A Plasma Pyrophosphate Cutoff Value for Diagnosing Pseudoxanthoma Elasticum.

Pseudoxanthoma elasticum (PXE) is a rare inherited systemic disease responsible for a juvenile peripheral arterial calcification disease. The clinical diagnosis of PXE is only based on a complex multi-organ phenotypic score and/or genetical analysis. Reduced plasma inorganic pyrophosphate concentration [PPi]p has been linked to PXE. In this study, we used a novel and accurate method to measure [PPi]p in one of the largest cohorts of PXE patients, and we reported the valuable contribution of a cutoff value to PXE diagnosis. Plasma samples and clinical records from two French reference centers for PXE (PXE Consultation Center, Angers, and FAVA-MULTI South Competent Center, Nice) were assessed. Plasma PPi were measured in 153 PXE and 46 non-PXE patients. The PPi concentrations in the plasma samples were determined by a new method combining enzymatic and ion chromatography approaches. The best match between the sensitivity and specificity (Youden index) for diagnosing PXE was determined by ROC analysis. [PPi]p were lower in PXE patients (0.92 ± 0.30 µmol/L) than in non-PXE patients (1.61 ± 0.33 µmol/L, p < 0.0001), corresponding to a mean reduction of 43 ± 19% (SD). The PPi cutoff value for diagnosing PXE in all patients was 1.2 µmol/L, with a sensitivity of 83.3% and a specificity of 91.1% (AUC = 0.93), without sex differences. In patients aged <50 years (i.e., the age period for PXE diagnosis), the cutoff PPi was 1.2 µmol/L (sensitivity, specificity, and AUC of 93%, 96%, and 0.97, respectively). The [PPi]p shows high accuracy for diagnosing PXE; thus, quantifying plasma PPi represents the first blood assay for diagnosing PXE.

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.

Reversible Kinetics in Multi-nucleotide Addition Catalyzed by S. cerevisiae RNA polymerase II Reveal Slow Pyrophosphate Release.

Eukaryotes express at least three nuclear DNA dependent RNA polymerases (Pols). Pols I, II, and III synthesize ribosomal (r) RNA, messenger (m) RNA, and transfer (t) RNA, respectively. Pol I and Pol III have intrinsic nuclease activity conferred by the A12.2 and C11 subunits, respectively. In contrast, Pol II requires the transcription factor (TF) IIS to confer robust nuclease activity. We recently reported that in the absence of the A12.2 subunit Pol I reverses bond formation by pyrophosphorolysis in the absence of added PPi, indicating slow PPi release. Thus, we hypothesized that Pol II, naturally lacking TFIIS, would reverse bond formation through pyrophosphorolysis. Here we report the results of transient-state kinetic experiments to examine the addition of nine nucleotides to a growing RNA chain catalyzed by Pol II. Our results indicate that Pol II reverses bond formation by pyrophosphorolysis in the absence of added PPi. We propose that, in the absence of endonuclease activity, this bond reversal may represent kinetic proofreading. Thus, given the hypothesis that Pol I evolved from Pol II through the incorporation of general transcription factors, pyrophosphorolysis may represent a more ancient form of proofreading that has been evolutionarily replaced with nuclease activity.

Inhibitive effect of trehalose and sodium pyrophosphate on oxidation and structural changes of myofibrillar proteins in silver carp surimi during frozen storage.

This work investigated the cryoprotective effect of trehalose (TH) and sodium pyrophosphate (SPP) alone and in combination on myofibrillar protein (MP) oxidation and structural changes in silver carp surimi during 90 days of frozen storage (-20 °C). TH combined with SPP was significantly more effective than single TH or SPP in preventing MP oxidation (P < 0.05), showing a higher SH content (6.05 nmol/mg protein), and a lower carbonyl (4.24 nmol/mg protein) and dityrosine content (1280 A.U.). SDS-PAGE results indicated that TH combined with SPP did not differ significantly from TH and SPP in inhibiting protein degradation but was more effective in inhibiting protein crosslinking. Moreover, all cryoprotectants could stabilise the secondary and tertiary structures and inhibit unfolded and aggregation of MP, with the combination of TH and SPP being the best. It's worth noting that TH combined with SPP had a synergistic effect on inhibiting the decrease in α-helix content and gel-forming ability, and the increase in surface hydrophobicity. Overall, TH combined with SPP could significantly inhibited MP oxidation and structural changes in surimi during frozen storage and improve the gel-forming ability, which was significantly better than single TH or SPP.

Kinetic analysis of T4 polynucleotide kinase via isothermal titration calorimetry.

T4 polynucleotide kinase (T4 PNK) phosphorylates the 5'-terminus of DNA and RNA substrates. It is widely used in molecular biology. Single nucleotides can serve as substrates if a 3'-phosphate group is present. In this study, the T4 PNK-catalyzed conversion of adenosine 3'-monophosphate (3'-AMP) to adenosine-3',5'-bisphosphate was characterized using isothermal titration calorimetry (ITC). Although ITC is typically used to study ligand binding, in this case the instrument was used to evaluate enzyme kinetics by monitoring the heat production due to reaction enthalpy. The reaction was initiated with a single injection of 3'-AMP substrate into the sample cell containing T4 PNK and ATP at pH 7.6 and 30 °C, and Michaelis-Menten analysis was performed on the reaction rates derived from the plot of differential power versus time. The Michaelis-Menten constant, KM, was 13 µM, and the turnover number, kcat, was 8 s-1. The effect of inhibitors was investigated using pyrophosphate (PPi). PPi caused a dose-dependent decrease in the apparent kcat and increase in the apparent KM under the conditions tested. Additionally, the intrinsic reaction enthalpy and the activation energy of the T4 PNK-catalyzed phosphorylation of 3'-AMP were determined to be -25 kJ/mol and 43 kJ/mol, respectively. ITC is seldom used as a tool to study enzyme kinetics, particularly for technically-challenging enzymes such as kinases. This study demonstrates that quantitative analysis of kinase activity can be amenable to the ITC single injection approach.

Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii.

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.

Nucleic Acid Plate Culture: Label-Free and Naked-Eye-Based Digital Loop-Mediated Isothermal Amplification in Hydrogel with Machine Learning.

Digital nucleic acid amplification enables the absolute quantification of single molecules. However, due to the ultrasmall reaction volume in the digital system (i.e., short light path), most digital systems are limited to fluorescence signals, while label-free and naked-eye readout remain challenging. In this work, we report a digital nucleic acid plate culture method for label-free, ultrasimple, and naked-eye nucleic acid analysis. As simple as the bacteria culture, the nanoconfined digital loop-mediated isothermal amplification was performed by using polyacrylamide (PAM) hydrogel as the amplification matrix. The nanoconfinement of PAM hydrogel with an ionic polymer chain can remarkably accelerate the amplification of target nucleic acids and the growth of inorganic byproducts, namely, magnesium pyrophosphate particles (MPPs). Compared to that in aqueous solutions, MPPs trapped in the hydrogel with enhanced light scattering characteristics are clearly visible to the naked eye, forming white "colony" spots that can be simply counted in a label-free and instrument-free manner. The MPPs can also be photographed by a smartphone and automatically counted by a machine-learning algorithm to realize the absolute quantification of antibiotic-resistant pathogens in diverse real samples.

Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters.

BACKGROUND: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis. RESULT: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu2+) ions. The off state of LA@AuAg_Cu2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu2+. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu2+ ions, and again quenched off state was retrieved due to interaction of free Cu2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples. SIGNIFICANCE: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

Thienopyrimidine-derived multifunctional fluorescence sensor for the detection of Cu2+, Fe3+, and PPi in different solvents.

Hydrazine substituted thienopyrimidine, a new fluorophore, was used to synthesize a novel Schiff base R1 as a chemosensor via the condensation with p-formyltriphenylamine, and the structure was confirmed using nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS) analysis. When treated with Cu2+ in dimethylsulfoxide (DMSO)/H2O buffer, R1 showed a phenomenon of fluorescence quenching, which was reversible with the action of ethylenediaminetetraacetic acid (EDTA). When treated with Fe3+ in dimethylformamide (DMF)/H2O buffer, R1 exhibited the same phenomenon, but fluorescence was recovered with inorganic pyrophosphate (PPi) quantitatively. The complexation ratios for R1-Cu2+ and R1-Fe3+ were both 1:2, which were manifested by MS titrations and corresponding Job's plots. The limits of detection of R1 for Cu2+ and Fe3+ were 3.11 × 10-8 and 1.24 × 10-7 M, respectively. The sensing mechanism of R1 toward Cu2+ and Fe3+ was confirmed using density functional theory calculations and electrostatic potential analysis. Test strips of R1 were fabricated successfully for on-site detection of Cu2+ and Fe3+. In addition, R1 was applied to recognize Cu2+ and Fe3+ in actual water samples with satisfactory recovery.

Efficient production of (S)-limonene and geraniol in Saccharomyces cerevisiae through the utilization of an Erg20 mutant with enhanced GPP accumulation capability.

Monoterpenes and monoterpenoids such as (S)-limonene and geraniol are valuable chemicals with a wide range of applications, including cosmetics, pharmaceuticals, and biofuels. Saccharomyces cerevisiae has proven to be an effective host to produce various terpenes and terpenoids. (S)-limonene and geraniol are produced from geranyl pyrophosphate (GPP) through the enzymatic actions of limonene synthase (LS) and geraniol synthase (GES), respectively. However, a major hurdle in their production arises from the dual functionality of the Erg20, a farnesyl pyrophosphate (FPP) synthase, responsible for generating GPP. Erg20 not only synthesizes GPP by condensing isopentenyl pyrophosphate (IPP) with dimethylallyl pyrophosphate but also catalyzes further condensation of IPP with GPP to produce FPP. In this study, we have tackled this issue by harnessing previously developed Erg20 mutants, Erg20K197G (Erg20G) and Erg20F96W, N127W (Erg20WW), which enhance GPP accumulation. Through a combination of these mutants, we generated a novel Erg20WWG mutant with over four times higher GPP accumulating capability than Erg20WW, as observed through geraniol production levels. The Erg20WWG mutant was fused to the LS from Mentha spicata or the GES from Catharanthus roseus for efficient conversion of GPP to (S)-limonene and geraniol, respectively. Further improvements were achieved by localizing the entire mevalonate pathway and the Erg20WWG-fused enzymes in peroxisomes, while simultaneously downregulating the essential ERG20 gene using the glucose-sensing HXT1 promoter. In the case of (S)-limonene production, additional Erg20WWG-LS was expressed in the cytosol. As a result, the final strains produced 1063 mg/L of (S)-limonene and 1234 mg/L of geraniol by fed-batch biphasic fermentations with ethanol feeding. The newly identified Erg20WWG mutant opens doors for the efficient production of various other GPP-derived chemicals including monoterpene derivatives and cannabinoids.

Lysosomal diacylglycerol pyrophosphate phosphatase is not essential in Trypanosoma brucei.

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.

Describing calcium pyrophosphate deposition: undoing the tower of Babel!

PURPOSE OF REVIEW: In 1977, McCarty astutely observed, 'The variety of names suggested for the condition associated with deposits of calcium pyrophosphate dihydrate crystals is exceeded only by the variations of its clinical presentation'. Fast forward to 2024, a standardized nomenclature for calcium pyrophosphate deposition (CPPD) is still lacking. This review aims to delineate the challenges in characterizing CPPD through nomenclature and imaging. RECENT FINDINGS: Despite the effort of nomenclature standardization in 2011 by the EULAR, confusion persists in the literature and clinical practice, with pseudo-forms and obscure abbreviations. The Gout, Hyperuricemia and Crystal-Associated Disease Network (G-CAN) has launched a project to redefine CPPD nomenclature and formulate a user-friendly language for effective communication with patients and other stakeholders. Additionally, recent advancements in imaging, have shed light on various aspects of the disorder. SUMMARY: Almost 60 years from the first description of a clinical manifestation related to calcium pyrophosphate crystals, a common language describing the disorder is still lacking. A redefined CPPD nomenclature, together with lay-friendly terminology, would significantly contribute to the uniformity of CPPD research, enhance public understanding and awareness and improve doctor-patient communication and therefore disease outcomes. Imaging can provide deep insights into CPPD elements, promoting comprehension of this disorder.

Phosphate type dependent phosphorylation on the interfacial and emulsion stabilizing behaviors of goose liver protein: Perspective of protein charging.

Elucidation on the emulsifying behaviors of goose liver protein (GLP) from interfacial perspective was scarce when protein charging was altered. This work aimed to elucidate the role of phosphorylation on the interfacial associative interaction and then emulsion stabilizing properties of GLP using three structurally relevant phosphates of sodium trimetaphosphate (STMP), sodium tripolyphosphate (STPP) and sodium pyrophosphate (TSPP). A monotonic increment of protein charging treated from STMP, STPP to TSPP caused progressively increased particle de-aggregation, surface hydrophobicity and structural flexibility of GLP. Compared with STMP and TSPP, STPP phosphorylation rendered the most strengthened interfacial equilibrium pressure (11.98 ± 0.24 mN/m) due to sufficient unfolding but moderated charging character conveyed. Desorption curve and interfacial protein microstructure indicated that STPP phosphorylation caused the highest interfacial connectivity between proteins adsorbed onto the same droplet, as was also verified by interfacial elastic modulus (10.3 ± 0.21 mN/m). STPP treated GLP also yielded lowest droplet size (8.16 ± 0.10 µm), flocculation (8.18%) and Turbiscan stability index (8.78 ± 0.36) of emulsion but most improved microrheological properties. Overall, phosphorylation functioned itself in fortifying the intradroplet protein-protein interaction but restraining the interdroplet aggregation, and STPP phosphorylation endowed the protein with most enhanced interfacial stabilization and emulsifying efficiency.

Comparison of diagnostic efficacy between 99mTc-methylene diphosphate SPECT/CT and MRI for bone and joint infections: a multicenter retrospective analysis.

Objective: There is currently no non-invasive examination that can fully determine the diagnosis of osteomyelitis. SPECT/CT tomographic fusion imaging can provide both local metabolic activity and anatomical information to determine the condition and location. This study evaluates the diagnostic efficacy of 99mTc-MDP SPECT/CT in bone infections, compared to MRI. Methods: In this multicenter retrospective study, 363 patients with suspected bone and joint infections or osteomyelitis were included. Participants underwent 99mTc-MDP SPECT/CT and/or MRI examinations, supplemented by pathogenic bacterial cultures and histopathological analysis. Results: Only SPECT/CT was tested in 169 patients, and only MRI was used in 116. 78 people have implemented both inspections and have detailed information. The diagnostic sensitivity and specificity of SPECT/CT for infection were 96% and 92% respectively, with an accuracy of 96%. For MRI, these figures were 88%, 84%, and 87% respectively. Conclusion: This represents the largest global study to date evaluating osteomyelitis and bone infection diagnosis using 99mTc-MDP SPECT/CT tomographic fusion imaging. The findings indicate that 99mTc-MDP SPECT/CT fusion imaging offers superior diagnostic accuracy compared to MRI. This is particularly evident in cases involving metallic implants and chronic infections. 99mTc-MDP SPECT/CT fusion imaging emerges as a highly suitable non-invasive diagnostic modality, facilitating enhanced clinical follow-up and treatment.

Rare Earth-Carbon Dots Nanocomposite-Modified Glass Nanopipettes: Electro-Optical Detection of Bacterial ppGpp.

As an important alarmone nucleotide, guanosine 3'-diphosphate-5'-diphosphate (ppGpp) can regulate the survival of bacteria under strict environmental conditions. Direct detection of ppGpp in bacteria with high sensitivity and selectivity is crucial for elucidating the role of ppGpp in bacterial stringent response. Herein, the terbium-carbon dots nanocomposite (CDs-Tb) modified glass nanopipet was developed for the recognition of ppGpp. The CDs-Tb in glass nanopipette preserved their fluorescence properties as well as the coordination capacity of Tb3+ toward ppGpp. The addition of ppGpp not only led to the fluorescence response of CDs-Tb but also triggered variations of surface charge inside the glass nanopipet, resulting in the ionic current response. Compared with nucleotides with similar structures, this method displayed good selectivity toward ppGpp. Moreover, the dual signals (fluorescence and ionic current) offered a built-in correction for potential interference. Apart from the high selectivity, the proposed method can determine the concentration of ppGpp from 10-13 to 10-7 M. Taking advantage of the significant analytical performance, we monitored ppGpp in Escherichia coli under different nutritional conditions and studied the relationship between ppGpp and DNA repair, which is helpful for overcoming antibiotic resistance and promoting the development of potential drugs for antibacterial treatment.

[Exploration of detection methods for free silica with different crystal forms in dust].

Objective: To investigate the differences and applicability of free silica detection methods of different crystal forms in dust, and to provide a basis for the selection of various methods. Methods: From December 2021 to June 2022, dust samples from 20 enterprises in different industries in 18 cities in Henan Province were randomly selected as the investigation objects. X-ray diffraction (XRD) method was used to analyze the samples and classify the samples. Based on GBZ/T 192.4-2007 "Determination of Dust in the Air of Workplace-Part 4: Content of Free Silica in Dust", pyrophosphate method and infrared spectrophotometry were used for quantitative determination. The measured results were analyzed by paired sample t test to evaluate the advantages and disadvantages of the two methods and their applicable scope. Results: The XRD results of 20 dust samples could be divided into α, ß, γ crystal types and the mixed type of α and γ. There was no significant difference between pyrophosphate method and infrared spectrophotometry (P=0.180). The pyrophosphate method results of ß, γ and α, γ mixed crystalline free silica were significantly higher than those of infrared spectrophotometry, and the difference was statistically significant (P<0.001) . Conclusion: Pyrophosphate method and infrared spectrophotometry are suitable for α-type free silica, while pyrophosphate method is suitable for ß, γ and α, γ mixed crystalline free silica.

Kcs1 and Vip1: The Key Enzymes behind Inositol Pyrophosphate Signaling in Saccharomyces cerevisiae.

The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae, renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae, highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.

Crystal structure and biophysical characterization of IspD from Burkholderia thailandensis and Mycobacterium paratuberculosis.

The methylerythritol phosphate (MEP) pathway is a metabolic pathway that produces the isoprenoids isopentyl pyrophosphate and dimethylallyl pyrophosphate. Notably, the MEP pathway is present in bacteria and not in mammals, which makes the enzymes of the MEP pathway attractive targets for discovering new anti-infective agents due to the reduced chances of off-target interactions leading to side effects. There are seven enzymes in the MEP pathway, the third of which is IspD. Two crystal structures of Burkholderia thailandensis IspD (BtIspD) were determined: an apo structure and that of a complex with cytidine triphosphate (CTP). Comparison of the CTP-bound BtIspD structure with the apo structure revealed that CTP binding stabilizes the loop composed of residues 13-19. The apo structure of Mycobacterium paratuberculosis IspD (MpIspD) is also reported. The melting temperatures of MpIspD and BtIspD were evaluated by circular dichroism. The moderate Tm values suggest that a thermal shift assay may be feasible for future inhibitor screening. Finally, the binding affinity of CTP for BtIspD was evaluated by isothermal titration calorimetry. These structural and biophysical data will aid in the discovery of IspD inhibitors.

Elucidation of productive alanine recognition mechanism by Escherichia coli alanyl-tRNA synthetase.

Alanyl-tRNA synthetase (AlaRS) incorrectly recognizes both a slightly smaller glycine and a slightly larger serine in addition to alanine, and the probability of incorrect identification is extremely low at 1/300 and 1/170, respectively. Alanine is the second smallest amino acid after glycine; however, the mechanism by which AlaRS specifically identifies small differences in side chains with high accuracy remains unknown. In this study, using a malachite green assay, we aimed to elucidate the alanine recognition mechanism of a fragment (AlaRS368N) containing only the amino acid activation domain of Escherichia coli AlaRS. This method quantifies monophosphate by decomposing pyrophosphate generated during aminoacyl-AMP production. AlaRS368N produced far more pyrophosphate when glycine or serine was used as a substrate than when alanine was used. Among several mutants tested, an AlaRS mutant in which the widely conserved aspartic acid at the 235th position (D235) near the active center was replaced with glutamic acid (D235E) increased pyrophosphate release for the alanine substrate, compared to that from glycine and serine. These results suggested that D235 is optimal for AlaRS to specifically recognize alanine. Alanylation activities of an RNA minihelix by the mutants of valine at the 214th position (V214) of another fragment (AlaRS442N), which is the smallest AlaRS with alanine charging activity, suggest the existence of the van der Waals-like interaction between the side chain of V214 and the methyl group of the alanine substrate.

Sole and Combined Application of Biodigestate, N, P, and K Fertilizers: Impacts on Soil Chemical Properties and Maize Performance.

The fertilizing effects of biodigestate produced from biogas plants on crop and soil productivity are very scarce. Hence, a field study was conducted in 2022 at the Teaching and Research Farm of Bowen University, Iwo, Osun State, Nigeria. The study evaluated the effects of biodigestate fertilizer, applied alone or in combination with urea, single superphosphate, or muriate of potash fertilizers at low (N1, K1, and P1) and high (N2, P2, and K2) rates on soil chemical properties, growth, and yield of maize (Zea mays (L.)). The treatments were biodigestate alone (D), D + N fertilizer (urea) at 60 kg·ha-1 (DN1), D + N at 120 kg·ha-1 (DN2), D + P fertilizer (single superphosphate) at 30 kg·ha-1 (DP1), D + P at 60 kg·ha-1 (DP2), D + K fertilizer (muriate of potash) at 30 kg·ha-1 (DK1), D + K 60 kg·ha-1 (DK2), D + N1 + P1 + K1 (DN1P1K1), D + N2 + P2 + K2 (DN2P2K2) (10), and control. The 10 treatments were arranged in a randomized complete block design and replicated three times. Results showed that both low and high rates of fertilizer application improved soil chemical properties, growth parameters, and yield of maize compared with the control. High fertilizer rates (N2, P2, and K2) significantly enhanced soil chemical properties and growth parameters, but lower rates (N1, P1, and K1) resulted in higher maize yield. DN1 fertilizer significantly increased maize yield compared with DN2, DP1, DP2, DK1, and DK2. Overall, the treatment of DN1P1K1 demonstrated the highest grain yield, likely due to optimal nutrient supply from N, P, and K fertilizers, along with an improved soil environment facilitated by the biodigestate. The study recommends a balanced and sustainable fertilizer application strategy of 60 kg·N·ha-1, 30 kg·P2O5·ha-1, and 30 kg·K·ha-1 with 2500 L·ha-1 of biodigestate to enhance maize production while minimizing cost and environmental impact. However, for those aiming for maize fodder production, a higher fertilizer rate of 120 kg·N·ha-1, 60 kg·P2O5·ha-1, and 60 kg·K·ha-1 with 2500 L·ha-1 of biodigestate is advised.