally His6-tagged UGT51 residues 722-1198 Escherichia coli srain BL21(DE3) CodonPlus by nickel affinity chromatography, tag cleavage by thrombin, followed by a secons step of nickel affinity chromatography, gel filtration, and ultrafiltration Saccharomyces cerevisiae

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.173 additional information substrate molecular docking and structure analysis of wild-type and mutant enzymes, sugar donor binding mechanism of UGT51, overview Saccharomyces cerevisiae ?
-
-
2.4.1.173 additional information substrate molecular docking and structure analysis of wild-type and mutant enzymes, sugar donor binding mechanism of UGT51, overview Saccharomyces cerevisiae ATCC 204508 ?
-
-
2.4.1.173 UDP-glucose + a sterol
-
 Saccharomyces cerevisiae UDP + a sterol 3-beta-D-glucoside
-
 ?
2.4.1.173 UDP-glucose + a sterol
-
 Saccharomyces cerevisiae ATCC 204508 UDP + a sterol 3-beta-D-glucoside
-
 ?
2.4.1.173 UDP-glucose + protopanaxadiol an unnatural substrate of enzyme mutant 7_1 Saccharomyces cerevisiae UDP + protopanaxadiol 3-beta-D-glucoside
-
 ?
2.4.1.173 UDP-glucose + protopanaxadiol an unnatural substrate of enzyme mutant 7_1 Saccharomyces cerevisiae ATCC 204508 UDP + protopanaxadiol 3-beta-D-glucoside
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.1.173 homodimer UGT51 forms a dimer in solution. The dimeric structure of UGT51 has two subunits packed against each other in a parallel fashion and adopts a butterfly shape. The full length of UGT51 contain 1198 amino acids, which can be divided into several domains: pleckstrin homology (PH) domain, GRAM domain and catalytic domain. The PH domain responsible interact with membrane lipids, the GRAM domain is essential for proper protein association with its target membrane, while the catalytic domain is essentiell for synthesis steroid glycosides. Each subunit displays the typical GT-B fold of GTs, comprising two distinct N- and C-terminal Rossman-fold domains having a similar topology and connected by a linker peptide and a long C-terminal helix. The N-terminal domain, a seven-stranded parallel beta-sheet flanked by nine alpha-helices, and the C-terminal domain, a six-stranded beta-sheet surrounded by five alpha-helices, are assumed to bind the aglycone and the nucleotide-sugar donor, respectively. The C-terminal helices alpha7 (residues 1129-1144) and alpha8 (residues 1147-1171) cross and interact with each Rossman-fold domain Saccharomyces cerevisiae

Synonyms

EC Number Synonyms Comment Organism
2.4.1.173 Atg26
-
 Saccharomyces cerevisiae
2.4.1.173 autophagy-related protein 26 UniProt Saccharomyces cerevisiae
2.4.1.173 sterol glycosyltransferase
-
 Saccharomyces cerevisiae
2.4.1.173 UDP-glycosyltransferase 51 UniProt Saccharomyces cerevisiae
2.4.1.173 Ugt51
-
 Saccharomyces cerevisiae

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.173 35
-
 assay at Saccharomyces cerevisiae

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.173 8
-
 assay at Saccharomyces cerevisiae

General Information

EC Number General Information Comment Organism
2.4.1.173 evolution enzyme UGT51 is a member of the GT1 family, GT-B fold group, comprising two Rossman-like domains Saccharomyces cerevisiae
2.4.1.173 metabolism UGT51 is not involved in autophagy-related pathways in Saccharomyces cerevisiae Saccharomyces cerevisiae
2.4.1.173 additional information a long hydrophobic cavity, 9.2 A in width and 17.6 A in length located at the N-terminal domain of UGT51, is suitable for the accommodation of sterol acceptor substrates. A short, conserved sequence of S847-M851 is identified at the bottom of the hydrophobic cavity, which might be the steroid binding site and play an important role for the UGT51 catalytic specificity towards sterols. Molecular docking simulations revealing the sugar acceptor specificity, overview. The N- and C-terminal domains predominantly dictate acceptor and donor specificities, respectively. The donor substrate binds in a deep inter-domain pocket and a hydrophobic crevice on the surface of the N-terminal domain, which is proposed to be the binding site of the aglycone substrate Saccharomyces cerevisiae
2.4.1.173 physiological function sterol glycosyltransferases (SGTs) catalyze the formation of a variety of glycosylated sterol derivatives and are involved in producing a plethora of bioactive natural products. Sterol glycosyltransferases as members of UGTs are involved in transferring a sugar from UDP-sugar to various SGs metabolites, including hormones and secondary metabolites Saccharomyces cerevisiae