Ktr1p is an alpha-1,2-mannosyltransferase of Saccharomyces cerevisiae. Comparison of the enzymic properties of soluble recombinant Ktr1p and Kre2p/Mnt1p produced in Pichia pastoris.
The yeast genome contains a KRE2/MNT1 family of nine related genes with amino acid similarity to the alpha 1,2-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 alpha 1,2-mannosyltransferase activity. The enzymes have an absolute requirement for Mn2+ and a similar K(m) for mannose (280-350 mM), methyl-alpha-mannoside (60-90 mM) and GDP-mannose (50-90 microM), but the Vmax was approx. 10 times higher for Kre2p/Mnt1p than for Ktr1p. The enzymes have similar substrate specificities and utilize mannose, methyl-alpha-mannoside, alpha-1,2-mannobiose and methyl-alpha-1,2-mannobiose, as well as Man15-30GlcNAc, derived from mnn2 mutant glycoproteins, as substrates. The enzymes do not utilize alpha-1,6-mannobiose, alpha-1,6-mannotriose, alpha-1,6-mannotetraose, mammalian Man9GlcNAc or yeast Man9-10GlcNAc. These results indicate that Kre2p/ Mnt1p and Ktr1p are capable of participating in both N-glycan and O-glycan biosynthesis.
Romero
P A
PA
McGill Cancer Centre, McGill University, Montréal, Québec, Canada.
Lussier
M
M
Sdicu
A M
AM
Bussey
H
H
Herscovics
A
A
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D008364:Mannosyltransferases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012995:Solubility; D013379:Substrate Specificity
D011994:Recombinant Proteins; D008364:Mannosyltransferases