Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser

Pig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc(2)-Man(9)-Glc(3) precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH2 as in beta,gamma -diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979-985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine P-methyl group by OST is also specific, with serine, L-threo-beta -hydroxynorvaline and L-beta -hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH3-CH(OH) group. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.