Search Results (19)

L-asparaginase (L-ASNase) remains a vital chemotherapeutic agent for acute lymphoblastic leukemia (ALL), primarily due to its mechanism of depleting circulating asparagine essential for leukemic cell proliferation. However, existing ASNases (including pegylated ones) face limitations including immunogenicity, rapid clearance, and off-target toxicities. Earlier, we have shown that the conjugation of L-ASNase with the polyamines and their copolymers results in significant enhancement of the antiproliferative activity due to accumulation in tumor cells. We suggested that this effect is probably mediated by polyamine transport system (PTS) receptors that are overexpressed in ALL cells. Here, we investigated the effect of competitive inhibitors of PTS receptors to the L-ASNase interaction with cancer cells (L5178Y, K562 and A549). L-ASNase from Rhodospirillum rubrum (RrA), Erwinia carotovora (EwA), and Escherichia coli (EcA) were conjugated with natural polyamines (spermine—spm, spermidine—spd, putrescine—put) and a synthetic branched polymer, polyethyleneimine 2 kDa (PEI2 ), using carbodiimide chemistry. Polyamine conjugation with L-ASNase significantly increased enzyme binding and cellular uptake, as quantified by fluorimetry and confocal microscopy. This increased cellular uptake translated into increased cytotoxicity of L-ASNase conjugates. The presence of competitive ligands to PTS receptors decreased the uptake of polyamine-conjugated enzymes-fatty acid derivatives of polyamines produced the strongest suppression. Simultaneously with this suppression, in some cases, competitive ligands to PTS significantly promoted the uptake of the native unconjugated enzymes, “equalizing” the cellular access for native vs conjugated ASNase. The screening for competing inhibitors of PTS receptor-mediated endocytosis revealed spermine and caproate/lipoate derivatives as the most potent inhibitors or antagonists, significantly reducing the cytostatic efficacy of polyamine-conjugated ASNases. The results obtained emphasize the complex, cell-type-dependent and inhibitor-specific nature of these interactions, which highlights the profound involvement of PTS in L-ASNase internalization and cytotoxic activity. These findings support the viability of polyamine conjugation as a strategy to enhance L-ASNase delivery and therapeutic efficacy by targeting the PTS. Full article

The underlying structural features of the mismatch between catalytic and cytostatic properties in L-asparaginase from Rhodospirillum rubrum (RrA) and three of its mutants were investigated. The rationale for selecting the specific mutations (RrA_{A64V, E67K}; RrA_{R118H, G120R}; RrA_{E149R, V150P, F151T}) is to elucidate the role of inter-subunit interaction in RrA and its impact on catalytic efficiency and stability. Bioinformatic modeling revealed a predominantly negative surface charge on RrA with limited positive charge clusters in the vicinity of the interface region. Thus, some negatively charged groups were replaced with positively charged ones to enhance the electrostatic interactions and stabilize the enzyme quaternary structure. RrA_{A64V, E67K} and RrA_{R118H, G120R} additionally contained an N-terminal 17-amino acid capsid peptide derived from the bacteriophage T7 (MASMTGGQQMGRGSSRQ), which could potentially affect the conformational stability of theenzymes. Circular dichroism (CD) spectroscopy was applied to the kinetic parameters analysis of Asn hydrolysis and showed that native RrA displayed a V_max of 30 U/mg and a K_M of 4.5 ± 0.5 mM. RrA_{E149R, V150P, and F151T} exhibited a substantially increased V_max of 57 U/mg. The catalytic efficiency of V_max/K_M also improved compared to the native enzyme: the V_max/K_M increased from approximately 7 U/mg × mM⁻¹ (for the native enzyme) to 9 U/mg × mM⁻¹ for Mut3. Other mutants exhibited less pronounced changes. Thermo-denaturation studies allowed us to determine the phase transition parameters of the RrA variants in comparison with commercial reference sample EcA. RrA_{A64V, E67K} and RrA_{R118H, G120R} exhibited the most favorable phase transition parameters, with melting temperatures (T_m) of 60.3 °C and 59.4 °C, respectively, exceeding that of the wild-type RrA (54.6 °C) and RrA_{E149R, V150P, F151T} (52 °C). The EcA demonstrated a slightly superior thermal stability, with a T_m of 62 °C. The mutations showed a significant effect on protein stability during trypsinolysis. Therefore, RrA_{E149R, V150P, F151T} showed higher resistance (45% activity remaining after 30 min of trypsin exposure) compared to the native RrA retained 20% activity. EcA preparations exhibited lower stability to trypsinolysis (losing over 90% activity in 15 min). The cytostatic effects were evaluated using MTT assays against K562 (leukemic) and A549 (lung carcinoma) cell lines. The MTT assays with K562 cells revealed that RrA_{E149R, V150P, F151T} (IC₅₀ of 10 U/mL) and RrA_{R118H, G120R} (IC₅₀ of 11.5 U/mL) exhibited superior antiproliferative activity compared to native enzymes RrA (IC₅₀ of 15 U/mL) and EcA (24 U/mL). RrA_{E149R, V150P, F151T} showed the most significant improvement in cytostatic activity. The results obtained indicate that the substitutions in RrA_{E149R, V150P, F151T} resulted in the improvement of the enzyme biocatalytic properties and an increase in the resistance to aggregation and trypsinolysis. This highlights the role of electrostatic interactions in stabilizing the oligomeric structure of the enzyme, which eventually translates into an improvement in cytostatic efficiency and antiproliferative forces. Full article

The genome of the mildly thermophilic hot spring purple sulfur bacterium, Allochromatium (Alc.) tepidum, contains a multigene pufBA family that encodes a series of α- and β-polypeptides, collectively forming a heterogeneous light-harvesting 1 (LH1) complex. The Alc. tepidum LH1, therefore, offers a unique model for studying an intermediate phenotype between phototrophic thermophilic and mesophilic bacteria, particularly regarding their LH1 Qy transition and moderately enhanced thermal stability. Of the 16 α-polypeptides in the Alc. tepidum LH1, six α1 bind Ca²⁺ to connect with β1- or β3-polypeptides in specific Ca²⁺-binding sites. Here, we use the purple bacterium Rhodospirillum rubrum strain H2 as a host to express Ca²⁺-bound and Ca²⁺-free Alc. tepidum LH1-only complexes composed of α- and β-polypeptides that either contain or lack the calcium-binding motif WxxDxI; purified preparations of each complex were then used to test how Ca²⁺ affects their thermostability and spectral features. The cryo-EM structures of both complexes were closed circular rings consisting of 14 αβ-polypeptides. The Q_y absorption maximum of Ca²⁺-bound LH1 (α1/β1 and α1/β3) was at 894 nm, while that of Ca²⁺-free (α2/β1) was at 888 nm, indicating that Ca²⁺ imparts a Q_y transition of 6 nm. Crucially for the ecological success of Alc. tepidum, Ca²⁺-bound LH1 complexes were more thermostable than Ca²⁺-free complexes, indicating that calcium plays at least two major roles in photosynthesis by Alc. tepidum—improving photocomplex stability and modifying its spectrum. Full article

Circadian rhythms, characterized by approximately 24 h cycles, play a pivotal role in enabling various organisms to synchronize their biological activities with daily variations. While ubiquitous in Eukaryotes, circadian clocks remain exclusively characterized in Cyanobacteria among Prokaryotes. These rhythms are regulated by a core oscillator, which is controlled by a cluster of three genes: kaiA, kaiB, and kaiC. Interestingly, recent studies revealed rhythmic activities, potentially tied to a circadian clock, in other Prokaryotes, including purple bacteria such as Rhodospirillum rubrum, known for its applications in fuel and plastic bioproduction. However, the pivotal question of how light and dark cycles influence protein dynamics and the expression of putative circadian clock genes remains unexplored in purple non-sulfur bacteria. Unraveling the regulation of these molecular clocks holds the key to unlocking optimal conditions for harnessing the biotechnological potential of R. rubrum. Understanding how its proteome responds to different light regimes—whether under continuous light or alternating light and dark cycles—could pave the way for precisely fine-tuning bioproduction processes. Here, we report for the first time the expressed proteome of R. rubrum grown under continuous light versus light and dark cycle conditions using a shotgun proteomic analysis. In addition, we measured the impact of light regimes on the expression of four putative circadian clock genes (kaiB1, kaiB2, kaiC1, kaiC2) at the transcriptional and translational levels using RT-qPCR and targeted proteomic (MRM-MS), respectively. The data revealed significant effects of light conditions on the overall differential regulation of the proteome, particularly during the early growth stages. Notably, several proteins were found to be differentially regulated during the light or dark period, thus impacting crucial biological processes such as energy conversion pathways and the general stress response. Furthermore, our study unveiled distinct regulation of the four kai genes at both the mRNA and protein levels in response to varying light conditions. Deciphering the impact of the diel cycle on purple bacteria not only enhances our understanding of their ecology but also holds promise for optimizing their applications in biotechnology, providing valuable insights into the origin and evolution of prokaryotic clock mechanisms. Full article

Numerous observations have supported the idea that various types of noncoding RNAs, including tRNA fragments (tRFs), are involved in communications between the host and its microbial community. The possibility of using their signaling function has stimulated the study of secreted RNAs, potentially involved in the interspecies interaction of bacteria. This work aimed at identifying such RNAs and characterizing their maturation during transport. We applied an approach that allowed us to detect oligoribonucleotides secreted by Prevotella copri (Segatella copri) or Rhodospirillum rubrum inside Escherichia coli cells. Four tRFs imported by E. coli cells co-cultured with these bacteria were obtained via chemical synthesis, and all of them affected the growth of E. coli. Their successive modifications in the culture medium and recipient cells were studied by high-throughput cDNA sequencing. Instead of the expected accidental exonucleolysis, in the milieu, we observed nonrandom cleavage by endonucleases continued in recipient cells. We also found intramolecular rearrangements of synthetic oligonucleotides, which may be considered traces of intermediate RNA circular isomerization. Using custom software, we estimated the frequency of such events in transcriptomes and secretomes of E. coli and observed surprising reproducibility in positions of such rare events, assuming the functionality of ring isoforms or their permuted derivatives in bacteria. Full article

This work aimed to study the structural features and mechanisms of thermoinactivation of hyperthermophilic L-asparaginase (L-ASNase) from archaea Thermococcus sibiricus (TsA) in comparison with bacterial L-ASNases from Melioribacter roseus (MrA) and Rhodospirillum rubrum (RrA). The catalytic parameters of L-asparagine hydrolysis under optimal conditions (pH 9) were determined for these enzymes by circular dichroism (CD) spectroscopy. TsA showed the highest activity among the studied L-ASNases (640 IU/mg at 90 °C). Thermo-inactivation kinetics were studied at temperatures close to the enzyme optimum: the first-order inactivation constants were 0.065 min⁻¹ (TsA), 0.011 min⁻¹ (MrA), and 0.026 min⁻¹ (RrA). In contrast to RrA and MrA, aggregation was detected as one of the thermoinactivation mechanisms for TsA. From the analysis of thermograms obtained with CD spectroscopy, the melting temperatures (Tm) for RrA, MrA, and TsA were determined as 50, 69, and 89 °C, respectively. A significant increase in the percentage of β-structures for TsA during heating (from 8 to 16%) indicating aggregation was observed in the interval from 70 to 100 °C. For RrA and MrA this value did not increase. Changes in the tertiary structure of the enzymes during heating were monitored by fluorescence spectroscopy. Thermal inactivation of RrA and MrA were accompanied by changes in the tertiary structure. For TsA, the observed denaturation enthalpy (ΔH) was 346 kJ/mol, which was 1.5–2 times higher than the same values for RrA and MrA. The study of the specific thermoinactivation mechanisms and structural- features in hyperthermophilic enzymes in comparison with mesophilic ones allows us to shed light on the molecular adaptation variants of the enzyme to function at high temperatures. Full article

L-asparaginase Rhodospirillum rubrum (RrA) is an enzyme (amidohydrolases; EC 3.5.1.1) that catalyzes the L-asparagine hydrolysis reaction to form L-aspartic acid. Due to the shortcomings of existing L-asparaginases from Esherichia coli (EcA) and Erwinia chrysanthemi (ErA), RrA may turn out to be a new promising drug for the treatment of leukemia. RrA has a low homology with EcA and ErA, which makes the enzyme potentially less immunogenic. RrA has pronounced antitumor activity on a number of leukemia cells. However, there is a need to improve the biocatalytic properties of the enzyme. So, in this study, the RrA conjugates with polyamines with different molecular architectures were developed to regulate the catalytic properties of the enzyme. Linear polyethyleneimine (PEI), branched polyethyleneimine, modified with polyethylene glycol (PEI-PEG), and spermine (Spm) were used to obtain conjugates with RrA. It was discovered by gel permeation chromatography that Spm allows the most active tetrameric form of RrA to be obtained and stabilized. Molecular docking was used to study the binding of spermine to RrA subunits. The activity of the RrA conjugates with Spm and PEI-PEG was 23–30% higher than the native enzyme. The pH optimum of the conjugates shifted from 9.0 to 8.5. The conjugates had higher stability: Spm and PEI-PEG reduced the inactivation constant (k_in) more than two-fold upon incubation at 53 °C. The conjugate RrA-PEI-PEG reduced the accessibility of trypsin to the protein surface and reduced k_in by eight times. The modification of RrA with polyamines made it possible to obtain enzyme preparations with improved biocatalytic properties. These conjugates represent interest for further study as potential therapeutic agents. Full article

Inorganic pyrophosphatases (PPases) catalyze an essential reaction, namely, the hydrolysis of PP_i, which is formed in large quantities as a side product of numerous cellular reactions. In the majority of living species, PP_i hydrolysis is carried out by soluble cytoplasmic PPase (S-PPases) with the released energy dissipated in the form of heat. In Rhodospirillum rubrum, part of this energy can be conserved by proton-pumping pyrophosphatase (H⁺-PPase^Rru) in the form of a proton electrochemical gradient for further ATP synthesis. Here, the codon-harmonized gene hppa^Rru encoding H⁺-PPase^Rru was expressed in the Escherichia coli chromosome. We demonstrate, for the first time, that H⁺-PPase^Rru complements the essential native S-PPase in E. coli cells. ¹³C-MFA confirmed that replacing native PPase to H⁺-PPase^Rru leads to the re-distribution of carbon fluxes; a statistically significant 36% decrease in tricarboxylic acid (TCA) cycle fluxes was found compared with wild-type E. coli MG1655. Such a flux re-distribution can indicate the presence of an additional method for energy generation (e.g., ATP), which can be useful for the microbiological production of a number of compounds, the biosynthesis of which requires the consumption of ATP. Full article

L-asparaginases (L-ASNases, EC 3.5.1.1) are a family of enzymes that are widely used for the treatment of lymphoblastic leukemias. L-ASNase from Rhodospirillum rubrum (RrA) has a low molecular weight, low glutaminase activity, and low immunogenicity, making it a promising enzyme for antitumor drug development. In our work, the complex formation and covalent conjugation of the enzyme with synthetic or natural polycationic polymers was studied. Among non-covalent polyelectrolyte complexes (PEC), polyethyleneimine (PEI) yielded the highest effect on RrA, increasing its activity by 30%. The RrA-PEI complex had increased stability to trypsinolysis, with an inactivation constant decrease up to 10-fold compared to that of the native enzyme. The covalent conjugation of RrA with chitosan-PEI, chitosan-polyethylene glycol (chitosan-PEG), and chitosan-glycol resulted in an increase in the specific activity of L-asparagine (up to 30%). RrA-chitosan-PEG demonstrated dramatically (by 60%) increased cytotoxic activity for human chronic myeloma leukemia K562 cells in comparison to the native enzyme. The antiproliferative activity of RrA and its conjugates was significantly higher (up to 50%) than for that of the commercially available EcA at the same concentration. The results of this study demonstrated that RrA conjugates with polycations can become a promising strategy for antitumor drug development. Full article

Lignocellulose and starch-based raw materials are often applied in the investigations regarding biohydrogen generation using dark fermentation. Management of the arising post-fermentation broth becomes a problem. The Authors proposed sequential processes, to improve the efficiency of both hydrogen generation and by-products management carried under model conditions. During the proposed procedure, the simple sugars remaining in broth are converted into organic acids, and when these products are used as substrates for the photo fermentation process. To enhance the broth management also conditions promoting Deep Eutectic Solvents (DES) precursors synthesis are simultaneously applied. Application of Box-Behnken design allows defining of the optimal conditions for conversion to DESs precursors. During the procedure hydrogen was obtained, the concentration of hydrogen in the photo fermentation reached up to 819 mL _H2/L _medium/7 d, depending on the broth type, i.e., when the broth was optimized for formic acid concentration. The DESs precursors were separated and engaged in DESs synthesis. To confirm the formation of the DESs, FT-IR analyses were performed. The Chemical Oxygen Demand of post-fermentation broths after dark fermentation optimized for formic acid was reduced by ca. 82%. The proposed procedure can be successfully used as a method of post-fermentation broth management. Full article

Biohydrogen production in small laboratory scale culture vessels is often difficult to perform and quantitate. One problem is that commonly used silicon tubing and improvised plastic connections used for constructing apparatus are cheap and easy to connect but are generally not robust for gases such as hydrogen. In addition, this type of apparatus presents significant safety concerns. Here, we demonstrate the construction of hydrogen-tight apparatus using a commercially available modular system, where plastic tubing and connections are made of explosion-proof dissipative plastic material. Using this system, we introduce a gas chromatograph calibration procedure, which can be easily performed without necessarily resorting to expensive commercial gas standards for the calibration of hydrogen gas concentrations. In this procedure, the amount of hydrogen produced by the reaction of sodium borohydride with water in a closed air-filled bottle is deduced from the observed decrease of the oxygen partial pressure, using the ideal gas law. Finally, the determined calibration coefficients and the gas-tight apparatus are used for the analysis of simultaneous oxygen consumption and hydrogen production of the purple photosynthetic bacterium, Rhodospirillum rubrum, during semi-aerobic growth in the dark. Full article

Rhodospirillum rubrum has a versatile metabolism, and as such can assimilate a broad range of carbon sources, including volatile fatty acids. These carbon sources are gaining increasing interest for biotechnological processes, since they reduce the production costs for numerous value-added compounds and contribute to the development of a more circular economy. Usually, studies characterizing carbon metabolism are performed by supplying a single carbon source; however, in both environmental and engineered conditions, cells would rather grow on mixtures of volatile fatty acids (VFAs) generated via anaerobic fermentation. In this study, we show that the use of a mixture of VFAs as carbon source appears to have a synergy effect on growth phenotype. In addition, while propionate and butyrate assimilation in Rs. rubrum is known to require an excess of bicarbonate in the culture medium, mixing them reduces the requirement for bicarbonate supplementation. The fixation of CO₂ is one of the main electron sinks in purple bacteria; therefore, this observation suggests an adaptation of both metabolic pathways used for the assimilation of these VFAs and redox homeostasis mechanism. Based on proteomic data, modification of the propionate assimilation pathway seems to occur with a switch from a methylmalonyl-CoA intermediate to the methylcitrate cycle. Moreover, it seems that the presence of a mixture of VFAs switches electron sinking from CO₂ fixation to H₂ and isoleucine production. Full article

The effect of singlet oxygen on light-harvesting (LH) complexes has been studied for a number of sulfur (S⁺) and nonsulfur (S⁻) photosynthetic bacteria. The visible/near-IR absorption spectra of the standard LH2 complexes (B800-850) of Allochromatium (Alc.) vinosum (S⁺), Rhodobacter (Rba.) sphaeroides (S⁻), Rhodoblastus (Rbl.) acidophilus (S⁻), and Rhodopseudomonas (Rps.) palustris (S⁻), two types LH2/LH3 (B800-850 and B800-830) of Thiorhodospira (T.) sibirica (S⁺), and an unusual LH2 complex (B800-827) of Marichromatium (Mch.) purpuratum (S⁺) or the LH1 complex from Rhodospirillum (Rsp.) rubrum (S⁻) were measured in aqueous buffer suspensions in the presence of singlet oxygen generated by the illumination of the dye Rose Bengal (RB). The content of carotenoids in the samples was determined using HPLC analysis. The LH2 complex of Alc. vinosum and T. sibirica with a reduced content of carotenoids was obtained from cells grown in the presence of diphenylamine (DPA), and LH complexes were obtained from the carotenoidless mutant of Rba. sphaeroides R26.1 and Rps. rubrum G9. We found that LH2 complexes containing a complete set of carotenoids were quite resistant to the destructive action of singlet oxygen in the case of Rba. sphaeroides and Mch. purpuratum. Complexes of other bacteria were much less stable, which can be judged by a strong irreversible decrease in the bacteriochlorophyll (BChl) absorption bands (at 850 or 830 nm, respectively) for sulfur bacteria and absorption bands (at 850 and 800 nm) for nonsulfur bacteria. Simultaneously, we observe the appearance of the oxidized product 3-acetyl-chlorophyll (AcChl) absorbing near 700 nm. Moreover, a decrease in the amount of carotenoids enhanced the spectral stability to the action of singlet oxygen of the LH2 and LH3 complexes from sulfur bacteria and kept it at the same level as in the control samples for carotenoidless mutants of nonsulfur bacteria. These results are discussed in terms of the current hypothesis on the protective functions of carotenoids in bacterial photosynthesis. We suggest that the ability of carotenoids to quench singlet oxygen (well-established in vitro) is not well realized in photosynthetic bacteria. We compared the oxidation of BChl850 in LH2 complexes of sulfur bacteria under the action of singlet oxygen (in the presence of 50 μM RB) or blue light absorbed by carotenoids. These processes are very similar: {[BChl + (RB or carotenoid) + light] + O₂} → AcChl. We speculate that carotenoids are capable of generating singlet oxygen when illuminated. The mechanism of this process is not yet clear. Full article

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is one of the best studied enzymes. It is crucial for photosynthesis, and thus for all of biosphere’s productivity. There are four isoforms of this enzyme, differing by amino acid sequence composition and quaternary structure. However, there is still a group of organisms, dinoflagellates, single-cell eukaryotes, that are confirmed to possess Rubisco, but no successful purification of the enzyme of such origin, and hence a generation of a crystal structure was reported to date. Here, we are using in silico tools to generate the possible structure of Rubisco from a dinoflagellate representative, Symbiodinium sp. We selected two templates: Rubisco from Rhodospirillum rubrum and Rhodopseudomonas palustris. Both enzymes are the so-called form II Rubiscos, but the first is exclusively a homodimer, while the second one forms homo-hexamers. Obtained models show no differences in amino acids crucial for Rubisco activity. The variation was found at two closely located inserts in the C-terminal domain, of which one extends a helix and the other forms a loop. These inserts most probably do not play a direct role in the enzyme’s activity, but may be responsible for interaction with an unknown protein partner, possibly a regulator or a chaperone. Analysis of the possible oligomerization interface indicated that Symbiodinium sp. Rubisco most likely forms a trimer of homodimers, not just a homodimer. This hypothesis was empowered by calculation of binding energies. Additionally, we found that the protein of study is significantly richer in cysteine residues, which may be the cause for its activity loss shortly after cell lysis. Furthermore, we evaluated the influence of the loop insert, identified exclusively in the Symbiodinium sp. protein, on the functionality of the recombinantly expressed R. rubrum Rubisco. All these findings shed new light onto dinoflagellate Rubisco and may help in future obtainment of a native, active enzyme. Full article