2.4.1.292: GalNAc-alpha-(1->4)-GalNAc-alpha-(1->3)-diNAcBac-PP-undecaprenol alpha-1,4-N-acetyl-D-galactosaminyltransferase
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Reaction
3 UDP-N-acetyl-alpha-D-galactosamine + = 3 UDP +
Synonyms
PglH, pglH2
ECTree
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Crystallization
Crystallization on EC 2.4.1.292 - GalNAc-alpha-(1->4)-GalNAc-alpha-(1->3)-diNAcBac-PP-undecaprenol alpha-1,4-N-acetyl-D-galactosaminyltransferase
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crystal structures of PglH in a binary complex with UDP-GalNAc and two ternary complexes containing a chemo-enzymatically generated lipid-linked oligosaccharide analogue and either UDP or synthetic, nonhydrolyzable UDP-CH2-GalNAc. PglH contains an amphipathic helix that has a dual role of facilitating membrane attachment and glycan counting. The helix contains three positively charged side chains that can bind the diphosphate group of the lipid-linked oligosaccharide substrate and thus limit the addition of GalNAc units to three
purified recombinant His-tagged PglH (native or the selenomethionine derivative) in three distinct states, including a binary complex with UDP-GalNAc and two ternary complexes containing a chemo-enzymatically generated LLO analogue and either UDP or synthetic, nonhydrolyzable UDP-CH2-GalNAc, apoenzyme by vapor diffusion in sitting or hanging drops at 20°C, using a reservoir containing 0.2M KI, 0.1M MOPS, pH 7.2, and 17% PEG 3350, the volume ratio of protein to reservoir is 1:1, for PglH with bound acceptor substrate, concentrated PglH is incubated with synthetic LLO at 0.6 mM and UDP or UDP-CH2-GalNAc at 2 mM (final concentrations) for 15 min, crystallization is performed by vapor diffusion in sitting drops or hanging drops at 20°C using a reservoir containing 0.2 M Ammonium acetate, 0.1 M ADA 6.3, and 21-22% PEG 3350, the volume ratio of protein to reservoir is 1:1, 1-4 days, X-ray diffraction structure determination and analysis at 2.3-3.3 A resolution