Classification, characterization and structural analysis of sugar nucleotidylyltransferase family of enzymes

Abstract S ugar N ucleotidyl T ransferases (SNTs) constitute a large family of enzymes that play important metabolic roles. Earlier, for one such SNT, termed N-acetylglucosamine-1-phosphate uridyltransferase- GlmU, we had established that two magnesium ions - Mg2+A and Mg2+B - catalyze the sugar-nucleotidyl transfer reaction. Despite a common structural framework that SNTs share, we recognized key differences around the active-site based on the analysis of available structures. Based on these differences, we had classified SNTs into two major groups, Group - I & II; and further, variation in ‘Mg2+A-stabilizing motifs’ led us to sub-classify them into five distinct sub-groups. Since group specific conservation of ‘Mg2+A-stabilizing motifs’ was based only for 45 available structures, here we validate this via an exhaustive analysis of 1,42,025 protein sequences. Previously, we had hypothesized that a metal-ion-catalyzed mechanism would be operative in all SNTs. Here, we validate it biochemically and establish that Mg2+ is a strict requirement for nucleotidyl transfer reactions in every group or sub-group and that a common metal ion dependent mechanism operates in SNTs. Further, mutating Mg2+A coordinating residue in each sub-group led to abolished catalysis, indicating an important role for both of these residues and suggest that SNTs employ variations over ‘a conserved catalytic mechanism mediated by Mg2+ ion(s)’, to bring about functional diversity. This would constitute a comprehensive study to establish the catalytic mechanism across the family of SNTs.