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2.4.1.40: glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase

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

Word Map on EC 2.4.1.40

Reaction

UDP-N-acetyl-alpha-D-galactosamine
+
glycoprotein-alpha-L-fucosyl-(1->2)-D-galactose
=
UDP
 +
glycoprotein-N-acetyl-alpha-D-galactosaminyl-(1->3)-[alpha-L-fucosyl-(1->2)]-D-galactose

Synonyms

A transferase, A-transferase, ABO, ABO(H) blood group A glycosyltransferase, alpha-(1,3)-N-acetylgalactosaminyltransferase, alpha-3-N-acetylgalactosaminyltransferase, bacterial GTA, BgtA, blood group A glycosyltransferase, blood group A glycosyltransferase 2, blood-group substance A-dependent acetylgalactosaminyltransferase, blood-group substance alpha-acetyltransferase, cis-AB-transferase, fucosylgalactose acetylgalactosaminyltransferase, glycosyltransferases A, GTA, histo-blood group A acetylgalactosaminyltransferase, histo-blood group A glycosyltransferase (Fucalpha1-2Galalpha1-3-N-acetylgalactosaminyltransferase), histo-blood group A transferase, histo-blood group ABO system transferase, More, UDP-GalNAc:Fucalpha1-2Galalpha1-3-N-acetylgalactosaminyltransferase, UDP-N-acetyl-D-galactosamine:alpha-L-fucosyl-1,2-D-galactose 3-N-acetyl-D-galactosaminyltransferase

ECTree

     2 Transferases
         2.4 Glycosyltransferases
             2.4.1 Hexosyltransferases
                2.4.1.40 glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase

Crystallization

Crystallization on EC 2.4.1.40 - glycoprotein-fucosylgalactoside alpha-N-acetylgalactosaminyltransferase

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CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
a large single crystal is subjected to H/D exchange prior to data collection and time-of-flight neutron diffraction data is collected to 2.5 A resolution at the Protein Crystallography Station to 85% overall completeness, with complementary X-ray diffraction data collected from a crystal from the same drop and extending to 1.85 A resolution
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
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 2.1 A resolution
enzyme adopts an open conformation in the absence of substrates. Binding 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
G176R/P234S/S235G/M266L/A268G-mutant with and without H-antigen, at 1.55 and 1.65 A resolution respectively
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. Depending on substrate concentrations in the Golgi apparatus an acceptor route and a donor route are possible. At high local concentrations of UDP-Gal or UDP-GalNAc binding of the nucleotide sugar to GTB or GTA would precede binding of the H-antigen. At low nucleotide sugar concentrations, it can be assumed that H-antigen binds first. In this latter case, the enzymes may discriminate between different types of H-antigens, preferring e. g. type-II over type-I H-antigens
of catalytic domain residues 63-354, with and without L-fucosyl-alpha-1,2-beta-galactosyl-O(CH2)7CH3-acceptor and UDP, at 1.35 and 1.8 A resolution respectively
purified recombinant GTA/GTB mutant chimeric enzymes, complexing with synthetic antigen disaccharides or UDP, hanging drop vapour diffusion method, different solutions for the different chimeric mutants, X-ray diffraction structure determinationand analysis at 1.41-1.75 A resolution, overview, structure modelling
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 mutant D302C. 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
wild-type and mutants E303A, E303C, E303D, E303Q