Pyrimidine metabolism in Pseudomonas acidovorans

1974 ◽  
Vol 20 (4) ◽  
pp. 427-433 ◽  
Author(s):  
Rod A. Kelln ◽  
R. A. J. Warren
Keyword(s):  
Biosynthetic Pathway ◽  
Wild Type Strain ◽  
De Novo ◽  
Nucleic Acid Metabolism ◽  
Aspartate Transcarbamylase ◽  
Bacteria Growth ◽  
Deoxycytidine Deaminase ◽  
Lag Period ◽  
Pseudomonas Acidovorans ◽  
Salvage Pathways

Pseudomonas acidovorans lacks a number of enzymes of the salvage pathways of nucleic acid metabolism, including uridine phosphorylase, purine nucleoside phosphorylase, cytidine (deoxycytidine) deaminase and thymidine phosphorylase, and probably uridine kinase and deoxycytidine kinase. Its growth is inhibited by adenosine and deoxyadenosine. The level of aspartate transcarbamylase is the same in extracts of P. acidovorans grown in minimal medium ± 25 μg uracil/ml, and the enzyme appears to be insensitive to nucleotides which affect this enzyme in other bacteria. Growth of two pyrimidine-requiring mutants of P. acidovorans is supported by uracil or cytosine but not by their nucleosides nor by intermediates of the de novo pyrimidine biosynthetic pathway. Concentrations of uracil greater than 50 μg/ml have the unusual effect of lengthening the lag period of the mutants. The wild-type strain is not inhibited by uracil.

Transcriptional regulation of the human CAD gene during myeloid differentiation

1987 ◽  
Vol 7 (5) ◽  
pp. 1961-1966
Author(s):  
G N Rao ◽  
E S Buford ◽  
J N Davidson
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Morphological Changes ◽  
Transcriptional Level ◽  
Carbamoyl Phosphate ◽  
Aspartate Transcarbamylase ◽  
Trans Retinoic Acid ◽  
Beta Actin ◽  
Cad Gene ◽  
Cad Protein

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

scholarly journals The biosynthetic origin of psychoactive kavalactones in kava

2018 ◽  
Author(s):  
Tomáš Pluskal ◽  
Michael P. Torrens-Spence ◽  
Timothy R. Fallon ◽  
Andrea De Abreu ◽  
Cindy H. Shi ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Biosynthetic Pathway ◽  
Chalcone Synthase ◽  
De Novo ◽  
Piper Methysticum ◽  
Heterologous Production ◽  
Traditional Use ◽  
Plant Biochemistry ◽  
Tailoring Enzymes

AbstractFor millennia, humans have used plants for medicinal purposes. However, our limited understanding of plant biochemistry hinders the translation of such ancient wisdom into modern pharmaceuticals1. Kava (Piper methysticum) is a medicinal plant native to the Polynesian islands with anxiolytic and analgesic properties supported by over 3,000 years of traditional use as well as numerous recent clinical trials2–5. The main psychoactive principles of kava, kavalactones, are a unique class of polyketide natural products known to interact with central nervous system through mechanisms distinct from those of the prescription psychiatric drugs benzodiazepines and opioids6,7. Here we reportde novoelucidation of the biosynthetic pathway of kavalactones, consisting of seven specialized metabolic enzymes. Based on phylogenetic and crystallographic analyses, we highlight the emergence of two paralogous styrylpyrone synthases, both of which have neofunctionalized from an ancestral chalcone synthase to catalyze the formation of the kavalactone scaffold. Structurally diverse kavalactones are then biosynthesized by subsequent regio- and stereo-specific tailoring enzymes. We demonstrate the feasibility of engineering heterologous production of kavalactones and their derivatives in bacterial, yeast, and plant hosts, thus opening an avenue towards the development of new psychiatric therapeutics for anxiety disorders, which affect over 260 million people globally8.

Two different thymidylate kinase gene homologues, including one that has catalytic activity, are encoded in the onion yellows phytoplasma genome

Microbiology ◽  
2003 ◽  
Vol 149 (8) ◽  
pp. 2243-2250 ◽  
Author(s):  
Shin-ichi Miyata ◽  
Kenro Oshima ◽  
Shigeyuki Kakizawa ◽  
Hisashi Nishigawa ◽  
Hee-Young Jung ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Genomic Library ◽  
De Novo ◽  
Bacterial Species ◽  
Point Mutations ◽  
Kinase Gene ◽  
Thymidylate Kinase ◽  
Pcr Products ◽  
Multiple Copies ◽  
Salvage Pathways

Thymidylate kinase (TMK) catalyses the phosphorylation of dTMP to form dTDP in both the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. Two homologues of bacterial thymidylate kinase genes were identified in a genomic library of the onion yellows (OY) phytoplasma, a plant pathogen that inhabits both plant phloem and the organs of insects. Southern blotting analysis suggested that the OY genome contained one copy of the tmk-b gene and multiple copies of the tmk-a gene. Sequencing of PCR products generated by amplification of tmk-a enabled identification of three other copies of tmk-a, although the ORF in each of these was interrupted by point mutations. The proteins, TMK-a and TMK-b, encoded by the two intact genes contained conserved motifs for catalytic activity. Both proteins were overexpressed as fusion proteins with a polyhistidine tag in Escherichia coli and purified, and TMK-b was shown to have thymidylate kinase activity. This is believed to be the first report of the catalytic activity of a phytoplasmal protein, and the OY phytoplasma is the first bacterial species to be found to have two intact homologues of tmk in its genome.

scholarly journals Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

2007 ◽  
Vol 71 (1) ◽  
pp. 36-47 ◽  
Author(s):  
Ying Xu ◽  
Bernard Labedan ◽  
Nicolas Glansdorff
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Single Gene ◽  
Metabolic Function ◽  
Gene Products ◽  
Arginine Biosynthesis ◽  
Acetyl Coenzyme A ◽  
Bacterial Phyla ◽  
Acetyl Group ◽  
Acetylglutamate Synthase

SUMMARY Major aspects of the pathway of de novo arginine biosynthesis via acetylated intermediates in microorganisms must be revised in light of recent enzymatic and genomic investigations. The enzyme N-acetylglutamate synthase (NAGS), which used to be considered responsible for the first committed step of the pathway, is present in a limited number of bacterial phyla only and is absent from Archaea. In many Bacteria, shorter proteins related to the Gcn5-related N-acetyltransferase family appear to acetylate l-glutamate; some are clearly similar to the C-terminal, acetyl-coenzyme A (CoA) binding domain of classical NAGS, while others are more distantly related. Short NAGSs can be single gene products, as in Mycobacterium spp. and Thermus spp., or fused to the enzyme catalyzing the last step of the pathway (argininosuccinase), as in members of the Alteromonas-Vibrio group. How these proteins bind glutamate remains to be determined. In some Bacteria, a bifunctional ornithine acetyltransferase (i.e., using both acetylornithine and acetyl-CoA as donors of the acetyl group) accounts for glutamate acetylation. In many Archaea, the enzyme responsible for glutamate acetylation remains elusive, but possible connections with a novel lysine biosynthetic pathway arose recently from genomic investigations. In some Proteobacteria (notably Xanthomonadaceae) and Bacteroidetes, the carbamoylation step of the pathway appears to involve N-acetylornithine or N-succinylornithine rather than ornithine. The product N-acetylcitrulline is deacetylated by an enzyme that is also involved in the provision of ornithine from acetylornithine; this is an important metabolic function, as ornithine itself can become essential as a source of other metabolites. This review insists on the biochemical and evolutionary implications of these findings.

scholarly journals Metabolic Reprogramming of Host Cells in Response to Enteroviral Infection

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 473 ◽  
Author(s):  
Mei-Ling Cheng ◽  
Kun-Yi Chien ◽  
Chien-Hsueh Lai ◽  
Guan-Jie Li ◽  
Jui-Fen Lin ◽  
...  
Keyword(s):  
Viral Replication ◽  
Host Cell ◽  
De Novo ◽  
Tricarboxylic Acid Cycle ◽  
Enterovirus 71 ◽  
Metabolic Reprogramming ◽  
Host Cells ◽  
Glutamine Supplementation ◽  
Aspartate Transcarbamylase ◽  
Functional Changes

Enterovirus 71 (EV71) infection is an endemic disease in Southeast Asia and China. We have previously shown that EV71 virus causes functional changes in mitochondria. It is speculative whether EV71 virus alters the host cell metabolism to its own benefit. Using a metabolomics approach, we demonstrate that EV71-infected Vero cells had significant changes in metabolism. Glutathione and its related metabolites, and several amino acids, such as glutamate and aspartate, changed significantly with the infectious dose of virus. Other pathways, including glycolysis and tricarboxylic acid cycle, were also altered. A change in glutamine/glutamate metabolism is critical to the viral infection. The presence of glutamine in culture medium was associated with an increase in viral replication. Dimethyl α-ketoglutarate treatment partially mimicked the effect of glutamine supplementation. In addition, the immunoblot analysis revealed that the expression of glutamate dehydrogenase (GDH) and trifunctional carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) increased during infection. Knockdown of expression of glutaminase (GLS), GDH and CAD drastically reduced the cytopathic effect (CPE) and viral replication. Furthermore, we found that CAD bound VP1 to promote the de novo pyrimidine synthesis. Our findings suggest that virus may induce metabolic reprogramming of host cells to promote its replication through interactions between viral and host cell proteins.

scholarly journals Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast

2019 ◽  
Author(s):  
Neta Agmon ◽  
Jasmine Temple ◽  
Zuojian Tang ◽  
Tobias Schraink ◽  
Maayan Baron ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Enzyme Regulation ◽  
Metabolite Level ◽  
De Novo Biosynthesis ◽  
Human Ortholog ◽  
Pathway Regulation ◽  
Traditional Approaches ◽  
Suppressor Analysis ◽  
Insight Into

Abstract Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the ‘humanized’ yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level ‘setpoints’ in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

Recovery of the photosynthetic apparatus from photoinhibition during dark incubation of the green alga Dunaliella salina

1999 ◽  
Vol 26 (7) ◽  
pp. 679 ◽  
Author(s):  
Jürgen E. W. Polle ◽  
Anastasios Melis
Keyword(s):  
Reaction Center ◽  
Dunaliella Salina ◽  
De Novo ◽  
Light Harvesting ◽  
Photosynthetic Apparatus ◽  
D1 Protein ◽  
Dark Incubation ◽  
Chl A ◽  
Lag Period ◽  
Stressed Cells

The light-independent recovery of the photosynthetic apparatus from photoinhibition was monitored upon a transition of irradiance-stressed Dunaliella salina Teod. to darkness. Upon dark incubation, the chlorophyll (Chl) a /Chl b ratio of the cells decreased promptly with a half-time of 2.5 h from about 12:1 to about 5:1. In contrast, dark incubation of control cells resulted in only a negligible change of the Chl a /Chl b ratio. During dark incubation of irradiance-stressed cells, the level of the Chl a and b light-harvesting proteins of photosystem II (PSII) increased, a change accompanied by alterations in the composition of these light-harvesting proteins. The amount of photodamaged PSII, measured from the relative amount of a 160 kDa protein complex which contains the photodamaged D1 reaction center protein, decreased during dark incubation after an initial lag period. Concomitantly, the amount of functional PSII, measured from the 32 kDa form of D1, increased slightly in the dark. The results show that, in the dark, photodamaged D1 is slowly removed upon degradation from the thylakoid membrane and replaced by a de novo synthesized D1 protein. The amount of reaction center proteins and number of photochemically active PSI centers increased in the dark. These results suggest that thylakoid membranes of irradiance-stressed D. salina exist in a state of dynamic flux. We conclude that several aspects of the D. salina recovery from photoinhibition are light independent.

Regulation of the purine salvage pathway in rat liver

1992 ◽  
Vol 262 (3) ◽  
pp. E344-E352 ◽  
Author(s):  
Y. A. Kim ◽  
M. T. King ◽  
W. E. Teague ◽  
G. A. Rufo ◽  
R. L. Veech ◽  
...  
Keyword(s):  
Rat Liver ◽  
De Novo ◽  
Equilibrium Constants ◽  
Purine Metabolism ◽  
Purine Salvage ◽  
Rate Limiting Step ◽  
Delta G ◽  
Salvage Pathways ◽  
Rate Limiting

The regulation of purine metabolism in rat liver has been examined under conditions that alter the flux through the pathway. Rats were given intraperitoneal injections of ethanol, sodium acetate, or sodium phosphate to attain body water concentrations of approximately 70, 20, and 10 mM, respectively. The livers were freeze-clamped after 30 min, and extracts were made for the analysis of metabolites, cofactors, purine bases, and nucleosides; homogenates were made for the measurement of the activities and kinetic parameters of seven enzymes that participate in purine salvage. The values of the equilibrium constants of nine reactions were determined in vitro and compared with the ratios of the reactants measured in liver. The changes in phosphoribosylpyrophosphate (PRPP), a key intermediate in both the de novo and salvage pathways of purine metabolism, were directly correlated with the changes in ribose 5-phosphate (ribose-5-P); ([PRPP] = 1.7[ribose-5-P] - 7.4 mumol/kg). Ribose-5-P concentrations in turn could be predicted from the liver content of fructose 6-phosphate and glyceraldehyde 3-phosphate by calculation from the known equilibria. The maximum velocities in the tissue of the seven enzymes measured were calculated from the measured substrate values in the liver and with consideration of other effectors of enzyme activity. PRPP synthetase was the least active of the enzymes measured, indicating a possible rate-limiting step. The delta G of the enzyme steps differed from equilibrium values by factors ranging from 4 (nucleoside phosphorylase) to 10(5) (PRPP synthetase and purine transferase reactions). The regulation of purine salvage appeared to depend on the levels of PRPP and ribose-5-P.

Characterization and chemical properties of phosphoribosylamine, an unstable intermediate in the de novo purine biosynthetic pathway

Biochemistry ◽  
1988 ◽  
Vol 27 (7) ◽  
pp. 2614-2623 ◽  
Author(s):  
F. J. Schendel ◽  
Y. S. Cheng ◽  
J. D. Otvos ◽  
S. Wehrli ◽  
JoAnne Stubbe
Keyword(s):  
Biosynthetic Pathway ◽  
De Novo ◽  
Chemical Properties ◽  
Unstable Intermediate ◽  
And Chemical Properties

scholarly journals UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts

2004 ◽  
Vol 279 (44) ◽  
pp. 45728-45736 ◽  
Author(s):  
Toshihisa Kotake ◽  
Daisuke Yamaguchi ◽  
Hiroshi Ohzono ◽  
Sachiko Hojo ◽  
Satoshi Kaneko ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Substrate Specificity ◽  
Molecular Mass ◽  
Glucuronic Acid ◽  
De Novo ◽  
Higher Plants ◽  
Maximum Activity ◽  
Broad Substrate Specificity ◽  
Salvage Pathways

UDP-sugars, activated forms of monosaccharides, are synthesized throughde novoand salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativumL.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5–7.5, and at 45 °C in the presence of 2 mmMg2+. The apparentKmvalues for Glc 1-phosphate andl-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively.PsUSPcDNA was cloned by reverse transcriptase-PCR.PsUSPappears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues fromArabidopsisand rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed inEscherichia colicatalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.

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