Form Of Enzyme Research Articles

Sialic Acids and Cancer: Pathophysiological Association between Metastatic Progress and Treatment

Abstract: Neoplasm metastasis is a multi-step process with a high rate of cancer mortality (>60%). Several complex pathogenesis pathways and key therapeutic targets are unclear to us now. To change this scenario, effective drug targets and underlying mechanisms should be found, and high-quality metastasis treatment should be supported. Aberrant tumor sialylation was proposed as a putative drug target candidate to bridge the gaps between metastatic spread and drug responses (genetic, molecular, and animal models). More recently, several promising therapeutic mechanisms and benefits against neoplasm metastasis have been observed by potential association for the target of higher levels and diverse forms of sialic acids (sia) analogues, antigens, glycan, sialylation enzymes, and conjugates. Subsequently, sia-related pathophysiology in cancer diagnosis, prognosis, and therapeutic responses has been reviewed. New algorithms, computation, experimental evaluations, and modern technology might see breakthroughs in therapeutic targets, responses, and immune regulation via sialylation enzymes, associated genes, different glycol conjugates, and other hallmarks of cancer.

Antioxidant and antidiabetic potential of Cystoseira myrica-mediated green metallic zinc nanoparticles using the algal extract: synthesis and characterization

Abstract Cystoseira myrica marine macroalgae (CSR) were used to produce metallic zinc nanoparticle composites by utilizing the phytochemicals naturally found in the algae. This involves homogenizing the residuals of CSR (10 g), zinc nitrate solution (5 M; 100 ml), and methanol liquid extract (100 ml) at 30 °C for 24 h of sonication and stirring, followed by filtration and drying. This resulted in a hybrid bio-composite (Zn/CSR), which demonstrated strong antioxidant and antidiabetic properties when compared to zinc oxide (ZnO) and CSR used separately. The Zn/CSR hybrid showed excellent antioxidant activity against common radicals such as DPPH (91.5 ± 1.66%), nitric oxide (90.4 ± 1.2%), ABTS (92.2 ± 1.9%), and O2 ·− (27.8 ± 1.12%) (p < 0.05), performing better than the standard antioxidant, ascorbic acid. Regarding its antidiabetic properties, the Zn/CSR composite significantly inhibited key enzymes involved in diabetes, including both commercial enzyme forms (α-amylase (80.3 ± 1.65%), α-glucosidase (96.6 ± 1.11%), amyloglucosidase (95.8 ± 1.3%)) and their crude intestinal forms (α-amylase (72.3 ± 1.5%), α-glucosidase (94.2 ± 1.7%)) (p < 0.05). This improvement increases the impact of the green CSR extract in reducing the agglomeration behaviors of the loaded metal and the formation of a capping layer from the phytochemicals on its surface, in addition to the beneficial effects of the CSR as substrate, which enhances the biological functions of the loaded metal and its interaction interfaces. The Zn/CSR composite also outperformed commercial miglitol drugs and slightly surpassed acarbose in effectiveness. Given the high cost and potential side effects of current medications, the Zn/CSR composite could be a cost-effective alternative for antioxidant and antidiabetic treatments. These findings also emphasize the role of CSR-derived phytochemicals and algae residues in enhancing the biological activity of the metal nanoparticles.

Virulence characterization of Staphylococcus spp. isolated from mozzarella cheeses and cheese-slicers

Different species of the Staphylococcus genus can be found as contaminants in foods and may carry virulence factors that could potentially make them The enterotoxigenic potential ofpathogenic or transfer genes to other bacteria. Therefore, the aim of the present study was to isolate and identify species of Staphylococcus genus isolated from sliced mozzarella cheeses and cold-cut slicers and characterize biofilm formation in vitro, presence of enterotoxins and antibiotic resistance genes, and β-lactamase production. Sampling was carried out in 44 retail markets and isolates identified on species level. Resistance genes blaZ and mecA, as well as enterotoxin genes sea, seb, sec, and sed, were identified using PCR. Phenotypic evaluation of biofilm formation and detection of β-lactamase enzyme were also analyzed. Nine species of coagulase-negative Staphylococcus (CNS) were identified from 85 bacterial isolates. Of this total, 67.0% were positive for the presence of one or both antimicrobial resistance genes blaZ and mecA, while the β-lactamase enzyme was detected in 52.9% isolates. Regarding biofilm production, 48.2% of isolates were positive and 51.8% were negative. However, none of the isolates were identified with the enterotoxin genes. The results demonstrate the potential risk of cross-contamination, as biofilm-producing CNS could persist in the cheese retail environment, highlighting the danger of resistance genes transferring to other commensal or pathogenic bacteria.

TRAF7 determines circadian period through ubiquitination and degradation of DBP.

D-site binding protein, DBP, is a clock-controlled transcription factor and drives daily rhythms of physiological processes through the regulation of an array of genes harboring a DNA binding motif, D-box. DBP protein levels show a circadian oscillation with an extremely robust peak/trough ratio, but it is elusive how the temporal pattern is regulated by post-translational regulation. In this study, we show that DBP protein levels are down-regulated by the ubiquitin-proteasome pathway. Analysis using 19 dominant-negative forms of E2 enzymes have revealed that UBE2G1 and UBE2T mediate the degradation of DBP. A proteomic analysis of DBP-interacting proteins and database screening have identified Tumor necrosis factor Receptor-Associated Factor 7 (TRAF7), a RING-type E3 ligase, that forms a complex with UBE2G1 and/or UBE2T. Ubiquitination analysis have revealed that TRAF7 enhances K48-linked polyubiquitination of DBP in cultured cells. Overexpression of TRAF7 down-regulates DBP protein level, while knockdown of TRAF7 up-regulates DBP in cultured cells. Knockout of TRAF7 in NIH3T3 cells have revealed that TRAF7 mediates the time-of-the-day-dependent regulation of DBP levels. Furthermore, TRAF7 has a period-shortening effect on the cellular clock. Together, TRAF7 plays an important role in circadian clock oscillation through destabilization of DBP.

Mechanistic basis of activation and inhibition of protein disulfide isomerase by allosteric antithrombotic compounds

BackgroundProtein disulfide isomerase (PDI) is a promising target for combating thrombosis. Extensive research over the past decade has identified numerous PDI-targeting compounds. However, limited information exists regarding how these compounds control PDI activity, which complicates further development. ObjectivesTo define the mechanism of action of 2 allosteric antithrombotic compounds of therapeutic interest, quercetin-3-O-rutinoside and bepristat-2a. MethodsA multipronged approach that integrates single-molecule spectroscopy, steady-state kinetics, single-turnover kinetics, and site-specific mutagenesis. ResultsPDI is a thiol isomerase consisting of 2 catalytic a domains and 2 inactive b domains arranged in the order a-b-b'-a'. The active sites CGHC are located in the a and a' domains. The binding site of quercetin-3-O-rutinoside and bepristat-2a is in the b' domain. Using a library of 9 Förster resonance energy transfer sensors, we showed that quercetin-3-O-rutinoside and bepristat-2a globally alter PDI structure and dynamics, leading to ligand-specific modifications of its shape and reorientation of the active sites. Combined with enzyme kinetics and mutagenesis of the active sites, Förster resonance energy transfer data reveal that binding of quercetin-3-O-rutinoside results in a twisted enzyme with reduced affinity for the substrate. In contrast, bepristat-2a promotes a more compact conformation of PDI, in which a greater enzymatic activity is achieved by accelerating the nucleophilic step of the a domain, leading to faster formation of the covalent enzyme–substrate complex. ConclusionThis work reveals the mechanistic basis underlying PDI regulation by antithrombotic compounds quercetin-3-O-rutinoside and bepristat-2a and points to novel strategies for furthering the development of PDI-targeting compounds into drugs.

Effect of Substituting of Amino Acids Residues Ser-911 and Thr-912 in the Plasma Membrane H+-ATPase of Yeast <i>Saccharomyces cerevisiae</i> on Its Activity and Distribution of Polyphosphates

The vital enzyme of yeast metabolism, plasma membrane H+-ATPase (PMA1), is phosphorylated during folding and functioning. The main phosphorylation sites are Ser911 and Thr-912 in the C-terminal regulatory domain. The work used wild and mutant forms of the enzyme with substitutions of these amino acid residues with Ala or Asp to determine their role in the functioning of the ATPase and the distribution of polyphosphates (polyP) by fractions. Growth parameters, ATP content, and in situ polyP distribution were determined on whole cells. To determine the ATPase activity in vitro, plasma membranes containing wild-type enzyme and mutant forms were isolated. Mutants S911D, T912D and S911D/T912A had ATPase activity close to the wild type; mutant S911A had increased activity. The growth rate of strains S911D, T912D and S911D/T912A was 2.0‒3.0 times lower than that of the wild type, and the growth rate of S911A was close to the wild type. Mutations S911D and S911D/T912A caused a decrease in ATP content by 2.0‒2.5 times. All substitutions affected the distribution of polyP by fractions. The effect depended on the chemical nature of the substitution: in the case of replacement with Asp, which changes the type of phosphosite, there was a decrease in the polyR1 fraction and an increase in the polyR2 fraction; when replaced with Ala, which removes phosphosite, the effect was the opposite. The content of the polyP3 fraction increased in all mutants. The data indicate that the residues of Ser911 and Thr-912 are important not only for the normal functioning of the PMA of H+-ATPase, but also for the regulation of phosphorus and energy metabolism.

Structural Insight into Wild Type of Arginine Deiminase Protein and its Three Mutated Versions in Complex with Arginine Molecule: A Computational Approach

Aims: This study aims to analyze the wild type of arginine deiminase and three mutant forms of it (G320L, A303R, and E304L) in complex with arginine molecule using Molecular Dynamics (MD) simulation to find the best enzyme form with a lower level of energy and more stability. Background: Arginine Deiminase (ADI) is an arginine-degrading enzyme that has an anticancer effect on some cancers, such as Hepatocellular Carcinoma (HCC) and melanoma. Methods: Homology models of native and mutant forms of ADI were generated and evaluated using the I-TASSER and other databases and prediction algorithms. All prepared enzyme structures via homology modeling were used as receptors in the molecular docking and MD simulation steps. Results: MD simulation results demonstrated that the G302L structure has the lowest value of Rg over MD simulation time, indicating its more appropriate intramolecular interactions than other structures. G302L has also established the most desirable interactions with arginine molecules during 80 ns MD simulation so that the G302L/ arginine complex has the maximum intermolecular hydrogen bonds. Arginine molecule in complex with G302L has the best position in the active site of ADI protein. Conclusion: The G302L mutant ADI from Mycoplasma hominis can be a good candidate for better stability in arginine deprivation therapy in arginine auxotrophic cancers.

New application of a dye-decolorizing peroxidase immobilized on magnetic nanoparticles for efficient simultaneous degradation of two mycotoxins

Nowadays the enzymatic approaches are the most promising strategies for mycotoxins detoxification in food stuffs. Herein, the dye-decolorizing peroxidase RhDypB from Rhodococcus jostii was studied for its ability to degrade two mycotoxins in both free and the immobilized enzyme forms. This enzyme was recombinantly expressed and purified, while Fe3O4 nanoparticles were prepared and modified with chitosan as the immobilization carrier. The immobilized enzyme Fe3O4@CS@RhDypB demonstrated degradation rate of 85.61 % toward aflatoxin B1, while it was firstly found to be able to degrade zearalenone with the rate of 86.52 %, at pH 4.0 on 30 °C. The degradation products were identified as aflatoxin Q1 and 15-OH-ZEN respectively. After 5 cycles of reuse, Fe3O4@CS@RhDypB still exhibited degradation rates of 38.50 % and 49.76 % toward the mycotoxins, indicating its high reusability. Moreover, Fe3O4@CS@RhDypB exhibited excellent stability after 10 days of storage. This work identified potential applications of nanoparticle-immobilized enzyme for biodegradation of mycotoxins in food industry.

Chemical Composition of Seeds in Soybean Glycine soja (Fabaceae) of Amur Oblast.

The wild soybean Glycine soja Sieb. et Zucc. is an ancestor of the cultivated soybean Glycine max (L.) Merr. and a source of many valuable genes missing in the G. max genome, including genes that determine stress resistance to adverse environmental factors. Biochemical parameters (protein, oil, ascorbic acid, carotene, higher fatty acids, and specific activities and multiple forms of enzymes of the oxidoreductase and hydrolase classes) were studied in five G. soja accessions from the collection of the All-Russian Institute of Soybean (КА-1413, КА-342, КBl-29, КBl-24, and Kеl-72). The accessions provide unique natural gene banks. Wild seeds were collected in three districts (Arkharinskii, Blagoveshchensk, and Belogorskii) of Amur Oblast. Based on superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), ribonuclease (RNase), acid phosphatase, esterase, and amylase (AML) activities and biochemical parameters of seeds, the G. soja accession KA-1413 was found to have higher contents of protein, oleic acid, and linolenic acid; a lower polyphenol oxidase specific activity; and higher activities of SODs, esterases, and RNases. The accession KA-1413 was therefore recommended to use as a source of dominant genes in breeding to increase the adaptive potential of new soybean varieties. A higher heterogeneity of multiple forms was observed for SOD, AML, RNase, and esterase, which can provide markers of adaptation to environmental conditions.

MaAzaR Influences Virulence of Metarhizium acridum against Locusta migratoria manilensis by Affecting Cuticle Penetration.

The entomopathogenic fungus (EPF) Metarhizium acridum is a typical filamentous fungus and has been used to control migratory locusts (Locusta migratoria manilensis). This study examines the impact of the Zn(II)2Cys6 transcription factor, MaAzaR, in the virulence of M. acridum. Disruption of MaAzaR (ΔMaAzaR) diminished the fungus's ability to penetrate the insect cuticle, thereby decreasing its virulence. The median lethal time (LT50) for the ΔMaAzaR strain increased by approximately 1.5 d compared to the wild-type (WT) strain when topically inoculated, simulating natural infection conditions. ΔMaAzaR compromises the formation, turgor pressure, and secretion of extracellular hydrolytic enzymes in appressoria. However, the growth ability of ΔMaAzaR within the hemolymph is not impaired; in fact, it grows better than the WT strain. Moreover, RNA-sequencing (RNA-Seq) analysis of ΔMaAzaR and WT strains grown for 20 h on locust hindwings revealed 87 upregulated and 37 downregulated differentially expressed genes (DEGs) in the mutant strain. Pathogen-host interaction database (PHI) analysis showed that about 40% of the total DEGs were associated with virulence, suggesting that MaAzaR is a crucial transcription factor that directly regulates the expression of downstream genes. This study identifies a new transcription factor involved in EPF cuticle penetration, providing theoretical support and genetic resources for the developing highly virulent strains.

When less is more: The association between the expression of polymorphic CYPs and AFB1-induced HCC.

An individual's genetic fingerprint is emerging as a pivotal predictor of numerous disease- and treatment-related factors. Single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes play key roles in an individual's exposure to a malignancy-associated risk, such as Aflatoxin B1 (AFB1)-induced hepatocellular carcinoma (HCC). This study aimed at reviewing literature on the polymorphisms that exist in CYP enzymes and their possible link with susceptibility to AFB1-induced HCC. A set of keywords associated with the study subject of interest was used to search the Google Scholar and the PubMed database. The last ten years' worth of research projects were included in the results filter. The research involved HCC patients and any connection between polymorphic forms of CYP enzymes and their susceptibility to AFB1-induced HCC, including older but significant data. Variations in CYP1A2 and CYP3A4 were reported to impact the rate and magnitude of AFB1 bio-activation, thus influencing an individual's vulnerability to develop HCC. In HCC patients, the activity of CYP isoforms varies, where increased activity has been reported with CYP2C9, CYP2D6, and CYP2E1, while CYP1A2, CYP2C8, and CYP2C19 exhibit decreased activity. CYP2D6*10 frequency has been discovered to differ considerably in HCC patients. Rs2740574 (an upstream polymorphism in CYP3A4 as detected in CYP3A4*1B) and rs776746 (which affects CYP3A5 RNA splicing), both of which influence CYP3A expression, thus impacting the variability of AFB1-epoxide adducts in HCC patients. CYP1A2 is the primary enzyme accountable for the formation of harmful AFBO globally. CYP3A4, CYP3A5, CYP3A7, CYP2B7, and CYP3A3 are also implicated in the bio-activation of AFB1 to mutagenic metabolites. It is thought that CYP3A4 is the protein that interacts with AFB1 metabolism the most. Polymorphic variants of CYP enzymes have a functional impact on the susceptibility to AFB1-induced HCC. Outlining such variation and their implications may provide deeper insights into approaching HCC in a more personalized manner for guiding future risk-assessment, diagnosis, and treatment.

In silico investigation of lipid-based compounds implicated in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, currently incurable neurodegenerative disease that progressively affects the upper motor neurons in the brain and lower motor neurons in the spinal cord. A significant portion of patients exhibit abnormalities in lipid metabolism, which appear to contribute to denervation and subsequent motor neuron degeneration. This study aimed to computationally identify lipid-based compounds associated with ALS and elucidate their pathological and therapeutic roles to facilitate effective ALS diagnosis and treatment. The methodologies employed encompassed rational database searches, pharmacokinetic and target predictions, as well as docking and molecular dynamics simulations (MDS). The findings from the disease-to-lipids analysis identified 20 lipid compounds correlated with ALS, primarily categorized as potential inhibitors within the functional classes of vitamins, antibiotics, organic acids, and phytochemicals. Pharmacokinetic predictions revealed that only four compounds exhibited permeability across the blood–brain barrier, namely 4-hydroxynonenal, resveratrol, rotenone, and valproic acid. Docking simulations indicated the highest binding affinity for 4-hydroxynonenal with alkaline ceramidase 2 (−4.516 kcal/mol), resveratrol with carbonic anhydrase 6 (−7.070 kcal/mol), rotenone with cytochrome P450 2C19 (−7.378 kcal/mol), and valproic acid with glutamate carboxypeptidase 2 (−5.629 kcal/mol). Furthermore, MDS and molecular mechanics/generalized born surface area calculations demonstrated the stability and binding energies of the complexes under simulated physiological conditions. In summary, further investigation is warranted to explore the synergistic effects of resveratrol and valproic acid in ALS, the mechanisms underlying 4-hydroxynonenal adduct-assisted aggregate formation of certain enzymes in ALS, and the impact of prolonged human exposure to rotenone from marine food sources (such as fish) on the development of ALS.

Iron and cardiovascular health: A review.

Iron is an essential element for the biological processes of living organisms, including the production of crucial oxygen-carrying proteins, formation of heme enzymes, and playing roles in electron transfer and oxidation-reduction reactions. It plays a significant role in various cardiovascular functions, including bioenergetics, electrical activity, and programmed cell death. Minor deficiencies of iron have been found to have negative impact on cardiovascular function in patients with heart failure (HF). The contractility of human cardiomyocytes is impaired by iron deficiency (ID), which results in reduced mitochondrial function and lower energy production, ultimately leading to cardiac function impairment, contributing to significant morbidity and mortality in patients with HF. This review discusses iron homeostasis within the human body, as well as ID pathophysiology and its role in HF. Focusing on therapeutic approaches including iron supplementation and/or repletion in patients with ID and HF, comparing results from recent clinical trials. Intravenous (IV) iron therapy has shown promising results in treating ID in HF patients. Large, randomized trials and meta-analysis, like Ferinject Assessment in patients with ID and chronic HF, AFFIRM-AHF, IRONMAN, and HEART-FID have demonstrated the efficacy of IV iron supplementation with IV ferric carboxymaltose or IV ferric derisomaltose in reducing hospitalizations and improving quality of life in patients with Heart Failure with reduced ejection fraction (HFrEF), New York Heart Association (NYHA) II-III. However, survival and mortality have demonstrated no improvement during acute exacerbations of HF or in outpatient management. The potential benefits of IV iron across the entire HF spectrum and its interaction with other HF therapies remain areas of interest for further research.

Chitosan decorated magnetic nanobiocatalyst of Bacillus derived α-amylase as a role model for starchy wastewater treatment, detergent additive and textile desizer

In this study, Bacillus tequilensis TB5 α-amylase from rice-milled by-products (rice bran and de-oiled rice bran) was successfully immobilized onto biologically synthesized magnetic nanoparticles fabricated with chitosan (MNP-Ch) and characterized via different biophysical techniques. Furthermore, the study emphasized incorporating this nanostructure framework (MNP@2mgchitosan_DORB-amy and MNP@3mgchitosan_RB-amy) to offer diverse applications, including enzymatic desizing, cleaning starchy stains, and treating synthetic starchy wastewater. An enzyme loading of > 90 % for both enzymes indicated increased binding sites due to the functional moieties of chitosan on the MNP. The Km was 0.28 and 0.31 mg/mL for the immobilized and free forms of DORB-amy, respectively, and 0.18 and 0.27 mg/mL for the immobilized and free forms of RB-amy, respectively. A low Km indicated an increased affinity of MNP-Ch-immobilized forms of enzymes toward the substrate. The performance of both immobilized enzymes improved at a wide range of pH and temperature, which may be attributed to the covalent binding of the enzyme on to the MNP-Ch. The nanobiocatalysts in the detergent act synergistically to fade the starchy stains. Furthermore, an 8–9 TEGEWA scale rating with > 11 % of starch removal was obtained through the biodesizing of starch-sized cotton fabric. The nanobiocatalyst efficiently decomposed starch and liberated 650–670 mg/L of reducing sugar from the synthetic wastewater, therefore offering promising opportunities for its exploration in a wastewater treatment plant. Thus, the study recommends the potential exploration of sturdy matrices like MNP to offer remarkable applications with maximum operational stability, easier recovery, and higher efficiency.

Activity-Based Protein Profiling of RHBDL4 Reveals Proteolysis of the Enzyme and a Distinct Inhibitor Profile.

Rhomboid proteases have fascinated scientists by virtue of their membrane-embedded active sites and proposed involvement in physiological and disease pathways. The human rhomboid protease RHBDL4 has generated particular interest due to its role in endoplasmic reticulum-associated protein degradation and upregulation in several cancers; however, chemical tools for studying this enzyme are currently lacking. Here, we describe the development of an activity-based protein profiling (ABPP) assay for RHBDL4. We have employed this assay to determine that human RHBDL4 undergoes proteolytic processing in cells to produce multiple active proteoforms with truncated C-termini. We have also used this assay to identify chemical scaffolds capable of inhibiting RHBDL4 activity and have observed distinct inhibitor preferences between RHBDL4 and a second human rhomboid protease PARL. Our work demonstrates the power of ABPP technology to characterize active forms of enzymes that might otherwise elude detection and the potential to achieve selective inhibition among the human rhomboid proteases.

3-Benzylaminomethyl Lithocholic Acid Derivatives Exhibited Potent and Selective Uncompetitive Inhibitory Activity Against Protein Tyrosine Phosphatase 1B (PTP1B).

Protein tyrosine phosphatase 1B (PTP1B) is a promising drug target for treating type 2 diabetes (T2DM) and obesity. As a result, developing new therapies that target PTP1B is an attractive strategy for treating these diseases. Herein, we detail the synthesis of 15 lithocholic acid (LA) derivatives, each containing different benzylaminomethyl groups attached to the C3 position of the steroid skeleton. The derivatives were assessed against two forms of PTP1B enzyme (hPTP1B1-400 and hPTP1B1-285), and the most potent compounds were then tested against T-cell protein tyrosine phosphatase (TCPTP) to determine their selectivity. The results showed that compounds 6m and 6n were more potent than the reference compounds (ursolic acid, chlorogenic acid, suramin, and TCS401). Additionally, both compounds exhibited greater potency over hPTP1B1-400. Furthermore, enzyme kinetic studies on hPTP1B1-400 revealed that these two lithocholic acid derivatives have an uncompetitive inhibition against hPTP1B1-400 with K i values of 2.5 and 3.4 μM, respectively. Interestingly, these compounds were around 75-fold more selective for PTP1B over TCPTP. Finally, docking studies and molecular dynamics simulations (MDS) were conducted to determine how these compounds interact with PTP1B. The docking studies revealed hydrophobic and H-bond interactions with amino acid residues in the unstructured region. MDS showed that these interactions persisted throughout the 200 ns simulation, indicating the crucial role of the unstructured zone in the biological activity and inhibition of PTP1B.

Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism.

Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.

The inorganic pyrophosphatases of microorganisms: a structural and functional review

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure–function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.

Exploring variations in glycolytic and gluconeogenic enzymes and isoforms across breast cancer cell lines and tissues

It is well established that tumour cells undergo metabolic changes to acquire biological advantage over normal cells with activation of the glycolytic pathway, a process termed “Warburg effect”. Enzyme isoforms are alternative enzymatic forms with the same function but with different biochemical or epigenetic features. Moreover, isoforms may have varying impacts on different metabolic pathways. We challenge ourselves to analyse the glycolytic and gluconeogenic enzymes and isoforms in breast cancer, a complex and heterogeneous pathology, associated with high incidence and mortality rates especially among women.We analysed epithelial and tumour cell lines by RT-PCR and compared values to a publicly available database for the expression profile of normal and tumour tissues (Gepia) of enzymes and enzymatic isoforms from glycolytic and gluconeogenic pathways. Additionally, GeneMANIA was used to evaluate interactions, pathways, and attributes of each glycolytic/gluconeogenic steps.The findings reveal that the enzymes and enzymatic isoforms expressed in cell culture were somewhat different from those in breast tissue. We propose that the tumor microenvironment plays a crucial role in the expression of glycolytic and gluconeogenic enzymes and isoforms in tumour cells. Nonetheless, they not only participate in glycolytic and gluconeogenic enzymatic activities but may also influence other pathways, such as the Pentose-Phosphate-Pathway, TCA cycle, as well as other carbohydrate, lipid, and amino acid metabolism.

Partial denitrification in rope-type biofilm reactors: Performance, kinetics, and microflora using internal vs. external carbon sources

Stable nitrite accumulation through partial denitrification (PDN) represents an efficient pathway to support the anammox process, but limited studies explored the internal wastewater carbon sources and biofilm processes. This study assessed the viability of the PDN process, biofilm community evolution, and functional enzyme formation in rope-type biofilm media reactors using primary effluent (PE) and anaerobically pretreated wastewater carbon sources for the first time. Comparison was made with external carbon (acetate) under varied pH and biofilm thicknesses, maintaining a favourable sCOD: NO3-N ratio of 3. The wastewater’s internal carbon resulted in thinner biofilms; nevertheless, modest nitrite accumulation (0.24 g/m2/d) occurred only at elevated pH. The highest nitrite accumulation (0.79 g/m2/d) was exhibited in the biofilm thickness-controlled acetate-fed reactor, featuring porous biofilms dominated by denitrifier Thauera (10.24 %) and imbalance between Nar, Nap, and Nir reductases. Using internal wastewater carbon sources offers a sustainable avenue for adopting the PDN process in full-scale application.