A novel mono-branched lipid phosphate acts as a substrate for dolichyl phosphate mannose synthetase.
Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-3H]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [3H]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.
Wilson
I B
IB
Dyson Perrins Laboratory, University of Oxford, U.K.
Taylor
J P
JP
Webberley
M C
MC
Turner
N J
NJ
Flitsch
S L
SL
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D007536:Isomerism; D008364:Mannosyltransferases / Q000378:metabolism*; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism*; D010766:Phosphorylation; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D010713:Phosphatidylcholines; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase