r5p  (Biosynth Carbosynth)

Journal: bioRxiv

Article Title: Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow

doi: 10.1101/2024.08.15.607825

Figure Lengend Snippet: (a) Pyruvate kinases across a phylogenetic tree built from 113 bacterial reference genomes. Red dots indicate the presence of ECTD. Detailed species list is presented in Supplemental Table 1. (b) Size-exclusion chromatography of wild type (WT, green trace) and ΔECTD pyruvate kinase (blue trace) with apparent molecular masses of approximately 239 and 191 kDa, respectively. These correspond to the theoretical masses of 258.0 (monomeric mass of 64.5 kDa) and 212.8 kDa (monomeric mass of 53.2 kDa) for homoterameric wild type and ΔECTD pyruvate kinase. The black trace represents the average partition coefficients and linear regression thereof of a protein standard containing thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), and RNase A (13.7 kDa). (c) B. subtilis pyruvate kinase wild type the ΔECTD variant in the presence of indicated concentrations of ATP. Pyruvate kinase activity (the initial velocity of the reaction V 0 ) is normalized against its activity in the absence of ATP. Error bars represent standard error of the mean of triplicates, unless otherwise stated. (d-e) Three dimensional plots of B. subtilis wild type (d) and ΔECTD (e) pyruvate kinase activities in the presence of combinations of different concentrations of the substrate PEP and activator R5P. (f-i) The in vitro enzymatic activities of wild type and ΔECTD pyruvate kinases from several representative Bacillota species, as indicated, at increasing concentrations of the substrate PEP (x-axis). Data were fitted to a nonessential activation equation (f) or an allosteric sigmoidal equation (g-i) to obtain the fitting curves (solid curves).

Article Snippet: When indicated, 1 mM AMP (Millipore Sigma) and/or 1 mM R5P (Biosynth International) and/or 5 mM ATP (Millipore Sigma) were added.

Techniques: Size-exclusion Chromatography, Variant Assay, Activity Assay, In Vitro, Activation Assay

Journal: bioRxiv

Article Title: Allosteric Regulation of Pyruvate Kinase Enables Efficient and Robust Gluconeogenesis by Preventing Metabolic Conflicts and Carbon Overflow

doi: 10.1101/2024.08.15.607825

Figure Lengend Snippet: (a) Schematics of gluconeogenesis, part of the pentose phosphate pathway, and the TCA cycle. Important target metabolites are indicated. G6P: glucose 6-phosphate; FBP: fructose 1,6-bisphosphate; R5P: ribose 5-phosphate; S7P: sedoheptulose 7-phosphate; BPG: 1,3- bisphosphoglycerate; 3PG: 3-phosphoglycerate. (b) Z score of metabolites levels in wild type cells grown in glycolytic or gluconeogenic carbon sources. (c)-(d) Z score of metabolites levels in wild type and pyk Δ ectd cells grown in gluconeogenic carbon sources (c) malate and (d) pyruvate. (e)-(j) Relative abundance of metabolites upstream and downstream of pyruvate kinase. Relative abundances are calculated based on internal control, except for S7P, BPG and 3PG. See Materials and Methods: Metabolic analysis by LC-MS. (k)-(l) Cumulative drops in Gibbs free energies of gluconeogenesis for (k) wild type and (l) pyk Δ ectd cells. MDF: max-min driving force; F6P: fructose 6-phosphate; DHAP: dihydroxyacetone phosphate; G3P: glyceraldehyde 3-phosphate; 2PG: 2-phosphoglycerate. (m) The calculated forward flux ratio, reverse flux ratio and net flux ratio of the bottleneck reaction in gluconeogenesis for wild type and pyk Δ ectd cells grown in pyruvate. (n) A summary diagram of pyruvate kinase regulation and the physiological consequences of pyruvate kinase dysregulation during gluconeogenesis. (1): thermodynamic feasibility of gluconeogenesis; (2): glyphosate resistance; (3): pyruvate overflow; (4): the PEP-pyruvate-OAA futile cycle.

Article Snippet: When indicated, 1 mM AMP (Millipore Sigma) and/or 1 mM R5P (Biosynth International) and/or 5 mM ATP (Millipore Sigma) were added.

Techniques: Control, Liquid Chromatography with Mass Spectroscopy

Journal: Journal of the American Chemical Society

Article Title: Evidence for the Chemical Mechanism of RibB (3,4-Dihydroxy-2-butanone 4-phosphate Synthase) of Riboflavin Biosynthesis

doi: 10.1021/jacs.2c03376

Figure Lengend Snippet: Structures of RibB with sugar phosphates that are substrate analogues show only one metal ion in the active site, and the sugar phosphates bind in an elongated fashion. The RibB structure with a variety of sugar phosphate molecules with metal coordinating residues, Glu39 and His154 shown as wheat colored. (A) RibB was crystallized in the absence of sugar phosphate in an open conformation such that the active site loop containing Glu39 coordinating the metal ion is not visible in this image. (B) Addition of d -Ru5P (yellow sticks) to the crystal orders the active site loop of RibB with an extended conformation of the sugar phosphate, as seen in previous structures. The substrates (C) d -R5P (blue) and (D) d -Xy5P (dark red) have the sugar phosphates in a similar elongated conformation and contain one Mn 2+ ion in the active site. (E) l -Xy5P (orange) is not a substrate for RibB and binds in the active site in a twisted conformation and coordinates two Mn 2+ metals. Manganese ions are depicted as purple spheres. The maps are Polder maps contoured at 3 σ.

Article Snippet: A major change to the assay included using the actual sugar phosphates as substrates as opposed to the addition of pentose phosphate isomerase to generate d -ribulose 5-phosphate during assay incubation. d -ribulose 5-phosphate ( d -Ru5P), d -ribose 5-phosphate ( d -R5P), d -xylulose 5-phosphate ( d -Xy5P), and l -xylulose 5-phosphate ( l -Xy5P) (Sigma-Aldrich) were dissolved in 50 mM Tris-HCl (pH 7.5) to a concentration of 90 mM.

Techniques: