Intracellular metabolomic profiling of Picochlorum sp. under diurnal conditions mimicking outdoor light, temperature, and seasonal variations.

Introduction: This study focuses on metabolic profiling of a robust marine green algal strain Picochlorum sp. MCC39 that exhibits resilient growth under diverse outdoor open pond conditions. Given its potential for producing high-value chemicals through metabolic engineering, understanding its metabolic dynamics is crucial for pathway modification.

PYK-SubstitutionOME: an integrated database containing allosteric coupling, ligand affinity and mutational, structural, pathological, bioinformatic and computational information about pyruvate kinase isozymes.

Interpreting changes in patient genomes, understanding how viruses evolve and engineering novel protein function all depend on accurately predicting the functional outcomes that arise from amino acid substitutions. To that end, the development of first-generation prediction algorithms was guided by historic experimental datasets. However, these datasets were heavily biased toward substitutions at positions that have not changed much throughout evolution (i.

H/D Exchange Characterization of Silent Coupling: Entropy-Enthalpy Compensation in Allostery.

The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a ΔH and a TΔS component. In the scenario in which TΔS completely compensates ΔH, no allosteric influence of effector binding on substrate affinity is observed.

Evaluation of freely available software tools for untargeted quantification of C isotopic enrichment in cellular metabolome from HR-LC/MS data.

C Metabolic Flux Analysis (C-MFA) involves the quantification of isotopic enrichment in cellular metabolites and fitting the resultant data to the metabolic network model of the organism. Coverage and resolution of the resultant flux map depends on the total number of metabolites and fragments in which C enrichment can be quantified accurately. Experimental techniques for tracking C enrichment are evolving rapidly and large volumes of data are now routinely generated through the use of Liquid Chromatography coupled with High-Resolution Mass Spectrometry (HR-LC/MS).

Mass Isotopologue Distribution of dimer ion adducts of intracellular metabolites for potential applications in 13C Metabolic Flux Analysis.

13C Metabolic Flux Analysis (13C-MFA) is a powerful tool for quantification of carbon flux distribution in metabolic pathways. However, the requirement to obtain accurate labeling patterns, especially for compounds with low abundance, poses a challenge. Chromatographic separation and high sensitivity of the modern mass spectrometers (MS) alleviate this problem to a certain extent.

Elevated carbon dioxide levels lead to proteome-wide alterations for optimal growth of a fast-growing cyanobacterium, Synechococcus elongatus PCC 11801.

The environmental considerations attributing to the escalation of carbon dioxide emissions have raised alarmingly. Consequently, the concept of sequestration and biological conversion of CO by photosynthetic microorganisms is gaining enormous recognition. In this study, in an attempt to discern the synergistic CO tolerance mechanisms, metabolic responses to increasing CO concentrations were determined for Synechococcus elongatus PCC 11801, a fast-growing, novel freshwater strain, using quantitative proteomics.

An improved method for extraction of polar and charged metabolites from cyanobacteria.

A key requirement for 13C Metabolic flux analysis (13C-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The 13C isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz.

SWATH Tandem Mass Spectrometry Workflow for Quantification of Mass Isotopologue Distribution of Intracellular Metabolites and Fragments Labeled with Isotopic C Carbon.

Accurate quantification of mass isotopologue distribution (MID) of metabolites is a prerequisite for C-metabolic flux analysis. Currently used mass spectrometric (MS) techniques based on multiple reaction monitoring (MRM) place limitations on the number of MIDs that can be analyzed in a single run. Moreover, the deconvolution step results in amplification of error.

Metabolic model of Synechococcus sp. PCC 7002: Prediction of flux distribution and network modification for enhanced biofuel production.

Flux Balance Analysis was performed with the Genome Scale Metabolic Model of a fast growing cyanobacterium Synechococcus sp. PCC 7002 to gain insights that would help in engineering the organism as a production host. Gene essentiality and synthetic lethality analysis revealed a reduced metabolic robustness under genetic perturbation compared to the heterotrophic bacteria Escherichia coli.

Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry.

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e.

Energetic coupling between an oxidizable cysteine and the phosphorylatable N-terminus of human liver pyruvate kinase.

During our efforts to characterize the regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for phosphoenolpyruvate (PEP) becomes reduced several days after cell lysis. A 1.8 Å crystallographic structure of L-PYK with the S12D mimic of phosphorylation indicates that Cys436 is oxidized, the first potential insight into explaining the effect of "aging".

HDXFinder: automated analysis and data reporting of deuterium/hydrogen exchange mass spectrometry.

Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data.

Allosteric regulation of human liver pyruvate kinase by peptides that mimic the phosphorylated/dephosphorylated N-terminus.

An advantage of studying allosteric regulation over covalent modification is that allostery allows the experimentalist to vary the concentration of effector, thereby allowing independent quantification of effector binding and allosteric coupling. In turn, this capacity allows the use of effector analogues to determine which regions of the effector contribute to effector binding and which contribute to allosteric regulation. Like many other proteins, human liver pyruvate kinase (hL-PYK) is regulated by phosphorylation.

Monitoring allostery in D2O: a necessary control in studies using hydrogen/deuterium exchange to characterize allosteric regulation.

There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g.

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis.

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation.