ABSTRACT
Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme-phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity.
Subject(s)
Mannosyltransferases/chemistry , Saccharomyces cerevisiae/enzymology , Amino Acid Sequence , Binding Sites , Energy Transfer , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/chemistry , Humans , Kinetics , Molecular Sequence Data , Sequence Homology, Amino Acid , Spectrometry, FluorescenceABSTRACT
Derivatives of dolichyl phosphate (Dol-P) with 2-aminopyridine or 1-aminonaphtalene fluorophore groups at the omega-end of the chain were synthesized. These products serve as substrates for recombinant yeast Dol-P-mannose synthase. Fluorescence resonance energy transfer between a Trp residue of the enzyme and the 1-aminonaphtalene group of the Dol-P analogue was demonstrated.
Subject(s)
Dolichol Phosphates/chemical synthesis , Fluorescent Dyes/chemical synthesis , Glycoproteins/chemistry , 1-Naphthylamine/chemistry , Aminopyridines/chemistry , Kinetics , Mannosyltransferases/metabolism , Substrate Specificity , Yeasts/enzymologyABSTRACT
Two phosphate-modified analogues of dolichyl phosphate were evaluated as substrates or inhibitors of the reactions catalyzed by mammalian microsomal enzymes. Dolichyl H-phosphonate could serve as an efficient acceptor for mannosyl and glucosyl transfer. The reaction products were chromatographically different from those formed from dolichyl phosphate. Lower activity of the H-phosphonate was observed for the reaction of N-acetylglucosaminyl phosphate transfer from UDP-GlcNAc. Dolichyl sulphate was shown not to serve as a substrate for the transfer of mannosyl (from GDP-Man), glucosyl (from UDP-Glc) or N-acetylglucosaminyl phosphate (from UDP-GlcNAc) residues in the presence of rat liver microsomes. Weak inhibitory properties of this analogue were demonstrated.
Subject(s)
Dolichol Phosphates/metabolism , Sulfates/metabolism , Animals , Brain/enzymology , Chromatography, Thin Layer , Dose-Response Relationship, Drug , Glycosylation , Glycosyltransferases/metabolism , Hydrogen-Ion Concentration , Microsomes, Liver/enzymology , Rats , Rats, Wistar , Temperature , Time FactorsABSTRACT
A novel antiviral drug with immunomodulatory activity (Phosprenyl) is presented. The main active ingredient of the preparation is polyprenyl phosphates. This medicine is highly efficient against a number of viruses, including HIV in vitro, and tick-borne encephalitis and rabies viruses in the experimental models in vivo. In veterinary practice Phosprenyl is now regarded as an effective therapeutic means for treatment of canine distemper, hepatitis, enteritis, etc.
Subject(s)
Adjuvants, Immunologic , Antiviral Agents , Dog Diseases/drug therapy , Polyisoprenyl Phosphates , Virus Diseases/veterinary , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Adjuvants, Immunologic/therapeutic use , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Clinical Trials as Topic , Cricetinae , Distemper/drug therapy , Dogs , Dolichol Phosphates/physiology , Drug Evaluation, Preclinical , Enteritis/drug therapy , Enteritis/veterinary , Hepatitis, Viral, Animal/drug therapy , Mice , Molecular Structure , Parvoviridae Infections/drug therapy , Parvoviridae Infections/veterinary , Polyisoprenyl Phosphates/chemistry , Polyisoprenyl Phosphates/pharmacology , Polyisoprenyl Phosphates/therapeutic use , Rats , Virus Diseases/drug therapyABSTRACT
A series of polyprenyl phosphates with modified structure of polyprenyl residue was prepared through phosphorylation of polyprenyl trichloroacetimidates with phosphoric acid. Interaction of polyprenols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile was found to represent a very efficient, simple and general method for the synthesis of polyprenyl phosphates. A procedure was developed for smooth conversion of polyprenyl pyrophosphates into the monophosphates through hydrolysis in the presence of 4-dimethylaminopyridine. The polyprenyl phosphates prepared were studied as substrates for the enzymes of Salmonella anatum O-specific polysaccharide biosynthesis. Correct stereochemistry of alpha- and beta-isoprenic units was found to be essential for substrate efficiency. At the more remote positions of the hydrocarbon chain just the presence of isoprenic units of any configuration seems necessary. Some changes in position of the phosphate group may be permissible without significant loss of substrate properties.
