Squalene synthetase. Solubilization and partial purification of squalene synthetase, copurification of presqualene pyrophosphate and squalene synthetase activities

Squalene synthetase (farnesyldiphosphate:farnesyldiphosphate farnesyltransferase, EC 2.5.1.21) is an intrinsic microsomal protein that catalyzes the synthesis of squalene from farnesyl pyrophosphate via the intermediate presqualene pyrophosphate. We have solubilized this enzyme from yeast with a mixture of the detergents N-octyl beta-D-glucopyranoside and Lubrol PX. Approximately 50-fold purification of the solubilized activities has been achieved by chromatography on DEAE-cellulose and hydroxylapatite and by isoelectric focusing. The most highly purified preparation has one major band of protein with a molecular weight of 53,000 as estimated by electrophoresis under denaturing conditions. The enzyme may also have been modified by proteolysis during isolation since a 47,000 molecular weight species was also found. The two activities, presqualene pyrophosphate synthetase and squalene synthetase, copurified during isolation.     

Squalene synthetase. I. Dissociation and reassociation of enzyme complex

Squalene synthetase, purified to near homogeneity from baker's yeast, has been resolved into two components of different molecular weight. One of these catalyzes the conversion of farnesyl pyrophosphate to squalene and the other catalyzes the first partial reaction of squalene synthesis, namely the formation of presqualene pyrophosphate. Each of these components is converted in part to the other under appropriate conditions of incubation.     

Stereochemistry of the biosynthesis of presqualene alcohol

Current hypotheses of the biosynthesis of presqualene pyrophosphate were tested by the examination of presqualene alcohol biosynthesized from [1R,5R,9R-1,5,9-D₃]farnesyl pyrophosphate and from [1-¹⁸O]farnesyl pyrophosphate. Nuclear magnetic resonance spectrometry showed that the octet of the two cyclopropylcarbinyl protons seen in the spectrum of protio-presqualene alcohol, centered at τ 6.35, was replaced by a broad doublet of one proton (τ, 6.23; J, 6.2 Hz), which became sharpened after deuterium decoupling and was reduced to a singlet after deuterium and proton decoupling. Also the doublet of a single olefinic proton adjacent to the cyclopropane ring, seen in the spectrum of protio-presqualene alcohol at τ 5.08 (J, 8.5 Hz), was reduced to a broad singlet. The presqualene alcohol biosynthesized from the [1-¹⁸O]farnesyl pyrophosphate contained the same isotopic concentration as its precursor. The observations, taken together with previous results, are interpreted to mean that the pyrophosphate-bearing group of one farnesyl pyrophosphate molecule appears without chhnge of configuration, and without previous cleavage of the CO bond of farnesyl pyrophosphate, in presqualene pyrophosphate and that the pro-R hydrogen atom at C-1 of the second farnesyl pyrophosphate molecule appears at C-3 of the cyclopropane ring anti to the vinylic substituent. The observations support the view that presqualene pyrophosphate is not an artifact, but a true intermediate in the biosynthesis of squalene.     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Presqualene alcohol,squalene, and sterol biosynthesis from bifarnesol

To cast light on the overall biosynthetic conversion of farnesol pyrophosphate to presqualene alcohol pyrophosphate (PSA), the biochemical precursor of squalene as well as all sterols, radiolabeled bifarnesol (1) was prepared and fed toGibberella fujikuroi. The diol (1), acting as a surrogate for a previously suggested phosphorylated version of1, was converted to radiolabeled presqualene alcohol and squalene, as well as various sterols, including lanosterol and24-β-methylcholesta-5,7,9(11),22-tetraen-3β-ol, previously isolated from the same fungus. The results are interpreted to imply that a phosphorylated version of1 may act as a bone fide intermediate in the biosynthesis of PSA, thereby rendering unlikely any type of concerted farnesyl/presqualene pyrophosphate change.     

The mechanism of ammonia assimilation in nitrogen fixing bacteria

Summary Enzymatic and genetic evidence are presented for a new pathway of ammonia assimilation in nitrogen fixing bacteria: ammonium glutamine glutamate. This route to the important glutamate-glutamine family of amino acids differs from the conventional pathway, ammonium glutamate glutamine, in several respects. Glutamate synthetase [(glutamine amide-2-oxoglutarate aminotransferase) (oxidoreductase)], which is clearly distinct from glutamate dehydrogenase, catalyzes the reduced pyridine nucleotide dependent amination of -ketoglutarate with glutamine as amino donor yielding two molecules of glutamate as product. The enzyme is completely inhibited by the glutamine analogue DON, whereas glutamate dehydrogenase is not affected by this inhibitor; the glutamate synthetase reaction is irreversible. Glutamate synthetase is widely distributed in bacteria; the pyridine nucleotide coenzyme specificity of the enzyme varies in many of these species.The activities of key enzymes are modulated by environmental nitrogenous sources; for example, extracts of N₂-grown cells of Klebsiella pneumoniae form glutamate almost exclusively by this new route and contain only trace amounts of glutamate dehydrogenase activity whereas NH₃-grown cells possess both pathways. Also, the biosynthetically active form of glutamine synthetase with a low K _m for ammonium predominates in the N₂-grown cell.Several mutant strains of K. pneumoniae have been isolated which fail to fix nitrogen or to grow in an ammonium limited environment. Extracts of these strains prepared from cells grown on higher levels of ammonium have low levels of glutamate synthetase activity and contain the biosynthetically inactive species of glutamine synthetase along with high levels of glutamate dehydrogenase. These mutants missing the new assimilatory pathway have serious defects in their metabolism of many inorganic and organic nitrogen sources; utilization of at least 20 different compounds is effected. We conclude that the new ammonia assimilatory route plays an important role in nitrogenous metabolism and is essential for nitrogen fixation.Abbreviation DON 6-diazo-5-oxo-l-norleucine     

Synthesis of dehydrosqualene in microsomal fraction of Saccharomyces cerevisiae.

When the microsomal fraction of Saccharomyces cerevisiae was incubated with farnesyl pyrophosphate or presqualene pyrophosphate in the presence of Mn²⁺, dehydrosqualene was formed. Incubation of the reaction mixture in the presence of NADPH gave squalene, not dehydrosqualene, as the product. Little dehydrosqualene was formed when Mn²⁺ was replaced with Mg²⁺. These observations suggest that dehydrosqualene formation is closely associated with squalene synthesis in yeast, which synthesizes neither carotenes nor related pigments.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Isoprenoid biosynthesis in rat liver peroxisomes. Characterization of cis-prenyltransferase and squalene synthetase.

Isolated peroxisomes were able to utilize [3H]isopentenyl diphosphate to synthesize farnesyl diphosphate, which then was utilized as substrate by both the peroxisomal squalene synthetase and cis-prenyltransferase. The specific activity of squalene synthetase in peroxisomes was as high as in microsomes, i.e. 160 pmol/mg of protein/min. If NADPH was omitted from the assay medium, presqualene diphosphate accumulated, which indicates that the reaction occurs in two steps, as in microsomes. In the presence of NADPH, incorporation from [3H]farnesyl diphosphate was stimulated 3-fold, and the major products were squalene and cholesterol. The specific activity of cis-prenyl-transferase in peroxisomes was 4-fold higher than in microsomes, i.e. 456 pmol of isopentenyl diphosphate incorporated/mg of protein/h. There were two major products formed from farnesyl diphosphate and [3H] isopentenyl diphosphate, i.e. trans,trans,cis-geranylgeranyl diphosphate and long chain polyprenyl diphosphates. The polyprenyl diphosphates had the same chain length distribution as that of dolichol derivatives in rat liver, with the dominating polyisoprenes being C90 and C95. In contrast to the microsomal enzyme, peroxisomal cis-prenyltransferase did not require detergents for optimal activity. The enzyme was associated primarily with the peroxisomal membrane after sonication of the peroxisomes.     

Squalene synthetase. Solubilization from yeast microsomes of a phospholipid-requiring enzyme.

Squalene synthetase was solubilized from yeast microsomal membranes with deoxycholate. Solubilized enzyme was associated with one or more proteins with s20, w = 3.3 S, Stokes' radius = 40 A, and computed molecular weight = 54,500. In the presence of detergent the enzyme was catalytically inactive and unstable to heat. When detergent was removed with cholestyramine resin, both phases of squalene synthesis (farnesyl pyrophosphate leads to presqualene pyrophosphate leads to squalene) were recovered, and the enzyme was reaggregated to form sedimentable particles with a density of approximately 1.16 g/ml. Both activities were lost to variable extent upon chromatography over Sephadex G-200 in the presence of 0.2% deoxycholate, but could be recovered if phosphatidylcholine or phosphatidylethanolamine (but not phosphatidylserine or phosphatidylinositol) were added to fractions before removal of detergent. There was an apparently absolute requirement for phospholipid by the enzyme. The proteins catalyzing the two phases of squalene synthesis could not be resolved from one another and behaved in an identical fashion throughout a variety of manipulations.     

Possible complex translocation t(9; 14; 13)(q12;pl?;q31) in mother of a child with 9p-trisomy syndrome

Summary A mentally retarded boy with trisomy 9p is described. This trisomy arose through aberrant segregation of translocation chromosome during meiosis in his mother, who has a complex translocation involving chromosomes 9, 13, and 14. Based on both G-, Q-banding, and DNA replication patterns, the patient's karyotype was identified as 47,XY,-13, +(9;13) (9pter9q12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter). We suppose his mother's karyotype to be 46,XX,-9,-13,-14,+t(9;13) (9pterq12::13q3113qter), +t(13;14) (13pter13q31::14pl?14pter), +t(9;14) (9qter9q12::14pl?14qter). His phenotypically normal brother and sister are also carriers, having the same translocation chromosome as their mother. Clinical findings of the patient included peculiar face with hypertelorism, prominent nasal bridge and deformed helix, marked delay of osseous development, hypoplastic phalangia in fingers and toes, dysplastic nails and absence of digital triradii.     

Pyrimidine dimers as pre-mutational lesions in Escherichia coli WP2 Hcr

Summary Mutation to prototrophy in E. coli WP2 Hcr^- induced by far-UV radiation (F-UV) in an intermediate dose range follows dose-squared kinetics. In a comparable dose-range with near-UV radiation in the presence of acetophenone (N-UV+Acph) mutation induction follows kinetics which are linearly related to dose. The difference in response to the two types of irradiation is a more general one in that it is the same for true revertants, for suppressor mutants, and for several markers.Double-irradiation experiments together with treatment by photoreactivating light (PR) after the first irradiation (i.e. F-UVPRF-UV; N-UV+AcphPRN-UV+Acph; N-UV+AcphF-UV; N-UV+AcphPRF-UV) seem to indicate the following: a) the dose-squared kinetics for F-UV are due to the necessary co-operation of at least two types of pre-mutational lesions, only one of which is photoreversible; b) N-UV+Acph also produces these photoreversible lesions in addition to such photoreversible ones which do not require the co-operation of other types; the production of the former is not indicated by the appearance of visible mutants because the non-photoreversible type, whose co-operation is required to give rise to such mutants, is not produced.     

Mechanism of squalene biosynthesis: evidence against the involvement of free nerolidyl pyrophosphate

Several mechanisms that utilize farnesyl pyrophosphate and nerolidyl pyrophosphate as condensing substrates have been postulated for the asymmetric condensation reaction in squalene biosynthesis. Although there is ample evidence that farnesyl pyrophosphate is a substrate for this reaction, there has been no information concerning the role of nerolidyl pyrophosphate. We have made the following observations that demonstrate that nerolidyl pyrophosphate cannot be a free intermediate in squalene biosynthesis. (a) There is no significant interconversion of farnesyl pyrophosphate and nerolidyl pyrophosphate in a squalene-synthesizing system from yeast. (b) Nerolidyl-1-(3)H(2) pyrophosphate is not converted to squalene in the presence or absence of farnesyl pyrophosphate. (c) The addition of unlabeled nerolidyl pyrophosphate to incubation mixtures does not alter the relative loss of alpha-hydrogens from farnesyl pyrophosphate during its conversion to squalene. The synthesis of nerolidyl-1-(3)H(2) pyrophosphate is described. Chromatographic methods for the separation of pyrophosphate esters of triprenols and terpenols are included.     

Direct evidence of structural changes in reaction centers of Rb. sphaeroides containing suppressor mutations for Asp L213 → Asn: A FTIR study of QB photoreduction

In bacterial reaction centers (RCs), changes of protonation state of carboxylic groups, of quinone-protein interactions as well as backbone rearrangements occuring upon Q_B photoreduction can be revealed by FTIR difference spectroscopy. The influence of compensatory mutations to the detrimental Asp L213 Asn replacement on Q_B ^–/Q_B FTIR spectra of Rb. sphaeroides RCs was studied in three double mutants carrying a Asn M44 Asp, Arg M233 Cys, or Arg H177 His suppressor mutation. The proton uptake by Glu L212 upon Q_B ^– formation, as reflected by the positive band at 1728 cm^–1, is increased in the Asn M44 Asp and Arg H177 His suppressor RCs with respect to native RCs, and remains comparable to that observed in Asp L213 Asn mutant RCs. Only the Arg M233 Cys suppressor mutation affected the 1728 cm^–1 band, reducing its amplitude to near native level. Thus, there is no clear correlation between the apparent extent of proton uptake by Glu L212 and the recovery of the proton transfer RC function. In all of the mutant spectra, several protein (amide I and amide II) and quinone anion (C...O/C...C) modes are perturbed compared to the spectrum of native RCs. These IR data show that all of the compensatory mutations alter the semiquinone-protein interactions and the backbone providing direct evidence of structural changes accompanying the restoration of efficient proton transfer in RCs containing the Asp L213 Asn lesion.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

A pyrophosphate bridge links the pyruvate-containing secondary cell wall polymer of Paenibacillus alvei CCM 2051 to muramic acid

The peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed:[(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(13)]_ñ11-(Pyr4,6)--D-Manp NAc-(14)--D-Glcp NAc-(1O)-PO₂-O-PO₂-(O6)-MurNAc-Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. ³¹P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex.In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.     

Polyisoprenoid amphiphilic compounds as inhibitors of squalene synthesis and other microsomal enzymes.

Analogues of farnesyl pyrophosphate containing a farnesyl moiety and a variety of amine residues replacing the pyrophosphate have been synthesized. Most of these compounds were effective inhibitors of the synthesis of squalene and presqualene pyrophosphate from farnesyl pyrophosphate. 50% inhibition was obtained at concentrations between 50 and 100 micron. These analogues also inhibited other microsomal enzymes so they apparently function as general inhibitors of microsomal enzymes.     

Mutations in a sequence near the N-terminus of the small subunit alter the CO2/O2specificity factor for ribulose bisphosphate carboxylase/oxygenase

Seven unique substitutions have been introduced by site-directed mutagenesis into the first conserved region of the small subunit of ribulose bisphosphate carboxylase/oxygenase from Anacystis nidulans 6301. After expression of each large, altered-small subunit gene tandem in Escherichia coli, two substitutions in the small subunit tyr17asp17 (Y17D) and arg10gly10 (R10G) result in little or no carboxylase activity. For the latter substitution, no L₈S₈enzyme complex could be detected suggesting that this mutation prevents assembly. Mutant enzymes containing the following substitutions R11G, T14A, S16A, Y17D and P19A have CO₂/O₂specificity factors ( values) of 40, 35, 18, 39 and 44, respectively, compared with that of 44 for wild-type recombinant enzyme whereas P20A has full carboxylase activity and a value of 55.     

Prevalence of common mutations in the arylsulphatase A gene in metachromatic leukodystrophy patients diagnosed in Britain

The frequency of two common disease-associated mutations in the arylsulphatase A (ASA) gene, and of a mutation causing ASA pseudodeficiency, have been established in metachromatic leukodystrophy patients diagnosed in our laboratory. A total of 37 mutant genes have been analysed. The GA change destroying the splice donor site of exon 2 is generally associated with more severe disease and was found in 43.2% of mutant ASA genes. The CT transition causing a proline to leucine substitution at position 426 in exon 8 (P426L) is associated with later onset disease, and was found in 16.2% of mutant genes. The AG transition leading to loss of a polyadenylation signal associated with ASA pseudodeficiency was present at a frequency of 7.5% in the patients and heterozygotes studied.