Asp141 and the Hydrogen-Bond Chain Asp141-Asn109-Asp33 Are Respectively Essential for GT80 Sialyltransferase Activity and Structural Stability.

ABSTRACT

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. In this study, the activity of a putative sialyltransferase (Pm0160) harboring an inherent mutation D141Y in the conserved DDG motif, which has been identified in GT52 and GT80 families, was restored by reverse mutation. More interestingly, a hydrogen-bond chain was found to form between three conserved residues (Asp141, Asn109, and Asp33) of GT80 sialyltransferases based on recently determined crystal structures. Our mutagenesis experiments demonstrated that the hydrogen-bond chain connecting the general base Asp141 with Nß4, Nß1, and Nα1 plays an essential role in maintaining protein structural stability other than keeping the general base Asp141 in a productive orientation for sialic acid transfer.