THE POTENTIAL DOLICHOL RECOGNITION SEQUENCE OF BETA-1,4-MANNOSYLTRANSFERASE IS NOT REQUIRED FOR ENZYMATIC-ACTIVITY USING NYL-PYROPHOSPHORYL-ALPHA-N,N'-DIACETYLCHITOBIOSIDE AS ACCEPTOR
L. Revers et al., THE POTENTIAL DOLICHOL RECOGNITION SEQUENCE OF BETA-1,4-MANNOSYLTRANSFERASE IS NOT REQUIRED FOR ENZYMATIC-ACTIVITY USING NYL-PYROPHOSPHORYL-ALPHA-N,N'-DIACETYLCHITOBIOSIDE AS ACCEPTOR, Biochemical journal, 299, 1994, pp. 23-27
The ALG1 gene of Saccharomyces cerevisiae encodes beta-1,4-mannosyltra
nsferase, an essential membrane-associated enzyme involved in the asse
mbly of dolichyl-linked oligosaccharide precursors for N-glycosylation
[Albright and Robbins (1990) J. Biol. Chem. 265, 7042-7049], which ca
talyses the transfer of a mannose residue from GDP-mannose to hylpyrop
hosphoryl-alpha-N,N'-diacetylchitobioside; it also possesses a putativ
e transmembrane domain, bearing an 11-amino-acid consensus sequence, w
hich has been proposed to mediate dolichol recognition. Here we report
the construction and bacterial expression of a mutant beta-1,4-mannos
yltransferase derived from ALG1, which carries a 34-amino-acid deletio
n resulting in the absence of the entire N-terminal transmembrane doma
in. This truncated enzyme has an apparent K-m value of 17 mu M for yl-
pyrophosphoryl-alpha-N,N'-diacetylchitobioside, a known acceptor for b
eta-1,4-mannosyltransferase [Flitsch, Pinches, Taylor and Turner (1992
) J. Chem. Sec., Perkin Trans. 1, 2087-2093]. The intact enzyme, expre
ssed in the same system, has an apparent K-m value of 25 mu M. These f
igures are in good agreement with previously reported values for wild-
type beta-1,4-mannosyltransferase 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 ve
rifies 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.