SphinGOMAP--a web-based biosynthetic pathway map of sphingolipids and glycosphingolipids.
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Databases as Topic↗
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Role of ethylene in the biosynthetic pathway of aliphatic ester aroma volatiles in Charentais Cantaloupe melons.
Compared to other melon types, Cantaloupe Charentais melons are highly aromatic with a major contribution to the aroma being made by aliphatic and branched esters. Using a transgenic line in which the synthesis of the plant hormone ethylene has been considerably lowered by antisense ACC oxidase mRNA (AS), the aliphatic ester pathway steps at which ethylene exerts its regulatory role were found. The data show that the production of aliphatic esters such as hexyl and butyl acetate was blocked in AS fruit and could be reversed by ethylene. Using fruit discs incubated in the presence of various precursors, the steps at which ester formation was inhibited in AS fruit was shown to be the reduction of fatty acids and aldehydes, the last step of acetyl transfer to alcohols being unaffected. However, treating AS fruit with the ethylene antagonist 1-methylcyclopropene resulted in about 50% inhibition of acetyl transfer activity, indicating that this portion of activity was ethylene-dependent and this was supported by the low residual ethylene concentration of AS fruit discs (around 2 microl l(-1)). In conclusion, the reduction of fatty acids and aldehydes appears essentially to be ethylene-dependent, whilst the last step of alcohol acetylation has ethylene-dependent and ethylene-independent components, probably corresponding to differentially regulated alcohol acetyltransferases.
Acetates↗
Light and an exogenous transcription factor qualitatively and quantitatively affect the biosynthetic pathway of condensed tannins in Lotus corniculatus leaves.
The effects of increasing light and of a heterologous bHLH transcription factor on the accumulation of condensed tannins (CT) were investigated in leaves of Lotus corniculatus, a model legume species which accumulates these secondary metabolites in leaves as well as reproductive tissues. Light and expression of the transgene increased the level of CT in a synergistic way. To monitor how the changes in accumulation of condensed tannins were achieved, the level of expression of four key genes in the flavonoid pathway was estimated by real-time RT-PCR analysis. Early genes of the pathway (PAL and CHS) were affected less in their expression and so appeared to be less involved in influencing the final level of CT than later genes in the pathway (DFR and ANS). Steady-state levels of DFR and ANS transcripts showed a strong positive correlation with CT and these genes might be considered the first rate-limiting steps in CT biosynthesis in Lotus leaves. However, additional factors mediated by light are limiting CT accumulation once these genes are up-regulated by the transgene. Therefore, the increment of the steady-state mRNA level for DFR and ANS might not be sufficient to up-regulate condensed tannins in leaves. The real-time RT-PCR approach adopted showed that members within the CHS and DFR gene families are differentially regulated by the exogenous bHLH gene and light. This finding is discussed in relation to the approaches for controlling CT biosynthesis and for studying the expression profile of multi-gene families.
Amino Acid Sequence↗
Biosynthetic pathway of ribothymidine in B. subtilis and M. lysodeikticus involving different coenzymes for transfer RNA and ribosomal RNA.
Ribothymidine (m5u) in tRNAs of M. lysodeikticus is not derived from methionine. The results indicate that as in tRNAs of B. subtilis a tetrahydrofolate derivative is involved in the formation of m5U, whereas methionine serves as precursor in the biosynthesis of m7G, m1A and m6A. Ribothymidine also occurs in 23S rRNA of B. subtilis and M. lysodeikticus. Approximately 2-3 moles of m5U residues were found per mole of 23S rRNA. In contrast to m5U residues present in tRNAs of B. subtilis and M. lysodeikticus, ribothymidine in 23S rRNA of these organisms and of E. coli is synthesized via S-adenosylmethionine. m6A and m1G, present in E. coli rRNAs, were not detected in rRNAs of (methyl-14C) methionine labeled B. subtilis and M. lysodeikticus.
Adenosine↗
Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods.
A novel protein superfamily with over 600 members was discovered by iterative profile searches and analyzed with powerful bioinformatics and information visualization methods. Evidence exists that these proteins generate a radical species by reductive cleavage of S:-adenosylmethionine (SAM) through an unusual Fe-S center. The superfamily (named here Radical SAM) provides evidence that radical-based catalysis is important in a number of previously well- studied but unresolved biochemical pathways and reflects an ancient conserved mechanistic approach to difficult chemistries. Radical SAM proteins catalyze diverse reactions, including unusual methylations, isomerization, sulfur insertion, ring formation, anaerobic oxidation and protein radical formation. They function in DNA precursor, vitamin, cofactor, antibiotic and herbicide biosynthesis and in biodegradation pathways. One eukaryotic member is interferon-inducible and is considered a candidate drug target for osteoporosis; another is observed to bind the neuronal Cdk5 activator protein. Five defining members not previously recognized as homologs are lysine 2,3-aminomutase, biotin synthase, lipoic acid synthase and the activating enzymes for pyruvate formate-lyase and anaerobic ribonucleotide reductase. Two functional predictions for unknown proteins are made based on integrating other data types such as motif, domain, operon and biochemical pathway into an organized view of similarity relationships.
Amino Acid Sequence↗
Impairment of glutathione biosynthetic pathway in uraemia and dialysis.
BACKGROUND: Glutathione (GSH), the predominant intracellular antioxidant, reportedly has been shown to be decreased in chronic renal failure patients, which renders these patients more susceptible to oxidative damage by free radicals. To our knowledge, the ability of erythrocytes to normalize the GSH level by de novo synthesis in uraemic and dialysis patients has not been studied previously. The main goal of the present study was to measure the activities of the enzymes that are responsible for de novo GSH generation, namely gamma-glutamylcysteine synthetase (gamma-GCS) and glutathione synthetase (GSH-S), in erythrocytes from uraemic and dialysis patients. METHODS: Erythrocyte total GSH level and gamma-GCS and GSH-S activities as well as plasma malondialdehyde (MDA) levels were measured in 19 non-dialysis patients (ND), 34 haemodialysis patients (HD), 22 continuous ambulatory peritoneal dialysis patients (CAPD) and 21 normal healthy controls. The effect of a single haemodialysis session was determined in 16 HD patients. RESULTS: Significant decreases in GSH levels and gamma-GCS activity but not GSH-S were observed in ND, HD and CAPD patients compared with controls. However, GSH levels as well as gamma-GCS and GSH-S activities were not different among the ND, HD and CAPD patients. The decrease in GSH was strongly and positively correlated with the decrease in gamma-GCS in ND, HD and CAPD patients (r = 0.717, P<0.001; r = 0.854, P<0.001; and r = 0.603, P<0.01, respectively). In addition, plasma MDA was negatively correlated with gamma-GCS in ND, HD and CAPD patients (r = 0.721, P<0.001; r = 0.560, P<0.01; and r = 0.585, P<0.01, respectively). A single dialysis session had no effect on GSH level or on gamma-GCS and GSH-S activities. Only a significant reduction in MDA was observed at the end of dialysis. CONCLUSIONS: The activity of the rate-limiting enzyme in GSH biosynthesis, gamma-GCS, was significantly decreased in uraemic and dialysis patients, which explains, at least in part, frequent reports of reduced GSH levels in these patients. The decrease in gamma-GCS activity may have been secondary to inhibitory effects from uraemic factors that are not removed by standard dialysis. However, this assumption does not exclude the possibility of down-regulation of gamma-GCS protein expression and further studies in this context are recommended.
Adult↗
The structure of sugar chains of Japanese quail ovomucoid. The occurrence of oligosaccharides not expected from the classical biosynthetic pathway for N-glycans; a method for the assessment of the structure of glycans present in picomolar amounts.
While the structure of the major oligosaccharide of Japanese quail ovomucoid was reported earlier (Hase, S. et al. (1982) J. Biochem. 91, 735-737), the structures of the minor oligosaccharide units were investigated for the first time in the present studies. For this purpose, the glycans of the protein were liberated from the polypeptide chain by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides obtained were coupled with 2-aminopyridine, and then the resulting fluorescent derivatives were purified by Bio-Gel P-2 column chromatography and reversed-phase HPLC. The chemical structures of two minor oligosaccharide units were determined with the aid of exoglycosidases, and by methylation analysis and Smith degradation. The results demonstrated that the ovomucoid contains the following two monoantennary glycans: Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc. The latter structure was not predicted by the classical metabolic pathway for the N-glycans to be formed. The structures of three additional minor heterosaccharides were deduced from their elution positions on HPLC together with the results of determination of their molecular sizes and the HPLC elution positions of their enzymatic degradation products. It is noteworthy that for the latter procedure for the estimation of the structures of oligosaccharides only minute quantities of glycans (several hundreds pmol) are required.
Amino Acids↗
Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes.
Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.
Amino Acid Sequence↗
Chromosomal insertion of phenazine-1-carboxylic acid biosynthetic pathway enhances efficacy of damping-off disease control by Pseudomonas fluorescens.
A disarmed Tn5 vector (pUT::Ptac-phzABCDEFG) was used to introduce a single copy of the genes responsible for phenazine-1-carboxylic acid (PCA) biosynthesis into the chromosome of a plant-growth-promoting rhizobacterium Pseudomonas fluorescens. The PCA gene cluster was modified for expression under a constitutive Ptac promoter and lacked the phzIR regulators. PCA-producing variants significantly improved the ability of the wild-type P. fluorescens to reduce damping-off disease of pea seedlings caused by Pythium ultimum, even under conditions of heavy soil infestation. Under conditions of oxygen limitation that are typical of the rhizosphere, PCA production per cell in vitro was greater than that recorded in fast-growing, nutrient-rich cultures. Similarly, when the in vitro nutrient supply was limited, P fluorescens::phz variants that produced the most PCA effectively competed against P. ultimum by suppressing mycelial development. Soil-based bioassays confirmed that the level of PCA biosynthesis correlated directly with the efficacy of biological control and the persistence of inocula in soil microcosms. They also showed that soil pretreatment with bacteria provides a suitable method for plant protection by reducing infection, effectively decontaminating the soil. These data demonstrate that the insertion of a single chromosomal copy of the genes for a novel antifungal compound, PCA, enhances the ecological fitness of a natural isolate already adapted to the rhizosphere and capable of suppressing fungal disease.
DNA Transposable Elements↗
Differentiation of steroid biosynthetic pathways in developing testes.
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Aging↗
Phospholipases modulate the rat testicular androgen biosynthetic pathway in vitro.
The role of membrane phospholipids in testicular androgen biosynthesis was investigated by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Androgen biosynthesis in untreated rat testicular microsomes was examined by monitoring the temporal appearance of pregnenolone metabolites and was found to proceed through the 4-ene route. When phospholipase A2 was included, the 5-ene steroids 17-hydroxypregnenolone and dehydroepiandrosterone (DHEA) were formed in greater quantities, and the production of 4-ene steroids was reduced indicating that the conversion of 5-ene steroids to the 4-ene configuration was inhibited by phospholipase A2 treatment. Phospholipase C, in addition to inhibiting this step, also inhibited the conversion of C21 steroids to C19 steroids. When the enzymatic steps were measured individually, phospholipase A2 inhibited 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) with an ED50 of 73 mU/ml but had no effect on the activities of 17-hydroxylase, C-17, 20 lyase, or 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD). However, though phospholipase C treatment inhibited 3 beta-HSD-Isomerase, it caused less inhibition (the ED50 value was 149 mU/ml). Furthermore, 17-hydroxylase and C-17, 20 lyase activities were also inhibited by phospholipase C treatment (ED50 values were 410 and 343 mU/ml, respectively), but no effect on 17 beta-HSD was observed. The differences in the apparent phospholipid requirements of the steroidogenic enzymes provides the possibility that the metabolic fate of pregnenolone may be regulated by changes in the phospholipid composition of the microenvironment.
17-Hydroxysteroid Dehydrogenases↗
Growth of Paracoccus denitrificans on [2,3-13C]succinate and [1,4-13C]succinate. III. Biosynthetic pathways.
The biosynthesis in vivo of a number of amino acids, sugars, and purines in Paracoccus denitrificans grown on either [2,3-13C]succinate or [1,4-13C]succinate was investigated by using gas chromatography-mass spectrometry. The distribution of label in the TCA-cycle-related amino acids indicated that carbon intermediates of energy metabolism were utilized as precursors for the biosynthesis of these amino acids in vivo. The biosynthesis of glycine, serine, phenylalanine and glycerol from labelled succinate in vivo were consistent with phosphoenol pyruvate as an intermediate. A mechanism for the formation of C4, C5 and C6 sugars without the use of fructose-1,6-bisphosphate aldolase (which has not been detected in P. denitrificans) is proposed. The 13C-enrichments of ribose in the bacterium indicate that there are at least three routes of ribose biosynthesis operating during growth on labelled succinate. The probability distribution of labelled purine molecules was successfully predicted for adenine, guanine and adenosine, thus confirming their generally accepted route of biosynthesis in vivo.
Amino Acids↗
Isolation of deacetoxycephalosporin C from fermentation broths of Penicillium chrysogenum transformants: construction of a new fungal biosynthetic pathway.
Deacetoxycephalosporin C (DAOC), a precursor of cephalosporins excreted by Cephalosporium and Streptomyces species, has been produced in Penicillium chrysogenum transformed with DNA containing a hybrid penicillin N expandase gene (cefEh) and a hybrid isopenicillin N epimerase gene (cefDh). DAOC from a P. chrysogenum transformant was identified by ultraviolet light (UV), high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and mass spectrum analyses. P. chrysogenum transformed with DNA containing cefEh without cefDh did not produce DAOC. Untransformed P. chrysogenum produced penicillin V (phenoxymethylpenicillin) but not DAOC. Transformants also produced penicillin V but, in general, less than untransformed P. chrysogenum. The cefEh and cefDh genes were constructed by replacing the open reading frame (ORF) of cloned P. chrysogenum pcbC and penDE genes with the ORF of the Streptomyces clavuligerus expandase gene, cefE, and the ORF of the Streptomyces lipmanii epimerase gene, cefD, respectively. Analyses of representative transformants suggested that production of DAOC occurred via cefEh and cefDh genes stably integrated in the P. chrysogenum genome. DNA from untransformed P. chrysogenum did not hybridize to cefE or cefD gene probes.
Amino Acid Isomerases↗
Mutants of Saccharomyces cerevisiae partially defective in the last steps of the haem biosynthetic pathway: isolation and genetical characterization.
A novel method for the isolation of Saccharomyces cerevisiae mutants partially defective in haem synthesis is described. Mutant clones were identified by their fluorescence under UV light due to the accumulation of porphyrins in cells, and by their ability to grow on nonfermentable carbon sources due to their preserved haemoprotein synthesis. Thirteen such mutants were obtained by this procedure. The defects in haem synthesis and accumulation of porphyrins in all the mutants were confirmed by spectrophotometric analysis. Complementation tests with biochemically defined, haem-less strains showed that in seven mutants uroporphyrinogen decarboxylase was affected and that in three mutants the defect concerned ferrochelatase. The defects in the remaining three mutants were not defined.
Genotype↗
Purification and some properties of carboxynorspermidine synthase participating in a novel biosynthetic pathway for norspermidine in Vibrio alginolyticus.
Carboxynorspermidine synthase, mediates the nicotinamide-nucleotide-linked reduction of the Schiff base H2N(CH2)3N = CHCH2CH(NH2)COOH. This is formed from L-aspartic beta-semialdehyde (ASA) and 1,3-diaminopropane (DAP) and is reduced to carboxynorspermidine [H2N(CH2)3NH(CH2)2CH(NH2)COOH], an intermediate in the novel pathway for norspermidine (NSPD) biosynthesis. The enzyme was purified to apparent homogeneity from Vibrio alginolyticus and characterized. The overall purification was about 1800-fold over the crude extract, with a yield of 33%. The enzyme displayed an apparent Mr of 93500 +/- 1000 by gel filtration and 45100 +/- 500 by SDS-PAGE, indicating that the native form is probably composed of two subunits of similar size. The specific activity of the purified enzyme was 31.0 mumol carboxynorspermidine produced min-1 (mg protein)-1. The enzyme was activated by dithiothreitol, and inhibited by SH-reactive compounds. The pH and temperature optima were 7.25-7.5 and 37 degrees C, respectively. The Km value for the Schiff base was 4.68 mM, measured by varying the ASA concentration while keeping the DAP concentration constant. Putrescine was slightly active as a substrate, forming carboxyspermidine (at about 7% of the rate of DAP), but ethylenediamine, cadaverine and D-ASA were inert. The Km value for NADPH was 1.51 mM. NADH was a much poorer cofactor than NADPH. When V. alginolyticus was grown in the presence of 5 mM-NSPD, the specific activity of this enzyme was reduced by approximately 70%. NSPD also repressed two other enzymes responsible for its biosynthesis, 2,4-diaminobutyrate decarboxylase and carboxynorspermidine decarboxylase.
Chromatography, Gel↗
Streptomyces akiyoshiensis differs from other gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids.
A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39,000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd(-)-complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (Mr 35484), was identified as the gene for Asd by comparing its sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the S. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the S. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in S. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.
Amino Acid Sequence↗
Poly(glucosyl-N-acetylgalactosamine 1-phosphate), a wall teichoic acid of Bacillus subtilis 168: its biosynthetic pathway and mode of attachment to peptidoglycan.
The ggaAB operon of Bacillus subtilis 168 encodes enzymes responsible for the synthesis of poly(glucosyl N-acetylgalactosamine 1-phosphate) [poly(GlcGalNAc 1-P)], a wall teichoic acid (WTA). Analysis of the nucleotide sequence revealed that both GgaA and GgaB contained the motif characteristic of sugar transferases, while GgaB was most likely to be bifunctional, being endowed with an additional motif present in glucosyl/glycerophosphate transferases. Transcription of the operon was thermosensitive, and took place from an unusually distant sigma(A)-controlled promoter. The incorporation of the poly(GlcGalNAc 1-P) precursors by various mutants deficient in the synthesis of poly(glycerol phosphate), which is the most abundant WTA of strain 168, revealed that both WTAs were most likely to be attached to peptidoglycan (PG) through the same linkage unit (LU). The incorporation of poly(GlcGalNAc 1-P) precursors by protoplasts confirmed the existence of this LU, and provided further evidence that incorporation takes place at the outer surface of the protoplast membrane. The data presented here strengthen the view that biosynthesis of the LU, and the hooking of the LU-endowed polymer to PG, offer distinct widespread targets for antibiotics specific to Gram-positive bacteria.
Bacillus subtilis↗
Beta turns in early evolution: chirality, genetic code, and biosynthetic pathways.
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Amino Acids↗