On the Origins of Enzymes: Phosphate-Binding Polypeptides Mediate Phosphoryl Transfer to Synthesize Adenosine Triphosphate

Reactions involving the transfer of a phosphoryl (−PO32–) group are fundamental to cellular metabolism. These reactions are catalyzed by enzymes, often large and complex, belonging to the phosphate-binding loop (P-loop) nucleoside triphosphatase (NTPase) superfamily. Due to their critical importance in life, it is reasonable to assume that phosphoryl-transfer reactions were also crucial in the pre-LUCA (last universal common ancestor) world and mediated by precursors that were simpler, in terms of their sequence and structure, relative to their modern-day enzyme counterparts. Here, we demonstrate that short phosphate-binding polypeptides (∼50 residues) comprising a single, ancestrally inferred, P-loop or Walker A motif mediate the reversible transfer of a phosphoryl group between two adenosine diphosphate molecules to synthesize adenosine triphosphate and adenosine monophosphate. This activity, although rudimentary, bears resemblance to that of adenylate kinase (a P-loop NTPase enzyme). The polypeptides, dubbed as “P-loop prototypes”, thus relate to contemporary P-loop NTPases in terms of their sequence and function, and yet, given their simplicity, serve as plausible representatives of the early “founder enzymes” involved in proto-metabolic pathways

Supplemental_Table_1 - Cost-Effectiveness of Percutaneous Lymphatic Embolization for Management of Plastic Bronchitis

Supplemental_Table_1 for Cost-Effectiveness of Percutaneous Lymphatic Embolization for Management of Plastic Bronchitis by Jamaal L. Benjamin, Jack Rychik, Jordan A. Johnstone, Gregory J. Nadolski and Maxim Itkin in World Journal for Pediatric and Congenital Heart Surgery</p

DataSheet_1_2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.pdf

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules’ outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.</p

KYNA salvages H9C2 cell viability upon exposure to ischemic conditions.

(A) Dose response for H9C2 cell viability grown in the presence of KYNA followed by exposure to normoxic or anoxic conditions, as determined by flow cytometry. Each box represents values derived from three independent experiments. X- Denotes mean values, horizontal lines stands for the median values. (B) Representetative flow cytometry captures showing H9C2 viability. (C) Reduced cell surface expression of GPR35 following exposure to KYNA, determind by flow cytometry (two independent experiments with two technical replicates).</p

Fig 1 -

(A) Creatine and urea levels provided by the metabolomics profile of the plasma (raw data). (B) Venn diagram yielding 26 differentially-expressed cardiac and plasma metabolites at both time points: 1 day and 1 week relative to sham.</p

Cardiac and plasma levels of KYNA, TRP and KYN following AKI (raw data).

Cardiac and plasma levels of KYNA, TRP and KYN following AKI (raw data).</p

KYNA reduces the anoxic-mediated oxidative damage to the mitochondria of H9C2 cells.

(A) Representative images of cytoplasm (Calcein-Green), superoxide levels (MitoSOXTM) and nuclei (Hoechst) staining. (B) Box plots demonstrating median and sample scattering by the combination of Calcein Green and MitoSOX staining after Z-score normalization (30 wells per treatment). The statistical analysis (One-way ANOVA) is given in the text.</p

Parameters which exhibit the rescue of H9C2 cells grown under anoxia in box plots graphs.

(A) Quantification of Mito Activity and Mito fraction as documented in Fig 4. (B) Box plots graph representation of MitoTracker Deep Red intensity, MitoTracker red intensity, and cell intensity. (C) Box plots graph re-presentation of mitochondria elongation and area (24–30 replicates per arm). (TIF)</p

KYNA enhances cardiac recovery following AMI.

Female BALB/C mice were treated with KYNA (250 mg/Lit) diluted in their drinking water (n = 9) versus regular tap water (n = 10). (A) KYNA treatment resulted in an elevated LV EF (%), SV (μl), and CO (ml/min) measurements relative to vehicle-treated control animals on day 30 relative to day 1. Statistical difference between day 1 and day 30 was determined by paired two-tailed Student’s T-test. X- Denotes mean values, horizontal lines stands for the median values. (B) Concamitantly, KYNA reduced the percentage of collagen deposition in the heart according to Picro Sirius Red staining. i. representative captures of the LV sections. ii. Box plot demonstrating the collagen content out of the total LV section (n = 6/ arm). Significance was determined by two-tailed Student’s T-test.</p

KYNA rescues H9C2 cells from anoxia-driven cell death and mitochondrial disruption.

(A) A PCA analysis demonstrating a clear separation between experimental conditions by the combination of mitochondrial features (24–30 wells per treatment). (B) Representative images of mitochondrial content (MitoTracker Green FM) and function (MitoTracker Deep Red FM) as well as nuclei (Hoechst) and cytoplasm (Calcein-Red-Orange) staining. The merged column represents the merging of Hoechst and the two MitoTracker dyes. (C) Box plot graphs demonstrating median and sample scattering of the values (24–30 wells per treatment) affiliated cell count, mitochondrial fraction and mitochondrial activity after Z-score-based normalization.</p