Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide. | Semantic Scholar (2024)

The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Structural function investigations have shown that donor specificity is attributed to the last two amino acids, andMutants have also been produced with greatly enhanced turnover rates as well as hybrid A/B enzymes that catalyse both reactions efficiently.

The first structural information of a dual specificity cis-AB blood group glycosyltransferase in complex with a synthetic UDP-GalNAc derivative is reported, and the GalNAc moiety adopts an unusual yet catalytically productive conformation in the binding pocket, which is different from the “tucked under” conformation previously observed for the UDP- Gal donor.

Enzyme kinetics and high resolution structural studies of mutant enzymes based on the O2 blood group transferase reveal that whereas the P74S mutation in the stem region of the protein does not appear to play a role in enzyme inactivation, the G268R mutation completely blocks the donor GalNAc-binding site leaving the acceptor binding site unaffected.

The increases observed in k cat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

An expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes is created, enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover.

The experiments revealed that modulation of enzymatic activity by metal ions critically depends on the total enzyme concentration, raising the question as to which of the bivalent metal cations Mg2+ and Mn2+ is more relevant under physiological conditions.

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The purified alpha-3-N-acetylgalactosaminyltransferase had a weak capacity to transfer D-galactose from UDP-D-Galactose to similar acceptors to make blood-group-B determinants.

The basis of differential affinity of these glycosyltransferases to nucleotide-sugar (UDP-GalNAc or UDP-Gal) is established and new enzymes are created which catalyze the transfer of both GalNAc and Gal, and may provide an explanation of the rare cis-AB phenotype.

A critical single-base deletion was found in the 0 gene, which results in an entirely different, inactive protein incapable of modifying the H antigen, and this work presents a molecular basis for the ABO genotypes.

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