Biochemical Journal

Research article

Ktr1p is an α-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris

Pedro A. ROMERO, Marc LUSSIER, Anne-Marie SDICU, Howard BUSSEY, Annette HERSCOVICS


The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the α1,2-mannosyltransferase Kre2p/Mnt1p, the only member of this family whose enzymic properties have been studied. In this study, the enzymic properties of Ktr1p, another member of this family, were studied and compared to those of Kre2p/Mnt1p. Recombinant soluble forms of Kre2p/Mnt1p and Ktr1p lacking their N-terminal regions were expressed as secreted proteins from the methylotrophic yeast Pichia pastoris. After induction with methanol, the medium contained approx. 40 and 400 mg/l of soluble recombinant Kre2p/Mnt1p and Ktr1p respectively. Both recombinant proteins were shown to exhibit α1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar Km for mannose (280Ő350 mM), methyl-α-mannoside (60Ő90 mM) and GDP-mannose (50Ő90 ƁM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-α-mannoside, α-1,2-mannobiose and methyl-α-1,2-mannobiose, as well as Man15Ő30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize α-1,6-mannobiose, α-1,6-mannotriose, α-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9Ő10GlcNAc. These results indicate that Kre2p/Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.