Characterization of the C-terminal extension of carboxysomal carbonic anhydrase from Synechocystis sp. PCC6803

2002 ◽  
Vol 29 (3) ◽  
pp. 183 ◽  
Author(s):  
Anthony K.-C. So ◽  
Swan S.-W. Cot ◽  
George S. Espie
Keyword(s):  
Carbonic Anhydrase ◽  
Protein Interaction ◽  
Recombinant Dna ◽  
Carbonic Anhydrases ◽  
Recombinant Dna Technology ◽  
Higher Plant ◽  
High Sequence Identity ◽  
Synechocystis Pcc6803 ◽  
Plant Enzymes ◽  
Genes Encoding

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18O between 13C18O2and H216O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

Nerve-Derived Trophic Factors and DNA Elements Controlling Expression of Genes Encoding Synaptic Proteins in Skeletal Muscle Fibers

1998 ◽  
Vol 23 (4) ◽  
pp. 366-376 ◽  
Author(s):  
Bernard J. Jasmin ◽  
Anthony O. Gramolini ◽  
Feisal A. Adatia ◽  
Lindsay Angus ◽  
Céline Boudreau-Larivière ◽  
...  
Keyword(s):  
Neuromuscular Junction ◽  
Transcriptional Activation ◽  
Recombinant Dna ◽  
Signal Transduction Pathway ◽  
Regulatory Elements ◽  
Postsynaptic Membrane ◽  
Synaptic Proteins ◽  
Trophic Factors ◽  
Recombinant Dna Technology ◽  
Genes Encoding

The neuromuscular junction represents an excellent model system for studying various critical issues in neurobiology at the molecular, cellular, and physiological levels. Our understanding of the basic events underlying synpase formation, maintenance, and plasticity has progressed considerably over the last few years primarily because of the numerous studies that have focused on this synapse and used sophisticated recombinant DNA technology. Recent data indicate that myonuclei located in the vicinity of the postsynaptic membrane are in a differential state of transcription compared to nuclei of the extrasynaptic sarcoplasm. Thus, renewal of postsynaptic membrane proteins appears to occur via a mechanism involving the local transcriptional activation of genes encoding these specialized proteins and extracellular cues originating from motoneurons. Such interaction between presynaptic nerve terminals and the postsynaptic sarcoplasm indicates that the entire signal transduction pathway is compartmentalized at the level of the neuromuscular junction. Expression of these genes appears less coregulated than originally anticipated, indicating that maintenance of the postsynaptic membrane requires the contribution of multiple extracellular signals, which ultimately urge target transcription factors to distinct DNA regulatory elements via various second messenger systems. Key words: neuromuscular junction, synapse, acetylcholinesterase, utrophin, agrin, CGRP, promoter, mRNA

Carbonic anhydrase in eukaryotic algae: characterization, regulation, and possible function during photosynthesis

1998 ◽  
Vol 76 (6) ◽  
pp. 962-972 ◽  
Author(s):  
Dieter Sültemeyer
Keyword(s):  
Carbonic Anhydrase ◽  
Carbon Concentration ◽  
Inorganic Carbon ◽  
Molecular Level ◽  
Carbonic Anhydrases ◽  
Ph Values ◽  
Genes Encoding ◽  
Inorganic Carbon Concentration ◽  
Macro Algae ◽  
Made In

Carbonic anhydrase (CA) speeds up the equilibrium between CO2 and HCO3- at physiological pH values and has been detected in almost every species of the animal and plant kingdoms. Among eucaryotic micro- and macro-algae the enzyme is widely distributed and plays an important role in photosynthetic CO2 fixation. In some cases, different forms of carbonic anhydrases located extracellularly and intracellularly have been found to occur in the same cell. The expression of the genes encoding these CA isoforms are under the control of the inorganic carbon concentration in the medium, as the activities increase with decreasing the inorganic carbon content. Considerable progress has been made in recent years in isolating and characterizing the various forms of carbonic anhydrases on a biochemical and molecular level. Most of the data have been collected for microalgae like Chlamydomonas reinhardtii (Dangeard), while the situation in macroalgae is still descriptive. Therefore, this review summarizes the recent development with an emphasis on microalgae carbonic anhydrases.Key words: carbonic anhydrase, CO2 concentrating mechanism, macroalgae, microalgae, photosynthesis.

Isolation, Partial Purification and Characterization of a Novel Restriction Enzyme from Pseudomonas Anguilliseptica

2021 ◽  
Author(s):  
Swarna Nirosha Jayasinghe Pathirana ◽  
Don Anushka Sandaruwan Elvitigala ◽  
Chandrika Malkanthi Nanayakkara ◽  
Prashanth Suravajhala ◽  
Sanath Rajapakse ◽  
...  
Keyword(s):  
16S Rrna ◽  
Recombinant Dna ◽  
Bacterial Species ◽  
Evolutionary Relationship ◽  
Restriction Enzymes ◽  
Partial Purification ◽  
Recombinant Dna Technology ◽  
Recognition Sequence ◽  
16S Rrna Analysis ◽  
Genes Encoding

Abstract Type II restriction enzymes (REs) which can cleave double stranded DNA in a sequence specific manner have many applications in recombinant DNA technology and are considered the work horses of molecular biology. Soil and water samples were screened for isolation of bacteria, harboring restriction enzymes. Cell lysates of isolated bacteria were incubated with unmethylated λ DNA, followed by analysis by agarose gel electrophoresis. The presence of distinct banding patterns indicated the presence of REs. Nine putative isolates harboring REs were morphologically and molecularly characterized using 16S rRNA analysis and belonged to four different genera (Acinetobacter, Lysinibacillus, Pseudomonas, and Brevibacillus). A HindIII like restriction digestion profile was observed in a lysate of a soil bacterium belonging to genus Pseudomonas. Based on 16S rRNA analysis, the bacterial species was identified as P. angulliseptica. The enzyme was partially purified and optimum conditions for enzyme activity and its recognition sequence were determined. The enzyme showed optimum activity at 40 0C and was stable at 40 °C for 20 minutes without the DNA substrate. The Recognition sequence of the enzyme was determined and found to be 5’AAGCT 3’ indicating it to be an isoschizomer of HindIII. The whole genome of the Pseudomonas species was sequenced and the coding sequence of the gene for the putative HindIII isoschizomer was identified together with other genes encoding putative REs. The gene coding for the HindIII isoschizomer was analyzed in silico and its homology and evolutionary relationship to other known isoschizomers of HindIII were determined. The enzyme was tentatively designated as PanI.

scholarly journals Enhancing the Sweetness of Yoghurt through Metabolic Remodeling of Carbohydrate Metabolism in Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

2016 ◽  
Vol 82 (12) ◽  
pp. 3683-3692 ◽  
Author(s):  
Kim I. Sørensen ◽  
Mirjana Curic-Bawden ◽  
Mette P. Junge ◽  
Thomas Janzen ◽  
Eric Johansen
Keyword(s):  
Lactic Acid ◽  
Recombinant Dna ◽  
Streptococcus Thermophilus ◽  
Lactobacillus Delbrueckii ◽  
Phosphotransferase System ◽  
Recombinant Dna Technology ◽  
Kinase Gene ◽  
Content Type ◽  
Metabolic Remodeling ◽  
Genes Encoding

ABSTRACTStreptococcus thermophilusandLactobacillus delbrueckiisubsp.bulgaricusare used in the fermentation of milk to produce yoghurt. These species normally metabolize only the glucose moiety of lactose, secreting galactose and producing lactic acid as the main metabolic end product. We used multiple serial selection steps to isolate spontaneous mutants of industrial strains ofS. thermophilusandL. delbrueckiisubsp.bulgaricusthat secreted glucose rather than galactose when utilizing lactose as a carbon source. Sequencing revealed that theS. thermophilusstrains had mutations in thegalKTEMpromoter, the glucokinase gene, and genes encoding elements of the glucose/mannose phosphotransferase system (PTS). These strains metabolize galactose but are unable to phosphorylate glucose internally or via the PTS. TheL. delbrueckiisubsp.bulgaricusmutants had mutations in genes of the glucose/mannose PTS and in the pyruvate kinase gene. These strains cannot grow on exogenous glucose but are proficient at metabolizing internal glucose released from lactose by β-galactosidase. The resulting strains can be combined to ferment milk, producing yoghurt with no detectable lactose, moderate levels of galactose, and high levels of glucose. Since glucose tastes considerably sweeter than either lactose or galactose, the sweetness of the yoghurt is perceptibly enhanced. These strains were produced without the use of recombinant DNA technology and can be used for the industrial production of yoghurt with enhanced intrinsic sweetness and low residual levels of lactose.IMPORTANCEBased on a good understanding of the physiology of the lactic acid bacteriaStreptococcus thermophilusandLactobacillus delbrueckiisubsp.bulgaricus, we were able, by selecting spontaneously occurring mutants, to change dramatically the metabolic products secreted into the growth medium. These mutants consume substantially more of the lactose, metabolize some of the galactose, and secrete the remaining galactose and most of the glucose back into the milk. This allows production of yoghurt with very low lactose levels and enhanced natural sweetness, because humans perceive glucose as sweeter than either lactose or galactose.

Enzymatic Synthesis of 15N-L-aspartic Acid Using Recombinant Aspartase from Escherichia Coli K12

2008 ◽  
Vol 59 (11) ◽  
Author(s):  
Iulia Lupan ◽  
Sergiu Chira ◽  
Maria Chiriac ◽  
Nicolae Palibroda ◽  
Octavian Popescu
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Enzymatic Synthesis ◽  
Large Scale ◽  
Recombinant Dna ◽  
Industrial Enzymes ◽  
Recombinant Dna Technology ◽  
Bacterial Fermentation ◽  
Natural Protein ◽  
Novel Method

Amino acids are obtained by bacterial fermentation, extraction from natural protein or enzymatic synthesis from specific substrates. With the introduction of recombinant DNA technology, it has become possible to apply more rational approaches to enzymatic synthesis of amino acids. Aspartase (L-aspartate ammonia-lyase) catalyzes the reversible deamination of L-aspartic acid to yield fumaric acid and ammonia. It is one of the most important industrial enzymes used to produce L-aspartic acid on a large scale. Here we described a novel method for [15N] L-aspartic synthesis from fumarate and ammonia (15NH4Cl) using a recombinant aspartase.

Are Carbonic Anhydrases Suitable Targets to Fight Protozoan Parasitic Diseases?

2019 ◽  
Vol 25 (39) ◽  
pp. 5266-5278 ◽  
Author(s):  
Katia D'Ambrosio ◽  
Claudiu T. Supuran ◽  
Giuseppina De Simone
Keyword(s):  
Drug Resistance ◽  
Animal Models ◽  
Carbonic Anhydrase ◽  
Drug Target ◽  
Treatment Options ◽  
Parasitic Diseases ◽  
Carbonic Anhydrases ◽  
Extensive Drug Resistance ◽  
Protozoan Infections

Protozoans belonging to Plasmodium, Leishmania and Trypanosoma genera provoke widespread parasitic diseases with few treatment options and many of the clinically used drugs experiencing an extensive drug resistance phenomenon. In the last several years, the metalloenzyme Carbonic Anhydrase (CA, EC 4.2.1.1) was cloned and characterized in the genome of these protozoa, with the aim to search for a new drug target for fighting malaria, leishmaniasis and Chagas disease. P. falciparum encodes for a CA (PfCA) belonging to a novel genetic family, the η-CA class, L. donovani chagasi for a β-CA (LdcCA), whereas T. cruzi genome contains an α-CA (TcCA). These three enzymes were characterized in detail and a number of in vitro potent and selective inhibitors belonging to the sulfonamide, thiol, dithiocarbamate and hydroxamate classes were discovered. Some of these inhibitors were also effective in cell cultures and animal models of protozoan infections, making them of considerable interest for the development of new antiprotozoan drugs with a novel mechanism of action.

scholarly journals Protein-Engineered Polymers Functionalized with Antimicrobial Peptides for the Development of Active Surfaces

2021 ◽  
Vol 11 (12) ◽  
pp. 5352
Author(s):  
Ana Margarida Pereira ◽  
Diana Gomes ◽  
André da Costa ◽  
Simoni Campos Dias ◽  
Margarida Casal ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Peptides ◽  
Recombinant Dna ◽  
Biological Properties ◽  
Resistant Bacteria ◽  
Recombinant Dna Technology ◽  
Free Standing ◽  
Microbial Cell Factories ◽  
Cell Factories ◽  
Antimicrobial Materials

Antibacterial resistance is a major worldwide threat due to the increasing number of infections caused by antibiotic-resistant bacteria with medical devices being a major source of these infections. This suggests the need for new antimicrobial biomaterial designs able to withstand the increasing pressure of antimicrobial resistance. Recombinant protein polymers (rPPs) are an emerging class of nature-inspired biopolymers with unique chemical, physical and biological properties. These polymers can be functionalized with antimicrobial molecules utilizing recombinant DNA technology and then produced in microbial cell factories. In this work, we report the functionalization of rPBPs based on elastin and silk-elastin with different antimicrobial peptides (AMPs). These polymers were produced in Escherichia coli, successfully purified by employing non-chromatographic processes, and used for the production of free-standing films. The antimicrobial activity of the materials was evaluated against Gram-positive and Gram-negative bacteria, and results showed that the polymers demonstrated antimicrobial activity, pointing out the potential of these biopolymers for the development of new advanced antimicrobial materials.

scholarly journals Synthesis and Human Carbonic Anhydrase I, II, IX, and XII Inhibition Studies of Sulphonamides Incorporating Mono-, Bi- and Tricyclic Imide Moieties

2021 ◽  
Vol 14 (7) ◽  
pp. 693
Author(s):  
Kalyan K. Sethi ◽  
KM Abha Mishra ◽  
Saurabh M. Verma ◽  
Daniela Vullo ◽  
Fabrizio Carta ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Carbonic Anhydrase ◽  
Docking Studies ◽  
Carbonic Anhydrases ◽  
Molecular Docking Studies ◽  
Structure Activity Relationships ◽  
Structure Activity ◽  
Carbonic Anhydrase I ◽  
Human Carbonic Anhydrase ◽  
Inhibition Studies

New derivatives were synthesised by reaction of amino-containing aromatic sulphonamides with mono-, bi-, and tricyclic anhydrides. These sulphonamides were investigated as human carbonic anhydrases (hCAs, EC 4.2.1.1) I, II, IX, and XII inhibitors. hCA I was inhibited with inhibition constants (Kis) ranging from 49 to >10,000 nM. The physiologically dominant hCA II was significantly inhibited by most of the sulphonamide with the Kis ranging between 2.4 and 4515 nM. hCA IX and hCA XII were inhibited by these sulphonamides in the range of 9.7 to 7766 nM and 14 to 316 nM, respectively. The structure–activity relationships (SAR) are rationalised with the help of molecular docking studies.

Polypharmacology of epacadostat: a potent and selective inhibitor of the tumor associated carbonic anhydrases IX and XII

2019 ◽  
Vol 55 (40) ◽  
pp. 5720-5723 ◽  
Author(s):  
Andrea Angeli ◽  
Marta Ferraroni ◽  
Alessio Nocentini ◽  
Silvia Selleri ◽  
Paola Gratteri ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Selective Inhibitor ◽  
Carbonic Anhydrase Inhibitor ◽  
Carbonic Anhydrases ◽  
Indoleamine 2,3 Dioxygenase

Epacadostat (EPA), a selective indoleamine-2,3-dioxygenase 1 (IDO1) inhibitor, has been investigatedin vitroas a human (h) Carbonic Anhydrase Inhibitor (CAI).

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