Functional characterization of the Candida albicans MNT1 mannosyltransferase expressed heterologously in Pichia pastoris.
The alpha1,2-mannosyltransferase gene MNT1 of the human fungal pathogen Candida albicans has been shown to be important for its adherence to various human surfaces and for virulence (Buurman, E. T. , Westwater, C., Hube, B., Brown, A. P. J., Odds, F. C., and Gow, N. A. R. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7670-7675). The CaMnt1p is a type II membrane protein, which is part of a family of proteins that are important for both O- and N-linked mannosylation in fungi and which represent a distinct subclass of glycosyltransferase enzymes. Here we use heterologous expression of CaMNT1 in the methylotrophic yeast Pichia pastoris to characterize the properties of the CaMnt1p enzyme as an example of this family of enzymes and to identify key amino acid residues required for coordination of the metal co-factor and for the retaining nucleophilic mechanism of the transferase reaction. We show that the enzyme can use both Mn(2+) and Zn(2+) as metal ion co-factors and that the reaction catalyzed is specific for alpha-methyl mannoside and alpha1,2-mannobiose acceptors. The N-terminal cytoplasmic tail, transmembrane domains, and stem regions were shown to be dispensable for activity, whereas truncations to the C-terminal catalytic domain destroyed activity without markedly affecting transcription of the truncated gene.
Thomson
L M
LM
Department of Molecular and Cell Biology, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom.
Bates
S
S
Yamazaki
S
S
Arisawa
M
M
Aoki
Y
Y
Gow
N A
NA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology*; D003001:Cloning, Molecular; D006801:Humans; D008364:Mannosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; C068701:KRE2 protein, S cerevisiae; D008364:Mannosyltransferases