Trehalose counteracts the dissociation of tetrameric rabbit lactate dehydrogenase induced by acidic pH conditions

The lactate dehydrogenase from rabbit skeletal muscle (rbLDH) is a tetrameric enzyme, known to undergo dissociation when exposed to acidic pH conditions. Moreover, it should be mentioned that this dissociation translates into a pronounced loss of enzyme activity. Notably, among the compounds able to stabilize proteins and enzymes, the disaccharide trehalose represents an outperformer. In particular, trehalose was shown to efficiently counteract quite a number of physical and chemical agents inducing protein denaturation. However, no information is available on the effect, if any, exerted by trehalose against the dissociation of protein oligomers. Accordingly, we thought it of interest to investigate whether this disaccharide is competent in preventing the dissociation of rbLDH induced by acidic pH conditions. Further, we compared the action of trehalose with the effects triggered by maltose and cellobiose. Surprisingly, both these disaccharides enhanced the dissociation of rbLDH, with maltose being responsible for a major effect when compared to cellobiose. On the contrary, trehalose was effective in preventing enzyme dissociation, as revealed by activity assays and by Dynamic Light Scattering (DLS) experiments. Moreover, we detected a significant decrease of both K0.5 and Vmax when the rbLDH activity was tested (at pH 7.5 and 6.5) as a function of pyruvate concentration in the presence of trehalose. Further, we found that trehalose induces a remarkable increase of Vmax when the enzyme is exposed to pH 5. Overall, our observations suggest that trehalose triggers conformational rearrangements of tetrameric rbLDH mirrored by resistance to dissociation and peculiar catalytic features

Structural and catalytic insights into HoLaMa, a derivative of Klenow DNA polymerase lacking the proofreading domain

We report here on the stability and catalytic properties of the HoLaMa DNA polymerase, a Klenow sub-fragment lacking the 3\u2019-5\u2019 exonuclease domain. HoLaMa was overexpressed in Escherichia coli, and the enzyme was purified by means of standard chromatographic techniques. High-resolution NMR experiments revealed that HoLaMa is properly folded at pH 8.0 and 20C. In addition, urea induced a cooperative folding to unfolding transition of HoLaMa, possessing an overall thermodynamic stability and a transition midpoint featuring \u394G and C M equal to (15.7 \ub1 1.9) kJ/mol and (3.5 \ub1 0.6) M, respectively. When the catalytic performances of HoLaMa were compared to those featured by the Klenow enzyme, we did observe a 10-fold lower catalytic efficiency by the HoLaMa enzyme. Surprisingly, HoLaMa and Klenow DNA polymerases possess markedly different sensitivities in competitive inhibition assays performed to test the effect of single dNTPs

Human lactate dehydrogenase A undergoes allosteric transitions under pH conditions inducing the dissociation of the tetrameric enzyme

The aerobic energetic metabolism of eukaryotic cells relies on the glycolytic generation of pyruvate, which is subsequently channelled to the oxidative phosphorylation taking place in mitochondria. However, under conditions limiting oxidative phosphorylation, pyruvate is coupled to alternative energetic pathways, e.g. its reduction to lactate catalyzed by lactate dehydrogenases (LDHs). This biochemical process is known to induce a significant decrease in cytosolic pH, and is accordingly denoted lactic acidosis. Nevertheless, the mutual dependence of LDHs action and lactic acidosis is far from being fully understood. Using human LDH-A, here we show that when exposed to acidic pH this enzyme is subjected to homotropic allosteric transitions triggered by pyruvate. Conversely, human LDH-A features Michaelis–Menten kinetics at pH values equal to 7.0 or higher. Further, citrate, isocitrate, and malate were observed to activate human LDH-A, both at pH 5.0 and 6.5, with citrate and isocitrate being responsible for major effects. Dynamic light scattering (DLS) experiments revealed that the occurrence of allosteric kinetics in human LDH-A is mirrored by a consistent dissociation of the enzyme tetramer, suggesting that pyruvate promotes tetramer association under acidic conditions. Finally, using the human liver cancer cell line HepG2 we isolated cells featuring cytosolic pH equal to 7.3 or 6.5, and we observed a concomitant decrease in cytosolic pH and lactate secretion. Overall, our observations indicate the occurrence of a negative feedback between lactic acidosis and human LDH-A activity, and a complex regulation of this feedback by pyruvate and by some intermediates of the Krebs cycle

High-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2

Objectives: The aim of the present work was to identify a time-saving, effective, and low-cost strategy to produce in Escherichia coli a protein chimera representing a fusion anti-SARS-CoV-2 candidate vaccine, consisting of immunogenic and antigenic moieties. Results: We overexpressed in E. coli BL21(DE3) a synthetic gene coding for CRM197-RBD, and the target protein was detected in inclusion bodies. CRM197-RBD was solubilized with 1 % (w/v) of the anionic detergent N-lauroylsarcosine (sarkosyl), the removal of which from the protein solution was conveniently accomplished with Amberlite XAD-4. The detergent-free CRM197-RBD was then separated from contaminating DNA using polyethylenimine (PEI), and finally purified from PEI by salting out with ammonium sulfate. Structural (CD spectrum) and functional (DNase activity) assays revealed that the CRM197-RBD chimera featured a native and active conformation. Remarkably, we determined a yield of purified CRM197-RBD equal to 23 mg per litre of culture. Conclusions: To produce CRM197-RBD, we devised the use of sarkosyl as an alternative to urea to solubilize the target protein from E. coli inclusion bodies, and the easy removal of sarkosyl by means of Amberlite XAD-4

Luminescent protein staining with Re(i) tetrazolato complexes

Within the general framework of our past and current studies dealing with the investigation of the photophysical properties and the biological behavior of the family of tetrazolato and tetrazole Re(i) complexes, we have endeavored to investigate their potential in the luminescent staining of proteins purified by acrylamide gel electrophoresis. With the aim to provide the first examples of luminescent Re(i) complexes to be exploited for this specific purpose, we have designed and prepared four new Re(i)-based species with the general formula fac-[Re(CO)3(N^N)(Tph)]2-/0, where Tph is the 5-(phenyl)tetrazolato anion and N^N is in turn represented by bathophenanthroline disulfonate (BPS), bathocuproine disulfonate (BCS) or by the SO3-free bathocuproine (BC). In this latter case, the neutral complex fac-[Re(CO)3(BC)(Tph)] served as a model species for the characterization of the former disulfonate complexes. Its cationic analogue fac-[Re(CO)3(BC)(Tph-Me)]+was also prepared by a straightforward methylation reaction. All complexes displayed bright phosphorescence in organic media and, relative to their water solubility, the dianionic species fac-[Re(CO)3(BPS)(Tph)]2-and fac-[Re(CO)3(BCS)(Tph)]2-were also highly emissive in aqueous solution. The sulfonate groups played a key role in promoting and significantly enhancing the luminescent staining performances of both the Re(i) complexes fac-[Re(CO)3(BPS)(Tph)]2-and fac-[Re(CO)3(BCS)(Tph)]2-for proteins. Highlighting a response superior to that of Coomassie Blue and comparable to the one obtained by the well-known silver staining method, these dianionic Re(i)-complexes could efficiently detect up to 50 ng of pure Bovine Serum Albumin (BSA), as well as all proteins found in a Standard Protein Marker mix and from a total protein extract. A lower but still good response for luminescent protein staining was surprisingly obtained by employing the-SO3-free neutral and cationic complexes fac-[Re(CO)3(BC)(Tph)] and fac-[Re(CO)3(BC)(Tph-Me)]+, respectively. These preliminary results open up new possibilities for the further widening of the use of Re(i)-based complexes as luminescent protein staining agents

The Mycobacterium tuberculosis protein tyrosine phosphatase MptpA features a pH dependent activity overlapping the bacterium sensitivity to acidic conditions

The Mycobacterium tuberculosis low-molecular weight protein tyrosine phosphatase (MptpA) is responsible for the inhibition of phagosome-lysosome fusion and is essential for the bacterium patho-genicity. This inhibition implies that M. tuberculosis is not exposed to a strongly acidic environment in vivo, enabling successful propagation in host cells. Remarkably, MptpA has been previously structurally and functionally investigated, with special emphasis devoted to the enzyme properties at pH 8.0. Considering that the virulence of M. tuberculosis is strictly dependent on the avoidance of acidic con-ditions in vivo, we analysed the pH-dependence of the structural and catalytic properties of MptpA. Here we show that this enzyme undergoes pronounced conformational rearrangements when exposed to acidic pH conditions, inducing a severe decrease of the enzymatic catalytic efficiency at the expense of phosphotyrosine (pTyr). In particular, a mild decrease of pH from 6.5 to 6.0 triggers a significant increase of K0.5 of MptpA for phosphotyrosine, the phosphate group of which we determined to feature a pKa2 equal to 5.7. Surface plasmon resonance experiments confirmed that MptpA binds poorly to pTyr at pH values < 6.5. Notably, the effectiveness of the MptpA competitive inhibitor L335-M34 at pH 6 does largely outperform the inhibition exerted at neutral or alkaline pH values. Overall, our observations indicate a pronounced sensitivity of MptpA to acidic pH conditions, and suggest the search for competitive in-hibitors bearing a negatively charged group featuring pKa values lower than that of the substrate phosphate group. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved

The Photosynthetic Apparatus and Its Regulation in the Aerobic Gammaproteobacterium Congregibacter litoralis gen. nov., sp. nov

BACKGROUND: There is accumulating evidence that in some marine environments aerobic bacteriochlorophyll a-producing bacteria represent a significant part of the microbial population. The interaction of photosynthesis and carbon metabolism in these interesting bacteria is still largely unknown and requires further investigation in order to estimate their contribution to the marine carbon cycle. METHODOLOGY/PRINCIPAL FINDINGS: Here, we analyzed the structure, composition and regulation of the photosynthetic apparatus in the obligately aerobic marine gammaproteobacterium KT71(T). Photoheterotrophically grown cells were characterized by a poorly developed lamellar intracytoplasmic membrane system, a type 1 light-harvesting antenna complex and a photosynthetic reaction center associated with a tetraheme cytochrome c. The only photosynthetic pigments produced were bacteriochlorophyll a and spirilloxanthin. Under semiaerobic conditions KT71(T) cells expressing a photosynthetic apparatus showed a light-dependent increase of growth yield in the range of 1.3-2.5 fold. The expression level of the photosynthetic apparatus depended largely on the utilized substrate, the intermediary carbon metabolism and oxygen tension. In addition, pigment synthesis was strongly influenced by light, with blue light exerting the most significant effect, implicating that proteins containing a BLUF domain may be involved in regulation of the photosynthetic apparatus. Several phenotypic traits in KT71(T) could be identified that correlated with the assumed redox state of growing cells and thus could be used to monitor the cellular redox state under various incubation conditions. CONCLUSIONS/SIGNIFICANCE: In a hypothetical model that explains the regulation of the photosynthetic apparatus in strain KT71(T) we propose that the expression of photosynthesis genes depends on the cellular redox state and is maximal under conditions that allow a balanced membrane redox state. So far, bacteria capable of an obligately aerobic, photosynthetic metabolism constitute a unique phenotype within the class Gammaproteobacteria, so that it is justified to propose a new genus and species, Congregibacter litoralis gen. nov, sp. nov., represented by the type strain KT71(T) ( = DSM 17192(T) = NBRC 104960(T))

Espressione genica e fisiologia cellulare

Il contributo analizza la regolazione dell'espressione genica descrivendo la costruzione e lo sviluppo del modello dell'operon lac

Espressione batterica di un gene artificiale per la produzione di CRM197 e derivati

La presente invenzione si riferisce al campo della produzione di proteine d\u2019interesse farmacologico mediante sequenze geniche artificiali, dette sequenze inserite in vettori d\u2019espressione, sovraespressione delle corrispondenti proteine in microorganismi trasformati con detti vettori d\u2019espressione e metodo di isolamento delle proteine espresse; in particolare si riferisce alla costruzione di un gene artificiale codificante il CRM197 intero e i suoi derivati, all\u2019 espressione di CRM197 e dei suoi derivati in Escherichia coli e ad un metodo di isolamento e purificazione della proteina CRM197