Functional assignment of motifs conserved in β1,3‐glycosyltransferases

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Abstract
The β1,3‐glycosyltransferase enzymes identified to date share several conserved regions and conserved cysteine residues, all being located in the putative catalytic domain. To investigate the importance of these motifs and cysteines for the enzymatic activity, 14 mutants of the murine β1,3‐galactosyltransferase‐I gene were constructed and expressed in Sf9 insect cells. Seven mutations abolished the galactosyltransferase activity. Kinetic analysis of the other seven active mutants revealed that three of them showed a threefold to 21‐fold higher apparent Km with regard to the donor substrate UDP‐galactose relative to the wild‐type enzyme, while two mutants had a sixfold to 7.5‐fold increase of the apparent Km value for the acceptor substrate N‐acetylglucosamine‐β‐p‐nitrophenol. Taken together, our results indicate that the conserved residues W101 and W162 are involved in the binding of the UDP‐galactose donor, the residue W315 in the binding of the N‐acetylglucosamine‐β‐p‐nitrophenol acceptor, and the domain including E264 appears to participate in the binding of both substrates.

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