Biosynthesis and characterization of large dolichyl diphosphate-linked oligosaccharides in Sacchromyces cerevisiae

Yeast membranes incorporate radioactivity from GDP[¹⁴C]mannose into various glycolipids. These can be separated by thin layer chromatography into at least seven components.The major component has been identified previously as dolichyl monophosphate mannose. Only one additional component is not sensitive to mild alkaline saponification, but is hydrolyzed instead under mild acidic conditios. This latter glycolipid has all the characteristics of a polyprenyl diphosphate oligosaccharide with a sugar moiety of more than 12 hexose units. It runs like dolichyl diphosphate derivatives on a DEAE column and evidence is presented that the lipid moiety is a polyprenol.When radioactive Dol-PP-di-N-acetylchitobiose is incubated with yeast membranes in the presence of non-radioactive GDPmannose a small amount of a larger lipid oligosaccharide is formed besides the previously-described Dol-PP-(GlcNAc₂ mannose. This oligosaccharide has all the properties of the glycolipid described above. Its formation is greatly increased when Triton is omitted from the incubation. Radioactivity of the polyprenyl diphosphate [¹⁴C]oligosaccharide is transferred to ethanol-insoluble material, most likely endogenous membrane glycoproteins.     

Formation of lipid-bound oligosaccharides in yeast

Incubation of a membrane fraction from Saccharomyces cerevisiae with UDP-N-acetyl [¹⁴C] glucosamine catalyzes the tranfer of N-acetylglucosamine to an endeenous lipid fraction as well as a methanol-insoluble polymer. The glycolipid was shown to separate into three compounds by thin-layer chromatography. The biosynthesis of two of them could clearly be stimulated by the addition of dolichol monophosphate to the incubation mixture. Evidence is presented that the substances are dolichol pyrophosphate derivatives: dolichol pyrophosphate N-acetylglucosamine and dolichol pyrophosphate di-N-acetylchitobiose. The formation of the chitobiose-containing lipid was increased by reincubation of the glycolipid with non-radioactive UDP-N-acetylglucosamine.The same particulate preparation transferred mannose from GDPmannose to dolichol pyrophosphate di-N-acetylchitobiose, giving rise to a lipid-bound oligosaccharide. Molecular weight determination of the oligosaccharide moiety gave a value of 780, which is consistent with a tetrasaccharide containing two mannose subunits attached to di-N-acetylchitobiose.The methanol-insoluble radioactive product obtained in the presence of UDP-N-acetyl[¹⁴C]glucosamine was transformed by pronase treatment to a large extent into dialyzable material. It is suggested that the glycolipids described serve as intermediates in the glycosylation of yeast mannoproteins.     

Studies on substrate specificity and activity regulating factors of trehalose-6-phosphate synthase of Saccharomyces cerevisiae

Purified trehalose-6-phosphate synthase (TPS) of Saccharomyces cerevisiae was effective over a wide range of substrates, although differing with regard to their relative activity. Polyanions heparin and chondroitin sulfate were seen to stimulate TPS activity, particularly when a pyrimidine glucose nucleotide like UDPG was used, rather than a purine glucose nucleotide like GDPG. A high V_max and a low K_m value of UDPG show its greater affinity with TPS than GDPG or TDPG. Among the glucosyl acceptors TPS showed maximum activity with G-6-P which was followed by M-6-P and F-6-P. Effect of heparin was also extended to the purification of TPS activity, as it helped to retain both stability and activity of the final purified enzyme. Metal co-factors, specifically MnCl₂ and ZnCl₂ acted as stimulators, while enzyme inhibitors had very little effect on TPS activity. Metal chelators like CDTA, EGTA stimulated enzyme activity by chelation of metal inhibitors. Temperature and pH optima of the purified enzyme were determined to be 40 °C and pH 8.5 respectively. Enzyme activity was stable at 0–40 °C and at alkaline pH.     

A Synthesis of Dolichyl Phosphorofluoridate

An improved method for the synthesis of dolichyl H-phosphonate was developed using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophosphite) as a reagent. Dolichyl phosphorofluoridate was for the first time synthesized from dolichyl H-phosphonate by its treatment with chlorotrimethylsilane,followed by oxidation with iodine in the presence of fluoride in pyridine.     

Evidence for multiple enzymes in the dolichol utilizing pathway of glycoprotein biosynthesis

A comparison has been made of the enzymes catalyzing the transfer of manose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented which suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not reverse of the N-acetylglucosamine or mannose transfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.     

Inhibition of glycosyltransferases by bis-(p-nitrophenyl)phosphate: general effect and relation to their membrane integration

The effect of bis-(p-nitrophenyl)phosphate on various glycosyltransferases involved in protein glycosylation (sialyl-, fucosyl-, galactosyl-, mannosyl- and glucosyltransferases) have been studied using crude enzyme preparations solubilized from rat spleen lymphocytes. Bis-(p-nitrophenyl)phosphate appears as a common inhibitor for every glycosyltransferase reaction utilizing sugar nucleotides as direct donors. In most cases 10 mM inhibitor is sufficient to obtain a 90 per cent inhibition. Kinetic studies achieved with a purified galactosyltransferase preparation reveal that bis-(p-nitrophenyl)phosphate exerts a competitive inhibition towards UDP-galactose binding. Concerning membrane-bound enzymes, the interaction of bis-(p-nitrophenyl)phosphate depends on its accessibility to the enzyme active site. This is shown by the different effect obtained with two UDP-Glc utilizing membrane-bound enzymes : UDP-Glc : phospho-dolichyl glucosyltransferase and UDP-Glc : ceramide glucosyltransferase : the first one not being affected but the second one being markedly inhibited under the same condition, although both are inhibited when the membrane environment is disturbed by detergent. Bis-(p-nitrophenyl)phosphate appears to be a tool to study membrane topology of glycosyltransferases.     

The first prenylation step in hyperforin biosynthesis

Prenylation reactions contribute considerably to the diversity of natural products. Polyprenylated secondary metabolites include hyperforin which is both quantitatively and pharmacologically a major constituent of the medicinal plant Hypericum perforatum (St. John's wort). Cell cultures of the related species Hypericum calycinum were found to contain a prenyltransferase activity which is likely to catalyze the first prenylation step in hyperforin biosynthesis. The enzyme was soluble and dependent on a divalent cation, with Fe2+ leading to maximum activity (Km=3.8 mM). The preferred prenyl donor was DMAPP (Km=0.46 mM) and the preferred prenyl acceptor was phlorisobutyrophenone (Km=0.52 mM). A broad pH optimum from 6.5 to 8.5 and a temperature optimum from 35 to 40 degrees C were observed. The formation of hyperforins in H. calycinum cell cultures was preceded by an increase in dimethylallyltransferase activity, with the maximum specific activity being 3.6 microkat/kg protein.     

Carbohydrate-deficient glycoprotein syndrome type 1: correction of the glycosylation defect by deprivation of glucose or supplementation of mannose

In the carbohydrate deficient glycoprotein syndrome (CDGS) type 1 glycoproteins with less and shorter N-linked oligosaccharides are synthesized due to a deficiency of phosphomannomutase. Glucose deprivation or mannose addition are shown to partially or fully correct the size of oligosaccharides incorporated into lipid linked oligosaccharides and nascent glycoproteins in skin fibroblasts from CDGS type 1 patients with a phosphomannomutase defect. The corrective effect is ascribed to regulatory mechanisms and/or metabolic pathways that bypass phosphomannomutase.     

The crystal structure of human isopentenyl diphosphate isomerase at 1.7 A resolution reveals its catalytic mechanism in isoprenoid biosynthesis

The essential Saccharomyces cerevisiae pre-messenger RNA splicing protein 24 (Prp24) has four RNA recognition motifs (RRMs) and facilitates U6 RNA base-pairing with U4 RNA during spliceosome assembly. Prp24 is a component of the free U6 small nuclear ribonucleoprotein particle (snRNP) but not the U4/U6 bi-snRNP, and so is thought to be displaced from U6 by U4/U6 base-pairing. The interaction partners of each of the four RRMs of Prp24 and how these interactions direct U4/U6 pairing are not known. Here we report the crystal structure of the first three RRMs and the solution structure of the first two RRMs of Prp24. Strikingly, RRM 2 forms extensive inter-domain contacts with RRMs 1 and 3. These contacts occupy much of the canonical RNA-binding faces (beta-sheets) of RRMs 1 and 2, but leave the beta-sheet of RRM 3 exposed. Previously identified substitutions in Prp24 that suppress mutations in U4 and U6 spliceosomal RNAs cluster primarily in the beta-sheet of RRM 3, but also in a conserved loop of RRM 2. RNA binding assays and chemical shift mapping indicate that a large basic patch evident on the surface of RRMs 1 and 2 is part of a high affinity U6 RNA binding site. Our results suggest that Prp24 binds free U6 RNA primarily with RRMs 1 and 2, which may remodel the U6 secondary structure. The beta-sheet of RRM 3 then influences U4/U6 pairing through interaction with an unidentified ligand.     

Membrane-bound geranylgeranyl diphosphate phosphatases: purification and characterization from Croton stellatopilosus leaves

Geranylgeranyl diphosphate phosphatase is an enzyme catalyzing the dephosphorylation of geranylgeranyl diphosphate (GGPP) to form geranylgeraniol (GGOH). The enzyme activity of GGPP phosphatase was detected in leaves of Croton stellatopilosus, a Thai medicinal plant containing plaunotol, a commercial anti-peptic acyclic diterpenoid. Enzymological studies of GGPP phosphatase in C. stellatopilosis leaves revealed that the enzyme is a membrane-bound protein that could be removed from 20,000g pellet by 0.1% Triton X-100 without significant loss of enzyme activity. The solubilized enzyme preparation was separated into two activity peaks, PI and PII, by BioGel A gel filtration chromatography. PI and PII were both partially purified and characterized. PI appeared to be a tetrameric enzyme with its native molecular mass of 232kDa and subunit size of 58kDa, whereas PII was a monomeric enzyme with a molecular mass of 30-34kDa. Both phosphatases utilized GGPP as the preferred substrate over farnesyl and geranyl diphosphates. The apparent K(m) values for GGPP of PI and PII appeared to be 0.2 and 0.1mM, respectively. Both activities were Mg(2+) independent and exhibited slightly acidic pH optima, 6.0-6.5 for PI and 6.5-7.0 for PII. The catalytic activities of PII was strongly inhibited by 1.0mM of Zn(2+), Mn(2+) and Co(2+), whereas that of PI was not affected. Both enzyme preparations were very stable upon storage at -20 degrees C for 45 days without significant loss of phosphatase activity. The presence of GGPP phosphatase enzymes in C. stellatopilosus is consistent with its putative involvement in the biosynthetic pathway of plaunotol although whether PI or PII is the actual enzyme involved in the pathway remains to be clarified.     

A subunit of decaprenyl diphosphate synthase stabilizes octaprenyl diphosphate synthase in Escherichia coli by forming a high-molecular weight complex

The length of the isoprenoid-side chain in ubiquinone, an essential component of the electron transport chain, is defined by poly-prenyl diphosphate synthase, which comprises either homomers (e.g., IspB in Escherichia coli) or heteromers (e.g., decaprenyl diphosphate synthase (Dps1) and D-less polyprenyl diphosphate synthase (Dlp1) in Schizosaccharomyces pombe and in humans). We found that expression of either dlp1 or dps1 recovered the thermo-sensitive growth of an E. coli ispB^R321A mutant and restored IspB activity and production of Coenzyme Q-8. IspB interacted with Dlp1 (or Dps1), forming a high-molecular weight complex that stabilized IspB, leading to full functionality.

Structured summary:

MINT-7385426:Dlp1 (uniprotkb:Q86YH6) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385083, MINT-7385058:IspB (uniprotkb:P0AD57) and IspB (uniprotkb:P0AD57) bind (MI:0407) by blue native page (MI:0276)MINT-7385413:Dlp1 (uniprotkb:O13851) and IspB (uniprotkb:P0AD57) physically interact (MI:0915) by blue native page (MI:0276)MINT-7385024:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dps1 (uniprotkb:O43091) by pull down (MI:0096)MINT-7385041:IspB (uniprotkb:P0AD57) physically interacts (MI:0915) with Dlp1 (uniprotkb:O13851) by pull down (MI:0096)MINT-7385388:IspB (uniprotkb:P0AD57) and Dps1 (uniprotkb:O43091) physically interact (MI:0915) by blue native page (MI:0276)     

On the feasibility of electron transfer to singlet oxygen from mitochondrial components

The incorporation of [¹⁴C]mannose from GDP-[¹⁴C]mannose into dolichyl mannosyl phosphate in rat liver microsomes showed a biphasic time-course; an initial rapid incorporation of mannose which ceased within 2 min and a much slower incorporation which continued for 30 min. In the presence of 0.18 mM (250 μg/ml) bacitracin, the rapid incorporation proceeded normally whereas the slow incorporation was inhibited by about 70%. Upon addition of dolichyl pyrophosphate, the microsomes catalyzed the dephosphorylation of the added compound which was also inhibited by bacitracin. The results, coupled with several other observations, suggest that the rapid reaction represents the transfer of mannose to endogenous dolichyl phosphate whereas the bacitracin-sensitive, slow reaction represents a more complex process in which the enzymatic dephosphorylation of dolichyl pyrophosphate is involved as a rate-limiting step.