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PubMed2000

Identification of possible signal transduction components mediating light induction of the Gsa gene for an early chlorophyll biosynthetic step in Chlamydomonas reinhardtii.

Light-induced expression of the Gsa gene encoding the heme and chlorophyll biosynthetic enzyme glutamate 1-semialdehyde aminotransferase in Chlamydomonas reinhardtii was previously shown to involve Ca2+ and calmodulin (CaM) (C. lm et al. 1996, Plant Cell 8: 2245-2253). To further analyze the signal transduction pathway for light-induced Gsa expression, the effects of several pharmacological agents were examined. Treatment of light-dark synchronized cells with the heterotrimeric G-protein agonist Mas-7 caused partial induction of Gsa in the dark. The phospholipase C inhibitor U73122 inhibited light induction of Gsa. Exposure of cells to light caused a sustained 3-fold increase in cellular D-inositol 1,4,5-trisphosphate (InsP3) concentration. KN-93, a specific inhibitor of Ca2+/CaM-dependent protein kinase II, inhibited light induction of Gsa. In contrast, cyclosporin A, a specific inhibitor of the Ca2+/CaM-dependent phosphoprotein phosphatase calcineurin, did not affect light induction of Gsa. These results, together with the earlier results, suggest the involvement of a canonical signal transduction pathway for light-regulated Gsa expression that involves a heterotrimeric G-protein activation, phospholipase C-catalyzed InsP3 formation, InsP3-dependent Ca2+ release, and activation of a downstream signaling pathway through a Ca2+/CaM-dependent protein kinase.

Animals↗
PubMed2005

DEG1, encoding the tRNA:pseudouridine synthase Pus3p, impacts HOT1-stimulated recombination in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, HOT1-stimulated recombination has been implicated in maintaining homology between repeated ribosomal RNA genes. The ability of HOT1 to stimulate genetic exchange requires RNA polymerase I transcription across the recombining sequences. The trans-acting nuclear mutation hrm3-1 specifically reduces HOT1-dependent recombination and prevents cell growth at 37 degrees . The HRM3 gene is identical to DEG1. Excisive, but not gene replacement, recombination is reduced in HOT1-adjacent sequences in deg1Delta mutants. Excisive recombination within the genomic rDNA repeats is also decreased. The hypo-recombination and temperature-sensitive phenotypes of deg1Delta mutants are recessive. Deletion of DEG1 did not affect the rate of transcription from HOT1 or rDNA suggesting that while transcription is necessary it is not sufficient for HOT1 activity. Pseudouridine synthase 3 (Pus3p), the DEG1 gene product, modifies the anticodon arm of transfer RNA at positions 38 and 39 by catalyzing the conversion of uridine to pseudouridine. Cells deficient in pseudouridine synthases encoded by PUS1, PUS2 or PUS4 displayed no recombination defects, indicating that Pus3p plays a specific role in HOT1 activity. Pus3p is unique in its ability to modulate frameshifting and readthrough events during translation, and this aspect of its activity may be responsible for HOT1 recombination phenotypes observed in deg1 mutants.

Alleles↗
PubMed1997

A simple signal element mediates transcription termination and mRNA 3' end formation in the DEG1 gene of Saccharomyces cerevisiae.

DEG1 is a weakly transcribed gene of Saccharomyces cerevisiae, closely associated with CEN6. We mapped its major poly(A) site only 24 nucleotides (nt) downstream of the stop codon, and only 26 nt upstream of the CDEI centromere element. The deletion of this 50 nt stretch completely abolishes formation of the mRNA 3' end. A shorter deletion of a 16 nt sequence in the 3'-untranslated region has the same effect on transcription termination and 3'-maturation function. A TATATA sequence within this 16 nt region is essential for both functions, while a TGTATA sequence has a weak compensating activity in 3' end maturation if the TATATA stretch is deleted. We assume that the 3' end formation signals of the DEG1 gene have this simple structure: a single essential element (TATATA, whether alone or with the few surrounding nucleotides), probably, but not necessarily, cooperating with the sequence at the poly(A) site. This simple structure differs from the emerging model for 3' end-processing signals in that (i) it is shorter: 24 nt long at the most, while the model suggests 39 nt; (ii) there is no element located downstream of the TATATA signal to position the poly(A) site; and (iii) unlike the other naturally occurring signals studied, no cooperation among multiple TATATA-like elements is observed. We found that the same TATATA sequence also directs transcription termination, irrespective of promoter strength, and presumably without the cooperation of a downstream polymerase II pausing site. Taken together, these findings support the hypothesis that the DEG1 3' end-forming signals are more condensed than in other yeast genes, probably because of their proximity to CEN6.

Base Sequence↗
PubMed1997

A suicide vector for allelic recombination involving the gene for glutamate 1-semialdehyde aminotransferase in the cyanobacterium Synechococcus PCC 7942.

Gabaculine (2,3-dihydro 3-amino benzoic acid) is a potent inhibitor of tetrapyrrole biosynthesis in organisms that use the C5 pathway for the synthesis of delta-aminolaevulinic acid. Glutamate semialdehyde aminotransferase (GSA-AT), the enzyme catalysing the formation of this key precursor of tetrapyrroles, is normally inhibited by concentrations of gabaculine in the order of 5 microM. However, in Synechococcus 6301 strain GR6, a cyanobacterium that is resistant to 100 microM gabaculine, this enzyme has undergone two changes in structure: a deletion of three amino acids from positions 5 to 7 and the substitution of isoleucine for methionine at position 248. To establish the effect in vivo of these specific changes in the gene for GSA-AT (hemL), a suicide vector (pHS7) containing an antibiotic cassette was constructed to achieve the replacement, by homologous recombination, of the wild-type hemL gene in the chromosome by a modified form of the gene. Recombinant strains of Synechococcus 7942 obtained using pHS7-hemLGR6 were indistinguishable from Synechococcus 6301 GR6 in terms of the resistance of growth and of chlorophyll accumulation to high concentrations of gabaculine, while a wild-type recombinant produced using pHS7-hemLWT had retained its sensitivity. Southern hybridisation using gene probes for hemL, ampr and cmr confirmed that chromosomal integration of the plasmids had occurred in both WT and GR6 recombinants. Growth and chlorophyll accumulation in equivalent strains with the hemL gene containing either the deletion or the transition characteristic of Synechococcus 6301 GR6 were inhibited by 10 microM gabaculine. Consequently, resistance in vivo to high concentrations of this compound is dependent on both the changes in gene/enzyme structure. This investigation has established the effectiveness of the suicide vector pHS7 for studying the effect in vivo of specific changes in the hemL gene. It has also demonstrated that replacement of the wild-type gene by that from Synechococcus 6301 GR6 is sufficient to confer resistance in vivo to high concentrations of gabaculine.

Alleles↗
PubMed1998

The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast.

The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis. A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140. To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophila melanogaster homolog, Nop60B, was identified. The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases. Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development. Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis. Nop60B mutants were generated and shown to be homozygous lethal. The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

Amino Acid Sequence↗
PubMed1999

Structure of the human Lanosterol synthase gene and its analysis as a candidate for holoprosencephaly (HPE1).

Holoprosencephaly (HPE) is the most common birth defect of the brain in humans. It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies. The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE. Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients. We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein. Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.

Base Sequence↗
PubMed2001

Adenosylmethionine-dependent iron-sulfur enzymes: versatile clusters in a radical new role.

Iron-sulfur clusters are widespread in biological systems and participate in a broad range of functions. These functions include electron transport, mediation of redox as well as non-redox catalysis, and regulation of gene expression. A new role for iron-sulfur clusters has emerged in recent years as a number of enzymes have been identified that utilize Fe-S clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. This Fe-S cluster-mediated radical catalysis includes the generation of stable protein-centered radicals as well as generation of substrate radical intermediates, with evidence suggesting a common mechanism involving an intermediate adenosyl radical. Although the mechanism of generation of the adenosyl radical intermediate is currently not well understood, it likely represents novel chemistry for iron-sulfur clusters. The purpose of this review is to present the current state of knowledge of this newly emerging group of Fe-S/AdoMet enzymes.

Acetyltransferases↗
PubMed2001

The iron-sulfur center of biotin synthase: site-directed mutants.

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by Mössbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and Mössbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.

Acetyltransferases↗
PubMed2006

A novel highly boron tolerant bacterium, Bacillus boroniphilus sp. nov., isolated from soil, that requires boron for its growth.

Three strains of gram-positive, motile, rod-shaped and boron (B)-tolerant bacterium were isolated from naturally B containing soil of Hisarcik area in the Kutahya Province, Turkey. The strains, designated as T-14A, T-15Z(T) and T-17s, produced spherical or ellipsoidal endospores in a terminal bulging sporangium. The strains required B for the growth and can tolerate more than 450 mM B. These also tolerated up to 7.0% (w/v) NaCl in the presence of 50 mM B in agar medium but grew optimally without NaCl. The temperature range for growth was 16-37 degrees C (optimal of 30 degrees C), whereas the pH range was 6.5-9.0 (optimal of 7.5-8.5). The DNA G + C content was 41.1-42.2 mol% and the predominant cellular fatty acid was iso-C(15:0). The major respiratory quinone system was detected as MK-7 and the diamino acid of the peptidoglycan was meso-diaminopimelic acid. Based on phenotypic and chemotaxonomic characteristics, phylogenetic analysis of 16S rRNA gene sequences data and DNA-DNA re-association values, we concluded that the three strains belong to a novel species of the genus Bacillus, the type strain of which is T-15Z(T) and for which we proposed the name, B. boroniphilus sp. nov. (DSM 17376(T) = IAM 15287(T) = ATCC BAA-1204(T)).

Bacillus↗
PubMed2003

Performance of a recombinant strain of Streptomyces lividans for bioconversion of penicillin G to deacetoxycephalosporin G.

We examined the performance of Streptomyces lividans strain W25 containing a hybrid expandase (deacetoxycephalosporin C synthase; DAOCS) gene, obtained by in vivo recombination between the expandase genes of S. clavuligerus and Nocardia lactamdurans for resting-cell bioconversion of penicillin G to deacetoxycephalosporin G. Strain W25 carried out a much more effective level of bioconversion than the previously used strain, S. clavuligerus NP1. The two strains also differed in the concentrations of FeSO(4) and alpha-ketoglutarate giving maximal activity. Whereas NP1 preferred 1.8 mM FeSO(4 )and 1.3 mM alpha-ketoglutarate, recombinant W25 performed best at 0.45 mM FeSO(4) and 1.9 mM alpha-ketoglutarate.

Biotransformation↗
PubMed2003

Metabolic engineering of Escherichia coli for improved 6-deoxyerythronolide B production.

Escherichia coli is an attractive candidate as a host for polyketide production and has been engineered to produce the erythromycin precursor polyketide 6-deoxyerythronolide B (6dEB). In order to identify and optimize parameters that affect polyketide production in engineered E. coli, we first investigated the supply of the extender unit ( 2S)-methylmalonyl-CoA via three independent pathways. Expression of the Streptomyces coelicolor malonyl/methylmalonyl-CoA ligase ( matB) pathway in E. coli together with methylmalonate feeding resulted in the accumulation of intracellular methylmalonyl-CoA to as much as 90% of the acyl-CoA pool. Surprisingly, the methylmalonyl-CoA generated from the matB pathway was not converted into 6dEB. In strains expressing either the S. coelicolor propionyl-CoA carboxylase (PCC) pathway or the Propionibacteria shermanii methylmalonyl-CoA mutase/epimerase pathway, methylmalonyl-CoA accumulated up to 30% of the total acyl-CoA pools, and 6dEB was produced; titers were fivefold higher when strains contained the PCC pathway rather than the mutase pathway. When the PCC and mutase pathways were expressed simultaneously, the PCC pathway predominated, as indicated by greater flux of (13)C-propionate into 6dEB through the PCC pathway. To further optimize the E. coli production strain, we improved 6dEB titers by integrating the PCC and mutase pathways into the E. coli chromosome and by expressing the 6-deoxyerythronolide B synthase (DEBS) genes from a stable plasmid system.

Acyl Coenzyme A↗
PubMed2004

Expression of a yeast acetyl CoA hydrolase in the mitochondrion of tobacco plants inhibits growth and restricts photosynthesis.

Acetyl Coenzyme A (acetyl CoA) is required in the mitochondria to fuel the operation of the Krebs cycle and within the cytosolic, peroxisomal and plastidial compartments wherein it acts as the immediate precursor for a wide range of anabolic functions. Since this metabolite is impermeable to membranes it follows that discrete pathways both for its synthesis and for its utilization must be present in each of these organelles and that the size of the various compartmented pools are independently regulated. To determine the specific role of acetyl CoA in the mitochondria we exploited a transgenic approach to introduce a yeast acetyl CoA hydrolase (EC 3.1.2.1.) into this compartment in tobacco plants. Despite the facts that the introduced enzyme was correctly targeted and that there were marked reductions in the levels of citrate and malate and an increase in the acetate content of the transformants, the transgenic plants surprisingly exhibited increased acetyl CoA levels. The lines were further characterised by a severe growth retardation, abnormal leaf colouration and a dramatic reduction in photosynthetic activity correlated with a marked reduction in the levels of transcripts of photosynthesis and in the content of photosynthetic pigments. The altered rate of photosynthesis in the transgenics was also reflected by a modified carbon partitioning in leaves of these lines, however, further studies revealed that this was most likely caused by a decreased source to sink transport of carbohydrate. In summary these results suggest that the content of acetyl CoA is under tight control and that alterations in the level of this central metabolite have severe metabolic and developmental consequences in tobacco.

Acetate-CoA Ligase↗
PubMed2000

Cloning and characterization of the Dictyostelium discoideum cycloartenol synthase cDNA.

Cycloartenol synthase converts oxidosqualene to cycloartenol, the first carbocyclic intermediate en route to sterols in plants and many protists. Presented here is the first cycloartenol synthase gene identified from a protist, the cellular slime mold Dictyostelium discoideum. The cDNA encodes an 81-kDa predicted protein 50-52% identical to known higher plant cycloartenol synthases and 40-49% identical to known lanosterol synthases from fungi and mammals. The encoded protein expressed in transgenic Saccharomyces cerevisiae converted synthetic oxidosqualene to cycloartenol in vitro. This product was characterized by 1H and 13C nuclear magnetic resonance and gas chromatography-mass spectrometry. The predicted protein sequence diverges sufficiently from the known cycloartenol synthase sequences to dramatically reduce the number of residues that are candidates for the catalytic difference between cycloartenol and lanosterol formation.

Amino Acid Sequence↗
PubMed2000

Rationally designed inhibitors as tools for comparing the mechanism of squalene-hopene cyclase with oxidosqualene cyclase.

The inhibition of squalene-hopene cyclase (SHC) (E.C. 5.4.99.-), an enzyme of bacterial membranes catalyzing the formation of pentacyclic sterol-like triterpenes, was studied by using different classes of compounds originally developed as inhibitors of oxidosqualene cyclase (OSC) (E.C. 5.4.99.7), the enzyme of eukaryotes responsible for the formation of tetracyclic precursors of sterols. The mechanism of cyclization of squalene by SHC, beginning with a protonation of the 2,3 double bond by an acidic residue of the enzyme, followed by a series of electrophilic additions of the carbocationic intermediates to the double bonds, is similar to the mechanism of cyclization of 2,3-oxidosqualene by OSC. The inhibitors studied included: (i) analogs of the carbocationic intermediates formed during cyclization, such as aza-analogs of squalene and 2,3-oxidosqualene; (ii) affinity-labeling inhibitors bearing a methylidene reactive group; and (iii) vinyldioxidosqualenes and vinylsulfide derivatives of the substrates. Comparison of the results obtained with the two enzymes, SHC and OSC, showed that many of the most effective inhibitors of OSC were also able to inhibit SHC, while some derivatives acted as specific inhibitors. Differences could be easily explained on the basis of the different substrate specificity of the two enzymes.

Animals↗
PubMed2001

Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases.

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in alpha-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 500% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.

Animals↗
PubMed2002

Subcellular localization of oxidosqualene cyclases from Arabidopsis thaliana, Trypanosoma cruzi, and Pneumocystis carinii expressed in yeast.

Cycloartenol synthase from Arabidopsis thaliana and lanosterol synthase from Trypanosoma cruzi and Pneumocystis carinii were expressed in yeast, and their subcellular distribution in the expressing cells was compared. Determination of enzymatic (oxidosqualene cyclase, OSC) activity and SDS-PAGE analysis of subcellular fractions proved that enzymes from T. cruzi and A. thaliana have high affinity for lipid particles, a subcellular compartment rich in triacylglycerols, and steryl esters, harboring several enzymes of lipid metabolism. In lipid particles of strains expressing the P. carinii enzyme, neither OSC activity nor the electrophoretic band at the appropriate M.W. were detected. Microsomes from the three expressing strains retained some OSC activity. Affinity of enzymes from A. thaliana and T. cruzi for lipid particles is similar to that of OSC of Saccharomyces cerevisiae, which is mainly located in this compartment. A different distribution of OSC in yeast cells suggests that they differ in some structural features critical for the interaction with the surface of lipid particles. Computer analysis supports the hypothesis of the structural difference since OSC from S. cerevisiae, A. thaliana, and T. cruzi lack or contain only one transmembrane spanning domain (a structural feature that makes a protein poorly inclined to associate with lipid particles), whereas OSC from P. carinii possesses six transmembrane domains. In the strain expressing cycloartenol synthase from A. thaliana, the accumulation of lipid particles largely exceeded that of the other strains.

Animals↗
PubMed2003

Conjugated methyl sulfide and phenyl sulfide derivatives of oxidosqualene as inhibitors of oxidosqualene and squalene-hopene cyclases.

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene-hopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.

Animals↗
PubMed2004

Umbelliferone aminoalkyl derivatives as inhibitors of oxidosqualene cyclases from Saccharomyces cerevisiae, Trypanosoma cruzi, and Pneumocystis carinii.

A series of umbelliferone aminoalkyl derivatives, previously studied as inhibitors of squalene-hopene cyclase, were tested as inhibitors of yeast (Saccharomyces cerevisiae) oxidosqualene cyclase (OSC) and OSC from Trypanosoma cruzi and Pneumocystis carinii expressed in yeast. Enzymes from these pathogens were included in this study to provide a preliminary screening for antiparasitic activity. Tests were carried out both on cell homogenates incubated with radiolabeled oxidosqualene and on spheroplasts incubated with radiolabeled acetate. Derivatives bearing a methylallylamino group were the most effective on all of the three enzymes. The P. carinii enzyme was the most susceptible to the action of the inhibitors, with IC50 values for almost all of them ranging from 0.1 to 1 microM. The T. cruzi enzyme was appreciably inhibited (IC50 4-5 microM) only by derivatives bearing a methylallylaminoalkyl flexible chain. Results identify a particularly promising new family of OSC inhibitors, for the development of novel antiparasitic agents.

Animals↗