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PubMed2006

Steroids, triterpenoids and molecular oxygen.

There is a close connection between modern-day biosynthesis of particular triterpenoid biomarkers and presence of molecular oxygen in the environment. Thus, the detection of steroid and triterpenoid hydrocarbons far back in Earth history has been used to infer the antiquity of oxygenic photosynthesis. This prompts the question: were these compounds produced similarly in the past? In this paper, we address this question with a review of the current state of knowledge surrounding the oxygen requirement for steroid biosynthesis and phylogenetic patterns in the distribution of steroid and triterpenoid biosynthetic pathways. The hopanoid and steroid biosynthetic pathways are very highly conserved within the bacterial and eukaryotic domains, respectively. Bacteriohopanepolyols are produced by a wide range of bacteria, and are methylated in significant abundance at the C2 position by oxygen-producing cyanobacteria. On the other hand, sterol biosynthesis is sparsely distributed in distantly related bacterial taxa and the pathways do not produce the wide range of products that characterize eukaryotes. In particular, evidence for sterol biosynthesis by cyanobacteria appears flawed. Our experiments show that cyanobacterial cultures are easily contaminated by sterol-producing rust fungi, which can be eliminated by treatment with cycloheximide affording sterol-free samples. Sterols are ubiquitous features of eukaryotic membranes, and it appears likely that the initial steps in sterol biosynthesis were present in their modern form in the last common ancestor of eukaryotes. Eleven molecules of O2 are required by four enzymes to produce one molecule of cholesterol. Thermodynamic arguments, optimization of function and parsimony all indicate that an ancestral anaerobic pathway is highly unlikely. The known geological record of molecular fossils, especially steranes and triterpanes, is notable for the limited number of structural motifs that have been observed. With a few exceptions, the carbon skeletons are the same as those found in the lipids of extant organisms and no demonstrably extinct structures have been reported. Furthermore, their patterns of occurrence over billion year time-scales correlate strongly with environments of deposition. Accordingly, biomarkers are excellent indicators of environmental conditions even though the taxonomic affinities of all biomarkers cannot be precisely specified. Biomarkers are ultimately tied to biochemicals with very specific functional properties, and interpretations of the biomarker record will benefit from increased understanding of the biological roles of geologically durable molecules.

Amino Acid Sequence↗

PubMed1997

Lysine-overproducing mutants of Saccharomyces cerevisiae baker's yeast isolated in continuous culture.

Saccharomyces cerevisiae baker's yeast mutants which produce 3 to 17 times as much lysine as the wild type, depending on the nitrogen source, have been selected. The baker's yeast strain was growth in a pH-regulated chemostat in minimal medium with proline as the nitrogen source, supplemented with increasing concentrations of the toxic analog of the lysine S-2-aminoethyl-L-cysteine (AEC). The lysine-overproducing mutants, which were isolated as AEC-resistant mutants, were also resistant to high external concentrations of lysine and to alpha-aminoadipate and seemed to be affected in the lysine biosynthetic pathway but not in the biosynthetic pathways of other amino acids. Lysine overproduction by one of the mutants seemed to be due to, at least, the loss of repression of the homocitrate synthase encoded by the LYS20 gene. The mutant grew slower than the wild type, and its dough-raising capacity was reduced in in vitro assays, probably due to the toxic effects of lysine accumulation or of an intermediate produced in the pathway. This mutant can be added as a food supplement to enrich the nutritive qualities of bakery products, and its resistance to alpha-aminoadipate, AEC, and lysine can be used as a dominant marker.

Amino Acids↗

PubMed1972

Genetic and physiological control of serine and glycine biosynthesis in Saccharomyces.

Two of the three known metabolic pathways to serine and glycine have been shown to be present in prototrophic yeast strains, i.e., the phosphorylated pathway from glycolytic intermediates and the glyoxylate pathway from tricarboxylic acid cycle intermediates. Two serine-glycine auxotrophs (ser1 and ser2) were found to be blocked in the phosphoglycerate pathway. The ser1 gene controls l-glutamate:phosphohydroxypyruvate transaminase biosynthesis, and the ser2 gene controls phosphoserine phosphatase biosynthesis. The other pathway to glycine, from isocitrate, is repressed by growth in glucose media, specifically, at isocitrate lyase and at the alanine:glyoxylate transaminase. This pathway is derepressed by growth to stationary phase in glucose media yielding high activity of these enzymes. The phosphorylated pathway appears to be the principal biosynthetic pathway to serine and glycine during growth on sugar media. Strains which are serine-glycine dependent in glucose media became capable of serine-glycine independent growth on acetate media. These results describe a method of physiological control of a secondary metabolic pathway allowing a single lesion in the principal biosynthetic pathway to produce auxotrophy. This may be termed conditional auxotrophy.

Acetates↗

PubMed2004

Control of pyrimidine biosynthesis in "Pseudomonas alkanolytica" ATCC 21034.

The control of de novo pyrimidine biosynthesis in the industrially important patent strain "Pseudomonas alkanolytica" ATCC 21034 was investigated. Uracil supplementation of succinate-grown "P. alkanolytica" cells produced the greatest depression of the de novo pyrimidine biosynthetic pathway enzyme activities. After the pyrimidine limitation of a "P. alkanolytica" orotate phosphoribosyltransferase mutant strain grown on succinate, the pyrimidine biosynthetic pathway enzyme activities were derepressed. The pyrimidine biosynthetic pathway enzyme aspartate transcarbamoylase in "P. alkanolytica" was inhibited by pyrophosphate, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP), and guanosine 5'-triphosphate (GTP).

Aspartate Carbamoyltransferase↗

PubMed1996

A novel involvement of the PurG and PurI proteins in thiamine synthesis via the alternative pyrimidine biosynthetic (APB) pathway in Salmonella typhimurium.

Thiamine is thought to be synthesized by two alternative pathways, one involving the first four enzymes of the purine pathway and a second that can function independently of the purine pathway. Insertion mutations in purG and purI prevent thiamine synthesis through the alternative pyrimidine biosynthetic (APB) pathway under aerobic but not anaerobic growth conditions. In contrast, point mutations in purG and purI caused one of three distinct phenotypes: Pur- Apb-, Pur- Apb+, or Pur+ Apb-. Analysis of these three mutant classes demonstrated two genetically separable functions for PurG and PurI in thiamine synthesis. In addition to their known enzymatic role in de novo purine synthesis, we propose that PurG and PurI play a novel, possibly nonenzymatic role in the APB pathway. Suppression analysis of Pur- Apb- mutants identified two new genetic loci involved in the APB pathway, apbB and apbD). We show here that mutations in apbB and apbD cause distinct, allele-specific suppression of the thiamine requirement of purG and purI mutants. Our results suggest that PurG and PurI and one or more components of the APB pathway may function as a complex needed for aerobic function of the APB pathway.

Alleles↗

PubMed1997

Developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana.

The GA1 gene of Arabidopsis thaliana encodes ent-kaurene synthase A (KSA), which catalyzes the first committed step in the biosynthetic pathway of the plant hormone gibberellin (GA). Its location in the GA biosynthetic pathway has led to speculation that KSA regulation is one of the controlling steps. However, because KSA activity is so low that it is only measurable in Arabidopsis siliques, GA1 promoter-GUS reporter gene fusions and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine the expression pattern of GA1. The results from this study indicate that GA1 gene expression is highly regulated during growth and development, and it is restricted to specific cell types at the sites of expression. GA1 promoter activity is highest in rapidly growing tissues, e.g. shoot apicas, root tips, developing flowers and seeds. It is also active in the vascular tissue of some non-growing organs, such as expanded leaves, suggesting that these leaves may be a site of GA synthesis for transport to other organs. It was also found that the first one or two introns in the GA1 gene are required for proper expression. Because of the high degree of regulation, GA1 may act as a gatekeeper, controlling the flow of metabolites into the GA biosynthetic pathway, while the levels of specific bioactive GAs are controlled by other downstream steps.

Alkyl and Aryl Transferases↗

PubMed2003

Genome-based analysis of biosynthetic aminotransferase genes of Corynebacterium glutamicum.

Due to broad and overlapping substrate specificities, aminotransferases remain the last uncharacterized enzymes from most amino acid biosynthetic pathways in Corynebacterium glutamicum. We report here a complete description of all aminotransferases participating in the biosynthesis of the branched-chain amino acids and phenylalanine in C. glutamicum. We used methods of profile analysis on the newly available genome sequence to systematically search for and characterize members of the four known aminotransferase classes. This led to the discovery of sixteen new, potential aminotransferase encoding genes in the C. glutamicum genome, eleven of which were subsequently characterized experimentally with respect to their participation in different amino acid biosynthetic pathways. Disruption by insertion mutagenesis of ilvE, encoding a branched-chain amino acid aminotransferase, confirmed its function in leucine and isoleucine biosynthesis. Two double mutants lacking both ilvE and genes classified as class I aminotransferases exhibited additional auxotrophic requirements for valine and phenylalanine, respectively. In C. glutamicum the branched-chain amino acid aminotransferase thus participates in four amino acid biosynthetic pathways, for which in case of valine and phenylalanine biosynthesis two additional enzymes with overlapping substrate specificity exist. The novel protein with aminotransferase activity in valine biosynthesis belongs to the very recently described MocR subfamily of GntR-type helix-turn-helix transcriptional regulators, is located upstream of a potential operon of a newly described pyridoxine biosynthetic pathway and when disrupted, gives rise to a pyridoxine auxotrophy. The theoretical and experimental data we present should further provide a solid platform for ongoing research and understanding of the network of aminotransferases which participate in amino acid biosynthesis in C. glutamicum.

Amino Acid Sequence↗

PubMed2006

Expression profiling of genes involved in paclitaxel biosynthesis for targeted metabolic engineering.

Taxus plant suspension cell cultures provide a sustainable source of paclitaxel (Taxol) for the treatment of many cancers. To develop an optimal bioprocess for paclitaxel supply, taxane biosynthetic pathway regulation must be better understood. Here we examine the expression profile of paclitaxel biosynthetic pathway genes by RNA gel blot analysis and RT-PCR in the Taxus cuspidata cell line P991 and compare with taxane metabolite levels. Upon methyl jasmonate (MJ) elicitation (100 microM), paclitaxel accumulates to 3.3 mg/L and cephalomannine to 2.2 mg/L 7 days after elicitation but neither are observed before this time. 10-deacetylbaccatin III accumulates to 3.3 mg/L and baccatin III to 1.2 mg/L by day 7 after elicitation. The early pathway enzyme genes GGPPS, TASY, and T5alphaH are up-regulated by MJ elicitation within 6 h and continue through 24 h before their abundances decrease. This study reveals the preference for one side of the biosynthetic pathway branch in early taxane synthesis, where transcripts coding for TalphaH are abundant after elicitation with MJ but transcripts encoding the two enzymes for the alternative branch (TDAT and T10betaH) are not highly expressed following elicitation. Transcripts encoding the enzymes DBBT and DBAT are up-regulated upon MJ elicitation. Their products, 10-deacetylbaccatin III and baccatin III, respectively, accumulate within 6 h of the initial increase in transcript abundance. Importantly, the steady-state levels of the two terminal enzyme transcripts (BAPT and DBTNBT) are much lower than transcripts of early pathway steps. These are potential steps in the pathway for targeted metabolic engineering to increase accumulation of paclitaxel in suspension cell culture.

Cells, Cultured↗

PubMed1989

Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway.

We have isolated and characterized a Dictyostelium discoideum gene (PYR1-3) encoding a multifunctional protein that carries the three first enzymatic activities of the de novo pyrimidine biosynthetic pathway. The PYR1-3 gene is adjacent to another gene of the pyrimidine biosynthetic pathway (PYR4); the two genes are separated by a 1.5-kb non-coding sequence and transcribed divergently. The PYR1-3 gene is transcribed to form a 7.5-kb polyadenylated mRNA. As with the other genes of the pyrimidine biosynthetic pathway, the PYR1-3 mRNA level is high during growth and decreases sharply during development. We have determined the nucleotide sequence of 63% of the coding region of the PYR1-3 gene. We have identified the activities of the protein encoded by the D. discoideum PYR1-3 gene by comparison of amino acid sequences with the products of genes of known function. The PYR1-3 gene contains four distinct regions that probably correspond to four domains in the protein. From the NH2 extremity to the COOH extremity, these domains are: glutamine amidotransferase, carbamoylphosphate synthetase, dihydroorotase and aspartate transcarbamylase. This organization is identical to the one found in the rudimentary gene of Drosophila. The evolutionary implications of this finding are discussed.

Amino Acid Sequence↗

PubMed1999

Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in catharanthus roseus

Two key genes in terpenoid indole alkaloid biosynthesis, Tdc and Str, encoding tryptophan decarboxylase and strictosidine synthase, respectively, are coordinately induced by fungal elicitors in suspension-cultured Catharanthus roseus cells. We have studied the roles of the jasmonate biosynthetic pathway and of protein phosphorylation in signal transduction initiated by a partially purified elicitor from yeast extract. In addition to activating Tdc and Str gene expression, the elicitor also induced the biosynthesis of jasmonic acid. The jasmonate precursor alpha-linolenic acid or methyl jasmonate (MeJA) itself induced Tdc and Str gene expression when added exogenously. Diethyldithiocarbamic acid, an inhibitor of jasmonate biosynthesis, blocked both the elicitor-induced formation of jasmonic acid and the activation of terpenoid indole alkaloid biosynthetic genes. The protein kinase inhibitor K-252a abolished both elicitor-induced jasmonate biosynthesis and MeJA-induced Tdc and Str gene expression. Analysis of the expression of Str promoter/gusA fusions in transgenic C. roseus cells showed that the elicitor and MeJA act at the transcriptional level. These results demonstrate that the jasmonate biosynthetic pathway is an integral part of the elicitor-triggered signal transduction pathway that results in the coordinate expression of the Tdc and Str genes and that protein kinases act both upstream and downstream of jasmonates.

Journal Article↗

PubMed2000

[Recent progress on the lupine alkaloids in leguminous plants growing mainly in Japan].

The existence of the lupine alkaloids in leguminous plants mainly growing in Japan has been thoroughly ascertained, and 106 kinds of lupine alkaloids, including 51 kinds of novel lupine-type alkaloids, have been isolated and characterized from 28 species belonging to the 9 genera of leguminous plants (Table 1). Among them (e.g. Fig. 1-2), a number of unusual types of alkaloids (e.g. Fig. 2) may be regarded as possible metabolites of the lupine alkaloids that coexist in the same plant, such as (+)-kuraramine-type, (-)-mamanine-type (Fig. 3), (-)-tsukushinamine-type (Fig. 5) and (+)-hupeol-type (Fig. 7) alkaloids, or as products of alternative biosynthetic pathway, such as tashiromine-type and (-)-camoensidine-type alkaloids in the Maackia species. The biosynthetic pathways (Fig. 8) by enzymes (Fig. 9) and some of biological activities (e.g. Table 2) of the lupine alkaloids have also been presented. The leguminous plants that accumulate the common lupine alkaloids may be divided into three main groups: plants which produce the matrine, the cytisine/sparteine, and the lupinine-type alkaloids. In addition, the Maackia species and a few other species produce rare bases. Some of more detailed chemical properties of the lupine alkaloids that have been isolated and studied in our laboratory, including a newly proposed biosynthetic pathway, biotechnological studies, a summary of biological activities, and a discussion of chemotaxonomic aspects of the leguminous plants which accumulate lupine alkaloids, have been reviewed by authors in English papers shown in References 1, 55, 65, and also reviewed by one of the authors (I.M.) in a Japanese papers shown in Ref. 66, in which many aspects of the active research history on (+)-matrine and its relatively alkaloids since 1892 in Japan are described.

Acyltransferases↗

PubMed2004

Chloroplast biogenesis 89: development of analytical tools for probing the biosynthetic topography of photosynthetic membranes by determination of resonance excitation energy transfer distances separating metabolic tetrapyrrole donors from chlorophyll a acceptors.

The thorough understanding of photosynthetic membrane assembly requires a deeper knowledge of the coordination and regulation of the chlorophyll (Chl) and thylakoid apoprotein biosynthetic pathways. As a working hypothesis we have recently proposed three different Chl-thylakoid apoprotein biosynthesis models: a single-branched Chl biosynthetic pathway (SBP)-single location model, a SBP-multilocation model, and a multibranched Chl biosynthetic pathway (MBP)-sublocation model. The detection of resonance excitation energy transfer between tetrapyrrole precursors of Chl, and several Chl-protein complexes, has made it possible to test the validity of the proposed Chl-thylakoid apoprotein biosynthesis models by resonance excitation energy transfer determinations. In this work, resonance excitation energy transfer techniques that allow the determination of distances separating tetrapyrrole donors from Chl-protein acceptors in green plants by using readily available electronic spectroscopic instrumentation are developed. It is concluded that the calculated distances are compatible with the MBP-sublocation model and incompatible with the operation of the SBP-single location Chl-protein biosynthesis model.

Chlorophyll↗

PubMed2000

Synthesis of mono- and di-fucosylated type I Lewis blood group antigens by Helicobacter pylori.

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast-atom bombardment MS provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one alpha(1,2) FucT and two alpha(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.

Amino Acid Sequence↗

PubMed2003

Early events of the exogenously provided L-Carnitine in murine macrophages, T- and B-lymphocytes: modulation of prostaglandin E1 and E2 production in response to arachidonic acid.

L-carnitine is an essential energy-providing compound to the cell since it transports long chain fatty acids through the mitochondrial membrane and delivers them to the beta-oxidation pathway for catabolism and/or entrance to biosynthetic pathways. Some of the early events taking place in immune cells after L-carnitine inoculation in vitro are defined in this report. Using arachidonic acid as a fatty acid source, we determined the utilization rate of L-carnitine by murine T-, B-lymphocytes and macrophages within two hours of cell culture, its effect on prostaglandin E1 and E2 production and the levels of beta-hydroxy-butyrate. The results show that although all immune cells consume a small portion of L-carnitine, beta-hydroxy-butyrate decreases upon addition of arachidonic acid and/or L-carnitine indicating that active biosynthetic pathways are induced. L-carnitine is shown to increase the arachidonic acid-induced production of prostaglandins E1 and E2 in macrophages, while their secretion from T- and B-lymphocytes is decreased. These findings indicate the L-carnitine may very rapidly alter the activation state of immune cells and lead to the development of various reactions, beneficial or not to the organism.

3-Hydroxybutyric Acid↗

PubMed2006

Activity screening of carrier domains within nonribosomal peptide synthetases using complex substrate mixtures and large molecule mass spectrometry.

For screening a pool of potential substrates that load carrier domains found in nonribosomal peptide synthetases, large molecule mass spectrometry is shown to be a new, unbiased assay. Combining the high resolving power of Fourier transform mass spectrometry with the ability of adenylation domains to select their own substrates, the mass change that takes place upon formation of a covalent intermediate thus identifies the substrate. This assay has an advantage over traditional radiochemical assays in that many substrates, the substrate pool, can be screened simultaneously. Using proteins on the nikkomycin, clorobiocin, coumermycin A1, yersiniabactin, pyochelin, and enterobactin biosynthetic pathways as proof of principle, preferred substrates are readily identified from substrate pools. Furthermore, this assay can be used to provide insight into the timing of tailoring events of biosynthetic pathways as demonstrated using the bromination reaction found on the jamaicamide biosynthetic pathway. Finally, this assay can provide insight into the role and function of orphan gene clusters for which the encoded natural product is unknown. This is demonstrated by identifying the substrates for two NRPS modules from the pksN and pksJ genes that are found on an orphan NRPS/PKS hybrid cluster from Bacillus subtilis. This new assay format is especially timely for activity screening in an era when new types of thiotemplate assembly lines that defy classification are being discovered at an accelerating rate.

Adenosine Triphosphate↗

PubMed2003

On the generation of novel anticancer drugs by recombinant DNA technology: the use of combinatorial biosynthesis to produce novel drugs.

Chemotherapeutic drugs for cancer treatment have been traditionally originated by the isolation of natural products from different environmental niches, by chemical synthesis or by a combination of both approaches thus generating semisynthetic drugs. In the last years, a number of gene clusters from several antitumor biosynthetic pathways, mainly produced by actinomycetes and belonging to the polyketides family, are being characterized. Genetic manipulation of these antitumor biosynthetic pathways will offer in the near future an alternative for the generation of novel antitumor derivatives and thus complementing current methods for obtaining novel anticancer drugs. Novel antitumor derivatives have been produced by targetted gene disruption and heterologous expression of single (or a few) gene(s) in another hosts or by combining genes from different, but structurally related, biosynthetic pathways ("combinatorial biosynthesis"). These strategies take advantage from the "relaxed substrate specificity" that characterize secondary metabolism enzymes.

Actinobacteria↗

PubMed2001

Biosynthesis of riboflavin.

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 4,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction, and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. Two reaction steps in the biosynthetic pathway catalyzed by 3,4-dihydroxy-2-butanone 4-phosphate synthase and riboflavin synthase are mechanistically very complex. The enzymes of the riboflavin pathway are potential targets for antibacterial agents.

Bacillus subtilis↗

PubMed2002

Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins.

Recent studies are beginning to implicate sphingolipids in the heat stress response. In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses. In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells. Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence. First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase. Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway. Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C. Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides. Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock. Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells.

Biological Transport↗