Structure-function analysis of the UDP-N-acetyl-D-galactosamine: Polypeptide N-acetylgalactosaminyltransferase - Essential residues lie in a predicted active site cleft resembling a lactose repressor fold

Mucin-type O-glycosylation is initiated by a family of UDP-GalNAc:polypepti de N-acetylgalactosaminyltransferases (ppGaNTases), Based on sequence relat ionships with divergent proteins, the ppGaNTases can be subdivided into thr ee putative domains: each putative domain contains a characteristic sequenc e motif. The 112-amino acid glycosyltransferase (1) under bar (GT1) motif r epresents the first half of the catalytic unit and contains a short asparta te-any residue-histidine (DXH) or aspartate-any residue-aspartate (DXD)-lik e sequence. Secondary structure predictions and structural threading sugges t that the GT1 motif forms a 5-stranded parallel beta-sheet flanked by 4 al pha-helices, which resembles the first domain of the lactose repressor. Fou r invariant carboxylates and a histidine residue are predicted to lie at th e C-terminal end of three beta-strands and line the active site cleft. Site -directed mutagenesis of murine ppGaNTase-T1 reveals that conservative muta tions at these 5 positions result in products with no detectable enzyme act ivity (D156Q, D209N, and H211D) or <1% activity (E127Q and E213Q). The seco nd half of the catalytic unit contains a DXXYXXWGGENXE motif (positions 310 -322) which is also found in beta 1,4-galactosyltransferases (termed the Ga l/GaLNAc-T motif), Mutants of carboxylates within this motif express either no detectable activity, 1% or 2% activity (F319Q, E322Q, and D310N, respec tively). Mutagenesis of highly conserved (but not invariant) carboxylates p roduces only modest alterations in enzyme activity. Mutations in the C-term inal 128-amino acid ricin-like lectin motif do not alter the enzyme's catal ytic properties.