Formation of 3-hexaprenyl-4-hydroxybenzoate by matrix-free mitochondrial membrane-rich preparations of yeast.

It has been shown that a 10 000 x g matrix-free mitochondrial membrane-rich preparation from commercial bakers' yeast is able to synthesize 3-all-transhexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate and isopentenyl pyrophosphate. The synthesis is Mg2+ dependent and is stimulated markedly by the primer for polyprenylpyrophosphate synthesis of 3-hexaprenyl-4-hydroxybenzoate from 4-hydroxybenzoate, isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate the priming function of 3,3-dimethylallyl pyrophosphate can be performed by either geranyl pyrophosphate (most efficient) or farnesyl pyrophosphate. At high Mg2+ concentrations, however, geranyl pyrophosphate and farnesyl pyrophosphate act mainly as sources of preformed side chains and 3-diprenyl- and 3-tripenyl-4-hydroxybenzoate, respectively, are produced. In the presence of a source of preformed polyprenyl pyrophosphates the membrane preparations catalysed the polyprenylation of methyl-4-hydroxybenzoate, 4-hydroxybenzaldehyde, 4-hydroxybenzylalcohol and 4-hydroxycinnamate. No evidence was obtained for the involvement of either 4-hydroxybenzoyl CoA or 4-hydroxybenzoyl-S-protein in the formation of 3-polyprenyl-4-hydroxybenzoates.     

Stimulation of polyprenyl 4-hydroxybenzoate transferase activity by sodium cholate and 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate

Polyprenyl 4-hydroxybenzoate transferase (Coq2p) plays a central role in ubiquinone biosynthesis. Coq2p mediates the conjugation of 4-hydroxybenzoate, the benzoquinone ring precursor, with the completed side chain. The activity is most easily assayed by measuring the rate of incorporation of 4-hydroxybenzoate as radiolabeled substrate into polyprenyl 4-hydroxybenzoate. The in vitro assay requires addition of a detergent into the reaction mixture to activate enzyme activity, and Triton X-100 is used for this purpose in the routine assay. We have found that both 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate and sodium cholate, but not sodium deoxycholate, lysophosphatidyl choline, or octylglucoside, significantly stimulate the activity over that measured with Triton X-100. High-performance liquid chromatography analysis of lipid extracts revealed that the increase of specific activity resulted in a similar increase in reaction product, this effect is due not merely to a better lipid extraction but also to the actual stimulation of enzyme activity. With our improved method, we were able to measure Coq2p activity with much greater sensitivity in both fresh and frozen/thawed mitochondria and in crude homogenates obtained from cultured cells. Our method will simplify evaluation of Coq2p activity in scarce biological materials, such as cells obtained from human tissue biopsies, and thus it will facilitate the biochemical characterization of ubiquinone deficiencies.     

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.     

Polyprenyl pyrophosphate–p-hydroxybenzoate polyprenyltransferase activity in mitochondria of broad-bean seeds and yeast (Short Communication)

Cell-free homogenates prepared from broad-bean seeds and yeast cells are capable of synthesizing 4-carboxy-2-polyprenylphenols from p-hydroxybenzoate and either isopentenyl pyrophosphate or protein-bound polyprenyl pyrophosphates (produced by incubating a Micrococcus lysodeikticus extract with isopentenyl pyrophosphate). The mitochondria contained all the polyprenyl pyrophosphate-p-hydroxybenzoate polyprenyltransferase activity; however, unlike the homogenates they could not synthesize a side chain from isopentenyl pyrophosphate and had to be provided with protein-bound polyprenyl pyrophosphates.     

Enzymatic synthesis of polyprenyl phosphosugars in Salmonella serotype C1

The cell envelopes of serogroup C1 Salmonella, viz. S. thompson and S. montevideo, catalyze the transfer of radiolabeled sugars from UDP-[14C]Glc and UDP-[14C]GlcNAc into the lipid-linked sugars. Using TLC and DEAE-cellulose chromatography, the radiolabeled products were identified as polyprenyl pyrophosphate N-acetylglucosamine (I), polyprenyl monophosphate N-acetylglucosamine and polyprenyl monophosphate glucose. The derivative (I) served as an acceptor for mannose transfer from GDP-Man with formation of Man1-2GlcNAc1PPPre. A similar reaction was observed after addition of synthetic GlcNAc1PPPre to the cell envelopes.     

Variable product specificity of solanesyl pyrophosphate synthetase

The distribution of polyprenyl pyrophosphates synthesized by the action of solanesyl pyrophosphate synthetase from Micrococcus luteus is dramatically changed depending on the Mg⁺⁺ concentration. When the metal ion concentration is higher than 5 mM, octaprenyl and solanesyl (nonaprenyl) pyrophosphate are synthesized predominantly. On the other hand, when the metal ion level is lower than 0.5 mM, a variety of polyprenyl pyrophosphates ranging in carbon chain length from C₁₅ to C₄₀ are formed. Heptaprenyl pyrophosphate is the longest of the products formed at 0.1 mM of Mg⁺⁺.     

Undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum: a dimeric protein

a++Undecaprenyl pyrophosphate synthetase has been purified from Lactobacillus plantarum. It catalyzes the formation of a C55 polyprenyl pyrophosphate having isoprene residues with cis stereochemistry. The enzyme was shown to be an acidic protein (pI = 5.1), which can be partially purified by preparative gel electrophoresis and Blue-agarose column chromatography. The Km's of the enzyme for its substrates t,t-farnesyl pyrophosphate and isopentenyl pyrophosphate were determined to be 0.13 and 1.92 microM, respectively. The molecular weight of the enzyme was estimated by molecular sieve chromatography and gradient centrifugation to be 56,000 +/- 4000. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the protein was composed of a dimer of 30,000-Da subunits. The enzyme was inactivated by the arginine-specific reagents phenylglyoxal, butanedione and, cyclohexanedione, but this inactivation was not prevented by either of the substrates.     

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.     

Synthesis of 4-carboxy-2-polyprenylphenols by a particulate fraction of baker's yeast

The preparation ofa cell-free homogenate and 10000 g particulate fraction with polyprenylpyrophosphate-p-hydroxybenzoate polyprenyltransferase activity from 0 to 7-day-old blocks of compressed baker's yeast is described. The synthesis of 4-carboxy-2-triprenylphenol from p-hydroxybenzoate and FPP by the particulate fraction has been studied in some detail. In particular it has been shown that the transferase catalysing the reaction is activated by Mg²⁺, has a pH optima of 7 and is inhibited by phosphate buffer. Intracellular distribution studies have established that in freshly grown cells of Saccharomyces carlsbergensis the greater part of the polyprenyl transferase activity is present in the mitochondria.     

Phenotypes of fission yeast defective in ubiquinone production due to disruption of the gene for p-hydroxybenzoate polyprenyl diphosphate transferase

Ubiquinone is an essential component of the electron transfer system in both prokaryotes and eukaryotes and is synthesized from chorismate and polyprenyl diphosphate by eight steps. p-Hydroxybenzoate (PHB) polyprenyl diphosphate transferase catalyzes the condensation of PHB and polyprenyl diphosphate in ubiquinone biosynthesis. We isolated the gene (designated ppt1) encoding PHB polyprenyl diphosphate transferase from Schizosaccharomyces pombe and constructed a strain with a disrupted ppt1 gene. This strain could not grow on minimal medium supplemented with glucose. Expression of COQ2 from Saccharomyces cerevisiae in the defective S. pombe strain restored growth and enabled the cells to produce ubiquinone-10, indicating that COQ2 and ppt1 are functional homologs. The ppt1-deficient strain required supplementation with antioxidants, such as cysteine, glutathione, and alpha-tocopherol, to grow on minimal medium. This suggests that ubiquinone can act as an antioxidant, a premise supported by our observation that the ppt1-deficient strain is sensitive to H(2)O(2) and Cu(2+). Interestingly, we also found that the ppt1-deficient strain produced a significant amount of H(2)S, which suggests that oxidation of sulfide by ubiquinone may be an important pathway for sulfur metabolism in S. pombe. Ppt1-green fluorescent protein fusion proteins localized to the mitochondria, indicating that ubiquinone biosynthesis occurs in the mitochondria in S. pombe. Thus, analysis of the phenotypes of S. pombe strains deficient in ubiquinone production clearly demonstrates that ubiquinone has multiple functions in the cell apart from being an integral component of the electron transfer system.     

Substrate specificity of cis-prenyltransferase in rat liver microsomes.

Long chain cis-prenyltransferase in rat liver microsomes was studied using various allylic isoprenoid substrates. Microsomes could utilize trans-geranyl pyrophosphate, but not cis-geranyl pyrophosphate for polyprenyl pyrophosphate synthesis. Both trans, trans-farnesyl pyrophosphate and trans,cis-farnesyl pyrophosphate were used as substrates with Km values of 24 and 5 microM, respectively. trans,trans,cis-Geranylgeranyl pyrophosphate could be used as substrate with an apparent Km of 36 microM. trans,trans,trans-Geranylgeranyl pyrophosphate was also utilized as substrate, but with a very low affinity. After pulse labeling for 4 min, using [3H]isopentenyl pyrophosphate and trans,trans-farnesyl pyrophosphate, the only product formed was trans,trans,cis-geranylgeranyl pyrophosphate, which, upon chasing, yielded polyprenyl pyrophosphate. Independent of the nature of the substrate used, even in the case of polyprenyl 12-pyrophosphate and all-trans-nonaprenyl pyrophosphate, the chain lengths of the products were identical, i.e. polyprenyl pyrophosphates with 15-18 isoprene residues. Microsomes were able to synthesize trans,trans-farnesyl pyrophosphate using trans-geranyl pyrophosphate as substrate. The results indicate that rat liver microsomes contain a farnesyl pyrophosphate synthase activity and that the reaction catalyzed by cis-prenyltransferase may consist of two individual steps, i.e. synthesis of trans,trans,cis-geranylgeranyl pyrophosphate and elongation of this product to long chain polyprenyl pyrophosphates.     

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.     

The synthesis of 3-nonaprenyl-4-hydroxybenzoate in rat liver mitochondria: the effect of Ca2 , Mg2+, chelators, and bacitracin on the activity of p-hydroxybenzoate-polyprenyl transferase

The formation of the first intermediate in ubiquinone-9 biosynthesis, 3-nonaprenyl-4-hydroxybenzoate (NPHB), by the enzyme p-hydroxybenzoate:polyprenyl transferase, has been studied in isolated rat liver mitochondria using solanesol pyrophosphate and p-hydroxybenzoate as the substrates. Phosphate buffer (100 mm) is inhibitory but at 20 mm inhibition is not apparent compared to other buffers at the same concentration. With various buffers at low concentration (20 mm) both EDTA and Mg²⁺ stimulate formation of NPHB while Ca²⁺ inhibits. Release of Ca²⁺ inhibition can be achieved by the addition of Mg²⁺, or EDTA, or EGTA, with EGTA being less effective than EDTA. When Mg²⁺, Ca²⁺, and EDTA are present together, a two- to threefold increase in activity of the enzyme is observed. The antibiotic bacitracin inhibits the synthesis of NPHB and the inhibition is increased when divalent cations are present. EGTA is more effective than EDTA in overcoming inhibition due to bacitracin. The possibility that these effects are partially due to alteration of mitochondrial membrane conformation as well as a direct effect on the enzyme is evaluated. The possible role of polyprenylphosphates in mitochondrial membrane function is discussed.     

Possible involvement of 3-hydroxymethylglutaryl-CoA reductase in determining the side-chain length of ubiquinone in rat heart.

The biosynthetic mechanism for determining the side-chain length of ubiquinone in rat heart mitochondria was investigated. The biosynthesis of nonaprenyl ubiquinone (UQ-9) and decaprenyl ubiquinone (UQ-10) in the mitochondria from rat hearts previously perfused with mevalonolactone was accelerated depending on the concentration of mevalonolactone. Furthermore the synthesis ratio between UQ-10 and UQ-9 (UQ-10/UQ-9) increased in accordance with the increasing concentration of mevalonolactone used. In addition, an enhancement of the synthesis ratio (UQ-10/UQ-9) was observed when the rats were treated with isoproterenol to increase the activity of 3-hydroxymethylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme which forms mevalonate. Moreover, the addition of isopentenyl pyrophosphate, which is a metabolite of mevalonate, elevated the synthetic ratios UQ-10/UQ-9 in intact mitochondria and decaprenyl pyrophosphate/solanesyl pyrophosphate in the partially purified polyprenyl pyrophosphate synthetase from rat heart. These results suggest that the HMG-CoA reductase could be involved as a determining factor of the side-chain length of ubiquinone in rat heart.     

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.     

Crystal structure of a novel polyisoprenoid-binding protein from Thermus thermophilus HB8

The isoprenoid quinones exist widely among prokaryotes and eukaryotes. They play essential roles in respiratory electron transport and in controlling oxidative stress and gene regulation. In the isoprenoid quinone biosynthetic pathway, polyprenyl pyrophosphates are used as isoprenoid side-chain precursors. Here we report the crystal structure of a novel polyprenyl pyrophosphate binding protein, TT1927b, from Thermus thermophilus HB8, complexed with its ligand. This protein belongs to the YceI-like family in the Pfam database, and its sequence homologs are present in a broad range of bacteria and archaea. The structure consists of an extended, eight-stranded, antiparallel beta-barrel. In the hydrophobic pore of the barrel, the protein binds the polyisoprenoid chain by hydrophobic interactions. Its overall structure resembles the lipocalin fold, but there is no sequence homology between TT1927b and the lipocalin family of proteins.     

Characterization of undecaprenyl pyrophosphate synthetase from Lactobacillus plantarum

A soluble long-chain polyprenyl pyrophosphate synthetase has been isolated from Lactobacillus plantarum and partially purified by DEAE-cellulose chromotography in 1% Triton X-100. This enzyme catalyzes the synthesis of polyprenyl pyrophosphate from farnesyl pyrophosphate and Δ³-isopentenyl pyrophosphate. The enzyme displays a requirement for farnesyl pyrophosphate and Triton X-series detergents. Treatment of polyprenyl pyrophosphate with C₅₅-isoprenyl pyrophosphate phosphatase (Micrococcus lysodeikticus) yielded polyprenyl monophosphate. Subsequent treatment of this product with a crude phosphatase from baker's yeast resulted in the formation of free polyprenol, which was characterized by thin layer chromatography and exhibited R_fs which corresponded to those of authentic undecaprenol isolated from Lactobacillus plantarum. Reverse phase cochromatography of the enzymically produced polyprenol and authentic undecaprenol indicated that the major enzymic products were undecaprenol and probably a longer chain polyprenol.     

Synthesis of a quaternary polyprenyl ammonium salt

Reaction of primary C(55)-allylic alcohol moraprenol (WT(3)C(7-9)-OH, a polyprenol from mulberry leaves) with triethylamine in the presence of phosphorus oxychloride leads to a quaternary ammonium chloride with a good yield (72%) and high cis-stereoselectivity of the terminal isoprene unit. Cationic polyprenyl derivatives may be useful for transfection and immunological studies.     

New cationic polyprenyl derivative proposed as a lipofecting agent

Cationic linear poly-cis-isoprenoid prepared from natural plant polyprenol in a mixture with dioleyl phosphatidylethanolamine was found to be an effective lipofection agent for eukaryotic cells. The transfecting activity is related to the poly-cis structure of the polyprenyl chain.     

The role of a protein factor in the effect of alpha-tocopherol on mitochondrial respiration

It is shown that alpha-tocopherol in vitro stimulates respiration of the liver mitochondria in E-hypovitaminosis rats only in the presence of the specific protein factor isolated from the liver cytosol. The action of alpha-tocopherol on mitochondria in the presence of NAD and a protein factor is not accompanied by an increase in the NADH level, that evidences for the absence of the direct redox interaction between NAD and tocopherol.