New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.     

Novel fluorescent analogues for transmembrane movement study of polyprenyl phosphates

In order to investigate the transmembrane movement of polyprenyl phosphate across biological membranes, NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled polyprenyl phosphate analogues were prepared. These analogues proved to be possible tools for a direct observation of the transmembrane flip-flop movement of polyprenyl phosphates by use of a sodium dithionite-quenching procedure.     

Characterization of the Saccharomyces cerevisiae cis-prenyltransferase required for dolichyl phosphate biosynthesis

The prenyltransferase involved in the biosynthesis of dolichyl phosphate has been characterized in Saccharomyces cerevisiae. Although the enzyme is predominantly membrane-bound, a significant percentage was found in the soluble fraction. The prenyltransferase preferentially utilizes farnesyl pyrophosphate as the allylic substrate and isopentenyl pyrophosphate as cosubstrate with half-maximal velocities obtained at 25 and 6.7 microM, respectively. The enzymatic activity is sensitive to sulfhydryl reagents and is inhibited by all detergents tested, except 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate at concentrations less than 5 mM. The product of the reaction has been characterized as an alpha-unsaturated polyprenyl pyrophosphate, containing 12-15 isoprene units, approximately two isoprene units shorter than the endogenous yeast dolichyl phosphate. The stereochemistry of addition of isoprene units by the prenyltransferase was shown to be cis by a comparison of the HPLC retention time for a pentadecaprenyl phosphate derived from the in vitro reaction product with that for an authentic mixture of alpha-cis- and alpha-trans-pentadecaprenyl phosphates.     

Membrane properties of branched polyprenyl phosphates, postulated as primitive membrane constituents

We have postulated earlier that the highly branched isoprenoid alkanes, which are distributed widely in many sediments, may have been derived from the corresponding branched polyprenyl phosphates, potentially present in biomembranes in primitive organisms. These polyprenyl-branched polyprenyl phosphates might be derived by a simple alkylation from non-substituted polyprenyl phosphates, which we postulate to be the precursors of all membrane terpenoids. We have now synthesized a series of 6-(poly)prenyl-substituted polyprenyl phosphates and studied the formation of vesicles from these phosphates, as a function of the substituted-chain length, the position of the double bond, and pH. Nine of the branched polyprenyl phosphates containing 20-30 C-atoms do form vesicles at a 'physiological' pH; the lipophilicity/hydrophilicity ratio is as expected an important factor. We have also studied the water permeability through membranes of these branched polyprenyl phosphate vesicles by our stopped-flow/light-scattering method. These highly branched polyprenyl phosphates can more effectively reduce the water permeability than non-substituted polyprenyl phosphates: the vesicles formed by the former are more stable against mechanical stress. This reinforces our hypothesis about the origin of the sedimentary polyprenyl-substituted polyprene hydrocarbons.     

Glycoprotein biosynthesis in Chlamydomonas. A mannolipid intermediate with the properties of a short-chain alpha-saturated polyprenyl monophosphate

A crude membrane preparation of the unicellular green alga Chlamydomonas reinhardii was found to catalyse the incorporation of D-[14C]mannose from GDP-D-[14C]-mannose into a chloroform/methanol-soluble compound and into a trichloroacetic acid-insoluble polymer fraction. The labelled lipid revealed the chemical and chromatographic properties of a short-chain (about C55-C65) alpha-saturated polyprenyl mannosyl monophosphate. In the presence of detergent both long-chain (C85-C105) dolichol phosphate and alpha-unsaturated undecaprenyl phosphate (C55) were found to be effective as exogenous acceptors of D-mannose from GDP-D-[14C]mannose to yield their corresponding labelled polyprenyl mannosyl phosphates. Exogenous dolichyl phosphate stimulated the incorporation of mannose from GDP-D-[14C]mannose into the polymer fraction 5-7-fold, whereas the mannose moiety from undecaprenyl mannosyl phosphate was not further transferred. Authentic dolichyl phosphate [3H]mannose and partially purified mannolipid formed from GDP-[14C]mannose and exogenous dolichyl phosphate were found to function as direct mannosyl donors for the synthesis of labelled mannoproteins. These results clearly indicate the existence of dolichol-type glycolipids and their role as intermediates in transglycosylation reactions of this algal system. Both the saturation of the alpha-isoprene unit and the length of the polyprenyl chain may be regarded as evolutionary markers.     

Glycosyltransfer by pea membranes from sugar nucleotides to added prenyl phosphates

Pea membranes supplied with GDP-[14C]mannose, UDP-N-[14C]acetylglucosamine or UDP-[14C]glucose catalyze the transfer of 14C-labeled sugars or sugar phosphates to endogenous lipid acceptors as well as to exogenously added dolichyl phosphates. Fully unsaturated polyprenyl phosphates were not used as effective acceptors by this system. Mannosyl-P-dolichol was formed most rapidly in the presence of long-chained dolichyl-P while mannosyl-PP-, glucosyl-PP- and GlcNAc-PP-dolichol were preferentially formed from relatively short-chained dolichyl phosphate acceptors. Glucosyl-PP- and mannosyl-PP-dolichol accumulated in the preparation without further metabolism, but GlcNAc-PP-dolichol was lengthened by addition of a second GlcNAc plus several [14C]mannose units to form an oligosaccharide fraction susceptible to the action of endoglycosidase H. This lipid-linked oligosaccharide could then be glycosylated in the presence of UDP-[14C]glucose to form a longer oligosaccharide. It is concluded that levels of endogenous dolichyl phosphates in pea membranes are rate-limiting for several of the key glycosyltransferases required for oligosaccharide assembly.     

Proteins containing reductively aminated disaccharides: immunochemical characterization.

Various polyprenyl phosphates were prepared by chemical phosphorylation of native and partially hydrogenated polyprenols. They were tested as lipid acceptors of sugars from nucleoside diphosphate sugars using a microsomal preparation from rat liver and membrane preparations from B. stearothermophilus, S. typhimurium, and Sh. flexneri. With the microsomal glycosyl transferase system, a demand for saturation of the α-isoprene residue of polyprenyl phosphate was observed; the chain length and cis/trans configuration of polyprenyl radical were less important. With bacterial glycosyl transferases, a demand for the unsaturated α-isoprene residue was observed. In B. stearothermophilus, the rate of synthesis of polyprenyl monophosphate glucose did not depend on the chain length of fully unsaturated polyprenyl phosphate. In S. typhimurium, C₅₅-polyprenyl phosphate was the most effective precursor of polyprenyl diphosphate galactose.     

CpsE from type 2 Streptococcus pneumoniae catalyzes the reversible addition of glucose-1-phosphate to a polyprenyl phosphate acceptor, initiating type 2 capsule repeat unit formation

The majority of the 90 capsule types made by the gram-positive pathogen Streptococcus pneumoniae are assembled by a block-type mechanism similar to that utilized by the Wzy-dependent O antigens and capsules of gram-negative bacteria. In this mechanism, initiation of repeat unit formation occurs by the transfer of a sugar to a lipid acceptor. In S. pneumoniae, this step is catalyzed by CpsE, a protein conserved among the majority of capsule types. Membranes from S. pneumoniae type 2 strain D39 and Escherichia coli containing recombinant Cps2E catalyzed incorporation of [14C]Glc from UDP-[14C]Glc into a lipid fraction in a Cps2E-dependent manner. The Cps2E-dependent glycolipid product from both membranes was sensitive to mild acid hydrolysis, suggesting that Cps2E was catalyzing the formation of a polyprenyl pyrophosphate Glc. Addition of exogenous polyprenyl phosphates ranging in size from 35 to 105 carbons to D39 and E. coli membranes stimulated Cps2E activity. The stimulation was due, in part, to utilization of the exogenous polyprenyl phosphates as an acceptor. The glycolipid product synthesized in the absence of exogenous polyprenyl phosphates comigrated with a 60-carbon polyprenyl pyrophosphate Glc. When 10 or 100 microM UMP was added to reaction mixtures containing D39 membranes, Cps2E activity was inhibited 40% and 80%, respectively. UMP, which acted as a competitive inhibitor of UDP-Glc, also stimulated Cps2E to catalyze the reverse reaction, with synthesis of UDP-Glc from the polyprenyl pyrophosphate Glc. These data indicated that Cps2E was catalyzing the addition of Glc-1-P to a polyprenyl phosphate acceptor, likely undecaprenyl phosphate.     

Synthesis of 2-deoxy-2-fluoro analogs of polyprenyl beta-D-arabinofuranosyl phosphates

Described is the synthesis of polyprenyl 2-deoxy-2-fluoro-beta-D-arabinofuranosyl phosphate derivatives, including an analog of decaprenyl beta-D-arabinofuranosyl phosphate, the donor species used by the arabinosyltransferases involved in mycobacterial cell-wall biosynthesis. The targets were synthesized via a route involving the synthesis of a protected beta-D-arabinofuranosyl phosphate derivative, its coupling with a polyprenyl trichloroacetimidate, and then deprotection of the resulting product. The use of arabinofuranosyl phosphates with the monosaccharide hydroxyl groups protected as either silyl ethers or benzoate esters was explored. Although the coupling yields between the phosphate and polyprenyl trichloroacetimidates were comparable with either type of protecting group, access to the benzoyl-protected derivative was more efficient and therefore gave the products in higher overall yield.     

In vivo biosynthesis of the saturated isoprene unit of dolichyl phosphate

Reduction of the e-isoprene unit of polyprenols to form dolichols was studied in vivo using³H-polyprenol derivatives as substrates and liposomes as carriers. Liposomes containing labeled polyprenol, polyprenyl phosphate, or polyprenyl pyrophosphate were injected through the portal vein into the livers of rats under anesthesia. Uptake and conversion of the labeled compounds to dolichol derivatives was studied at different intervals. The greatest conversion to dolichol derivatives was found with polyprenyl pyrophosphate and polyprenyl monophosphate, with 31% and 8% of the absorbed dose converted respectively. Less than 0.2% of the absorbed polyprenol was converted to dolichol derivatives. These results suggest that the substrate for the -isoprene reductase involved in dolichol biosynthesis is either polyprenyl monophosphate or polyprenyl pyrophosphate, or both.     

Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae

This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.

A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an α-saturated polyprenyl phosphate.

The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [¹⁴C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C₉₀ to C₁₀₅, C₉₅ and C₁₀₀ being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.

The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate.

     

Characterization and distribution of cis-prenyl transferase participating in liver microsomal polyisoprenoid biosynthesis.

The properties of rat liver cis-prenyl transferase, mediating the synthesis of polyisoprenoid pyrophosphate from trans,trans-farnesyl pyrophosphate and [3H]isopentenyl pyrophosphate were studied. The Km values for farnesyl pyrophosphate and isopentenyl pyrophosphate were found to be 25 microM and 4.4 microM, respectively. Appropriate conditions were established to measure the condensation reaction, which was linear during the first hour using 1 mg microsomal protein. Various detergents could solubilize the enzyme, but the presence of Triton X-100 was required during the incubation to obtain full activity. There was also an absolute requirement for Mg2+ and the pH maximum was 7.0. Inorganic phosphate, especially pyrophosphate, proved to be inhibitory. cis-Prenyl transferase is associated mainly with the cytoplasmic surface of rough microsomes and, to some extent, also with smooth I microsomes, but was almost absent from smooth II microsomes. At all localizations, the product is polyprenyl pyrophosphate and to some extent, also polyprenyl monophosphate. The isoprenoids formed contain 15-18 units in the presence of detergents and 16-20 units in the absence of detergents.     

The pssA gene encodes UDP-glucose: polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of Rhizobium leguminosarum exopolysaccharide

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum by. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation, with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.     

Substrate specificity of N-acetylglucosamine 1-phosphate transferase activity in Chinese hamster ovary cells.

The assembly pathway of the oligosaccharide chains of asparagine-linked glycoproteins in mammalian cells begins with the formation of GlcNAc-PP-dolichol in a reaction catalysed by the enzyme N-acetylglucosamine 1-phosphate transferase. We have investigated the efficiency of two lipid substrates for the transferase activity in an in vitro assay using Chinese hamster ovary (CHO) cell membranes as an enzyme source. Experiments were carried out with varying concentrations of dolichyl phosphate or its precursor, polyprenyl phosphate. We determined that enzyme activity was optimal at pH 9, where the enzyme exhibited a 3-fold higher Vmax and a 2-fold lower Km for the dolichol substrate. At pH 7.4, the Km and Vmax differences between the two lipids were 10-fold. Under all assay conditions tested, we found that GlcNAc-PP-lipid was the only product formed. We conclude from these results that dolichyl phosphate rather than polyprenyl phosphate is the preferred substrate for the transferase enzyme in CHO cells. This observation is significant in light of the fact that we have previously isolated CHO glycosylation mutants which fail to convert polyprenol into dolichol, and hence utilize polyprenyl derivatives for glycosylation reactions. Thus, these results contribute to our understanding of the glycosylation defects in the mutant cell lines.     

The <Emphasis Type="Italic">pssA</Emphasis> gene encodes UDP-glucose: Polyprenyl phosphate-glucosyl phosphotransferase initiating biosynthesis of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> exopolysaccharide

Symbiotic nitrogen-fixing bacteria Rhizobium leguminosarum bv. viciae VF39 secrete an acidic heteropolysaccharide, the biosynthesis of which involves the stage of polyprenyl diphosphate octasaccharide formation with its carbohydrate fragment corresponding to the repeating polymer unit. The amino acid analysis of the product of the pssA gene, we have earlier identified, showed its homology to bacterial polyisoprenyl phosphate hexose 1-phosphate transferases catalyzing the formation of phosphodiester bonds between polyprenyl phosphates and hexose 1-phosphates, whose donors are nucleotide sugars. The immunoblotting demonstrated that Rhizobium cells synthesize a protein with a molecular mass of 25 kDa, which implies the translation of the open reading frame occurring from the second initiating codon followed by the protein processing. It was shown that PssA is an integral membrane-bound protein involved in glucose 1-phosphate transfer from UDP-glucose to polyprenyl phosphate to form polyprenyl diphosphate glucose. These results suggest that the pssA gene encodes UDP-glucose:polyprenyl phosphate-glucosyl phosphotransferase.     

Chemoenzymatic synthesis of polyprenyl phosphates

Polyprenyl phosphates, including undecaprenyl phosphate and dolichyl phosphate, are essential intermediates in several important biochemical pathways including N-linked protein glycosylation in eukaryotes and prokaryotes and prokaryotic cell wall biosynthesis. Herein, we describe the evaluation of three potential undecaprenol kinases as agents for the chemoenzymatic synthesis of polyprenyl phosphates. Target enzymes were expressed in crude cell envelope fractions and quantified via the use of luminescent lanthanide-binding tags (LBTs). The Streptococcus mutans diacylglycerol kinase (DGK) was shown to be a very useful agent for polyprenol phosphorylation using ATP as the phosphoryl transfer agent. In addition, the S. mutans DGK can be coupled with two Campylobacter jejuni glycosyltransferases involved in N-linked glycosylation to efficiently biosynthesize the undecaprenyl pyrophosphate-linked disaccharide needed for studies of PglB, the C. jejuni oligosaccharyl transferase.     

Biosynthesis of carbohydrate-containing polymers in plants. I. Products formed with enzyme preparation from clover seedlings. Characterization of polyprenylphosphosugars

Membrane preparations from clover seedlings catalyzed the incorporation of monosaccharide residues from GDPMan, UDPGlc and UDPGal into glycolipids, lipid-oligosaccharides and polymers. The lipid-oligosaccharides were shown to be alpha-dihydropolyprenyl pyrophosphate derivatives. Incorporation of mannose residues into the lipid-oligosaccharides and the polymers was significantly stimulated by addition of UDPGlc, GDPGlc, UDPGal but not by UDPGlcNAc. Exogenic polyprenyl phosphates also stimulated the process; the formation of moraprenyl pyrophosphate oligosaccharides was demonstrated after addition of moraprenyl phosphate. The lipid-oligosaccharides were precursors of the polymers which were shown to be mainly glycoproteins. A solubilized preparation of mannosyl transferase from clover membranes was obtained and some properties of the enzyme were studied.     

Uptake and metabolism of polyprenols by animal cells cultured in vitro

Several mammalian, chicken, and mosquito cells grown in vitro take up tritiated dolichol supplied to the incubation medium. The extent of labelling varied markedly between different cell cultures. After 20 h incubation most of the dolichol taken up was unchanged and the major product of metabolism of dolichol was identified as its fatty acid esters. Green-monkey kidney cells were tested with 8 fully unsaturated and 6 alpha-saturated polyprenols ranging from C35 to C105. In general the uptake of alpha-saturated polyprenols (dolichol type, was higher. Considerable differences were found between the uptake of polyprenols of differing chain lengths. Less than 1% of the polyprenols taken up was converted into more polar product, mainly polyprenyl phosphates and polyprenyl phosphate sugars. The short-chain polyprenols, from C35 to C65, were metabolized more rapidly than the long-chain polyprenols, as judged from the amount of polar products and fatty acid esters of polyprenols.     

Biosynthesis of lipid-linked oligosaccharides by microsomes from virus induced Hepatoma MC-29. Dependence of some glycosyl transferases on dolichyl phosphates of different chain length

Dolichyl phosphates of various chain length ranging from 7 to 22 isoprene units were tested as lipid acceptors in transglycosylation reactions in chicken liver and Hepatoma MC-29. In the presence of exogenous dolichyl phosphate mixture (18 and 19 isoprene units) the synthesis of dolichyl pyrophosphate N-acetylglucosamine and dolichyl phosphate mannose increased 3 times both in the liver and Hepatoma MC-29, while the formation of dolichyl phosphate glucose was 4 fold higher in the liver and 6-fold higher in Hepatoma MC-29. In liver microsomes the maximum rate of the stimulation of glycosylation was achieved by exogenous dolichyl phosphates, containing 18 and 19 isoprene units, while glycosyl transferases in microsomes from Hepatoma MC-29 did not show any structural requirements to the chain length of dolichyl phosphates.     

Mechanism of reaction of acyl phosph(on)ates with the beta-lactamase of Enterobacter cloacae P99

A series of acyl phosph(on)ates has been prepared to more closely examine the details of the interactions of this class of molecule with beta-lactamases. In general, they were found to react with the class C beta-lactamase of Enterobacter cloacae P99 in two ways, by acylation and by phosphylation. The acyl-enzymes generated by the former reaction were transiently stable with half-lives of between 3 and 45 s, of comparable lifetime therefore to those generated by the inhibitory beta-lactams cefotaxime, cefuroxime, and cefoxitin. On the other hand, phosphylation led to a completely inactive enzyme. In general, the second-order rate constants for acylation (k(cat)/K(m)) were larger than for phosphylation (k(i)). As expected on chemical grounds, phosphylation was found to be relatively more effective for the phosphonates than the phosphates. The acyl phosphates were much more effective acylating agents however. The acylation reaction was found to be enhanced by hydrophobic substituents in both the acyl and leaving group moieties. Thus, the most reactive compound in this series was benzo[b]thiophene-2-carbonyl 2'-naphthyl phosphate with a K(m) value of 0.15 microM and a k(cat) of 0.2 s(-1); k(cat)/K(m) is therefore 1.3 x 10(6) s(-1) M(-1), making this compound the most specific acyclic acylation reagent for this beta-lactamase yet described. Significant substrate inhibition by this compound suggested that further binding regions may be available for exploitation in inhibitor design. A linear free energy analysis showed that the transition states for acylation of the beta-lactamase by aroyl phosphates are analogues of the corresponding aryl boronic acid adducts. Molecular modeling suggested that the aroyl phosphates react with the P99 beta-lactamase with the aroyl group in the side chain/acyl group site of normal substrates and the phosphate in the leaving group site. In this orientation, the phosphate leaving group interacts strongly with Lys 315.