Literature summary for 2.4.1.173 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 .

Search for more literature for 2.4.1.173

Cloned(Commentary)

Cloned (Comment)	Organism
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)

Crystallization (Comment)	Organism
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

Crystallization (Comment)

Organism

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

Protein Variants	Comment	Organism
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

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

Natural Substrates/ Products (Substrates)

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

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	Q06321	-	-
Saccharomyces cerevisiae ATCC 204508	Q06321	-	-

Purification (Commentary)

Purification (Comment)	Organism
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)

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

Subunits

Subunits	Comment	Organism
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

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

Temperature Optimum [°C]

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

pH Optimum

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

General Information

General Information	Comment	Organism
evolution	enzyme UGT51 is a member of the GT1 family, GT-B fold group, comprising two Rossman-like domains	Saccharomyces cerevisiae
metabolism	UGT51 is not involved in autophagy-related pathways in Saccharomyces cerevisiae	Saccharomyces cerevisiae
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
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