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Biosynthetic Pathway (biosynthetic + pathway)
Kinds of Biosynthetic Pathway Terms modified by Biosynthetic Pathway Selected AbstractsInhibition of Diamino Pelargonic Acid Aminotransferase, an Enzyme of the Biotin Biosynthetic Pathway, by Amiclenomycin: A Mechanistic StudyHELVETICA CHIMICA ACTA, Issue 11 2003Stéphane Mann The mechanism of action of amiclenomycin (1a), a naturally occuring inhibitor of diaminopelargonic acid aminotransferase, has been established. The enzyme catalyzes the formation of an aromatic adduct between the inhibitor and pyridoxal-5,-phosphate. The structure of the adduct, determined by mass spectrometry, is in agreement with the reported X-ray crystal structure. Kinetic parameters, characteristic of kcat inhibitors, have been observed, with a KI value of 2,,M and a kinact value of 0.4,min,1. The irreversibility of the inactivation observed, in spite of the absence of covalent bond between the inhibitor and the protein, reveals the high affinity of the adduct for the active site. Two other cis -1-amino-4-substituted-cyclohexa-2,5-dienes, 3a and 4a, were also found to efficiently inhibit the enzyme. The trans -isomers were either much less potent (1b) or inactive (3b and 4b). The aminocyclohexadiene moiety, which is, apparently, responsible for the inhibition, could constitute an original pharmacophore for the design of new herbicides. [source] An Environmental DNA-Derived Type,II Polyketide Biosynthetic Pathway Encodes the Biosynthesis of the Pentacyclic Polyketide Erdacin,ANGEWANDTE CHEMIE, Issue 34 2009Wüst, aber nicht leer: Cosmid-Klone mit Typ-II-Polyketidsynthase-Genen aus Umwelt-DNA (aus Wüstenboden) wurden auf die Produktion klonspezifischer Metabolite in Streptomyces hin untersucht. Ein rekombinanter Streptomyces stellte das Polyketid Erdacin (1) mit einer bislang unbekannten pentacyclischen Struktur her. [source] In vivo Mutational Analysis of the Mupirocin Gene Cluster Reveals Labile Points in the Biosynthetic Pathway: the "Leaky Hosepipe" MechanismCHEMBIOCHEM, Issue 9 2008Ji'en Wu Dr. Abstract A common feature of the mupirocin and other gene clusters of the AT-less polyketide synthase (PKS) family of metabolites is the introduction of carbon branches by a gene cassette that contains a ,-hydroxy-,-methylglutaryl CoA synthase (HMC) homologue and acyl carrier protein (ACP), ketosynthase (KS) and two crotonase superfamily homologues. In vivo studies of Pseudomonas fluorescens strains in which any of these components have been mutated reveal a common phenotype in which the two major isolable metabolites are the truncated hexaketide mupirocin H and the tetraketide mupiric acid. The structure of the latter has been confirmed by stereoselective synthesis. Mupiric acid is also the major metabolite arising from inactivation of the ketoreductase (KR) domain of module 4 of the modular PKS. A number of other mutations in the tailoring region of the mupirocin gene cluster also result in production of both mupirocin H and mupiric acid. To explain this common phenotype we propose a mechanistic rationale in which both mupirocin H and mupiric acid represent the products of selective and spontaneous release from labile points in the pathway that occur at significant levels when mutations block the pathway either close to or distant from the labile points. [source] Characterization of the ,-Methylaspartate-,-decarboxylase (CrpG) from the Cryptophycin Biosynthetic PathwayCHEMBIOCHEM, Issue 12 2007Zachary Q. Beck Dr. More pieces for the puzzle. The ,-methylaspartate-,-decarboxylase (CrpG) from the cryptophycin biosynthetic pathway was cloned, over-expressed, and purified. We found that CrpG decarboxylates (2S,3R)-3-methylaspartic acid to form 3-amino-2(R)-methylpropionic acid, which is subsequently incorporated into Unit C of cryptophycins (see scheme). [source] Improved Synthesis of Triketide ,-Lactones from the Pikromycin Biosynthetic Pathway.CHEMINFORM, Issue 36 2006Hae-Heon Yang Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source] Antituberculosis Agents and an Inhibitor of the para -Aminobenzoic Acid Biosynthetic Pathway from Hydnocarpus anthelminthica SeedsCHEMISTRY & BIODIVERSITY, Issue 8 2010Jun-Feng Wang Abstract Investigation on the extracts of Hydnocarpus anthelminthica seeds led to the isolation of three new compounds, anthelminthicins A,C (1,3, resp.), and two known ones, namely chaulmoogric acid (4) and ethyl chaulmoograte (5). Their structures were determined mainly by using spectroscopic techniques. The absolute configuration at the cyclopentenyl moiety of compound 2 was rationalized by quantum calculations. Base hydrolysis, followed by optical-rotation comparison, allowed assignment of the configuration of chaulmoogric-acid moiety of compounds 3 and 5. Biological assays revealed that compounds 1,5 significantly inhibit Mycobacterium tuberculosis (MTB) growth with MIC values of 5.54, 16.70, 4.38, 9.82, and 16.80,,M, respectively. Compound 3 was found to inhibit the pathway between chorismate and para -aminobenzoic acid (pAba) with a MIC value of 11.3,,M, representing a new example of pAba inhibitor isolated from a natural source. All compounds were not toxic to Candida albicans SC5314 at a concentration up to 100,,M. [source] Exploring the Phospholipid Biosynthetic Pathways of Aspergillus fumigatus by Computational Genome AnalysisENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 6 2005H. Do Abstract Aspergillus fumigatus causes a wide range of diseases that include mycotoxicosis, allergic reactions and systematic diseases (invasive aspergillosis) with high mortality rates. In recent years, considerable progress in the genome sequencing of this fungus has been made by an international consortium, which includes the Wellcome Trust Sanger Institute (UK) and the Institute for Genome Research (USA). A tenfold whole genome shotgun sequence assembly of A. fumigatus has been made publicly available. In this study, it was attempted to identify the genes related to the phospholipid biosynthesis from the A. fumigatus genome by a gene prediction program (GlimmerM) and to reconstruct the metabolic pathway for phospholipids of A. fumigatus. Fifteen genes related to phospholipid pathway were identified in the A. fumigatus genomic sequence. The open reading frames predicted by GlimmerM showed a high amino acid sequence similarity with the other fungal phospholipid biosynthetic genes and well-conserved functional domains. The obtained results also demonstrated that the reconstructed pathway of A. fumigatus in phospholipid biosynthesis was very similar to that of other fungi such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, and Neurospora crassa. Therefore it is postulated that the antifungal drugs targeted for the biosynthesis of phospholipids could also be effective against A. fumigatus. [source] Biosynthetic pathways of the pheromone of the Egyptian armyworm Spodoptera littoralisPHYSIOLOGICAL ENTOMOLOGY, Issue 4 2008LOURDES MUÑOZ Abstract Most insect pheromones comprise multicomponent blends of geometric or optical isomers, and one major question is how insects produce species-specific ratios of components for successful reproductive isolation. Key enzymes suggested to be involved in pheromone biosynthesis are acetyl-coenzyme A carboxylase and fatty acyl synthetase, chain-shortening enzymes, desaturases, elongases, reductases, oxidases, and alcohol acetyl transferases. The female pheromone composition of the Egyptian armyworm Spodoptera littoralis (Boisd.) is highly dependent on the origin of the strain. In this review, we present a summary of the different reported pheromone compositions of the moth, including from our recent studies on this subject, as well as the biosynthetic routes to the different components and the molecular approaches involved. In addition, the key role played in the proposed biosynthetic pathways by a number of important biosynthetic enzymes, such as chain shortening enzymes, desaturases and alcohol acetyl transferases, is outlined, as well as the latest developments on the inhibition of these enzymes. [source] The mitochondrial proteome: A dynamic functional program in tissues and disease states,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 5 2010Robert S. Balaban Abstract The nuclear DNA transcriptional programming of the mitochondria proteome varies dramatically between tissues depending on its functional requirements. This programming generally regulates all of the proteins associated with a metabolic or biosynthetic pathway associated with a given function, essentially regulating the maximum rate of the pathway while keeping the enzymes at the same molar ratio. This may permit the same regulatory mechanisms to function at low- and high-flux capacity situations. This alteration in total protein content results in rather dramatic changes in the mitochondria proteome between tissues. A tissues mitochondria proteome also changes with disease state, in Type 1 diabetes the liver mitochondrial proteome shifts to support ATP production, urea synthesis, and fatty acid oxidation. Acute flux regulation is modulated by numerous posttranslational events that also are highly variable between tissues. The most studied posttranslational modification is protein phosphorylation, which is found all of the complexes of oxidative phosphorylation and most of the major metabolic pathways. The functional significance of these modifications is currently a major area of research along with the kinase and phosphatase regulatory network. This near ubiquitous presence of protein phosphorylations, and other posttranslational events, in the matrix suggest that not all posttranslational events have functional significance. Screening methods are being introduced to detect the active or dynamic posttranslational sites to focus attention on sites that might provide insight into regulatory mechanisms. Environ. Mol. Mutagen., 2010. Published 2010 Wiley-Liss, Inc. [source] Secondary Metabolites of Phomopsis sp.EUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 18 2009XZ-2, an Endophytic Fungus from Camptothecaacuminate Abstract Eleven new metabolites, including nine lovastatin analogues [oblongolides N,V (1,2 and 5,11), which were defined as naphthalene-type fungal polyketides], one linear furanopolyketide (13) and a monoterpene named dihydroxysabinane (14), together with four known compounds including oblongolides B (3) and C (4), one linear furanopolyketide (12) and the sesterterpene terpestacin (15), were isolated from the endophytic fungal strain Phomopsis sp. XZ-26 of Camptotheca acuminate. Their structures were elucidated by spectroscopic analyses including HR-ESI-MS, 1H and 13C NMR, 2D NMR (HMQC, HMBC, 1H- 1H COSY and NOESY), and X-ray single-crystal analysis. The antimicrobial activities of 1,5, 8, 10 and 13,15 were evaluated, but none showed a substantial effect. Additionally, a hypothetical biosynthetic pathway for oblongolides was proposed.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] The Biosynthesis of 3-(trans -2-Nitrocyclopropyl)alanine, a Constituent of the Signal Metabolite HormaomycinEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 1 2005Melanie Brandl Abstract Feeding experiments with Streptomyces griseoflavus using deuterium-labeled racemic 3,3-[D2]- (6b), 4,4-[D2]- (6c), 5,5-[D2]- (6d), and 6,6-[D2]-lysine (6e), and 3-amino-5-(2-amino-1,1-dideuterioethyl)-4,5-dihydrofuran-2-one dihydrochloride (34·2HCl) were carried out in order to obtain detailed information about the hitherto unknown biosynthetic pathway from lysine to the unusual amino acid 3-(trans -2,-nitrocyclopropyl)alanine [(3-Ncp)Ala] (2), which is a building block of hormaomycin 1a. The corresponding lysine dihydrochlorides were prepared in 33, 24, 19, and 30% overall yield, respectively, along a new efficient general synthetic route applying an alkylation of the lithium enolate of O,Donnel's glycine equivalent 7 as a key step. In the attempted preparation of 5,5-[D2]-4-hydroxylysine (29), the respective ,-lactone (34·2 HCl) was obtained in five steps with 10% overall yield. The distribution of isotope labels in hormaomycins 1b,d led to the formulation of a reasonable cyclization mechanism of 2-amino-4-hydroxy-6-(hydroxyimino)hexanoic acid, an ,-oxime analogue of 4-hydroxylysine. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Topical ascorbic acid on photoaged skin.EXPERIMENTAL DERMATOLOGY, Issue 3 2003Clinical, topographical, ultrastructural evaluation: double-blind study vs. placebo Abstract:, Vitamin C is known for its antioxidant potential and activity in the collagen biosynthetic pathway. Photoprotective properties of topically applied vitamin C have also been demonstrated, placing this molecule as a potential candidate for use in the prevention and treatment of skin ageing. A topically applied cream containing 5% vitamin C and its excipient were tested on healthy female volunteers presenting with photoaged skin on their low-neck and arms in view to evaluate efficacy and safety of such treatment. A double-blind, randomized trial was performed over a 6-month period, comparing the action of the vitamin C cream vs. excipient on photoaged skin. Clinical assessments included evaluation at the beginning and after 3 and 6 months of daily treatment. They were performed by the investigator and compared with the volunteer self assessment. Skin relief parameters were determined on silicone rubber replicas performed at the same time-points. Cutaneous biopsies were obtained at the end of the trial and investigated using immunohistochemistry and electron microscopy. Clinical examination by a dermatologist as well as self-assessment by the volunteers disclosed a significant improvement, in terms of the ,global score', on the vitamin C-treated side compared with the control. A highly significant increase in the density of skin microrelief and a decrease of the deep furrows were demonstrated. Ultrastructural evidence of the elastic tissue repair was also obtained and well corroborated the favorable results of the clinical and skin surface examinations. Topical application of 5% vitamin C cream was an effective and well-tolerated treatment. It led to a clinically apparent improvement of the photodamaged skin and induced modifications of skin relief and ultrastructure, suggesting a positive influence of topical vitamin C on parameters characteristic for sun-induced skin ageing. [source] Substrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductaseFEBS JOURNAL, Issue 13 2007Ronnie A. Böck Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80,90%, and D31A and D31L by >,90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in Km for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in kcat/Km. Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in Km; 21-fold decrease in kcat/Km). Only minor variations in the Km for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. [source] Cloning, characterization and localization of a novel basic peroxidase gene from Catharanthus roseusFEBS JOURNAL, Issue 5 2007Santosh Kumar Catharanthus roseus (L.) G. Don produces a number of biologically active terpenoid indole alkaloids via a complex terpenoid indole alkaloid biosynthetic pathway. The final dimerization step of this pathway, leading to the synthesis of a dimeric alkaloid, vinblastine, was demonstrated to be catalyzed by a basic peroxidase. However, reports of the gene encoding this enzyme are scarce for C. roseus. We report here for the first time the cloning, characterization and localization of a novel basic peroxidase, CrPrx, from C. roseus. A 394 bp partial peroxidase cDNA (CrInt1) was initially amplified from the internodal stem tissue, using degenerate oligonucleotide primers, and cloned. The full-length coding region of CrPrx cDNA was isolated by screening a leaf-specific cDNA library with CrInt1 as probe. The CrPrx nucleotide sequence encodes a deduced translation product of 330 amino acids with a 21 amino acid signal peptide, suggesting that CrPrx is secretory in nature. The molecular mass of this unprocessed and unmodified deduced protein is estimated to be 37.43 kDa, and the pI value is 8.68. CrPrx was found to belong to a ,three intron' category of gene that encodes a class III basic secretory peroxidase. CrPrx protein and mRNA were found to be present in specific organs and were regulated by different stress treatments. Using a ,-glucuronidase,green fluorescent protein fusion of CrPrx protein, we demonstrated that the fused protein is localized in leaf epidermal and guard cell walls of transiently transformed tobacco. We propose that CrPrx is involved in cell wall synthesis, and also that the gene is induced under methyl jasmonate treatment. Its potential involvement in the terpenoid indole alkaloid biosynthetic pathway is discussed. [source] Guanosine diphosphate-4-keto-6-deoxy- d -mannose reductase in the pathway for the synthesis of GDP-6-deoxy- d -talose in Actinobacillus actinomycetemcomitansFEBS JOURNAL, Issue 23 2002Nao Suzuki The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy- d -talose. Guanosine diphosphate (GDP)-6-deoxy- d -talose is the activated sugar nucleotide form of 6-deoxy- d -talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy- d -talose synthetic enzymes, GDP-,- d -mannose 4,6-dehydratase and GDP-4-keto-6-deoxy- d -mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-,- d -mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy- d -talose is produced from GDP-,- d -mannose. This paper is the first report on the GDP-6-deoxy- d -talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy- d -mannose reductase in the synthesis of GDP-6-deoxy- d -talose. [source] ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activityFEBS JOURNAL, Issue 8 2002Nadia J. Kershaw The clinically used beta-lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ,,15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to >,95% purity) by a one-step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate , and , subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX--(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an ,2,2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the , subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post-translational modification of the , subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major , subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N -acetylornithine to glutamate, but not the formation of N -acetylglutamate from glutamate and acetyl-coenzyme A, nor (detectably) the hydrolysis of N -acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the , subunit. [source] Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCGFEMS MICROBIOLOGY LETTERS, Issue 1 2003Indrajit Sinha Abstract Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation. [source] Bactericidal and inhibitory effects of azole antifungal compounds on Mycobacterium smegmatisFEMS MICROBIOLOGY LETTERS, Issue 2 2000Colin J Jackson Abstract Azole antifungals are central to therapy and act by inhibiting a cytochrome P450, sterol 14-demethylase and blocking normal sterol synthesis. Our recent identification of a mycobacterial sterol biosynthetic pathway led us to probe the efficacy of a range of these compounds against Mycobacterium smegmatis. Several showed equivalent or greater inhibitory effects to those against Candida albicans, and bactericidal activity was demonstrated for four compounds, clotrimazole, econazole, miconazole and tebuconazole. The major drug used clinically, fluconazole, was ineffective. The results are discussed in the light of the world-wide spread of tuberculosis, including drug-resistant forms and the requirement for new drugs. [source] Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl geneFEMS YEAST RESEARCH, Issue 7 2007Loknath Gidijala Abstract We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host,vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine ,-lyase. With this new host,vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein,PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL,SKL produced in H. polymorpha displays normal enzymatic activities. [source] The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungiFEMS YEAST RESEARCH, Issue 4-5 2004Carlos Gancedo Abstract The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them. [source] Combined overexpression of genes of the ergosterol biosynthetic pathway leads to accumulation of sterols in Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 1 2003Markus Veen GC, gas chromatography; TLC, thin layer chromatography Abstract Genes of the post-squalene ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been overexpressed in a systematic approach with the aim to construct yeast strains that produce high amounts of sterols from a squalene-accumulating strain. This strain had previously been deregulated by overexpressing a truncated HMG-CoA reductase (tHMG1) in the main bottleneck of the early ergosterol pathway. The overexpression of the gene ERG1 (squalene epoxidase) induced a significant decrease of the direct substrate squalene, a high increase of lanosterol, and a small increase of later sterols. The overexpression of the ERG11 gene encoding the sterol-14,-demethylase resulted in a decrease of lanosterol and an increase of downstream sterols. When these two genes were simultaneously overexpressed, later sterols from zymosterol to ergosterol accumulated and the content of squalene was decreased about three-fold, indicating that these steps had limited the transformation of squalene into sterols. The total sterol content in this strain was three-fold higher than in a wild-type strain. [source] An Improved Preparation of D -Glyceraldehyde 3-Phosphate and Its Use in the Synthesis of 1-Deoxy- D -xylulose 5-PhosphateHELVETICA CHIMICA ACTA, Issue 9 2010Heng Li Abstract D -Glyceraldehyde 3-phosphate (=D -GAP; 2) was prepared by an improved chemical method (Scheme,2), and it was then employed to synthesize 1-deoxy- D -xylulose 5-phosphate (=DXP; 3) which is enzymatically one of the key intermediates in the MEP (4) terpenoid biosynthetic pathway (Scheme,1). The recombinant DXP synthase of Rhodobacter capsulatus was used to catalyze the condensation of D -glyceraldehyde 3-phosphate (2) and pyruvate (=2-oxopropanoate; 1) to produce the sugar phosphate 3 (Scheme,2). The simple two-step chemoenzymatic route described affords DXP (3) with more than 70% overall yield and higher than 95% purity. The procedure may also be used for the synthesis of isotope-labeled DXP (3) by using isotope-labeled pyruvate. [source] Two Novel Triterpenes from the Leaves of Ficus microcarpaHELVETICA CHIMICA ACTA, Issue 5 2004Yueh-Hsiung Kuo Two novel triterpenes, 29(20,19)abeolupane-3,20-dione (4) and 19,20-secoursane-3,19,20-trione (5), besides (3,)-3-hydroxy-29(20,19)abeolupan-20-one (2), lupenone, and , -amyrone (6), were isolated from the leaves of Ficus microcarpa and were characterized by spectroscopic means, including 2D-NMR techniques and chemical methods. Compound 4 is the second derivative having the 29(20,19)abeolupane skeleton, and 5 is a novel skeleton. A biosynthetic pathway to 5 is proposed (Scheme). [source] Pathogen evasion strategies for the major histocompatibility complex class I assembly pathwayIMMUNOLOGY, Issue 1 2008Antony N. Antoniou Summary Major histocompatibility complex (MHC) class I molecules bind and present short antigenic peptides from endogenously or exogenously derived sources to CD8+ cytotoxic T lymphocytes (CTL), with recognition of a foreign peptide normally targeting the cell for lysis. It is generally thought that the high level of MHC polymorphism, which is concentrated mostly within the peptide-binding groove, is driven by the ,evolutionary arms race' against pathogens. Many pathogens have developed novel and intriguing mechanisms for evading the continuous sampling of the intracellular and intercellular environments by MHC molecules, none more so than viruses. The characterization of immunoevasion mechanisms has improved our understanding of MHC biology. This review will highlight our current understanding of the MHC class I biosynthetic pathway and how it has been exploited by pathogens, especially viruses, to potentially evade CTL recognition. [source] Cloning and expression of a geranylgeranyl diphosphate synthase gene: insights into the synthesis of termite defence secretionINSECT MOLECULAR BIOLOGY, Issue 1 2007Masaru Hojo Abstract In Nasutitermes takasagoensis, a termite in which soldiers perform specialized chemical defence, Nts19-1 gene is highly expressed exclusively in soldier head. In this study, two types of transcripts for this gene were obtained, and the full-length cDNAs were determined by rapid amplification of cDNA ends (RACE). These transcripts were putative homologues of the geranylgeranyl diphosphate (GGPP) synthase gene, involved in the condensation of dimethylallyl diphosphate with isopentenyl diphosphate in the isoprenoid biosynthetic pathway. The genes were thus termed NtGGPPS1. GGPP is a precursor of diterpenes in plants. In situ hybridization localized NtGGPPS1 expression to the epidermal secretory cells of the frontal gland reservoir where many kinds of diterpenes are produced, suggesting that NtGGPPS1 is involved in the biosynthesis of defence secretion. [source] Glucosamine:fructose-6-phosphate aminotransferase: gene characterization, chitin biosynthesis and peritrophic matrix formation in Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 3 2002N. Kato Abstract Glucosamine:fructose-6-phosphate aminotransferase (GFAT) catalyses the formation of glucosamine 6-phosphate and is the first and rate-limiting enzyme of the hexosamine biosynthetic pathway. The final product of the hexosamine pathway, UDP- N -acetyl glucosamine, is an active precursor of numerous macromolecules containing amino sugars, including chitin in fungi and arthropods. Chitin is one of the essential components of insect cuticle and peritrophic matrix. The peritrophic matrix is produced in the midgut of mosquitoes in response to bloodfeeding, and may affect vector competence by serving as a physical barrier to pathogens. It is hypothesized that GFAT plays a regulatory role in biosynthesis of chitin and peritrophic matrix formation in insects. We cloned and sequenced the GFAT gene (AeGfat-1) and its 5, regulatory region from Aedes aegypti. There is no intron in AeGfat-1 and there are two potential transcription start sites. AeGfat-1 cDNA is 3.4 kb in length and its putative translation product is 75.4 kDa. The amino acid sequence of GFAT is highly conserved in lower and higher eukaryotes, as well as in bacteria. AeGfat-1 message is constitutively expressed but is gradually up-regulated in the midgut after bloodfeeding. The putative regulatory region of the gene contains the ecdysone response element, E74, and Broad complex motifs, similar to what is found in the glutamine synthetase gene in Ae. aegypti. Results suggest that Ae. aegypti GFAT-1 may have a regulatory role in chitin biosynthesis and peritrophic matrix formation, and probably is under the regulation of ecdysteroids. [source] Investigating the role of heparin sulfate proteoglycans in hereditary multiple exostoses (HME) tumourigenesisINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Z.M. Scholefield Introduction Heparin sulfate (HS) has long been implicated in the bone deformity hereditary multiple exostoses (HME), and it is now clear that HME is associated with mutations in the HS biosynthetic genes EXT1 and EXT2. Interestingly, HME is also associated with an increased risk of chondro- and osteo-sarcomas. Methods and results Preliminary analysis of GAG samples purified from fibroblasts of both HME and isolated non-HME exostoses patients reveal a dramatic shift in the ratio of CS : HS, with the HME and isolated cases having a much higher proportion of CS relative to normal controls. This is true in the case of both shed and cell surface material but is far more extreme in the latter, with the HS reducing from approximately 45% in the controls to less than 10% in HME patients. Initial analysis also reveals shortened chain length within these samples; indeed they often have two populations of chains present. Simple analysis of the total disaccharide composition of these samples demonstrates no significant differences against controls. However, detailed analysis of the subpopulations of chains (as determined by chain length) within these samples as well as cartilaginous samples from exostoses patients may provide further insight into the changes that occur within the biosynthetic pathway following disrupted EXT function. We are also carrying out immunocytochemistry with a variety of HS-specific antibodies with the aim to further investigate normal HS structure and localization. This is being carried out on human primary chondrocytes isolated from normal patients and also adult mesenchymal stem cells as they undergo differentiation into chondrocytes. HS has been identified in both these cell types, and it is hoped that the manipulation of these cells through RNAi of different enzymes of the HS biosynthetic pathway will provide a suitable model for studying what changes may occur in cellular HS structures over the initial differentiation process in the growth plate. Discussion Together, these investigations should provide a good model to allow us to determine the role of HS in chondrocyte differentiation and maturation in both normal and diseased states. [source] Structural Diversification of Macrolactones by Substrate-Flexible Cytochrome P450 MonooxygenasesADVANCED SYNTHESIS & CATALYSIS (PREVIOUSLY: JOURNAL FUER PRAKTISCHE CHEMIE), Issue 10 2005Kil Lee Abstract The substrate flexibilities of several cytochrome P450 monooxygenases involved in macrolide biosynthesis were investigated to test their potential for the generation of novel macrolides. PikC hydroxylase in the pikromycin producer Streptomyces venezuelae accepted oleandomycin as an alternative substrate and introduced a hydroxy group at the C-4 position, which is different from the intrinsic C-12 hydroxylation position in the natural substrate. This is the first report of C-4 hydroxylation activity of cytochrome P450 monooxygenase involved in the biosynthesis of 14-membered macrolides. EryF hydroxylase from the erythromycin biosynthetic pathway of Saccharopolyspora erythraea and OleP oxidase from the oleandomycin biosynthetic pathway of Streptomyces antibioticus also showed a certain degree of plasticity towards alternative substrates. In particular, EryF and OleP were found to oxidize a 12-membered macrolactone as an alternative substrate. These results demonstrate the potential usefulness of these enzymes to diversify macrolactones by post-PKS oxidations. [source] Biosynthesis of spectinomycin: heterologous production of spectinomycin and spectinamine in an aminoglycoside-deficient host, Streptomyces venezuelae YJ003JOURNAL OF APPLIED MICROBIOLOGY, Issue 1 2008L.P. Thapa Abstract Aims:, To obtain spectinomycin and spectinamine by heterologous expression into the biosynthetic deoxysugar (desosamine) gene-deleted host Streptomyces venezuelae YJ003. Methods and Results:, The 17-kb spectinomycin biosynthetic gene cluster from Streptomyces spectabilis ATCC 27741 was heterologously expressed into Streptomyces venezuelae YJ003. Furthermore, the speA, speB and spcS2 encoded in the spectinomycin biosynthetic gene cluster of cosmid pSPC8 were also heterologously characterized to be responsible for the production of spectinamine. Conclusions:, The results of this study indicated that pSPC8 contains all the genes necessary for the biosynthesis of spectinomycin. We also concluded that SpeA, SpeB and SpcS2 are sufficient for the biosynthesis of spectinamine. We also verified that SpeB and SpcS2 show dual character in the biosynthetic pathway of spectinomycin in Streptomyces spectabilis. Significance and Impact of the Study:, This is the report regarding the expression of a biosynthetic gene cluster that gives rise to the production of aminoglycoside antibiotics in Streptomyces venezuelae YJ003. Therefore, this work may serve as a foundation for further research on spectinomycin biosynthesis and other aminoglycosides. [source] Abnormal kinetic behavior of uroporphyrinogen decarboxylase obtained from rats with hexachlorobenzene-induced porphyriaJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 1 2005Gabriela Chaufan Abstract Uroporphyrinogen decarboxylase is an essential enzyme in all organisms and functions in the heme biosynthetic pathway, catalyzing the decarboxylation of the four acetate groups of uroporphyrinogen to form coproporphyrinogen. This work examines whether the four sequential decarboxylations occur at the same active site, and explores whether hexachlorobenzene-induced porphyria affects the behavior of the enzyme. For this purpose, kinetic competition studies were done with mixtures of uroporphyrinogen III and pentacarboxyporphyrinogen III. With the enzyme from normal rats, a constant velocity was obtained with all the mixtures, indicating that uroporphyrinogen and pentacarboxy-porphyrinogen react at the same active site, i.e. the first and fourth decarboxylations occur at the same site. In contrast, in experiments with enzyme from rats with hexachlorobenzene-induced porphyria, the total rate for mixtures was always lower than the reference rate; and a curve with a deep minimum was obtained, indicating that the two reactions occur at functionally different sites, but with cross-inhibition. This suggests that the modifications induced in the enzyme by hexachlorobenzene cause the two active sites to become nonequivalent and functionally different. The question is discussed how the hexachlorobenzene treatment may produce this abnormal kinetic behavior, and alternative hypotheses are considered. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:19,24, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20055 [source] | |||||||||||||||||||||||||||||||||||||