Publications

• GDPmannose dolicholphosphate mannosyltransferase of chicken liver mitochondria.

  Tomita Y., Motokawa Y.

  Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimul ... >> More

  Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimulated by MgCl2 (3 mM optimum) and exhibited a pH optimum at around 7.2. Bisubstrate kinetic analysis indicated that the enzyme follows a sequential mechanism. The Km values for GDPmannose and dolichyl phosphate were 0.43 and 14.3 microM, respectively. The purified enzyme was labile and lost its activity on storage at 0 degree C overnight or incubation at 30 degrees C or higher temperature. Inactivation could be prevented by the addition of heat-denatured mitochondrial extract. Further investigation revealed that phospholipids and dolichyl phosphate are responsible for the stabilization. Single addition of either phospholipid or dolichyl phosphate showed little activity, but the combination of these lipids enhanced the stabilizing activity greatly. Eight naturally occurring phospholipids were tested and found to be effective in combination with dolichyl phosphate. Among these, sphingomyelin was the most effective. Dolichol could partially substitute dolichyl phosphate but worked at higher concentrations. << Less

  Biochim Biophys Acta 842:176-183(1985) [PubMed] [EuropePMC]

• In vitro phosphorylation by cAMP-dependent protein kinase up-regulates recombinant Saccharomyces cerevisiae mannosylphosphodolichol synthase.

  Banerjee D.K., Carrasquillo E.A., Hughey P., Schutzbach J.S., Martinez J.A., Baksi K.

  DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphoryla ... >> More

  DPM1 is the structural gene for mannosylphosphodolichol synthase (i.e. Dol-P-Man synthase, DPMS) in Saccharomyces cerevisiae. Earlier studies with cDNA cloning and sequence analysis have established that 31-kDa DPMS of S. cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA). We have been studying the up-regulation of DPMS activity by protein kinase A-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S. cerevisiae in this study to advance our knowledge further. DPMS catalytic activity was indeed enhanced severalfold when the recombinant protein was phosphorylated in vitro. The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme. A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P). Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme. In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and enzyme efficiency (kcat/Km). SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS. To confirm the phosphorylation activation of recombinant DPMS, serine 141 in the consensus sequence was replaced with alanine by PCR site-directed mutagenesis. The S141A DPMS mutant exhibited more than half-a-fold reduction in catalytic activity compared with the wild type when both were analyzed after in vitro phosphorylation. Thus, confirming that S. cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine 141. << Less

  J. Biol. Chem. 280:4174-4181(2005) [PubMed] [EuropePMC]

• Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate. Partial purification and characterization of the solubilized enzyme.

  Babczinski P., Haselbeck A., Tanner W.

  The first mannosyl unit of manno-oligosaccharides of fungal mannoproteins is transferred in a dolichyl-phosphate-dependent reaction sequence to serine/threonine residues of the protein. The two membrane-bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubili ... >> More

  The first mannosyl unit of manno-oligosaccharides of fungal mannoproteins is transferred in a dolichyl-phosphate-dependent reaction sequence to serine/threonine residues of the protein. The two membrane-bound enzymes catalyzing this transfer in the yeast Saccharomyces cerevisiae have been solubilized by detergents. The enzyme transferring mannose from guanosine diphosphate mannose to dolichyl phosphate has been purified 18-fold when based on membrane protein and 140-fold when based on total cell protein. The enzyme transferring mannose from dolichyl phosphate mannose to protein has been purified 48-fold and 380-fold, respectively. A HCl-treated cell-wall mannoprotein from yeast served as acceptor protein for the second enzyme. The solubilized enzyme catalyzing the formation of dolichyl diphosphate mannose has a Km for guanosine diphosphate mannose of 7 x 10(-6) M and is saturated with about 0.15 mM yeast dolichyl phosphate. The metal requirement, pH-optima, and the detergent concentration necessary for optimal activity have been determined for both solubilized enzymes. << Less

  Eur J Biochem 105:509-515(1980) [PubMed] [EuropePMC]

• Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae.

  Haselbeck A.

  The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of colum ... >> More

  The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis. The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band. Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment. << Less

  Eur J Biochem 181:663-668(1989) [PubMed] [EuropePMC]

• Ppm1, a novel polyprenol monophosphomannose synthase from Mycobacterium tuberculosis.

  Gurcha S.S., Baulard A.R., Kremer L., Locht C., Moody D.B., Muhlecker W., Costello C.E., Crick D.C., Brennan P.J., Besra G.S.

  Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that ar ... >> More

  Dolichol monophosphomannose (DPM) is an ever-present donor of mannose (Man) in various eukaryotic glycosylation processes. Intriguingly, the related polyprenol monophosphomannose (PPM) is involved in the biosynthesis of lipomannan and lipoarabinomanan, key bacterial factors termed modulins that are found in mycobacteria. Based on similarities to known DPM synthases, we have identified and characterized the PPM synthase of Mycobacterium tuberculosis, now termed Mt-Ppm1. In the present study, we demonstrate that Mt-Ppm1 possesses an unusual two-domain architecture, by which the second domain is sufficient for PPM synthesis. However, when overexpressed separately in mycobacteria, domain 1 of Mt-Ppm1 appears to increase the synthesis of PPM. Interestingly, other mycobacteria such as M. smegmatis, M. avium and M. leprae produce two distinct proteins, which are similar to the two domains found in Mt-Ppm1. Using an in vitro assay, we also demonstrate that Mt-Ppm1 transfers Man from GDP-Man to a structurally diverse range of lipid monophosphate acceptors. The identification of the PPM synthase as a key enzyme in lipoarabinomannan biosynthesis now provides an attractive candidate for gene disruption to generate mutants for subsequent immunological studies. PPM synthase can also be exploited as a target for specific inhibitors of M. tuberculosis. << Less

  Biochem. J. 365:441-450(2002) [PubMed] [EuropePMC]