The potential dolichol recognition sequence of beta-1,4-mannosyltransferase is not required for enzymic activity using phytanyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside as acceptor.
The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltransferase, an essential membrane-associated enzyme involved in the assembly of dolichyl-linked oligosaccharide precursors for N-glycosylation [Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which catalyses the transfer of a mannose residue from GDP-mannose to dolichyl-pyrophosphoryl-alpha-N,N'- diacetylchitobioside; it also possesses a putative transmembrane domain, bearing an 11-amino-acid consensus sequence, which has been proposed to mediate dolichol recognition. Here we report the construction and bacterial expression of a mutant beta-1,4-mannosyltransferase derived from ALG1, which carries a 34-amino-acid deletion resulting in the absence of the entire N-terminal transmembrane domain. This truncated enzyme has an apparent Km value of 17 microM for phytanyl-pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for beta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992) J. Chem. Soc., Perkin Trans. 1, 2087-2093]. The intact enzyme, expressed in the same system, has an apparent Km value of 25 microM. These figures are in good agreement with previously reported values for wild-type beta-1,4-mannosyl-transferase incubated with the natural dolichyl-linked substrate. Gel-filtration chromatography (before and after beta-mannosidase digestion) of the products of both forms of the enzyme verifies the formation of Man beta 1-->4GlcNAc beta 1-->4GlcNAc. We therefore conclude that the putative dolichol recognition sequence is not necessary for recognition of the phytanyl analogue of its natural dolichol substrate and suggest it probably also is not needed for its natural substrate.
Revers
L
L
Dyson Perrins Laboratory, University of Oxford, U.K.
Wilson
I B
IB
Webberley
M C
MC
Flitsch
S L
SL
D001483:Base Sequence; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002462:Cell Membrane; D003001:Cloning, Molecular; D017931:DNA Primers; D004187:Disaccharides / Q000378:metabolism*; D004286:Dolichols / Q000378:metabolism*; D004926:Escherichia coli; D005800:Genes, Fungal; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D017931:DNA Primers; D004187:Disaccharides; D004286:Dolichols; D011994:Recombinant Proteins; C086865:phytanylpyrophosphoryl-N,N'-diacetylchitobioside; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase