The functional glycosyltransferase signature sequence of the human β1,3-glucuronosyltransferase is a XDD motif

Abstract

The human β1,3-glucuronosyltransferase I (GIcAT-I) is the key enzyme responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide of proteoglycans (GlcAβ1,3Galβ1,3Galβ1,4Xylβ 1-O-serine). We have investigated the role of aspartate residues Asp 194. Asp195-Asp196 corresponding to the glycosyltransferase DXD signature motif, in GlcAT-I function by UDP binding experiments, kinetic analyses, and site-directed mutagenesis. We presented the first evidence that Mn2+ is not only essential for GIcAT-I activity but is also required for cosubstrate binding. In agreement, kinetic studies were consistent with a metal-activated enzyme model whereby activation probably occurs via binding of a Mn2+·UDP-GlcA complex to the enzyme. Mutational analysis showed that the Asp194-Asp195-Asp 196 motif is a major element of the UDP/Mn2+ binding site. Furthermore, determination of the individual role of each aspartate showed that substitution of Asp195 as well as Asp196 to alanine strongly impaired GlcAT-I activity, whereas Asp194 replacement produced only a moderate alteration of the enzyme activity. These findings along with molecular modeling and three-dimensional structure comparison of the GlcAT-I catalytic center with that of the Bacillus subtilis glycosyltransferase SpsA provided evidence that the interactions of Asp195 with the ribose moiety of UDP and of Asp196 with the metal cation Mn 2+ were crucial for GlcAT-I function. Altogether, these results indicated that, similarly to the SpsA enzyme, the nucleotide binding site of GIcAT-I contains a XDD motif rather than a DXD motif.