Altered sugar donor specificity and catalytic activity of pteridine glycosyltransferases by domain swapping or site-directed mutagenesis
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CY-007 and CY-049 pteridine glycosyltransferases (PGTs) that differ in sugar donor specificity to catalyze either glucose or xylose transfer to tetrahydrobiopterin were studied here to uncover the structural determinants necessary for the specificity. The importance of the C-terminal domain and its residues 218and 258 that are different between the two PGTs was assessed via structure-guided domain swapping or single and dual amino acid substitutions. Catalytic activity and selectivity were altered in all the mutants (2 chimeric and 6 substitution) to accept both UDP-glucose and UDP-xylose. In addition, the wild type activities were improved 1.6-4.2 fold in 4 substitution mutants and activity was observed towards another substrate UDP-Nacetylglucosamine in all the substitution mutants from CY-007PGT. The results strongly support essential role of the C-terminal domain and the two residues for catalysis as well as sugar donor specificity, bringing insight into the structural features of the PGTs.
Abstract
CY-007 and CY-049 pteridine glycosyltransferases (PGTs) that differ in sugar donor specificity to catalyze either glucose or xylose transfer to tetrahydrobiopterin were studied here to uncover the structural determinants necessary for the specificity. The importance of the C-terminal domain and its residues 218and 258 that are different between the two PGTs was assessed via structure-guided domain swapping or single and dual amino acid substitutions. Catalytic activity and selectivity were altered in all the mutants (2 chimeric and 6 substitution) to accept both UDP-glucose and UDP-xylose. In addition, the wild type activities were improved 1.6-4.2 fold in 4 substitution mutants and activity was observed towards another substrate UDP-Nacetylglucosamine in all the substitution mutants from CY-007PGT. The results strongly support essential role of the C-terminal domain and the two residues for catalysis as well as sugar donor specificity, bringing insight into the structural features of the PGTs.
참고문헌 (15)
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