Search

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence-related to other ATP-dependent enzymes involved in lactam hydrolysis, like 5-oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP-dependent acetone- and acetophenone carboxylases. The caprolactamase was produced in E. coli, isolated by His-tag affinity chromatography, and subjected to functional and structural studies. Activity towards caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6-aminocaproic acid (6-ACA). Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0 ' = 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP-binding site in CapA and a Zn-coordinating site in CapB. Mutations in the ATP-binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A and H124A) were inactive and less thermostable than the wild-type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP-dependent lactam hydrolysis. This article is protected by copyright. All rights reserved.

Background Takotsubo syndrome (TS) is defined as transient left ventricular dysfunction, which is often related to an emotional or physically stressful event. We describe a case of TS in a lady with pyruvate carboxylase deficiency (PCD). Pyruvate carboxylase deficiency is rare condition with the majority of those affected demonstrating signs of failure to thrive, recurrent seizures, and metabolic acidosis. To our knowledge, this is the first documented case of TS in an individual with PCD. Case summary This 28-year-old female presented to the emergency department after a tonic-clonic seizure. For 4 days prior to the presentation, she had been suffering from cough and pyrexia. On Day 2, she developed abdominal pain associated with tachycardia and hypotension, and an elevated troponin (791 ng/L). The echocardiogram showed a severely impaired left ventricular systolic function, regional wall motion abnormalities (RWMAs), and a visually estimated left ventricular ejection fraction of 25–30%. Eight days following admission her clinical state significantly improved, with a reduction troponin to 60 ng/L. A repeat echocardiogram on Day 9 showed complete resolution of cardiac function with no RWMAs. Following this, she was discharged from hospital the next day with a diagnosis of TS. Discussion This is the first case report of TS in a patient with PCD. In this case, multiple aetiologies of TS such as emotional and physical stress, seizures, and acute infection were considered. This case also highlights that TS should be an important differential diagnosis in patients presenting with cardiac symptoms.

Anthropogenic carbon dioxide (CO2) emissions cause an imbalance in the global carbon cycle that consequently leads to global warming. Besides the indisputable role of CO2 as harmful greenhouse gas, this small molecule harbors great potential as a simple and accessible carbon source. To exploit this potential, biotechnological strategies need to be established to convert CO2 into value-added products, like fuels or antibiotics. It is therefore indispensable to identify and characterize efficient carboxylases. To date, the members of the enoyl-CoA carboxylase/reductase (Ecr) family account for the most efficient carboxylases found in nature. Their efficiency partly depends on the effective stabilization of the CO2 molecule within the active site. The conserved CO2-binding motif is characteristic for Ecrs. This work deals with the thorough study of the three-domain fusion enzyme propionyl-CoA synthase (Pcs) of Erythrobacter sp. NAP1. This complex enzyme comprises the Ecr family CO2-binding motif in its reductase domain, suggesting a potential carboxylase activity and therefore deserves detailed investigation. The first part sets a focus on the biochemical features of Pcs. Combined kinetic and structural analysis proposed that Pcs uses a highly synchronized catalytic mechanism to sequester its reactive intermediate acrylyl-CoA. X-ray crystallography revealed an enclosed reaction chamber that features all three active sites of the fusion enzyme. This allows for the catalysis of the three subsequent reactions within the chamber. Kinetic data supported the idea that conformational changes in the Pcs ligase domain regulate the opening and closing of the catalytic compartment. Additional structural elements in Pcs either mimic domains of neighboring protomers in standalone homologues that contribute essential residues for catalysis or seal the reaction chamber. The presumed carboxylation potential of the reductase domain was demonstrated albeit at a very low efficiency in Pcs wildtype. Rational design was used to implement the two principles of efficient carboxylation known from Ecrs into the Pcs reductase domain. Improved CO2-binding and shielding of the active site from water converted the reductase domain into a carboxylase domain. The engineered trifunctional, substrate-channeling carboxylase could prove advantageous in synthetic CO2-fixation pathways. In the second part of this work, light is shed on the physiological and ecological role of Pcs. While well described in the context of the autotrophic 3-hydroxypropionate bi-cycle in Chloroflexus aurantiacus, the presence of Pcs in the genome of several (potential photo-) heterotrophic microorganisms suggests an alternative function. The genome of the aerobic anoxygenic phototrophic bacterium Erythrobacter sp. NAP1 encodes homologous enzymes of a partial 3-hydroxypropionate bi-cycle able to convert acetyl-CoA and two bicarbonate molecules into succinyl-CoA. The two key enzymes, Pcs and malonyl-CoA reductase (Mcr), were shown to be upregulated when the cells were grown in the light. Hence, it was suggested that this pathway might be involved in the adjustment of photosynthesis-induced redox imbalance., Anthropogene Kohlenstoffdioxid (CO2) Emissionen stören das Gleichgewicht im globalen Kohlenstoffzyklus und führen schließlich zur Erderwärmung. Abgesehen von der Funktion als schädliches Treibhausgas, steckt in diesem kleinen Molekül großes Potential als einfache und zugängliche Kohlenstoffquelle. Um dieses Potential jedoch ausnutzen zu können, müssen biotechnologische Strategien entwickelt werden für die Umsetzung von CO2 in höherwertige Produkte, wie Treibstoff oder Antibiotika. Die Identifizierung und Charakterisierung von effizienten Carboxylasen ist daher unabdingbar. Die Familie der Enoyl-CoA Carboxylasen/Reduktasen (Ecr) umfasst die am schnellsten CO2-fixierenden Enzyme, die bisher in der Natur identifiziert wurden. Ihre Effizienz beruht unter anderem auf der erfolgreichen Stabilisierung des CO2-Moleküls im aktiven Zentrum des Enzyms. Das konservierte CO2-Bindungsmotiv ist charakteristisch für Ecrs. Diese Arbeit befasst sich mit der gründlichen Untersuchung des Fusionsenzyms Propionyl-CoA Synthase (Pcs) von Erythrobacter sp. NAP1, welches aus drei Domänen besteht. Die Reduktase-Domäne dieses komplexen Enzyms enthält das CO2-Bindungsmotiv der Ecr Familie, was eine potentielle Aktivität als Carboxylase vermuten lässt und daher genauer untersucht werden soll. Der erste Teil dieser Arbeit konzentriert sich auf die biochemischen Eigenschaften von Pcs. Anhand kinetischer und struktureller Analysen wurde vorgeschlagen, dass Pcs einen hoch-synchronisierten Katalysemechanismus nutzt, um das Entweichen des reaktiven Zwischenprodukts Acrylyl-CoA zu verhindern. Proteinkristallographie offenbarte eine geschlossene Reaktionskammer, welche alle drei aktiven Zentren des Fusionsenzyms enthält. Somit wird die Katalyse der drei aufeinanderfolgenden Reaktionen im Innern der Kammer ermöglicht. Kinetische Daten stützten die Idee, dass Konformationsänderungen in der Pcs Ligase-Domäne das Öffnen und Schließen der Kammer regulieren. Zusätzliche strukturelle Elemente in Pcs imitieren entweder Domänen benachbarter Protomere in eigenständigen Homologen, welche wesentliche Aminosäurereste zur Katalyse beitragen, oder verschließen die Reaktionskammer. Das vorgeschlagene Carboxylierungspotential der Reduktase-Domäne konnte nachgewiesen werden, wenn auch nur mit sehr geringer Effizienz im Pcs Wildtyp. Rationales Design wurde angewandt, um die zwei Prinzipien der effizienten Carboxylierung, welche aus den Ecrs bekannt sind, in der Reduktase-Domäne der Pcs anzuwenden. Verbesserte CO2-Bindung und Abschirmung des aktiven Zentrums gegen das Einströmen von Wasser überführten die Reduktase-Domäne in eine Carboxylase-Domäne. Die verbesserte trifunktionelle Carboxylase, welche den direkten Transfer reaktiver Zwischenprodukte von einem aktiven Zentrum zum nächsten ermöglicht, könnte sich in einem synthetischen Stoffwechselweg zur CO2-Fixierung als sehr erfolgreich erweisen. Der zweite Teil dieser Arbeit gibt Aufschluss über die physiologische und ökologische Rolle von Pcs. Pcs wurde ursprünglich im Kontext des autotrophen 3-Hydroxypropionat Bizyklus in Chloroflexus aurantiacus entdeckt und detailliert beschrieben. Die Verbreitung des pcs Gens in mehreren (potentiell photo-) heterotrophen Mikroorganismen suggeriert jedoch noch eine alternative Funktion. Im Genomdes aeroben anoxygenen phototrophen Bakteriums Erythrobacter sp. NAP1 sind neben pcs auch alle Gene des 3-Hydroxypropionat-Bizyklus Abschnittes vertreten, um Acetyl-CoA und zwei Bicarbonat-Moleküle in Succinyl-CoA umzuwandeln. Die zwei Schlüsselenzyme Pcs und Malonyl-CoA Reduktase (Mcr) waren in höheren Mengen vertreten, wenn die Zellen bei Licht wuchsen. Folglich wurde vorgeschlagen, dass dieser Stoffwechselweg in der Regulierung eines Redox-Ungleichgewichts beteiligt ist, welches durch photosynthetische Aktivität ausgelöst wird.

The structure of 2-ketopropyl coenzyme M oxidoreductase/carboxylase (2-KPCC) has been determined in a state in which CO2 is observed providing insights into the mechanism of carboxylation. In the substrate encapsulated state of the enzyme, CO2 is bound at the base of a narrow hydrophobic substrate access channel. The base of the channel is demarcated by a transition from a hydrophobic to hydrophilic environment where CO2 is located in position for attack on the carbanion of the ketopropyl group of the substrate to ultimately produce acetoacetate. This binding mode effectively discriminates against H2O and prevents protonation of the ketopropyl leaving group. Structured summary 2-KPCC binds to 2-KPCC by x-ray crystallography ( View interaction )

Mycolic acids are essential for the survival, virulence and antibiotic resistance of the human pathogen Mycobacterium tuberculosis. Inhibitors of mycolic acid biosynthesis, such as isoniazid and ethionamide, have been used as efficient drugs for the treatment of tuberculosis. However, the increase in cases of multidrug-resistant tuberculosis has prompted a search for new targets and agents that could also affect synthesis of mycolic acids. In mycobacteria, the acyl-CoA carboxylases (ACCases) provide the building blocks for de novo fatty acid biosynthesis by fatty acid synthase (FAS) I and for the elongation of FAS I products by the FAS II complex to produce meromycolic acids. By generating a conditional mutant in the accD6 gene of Mycobacterium smegmatis, we demonstrated that AccD6 is the essential carboxyltransferase component of the ACCase 6 enzyme complex implicated in the biosynthesis of malonyl-CoA, the substrate of the two FAS enzymes of Mycobacterium species. Based on the conserved structure of the AccD5 and AccD6 active sites we screened several inhibitors of AccD5 as potential inhibitors of AccD6 and found that the ligand NCI-172033 was capable of inhibiting AccD6 with an IC50 of 8 ìM. The compound showed bactericidal activity against several pathogenic Mycobacterium species by producing a strong inhibition of both fatty acid and mycolic acid biosynthesis at minimal inhibitory concentrations. Overexpression of accD6 in M. smegmatis conferred resistance to NCI-172033, confirming AccD6 as the main target of the inhibitor. These results define the biological role of a key ACCase in the biosynthesis of membrane and cell envelope fatty acids, and provide a new target, AccD6, for rational development of novel anti-mycobacterial drugs Fil: Kurth, Daniel German. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Gago, Gabriela Marisa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: de la Iglesia, Agustina Inés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Bazet Lyonnet, Bernardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina Fil: Lin, Ting Wang. University of California; Estados Unidos Fil: Morbidoni, Héctor Ricardo. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina Fil: Tsai, Shiou Chuan. University of California; Estados Unidos Fil: Gramajo, Hugo Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina

The involvement of a carboxylase in metabolism of C-2 alkenes by Ochrobactrum sp. strain TD and Pseudomonas putida strain AJ was examined. With resting cells of strain TD grown on vinyl chloride, ethene, and ethylene oxide, the maximum specific rate of ethylene oxide consumption decreased significantly in the absence of external CO2 in comparison to cells provided with room air or added CO2. The amount of 14CO2 incorporated into biomass by resting cells of strain TD grown on ethylene oxide increased more than 13-fold when the assay substrate was ethylene oxide versus acetate. These results indicate that strain TD uses a carboxylase. Similar experiments were performed with strain AJ with the results suggesting that a carboxylase is not involved. In this regard, strain AJ is more similar to various Mycobacterium isolates that also do not appear to use a carboxylase during metabolism of vinyl chloride and ethene., Department of Biomedical and Biomolecular Sciences, Sheffield Hallam University.

We report draft genome sequence of Haloterrigena turkmenica strain WANU15, isolated from Soda Lake. The draft genome size is 2,950,899 bp with a G + C content of 64% and contains 49 RNA sequence. The genome sequence can be accessed at DDBJ/EMBL/GenBank under the accession no. LKCV00000000. Keywords: Soda Lake, Haloterrigena turkmenica, Carboxylesterase, Carboxylase, Xylose isomerase, Whole genome sequencing

Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy.

Holocarboxylase synthetase (HCS), catalyzing the covalent attachment of biotin, is ubiquitously represented in living organisms. Indeed, the biotinylation is a post-translational modification that allows the transformation of inactive biotin-dependent carboxylases, which are committed in fundamental metabolisms such as fatty acid synthesis, into their active holo form. Among other living organisms, plants present a peculiarly complex situation. In pea, HCS activity has been detected in three subcellular compartments and the systematic sequencing of the Arabidopsis genome revealed the occurrence of two hcs genes (hcs1 and hcs2). Hcs1 gene product had been previously characterized at molecular and biochemical levels. Here, by PCR amplification, we cloned an hcs2 cDNA from Arabidopsis thaliana (Ws ecotype) mRNA. We observed the occurrence of multiple cDNA forms which resulted from the alternative splicing of hcs2 mRNA. Furthermore, we evidenced a nucleotide polymorphism at the hcs2 gene within the Ws ecotype, which affected splicing of hcs2 mRNA. This contrasted sharply with the situation at hcs1 locus. However, this polymorphism had no apparent effect on total HCS activity in planta. Finally, hcs2 mRNAs were found 4-fold less abundant than hcs1 mRNA and the most abundant hcs2 mRNA spliced variant should code for a truncated protein. We discuss the possible role of such a multiplicity of putative HCS proteins in plants and discuss the involvement of each of hcs genes in the correct realization of biotinylation.

Growth and photosynthesis of an alloplasmic tomato (cybrid), i.e. line AH47, containing the nuclear genome of the chilling-sensitive cytoplasmic albino mutant of L. esculentum Mill. 'Large Red Cherry' (LRC) and the plastome of a more chilling-tolerant high-altitude accession of the related wild species L, hirsutum Humb. & Bonpl. LA 1777, were investigated at an optimal (25/20 degrees C) and suboptimal (16/14 degrees C) day/night temperature regime and their performance compared with that of both euplasmic parents. The cybrid shoot had a similar biomass and development rate to the nuclear tomato (L. esculentum) parent at both temperature regimes. Compared with the biomass production of shoots grown at optimal temperature, the reduction in shoot biomass at suboptimal temperature was smaller for L. hirsutum than for L, esculentum and the cybrid. This difference was related to a stronger inhibition of leaf area expansion in L, esculentum and the cybrid in the suboptimal temperature regime than in L. hirsutum. Irrespective of the temperature regime under which the plants were grown, photosynthetic performance and leaf pigment, carbohydrate and soluble-protein contents of the cybrid resembled those of the nuclear parent. No advantages of the alien L. hirsutum chloroplast with respect to growth and photosynthesis-related characteristics were observed in the cybrid in the suboptimal temperature regime, indicating that the temperature sensitivity of the photosynthetic apparatus is regulated by nuclear genes, An adverse consequence of interspecific chloroplast transfer was the increased susceptibility to chill-induced photoinhibition of the cybrid. It is concluded that cybridization is not a useful tool for improving low-temperature tolerance of tomato. (C) 2000 Annals of Botany Company.

The expression of the cbb and gap–pgk operons of Xanthobacter flavus encoding enzymes of the Calvin cycle is regulated by the transcriptional regulator CbbR. In order to identify other genes involved in the regulation of these operons, a mutant was isolated with a lowered activity of a fusion between the promoter of the cbb operon and the reporter gene lacZ. This mutant was unable to grow autotrophically and had a reduced growth rate of medium supplemented with gluconate or succinate. The regulation of the gap–pgk operon in the mutant was indistinguishable from the wild-type strain, but induction of the cbb operon upon transition to autotrophic growth conditions was delayed. Complementation of the mutant with a genomic library of X. flavus resulted in the isolation of a 1∙1 kb ApaI fragment which restored autotrophic growth of the mutant. One open reading frame (ORF) was present on the ApaI fragment, which could encode a protein highly similar to triosephosphate isomerase proteins from other bacteria. Cell extracts of the mutant grown under glycolytic or gluconeogenic conditions had severely reduced triosephosphate isomerase activities. The ORF was therefore identified as tpi, encoding triosephosphate isomerase. The tpi gene is not linked to the previously identified operons encoding Calvin cycle enzymes and therefore represents a third transcriptional unit required for autotrophic metabolism.

coding for β-galactosidase to the promoterregion of a structural gene is a frequently used technique to study the expression of the gene.The rate of transcription, inferred from the β-galactosidase activity of the cells, is equated to‘intrinsic promoter strength' but, more importantly, also measures the action of

We quantified the ozone impact on levels of Zea mays L. cv. Chambord mRNAs encoding C4-phosphoenolpyruvate carboxylase (C4-PEPc), ribulose-l,5-bisphosphate carboxylase/oxygenase small and large subunits (Rubisco-SSU and Rubisco-LSU, respectively) and Rubisco activase (RCA) using real-time RT-PCR. Foliar pigment content, PEPc and Rubisco protein amounts were simultaneously determined. Two experiments were performed to study the ozone response of the 5th and the 10th leaf. For each experiment, three ozone concentrations were tested in open-top chambers: non-filtered air (NF, control) and non-filtered air containing 40 (+40) and 80 nL L-1 (+80) ozone. Regarding the 5th leaf, +40 atmosphere induced a loss in pigmentation, PEPc and Rubisco activase mRNAs. However, it was unable to notably depress carboxylase protein amounts and mRNAs encoding Rubisco. Except for Rubisco mRNAs, all other measured parameters from 5th leaf were depressed by +80 atmosphere. Regarding the 10th leaf, +40 atmosphere increased photosynthetic pigments and transcripts encoding Rubisco and Rubisco activase. Rubisco and PEPc protein amounts were not drastically changed, even if they tended to be increased. Level of C4-PEPc mRNA remained almost stable. In response to +80 atmosphere, pigments and transcripts encoding PEPc were notably decreased. Rubisco and PEPc protein amounts also declined to a lesser extent. Conversely, the level of transcripts encoding both Rubisco subunits and Rubisco activase that were not consistently disturbed tended to be slightly augmented. So, the present study suggests that maize leaves can respond differentially to a similar ozone stress.

The presence of vitamin K-dependent carboxylase was investigated in the microsomal fraction of 20 different types of bovine tissue. Except for muscle, veins, lymphocytes and bone membrane, carboxylase was found in all these preparations, albeit in varying amounts. No differences could be detected between these carboxylating systems with respect to their affinity for vitamin K and warfarin. Most of the endogenous substrates had some affinity towards antiprothrombin or antifactor X.

Bovine bone Gla-protein (B.G.P.) was prepared and decarboxylated into descarboxy-B.G.P. (d-B.G.P.). The latter was purified and identified as decarboxylated osteocalcin. Both crude and purified d-B.G.P. are good substrates for vitamin K-dependent carboxylase. Because the Km of this enzyme for d-B.G.P. is low, the latter is a better substrate than the frequently used pentapeptide F L E E L or exogenous protein substrates such as descarboxyprothrombin.

In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of proteinbound glutamic acid residues, is recycled by the action of one or more dithiol-dependent reductases. In vitro synthetic dithiols may serve as a cofactor for these enzymes, but the physiological reluctant has not yet been found. In this paper we report that in vitro the commercially available thioredoxin/thioredoxin reductase from E. coli can replace the synthetic dithiols during the various reactions of the vitamin K cycle. Based on the assumption that in vivo thioredoxin also plays a role in the regeneration of vitamin K hydroquinone from the epoxide, an extension of the generally accepted vitamin K cycle is proposed.

The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH.