Mutation of Arg(273) to Leu alters the specificity of the yeast N-glycan processing class I alpha 1,2-mannosidase

Class I alpha 1,2-mannosidases (glycosyl hydrolase family 47) involved in t he processing of N-glycans during glycoprotein maturation have different sp ecificities, Enzymes in the endoplasmic reticulum of yeast and mammalian ce lls remove a single mannose from Man(9)GlcNAc(2) to form Man(8)GlcNAc(2) is omer B (lacking the alpha 1, 2-mannose residue of the middle alpha 1, 3-arm ), whereas other alpha 1,2-mannosidases, including Golgi alpha 1,2-mannosid ases IA and IB, can convert Man(9)GlcNAc(2) to Man(5)GlcNAc(2). In the pres ent work, it is demonstrated that with a single mutation in its catalytic d omain (Arg(273) --> Leu) the yeast endoplasmic reticulum alpha 1,2-mannosid ase acquires the ability to transform Man(9)GlcNAc to Man(5)GlcNAc. High re solution proton nuclear magnetic resonance analysis of the products shows t hat the order of removal of mannose from Man(9)GlcNAc is different from tha t of other alpha 1,2-mannosidases that remove four mannose from Man(9)GlcNA c. These results demonstrate that Arg(273) is in part responsible for the s pecificity of the endoplasmic reticulum alpha 1,2-mannosidase and that smal l differences in non-conserved amino acids interacting with the oligosaccha ride substrate in the active site of class I alpha 1,2-mannosidases are res ponsible for the different specificities of these enzymes.