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2.4.1.37: fucosylgalactoside 3-alpha-galactosyltransferase

This is an abbreviated version!
For detailed information about fucosylgalactoside 3-alpha-galactosyltransferase, go to the full flat file.

Word Map on EC 2.4.1.37

Reaction

UDP-alpha-D-galactose
+
alpha-L-fucosyl-(1->2)-D-galactosyl-R
=
UDP
 +
alpha-D-galactosyl-(1->3)-[alpha-L-fucosyl(1->2)]-D-galactosyl-R

Synonyms

ABO(H) blood group B alpha-(1->3)-galactosyltransferase, ABO(H) blood group B alpha-1,3-galactosyltransferase, ABO(H) blood group B glycosyltransferase, ABO(H) blood-group glycosyltransferase B, alpha(1-3)galactosyltransferase, alpha-(1,3)-galactosyltransferase, alpha-(1-3)-galactosyltransferase, alpha-(1->3)-galactosyltransferase, alphaGal-T1, B glycosyltransferase, B transferase, blood group alpha-(1-3)-galactosyltransferase, blood group B alpha-(1,3)-galactosyltransferase, blood group B galactosyltransferase, blood group B glycosyltransferase, blood group B glycosyltransferase GTB, blood group B-glycosyltransferase, blood-group substance B-dependent galactosyltransferase, cis-AB-transferase, glycoprotein-fucosylgalactoside alpha-galactosyltransferase, glycosyltransferases B, GTB, histo-blood group ABO system transferase, histo-blood group B enzyme, histo-blood group B transferase, histo-blood substance B-dependent galactosyltransferase, More, UDP-galactose:fucoside alpha1,3-galactosyltransferase, UDP-galactose:O-alpha-L-fucosyl(1-2)D-galactose alpha-D-galactosyltransferase, UDPgalactose:glycoprotein-alpha-L-fucosyl-(1,2)-D-galactose 3-alpha-D-galactosyltransferase, [blood group substance] alpha-galactosyltransferase

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.1 Hexosyltransferases
                2.4.1.37 fucosylgalactoside 3-alpha-galactosyltransferase

Crystallization

Crystallization on EC 2.4.1.37 - fucosylgalactoside 3-alpha-galactosyltransferase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
assignment of all methyl resonance signals in Ala, Ile, Leu, Met and Val labeled samples of GTA and GTB by lanthanide-induced pseudocontact shifts and methyl-methyl NOESY. The fully closed state is not adopted in the presence of lanthanide ions
catalytic domain with and without H-antigen and UDP, at 1.32 and 1.65 A resolution
crystals of purified native enzyme are soaked with various combinations of UDP-GalNAc, UDP-Gal, UDP, and acceptor analogues alpha-L-fucosyl-1,2-beta-D-(3-deoxy)-galactosyl-O-R or alpha-L-fucosyl-1,2-beta-D-(3-amino)-galactosyl-O-R, ligands are solved in 7.5% PEG 4000, 15% glycerol, 75 mM N-[2-acetamido]-2-iminodiacetic acid, pH 7.5, 10 mM MnCl2, and 10 mM inhibitor, 3-4 days, X-ray diffraction structure determination and analysis at 1.6 A resolution
enzyme adopts an open conformation in the absence of substrates. Binding of the donor substrate UDP-Gal or of UDP induces a semiclosed conformation. In the presence of both donor and acceptor substrates, the enzymes shift towards a closed conformation with ordering of an internal loop and the C-terminal residues, which then completely cover the donor-binding pocket. The enzyme shows substantial plasticity and conformational flexibility. Residues Ile123 at the bottom of the UDP binding pocket, and Ile192 as part of the internal loop are significantly disturbed upon stepwise addition of UDP and H-disaccharide-O-CH3
enzyme soaked with acceptor analogs: galactose, lactose, N-acetyllactosamine, beta-D-Galp-O(CH2)8CO2CH3, alpha-L-Fucp-(1,2)-beta-D-Galp-O(CH2)7CH3, beta-D-Galp-(1,4)-beta-D-Glcp-OCH3, alpha-L-Fucp-(1,2)-beta-D-Galp-(1,3)-beta-D-GlcNAcp-O(CH2)7CH3, alpha-L-Fucp-(1,2)-beta-D-Galp-(1,4)-beta-D-GlcNAcp-O-(CH2)8CO2CH3
in complex with UDP and galactose, using 1% (w/v) PEG 4000, 4.5-5% (w/v) 2-methyl-2,4-pentanediol, 100 mM ammonium sulfate, 70 mM sodium chloride, 50 mM N-[2-acetamido]-2-iminodiacetic acid buffer pH 7.5, 30 mM sodium acetate buffer pH 4.6 and 5 mM MnCl2
Methyl-TROSY-based titration experiments in combination with zz-exchange experiments show dramatic changes of binding kinetics associated with allosteric interactions between donor-type and acceptor-type ligands. Binding of the acceptor substrates H-disaccharide, H-type II trisaccharide, and H-type VI trisaccharide affects the chemical shifts of the 13C-methyl groups of Met 266, Val 299, Leu 324, and Leu 329, which belong to the acceptor substrate binding pocket
mutant GTB C209A is crystallized in both the presence and the absence of mercury, 0.01 ml drops containing 6-8 mg/ml GTB, 70 mM N-(2-acetamido)-2-iminodiacetic acid, pH 7.5, 50 mM sodium acetate, pH 4.6, 40 mM NaCl, 5-8 mM MnCl2, 2.5% v/v 2-methyl-2,4-pentanediol, 5% v/v glycerol, 2% w/v PEG 4000 and 0.3-0.5 mM 3-chloromercuri-2-methoxypropylurea is suspended over 1 ml reservoir solution containing 50 mM N-(2-acetamido)-2-iminodiacetic acid, pH 7.5, 10 mM, MnCl2, 100 mM ammonium sulfate, 5% v/v MPD, 10% v/v glycerol and 8-10% w/v PEG 4000. Growing crystals of native GTB in the absence of mercury using protein concentrations of 6-8 mg /ml are unsuccessful, therefore crystals of the C209A mutant are grown from protein concentrations of over 30 mg/ml with the lowest observed concentration that yielded diffraction-quality crystals being 15 mg/ml 5-8 ml drops containing 1% PEG 4000, 4.5% MPD, 0.1 M ammonium sulfate, 0.07 M NaCl, 0.05 M N-(2-acetamido)-2-iminodiacetic acid, pH 7.5, and 5 mM CoCl2 are stored at 4-6°C for 3-5 days over a reservoir of 2.7% PEG 4000, 7% MPD, 0.32 M ammonium sulfate, 0.25 M NaCl and 0.2 M N-(2-acetamido)-2-iminodiacetic acid. Both sets of crystals are washed with mother liquor containing 6-7% PEG 4000, 70 mM N-(2-acetamido)-2-iminodiacetic acid, pH 7.5, 30 mM sodium acetate, pH 4.6, 40 mM ammonium sulfate, 29-30% glycerol and 9-10 mM MnCl2 or 5 mM CoCl2 for the heavy-metal derivative or native protein, respectively. X-ray diffraction structure determination and analysis at 1.8-2.4 A resolution
P234S-mutant, 1.55 and 1.65 A resolution, with and without H-antigen
structures of GTA, GTB and several chimeras determined by single-crystal X-ray diffraction demonstrate a range of susceptibility to the choice of cryoprotectant, in which the mobile polypeptide loops can be induced by glycerol to form the ordered closed conformation associated with substrate recognition and by MPD (hexylene glycol, 2-methyl-2,4-pentanediol) to hinder binding of substrate in the active site owing to chelation of the Mn2+ cofactor and thereby adopt the disordered open state
-
structures of isoforms GTA and GTB in complex with their respective trisaccharide products. A conflict exists between the transferred sugar monosaccharide and the beta-phosphate of the UDP donor. The mechanism of product release shows monosaccharide transfer to the H-antigen acceptor induces active site disorder and ejection of the UDP leaving group prior to trisaccharide egress
structures of wild-type and mutants D302A, D302C, D302L, R188K. Conserved active site residues Arg188 and Asp302 are critical for catalysis, and disruption of their hydrogen bond network through mutation can dramatically decrease enzymatic activity
study of substrate-induced conformational transitions of GTB. Acceptor binding is fast on the chemical-shift timescale with rather small chemical-shift perturbations in the range of less than approximately 20 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (below 10 Hz). Substrate binding drives the enzyme into the closed state required for catalysis
using 1% (w/v) polyethylene glycol 4000, 4.5-5% (w/v) 2-methyl-2,4-pentanediol, 100 mM ammonium sulfate, 70 mM sodium chloride, 50 mM N-[2-acetamido]-2-iminodiacetic acid buffer, pH 7.5, 30 mM sodium acetate buffer, pH 4.6, and 5 mM MnCl2
wild-type and mutant enzymes, X-ray diffraction structure determination and analysis at 1.99 A resolution, modelling
wild-type and mutants E303A, E303C, E303D, E303Q
wild-type enzyme and GTA/GTB chimeric enzyme mutants GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analogue substrates, hanging drop vapour diffusion method, method variantions, overview, X-ray diffraction structure determination and analysis at 1.43-1.75 A resolution