Glycogen metabolism in methanogens: A key pathway for metabolic response to nutrient availability.

Methanogens, which are found exclusively in the Archaea domain of life, have the potential to help solve future energy challenges by producing methane. As a result, their metabolism has attracted significant attention in recent years. Despite being unable to grow on sugars, they store glycogen, which raises intriguing questions about the role of this polymer in methanogen metabolism and the signals that trigger its degradation when methanogenic substrates are not available.

Deciphering Structural Traits for Thermal and Kinetic Stability across Protein Family Evolution through Ancestral Sequence Reconstruction.

Natural proteins are frequently marginally stable, and an increase in environmental temperature can easily lead to unfolding. As a result, protein engineering to improve protein stability is an area of intensive research. Nonetheless, since there is usually a high degree of structural homology between proteins from thermophilic organisms and their mesophilic counterparts, the identification of structural determinants for thermoadaptation is challenging.

Structural insights into a functional unit from an immunogenic mollusk hemocyanin.

Mollusk hemocyanins, among the largest known proteins, are used as immunostimulants in biomedical and clinical applications. The hemocyanin of the Chilean gastropod Concholepas concholepas (CCH) exhibits unique properties, which makes it safe and effective for human immunotherapy, as observed in animal models of bladder cancer and melanoma, and dendritical cell vaccine trials. Despite its potential, the structure and amino acid sequence of CCH remain unknown.

Metal Ions Can Modulate the Self-Assembly and Activity of Catalytic Peptide Amyloids.

Rational design of peptides has become a powerful tool to produce self-assembled nanostructures with the ability to catalyze different chemical reactions, paving the way to develop minimalistic enzyme-like nanomaterials. Catalytic amyloid-like assemblies have emerged among the most versatile and active, but they often require additional factors for activity. Elucidating how these factors influence the structure and activity is key for the design.

Unraveling the folding and dimerization properties of the human FoxP subfamily of transcription factors.

Human FoxP proteins share a highly conserved DNA-binding domain that dimerizes via three-dimensional domain swapping, although showing varying oligomerization propensities among its members. Here, we present an experimental and computational characterization of all human FoxP proteins to unravel how their amino acid substitutions impact their folding and dimerization mechanism. We solved the crystal structure of the forkhead domain of FoxP4 to then perform a comparison across all members, finding that their sequence changes impact not only the structural heterogeneity of their forkhead domains but also the protein-protein association energy barrier.

Kinetic characterization and phylogenetic analysis of human ADP-dependent glucokinase reveal new insights into its regulatory properties.

Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form.

Characterisation of kinetics, substrate inhibition and product activation by AMP of bifunctional ADP-dependent glucokinase/phosphofructokinase from Methanococcus maripaludis.

Methanogenic archaea have received attention due to their potential use in biotechnological applications such as methane production, so their metabolism and regulation are topics of special interest. When growing in a nutrient-rich medium, these organisms exhibit gluconeogenic metabolism; however, under starvation conditions, they turn to glycolytic metabolism. To date, no regulatory mechanism has been described for this gluconeogenic/glycolytic metabolic switch.

The Emergence of New Catalytic Abilities in an Endoxylanase from Family GH10 by Removing an Intrinsically Disordered Region.

Endoxylanases belonging to family 10 of the glycoside hydrolases (GH10) are versatile in the use of different substrates. Thus, an understanding of the molecular mechanisms underlying substrate specificities could be very useful in the engineering of GH10 endoxylanases for biotechnological purposes. Herein, we analyzed XynA, an endoxylanase that contains a (β/α)-barrel domain and an intrinsically disordered region (IDR) of 29 amino acids at its amino end.

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From .

Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored.

Crystal Structure of Agmatinase: Catalytic Mechanism and Residues Relevant for Substrate Specificity.

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation.

Structure of an ancestral ADP-dependent kinase with fructose-6P reveals key residues for binding, catalysis, and ligand-induced conformational changes.

ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFKs), specific glucokinases (ADP-GKs), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, none exhibits fructose-6-phosphate (F6P) at the active site.

Crystal structure and molecular dynamics simulations of a promiscuous ancestor reveal residues and an epistatic interaction involved in substrate binding and catalysis in the ATP-dependent vitamin kinase family members.

Enzymes with hydroxymethylpyrimidine/phosphomethylpyrimidine kinase activity (HMPPK) are essential in the vitamin B1 (thiamine pyrophosphate) biosynthesis and recycling pathways. In contrast, enzymes with pyridoxal kinase activity (PLK) produce pyridoxal phosphate (vitamin B6), an essential cofactor for various biochemical reactions. In the ATP-dependent vitamin kinases family, the members of PLK/HMPPK-like subfamily have both enzymatic activities.

Characterization of hydroxymethylpyrimidine phosphate kinase from mesophilic and thermophilic bacteria and structural insights into their differential thermal stability.

The hydroxymethylpyrimidine phosphate kinases (HMPPK) encoded by the thiD gene are involved in the thiamine biosynthesis pathway, can perform two consecutive phosphorylations of 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) and are found in thermophilic and mesophilic bacteria, but only a few characterizations of mesophilic enzymes are available. The presence of another homolog enzyme (pyridoxal kinase) that can only catalyze the first phosphorylation of HMP and encoded by pdxK gene, has hampered a precise annotation in this enzyme family. Here we report the kinetic characterization of two HMPPK with structure available, the mesophilic and thermophilic enzyme from Salmonella typhimurium (StHMPPK) and Thermus thermophilus (TtHMPPK), respectively.

Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon ().

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes ( [], integrin β2 (itβ2, also named ), [], [], []) were analyzed to identify SNPs by data mining.

Free radicals derived from γ-radiolysis of water and AAPH thermolysis mediate oxidative crosslinking of eGFP involving Tyr-Tyr and Tyr-Cys bonds: the fluorescence of the protein is conserved only towards peroxyl radicals.

The enhanced green fluorescent protein (eGFP) is one of the most employed variants of fluorescent proteins. Nonetheless little is known about the oxidative modifications that this protein can undergo in the cellular milieu. The present work explored the consequences of the exposure of eGFP to free radicals derived from γ-radiolysis of water, and AAPH thermolysis.

Protein topology determines substrate-binding mechanism in homologous enzymes.

During evolution, some homologs proteins appear with different connectivity between secondary structures (different topology) but conserving the tridimensional arrangement of them (same architecture). These events can produce two types of arrangements; circular permutation or non-cyclic permutations. The first one results in the N and C terminus transferring to a different position on a protein sequence while the second refers to a more complex arrangement of the structural elements.

ADP-Dependent Kinases From the Archaeal Order Adapt to Salt by a Non-canonical Evolutionarily Conserved Strategy.

Halophilic organisms inhabit hypersaline environments where the extreme ionic conditions and osmotic pressure have driven the evolution of molecular adaptation mechanisms. Understanding such mechanisms is limited by the common difficulties encountered in cultivating such organisms. Within the , for example, only the and the order include readily cultivable halophilic species.

ADP-dependent phosphofructokinases from the archaeal order Methanosarcinales display redundant glucokinase activity.

The genome of Methanosarcinales organisms presents both ADP-dependent glucokinase and phosphofructokinase genes. However, Methanococcoides burtonii has a truncate glucokinase gene with a large deletion at the C-terminal, where the catalytic GXGD motif is located. Characterization of its phosphofructokinase annotated protein shows that is a bifunctional enzyme able to supply the absence of the glucokinase activity.

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders , , and , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts.

Unusual dimerization of a BcCsp mutant leads to reduced conformational dynamics.

Unlabelled: Cold shock proteins (Csp) constitute a family of ubiquitous small proteins that act as RNA-chaperones to avoid cold-induced termination of translation. All members contain two subdomains composed of 2 and 3 β-strands, respectively, which are connected by a hinge loop and fold into a β-barrel. Bacillus caldolyticus Csp (BcCsp) is one of the most studied members of the family in terms of its folding, function, and structure.

Structural and functional analysis of the ASM p.Ala359Asp mutant that causes acid sphingomyelinase deficiency.

Niemann-Pick disease (NPD) type A and B are recessive hereditary disorders caused by deficiency in acid sphingomyelinase (ASM). The p.Ala359Asp mutation has been described in several patients but its functional and structural effects in the protein are unknown.

Emergence of pyridoxal phosphorylation through a promiscuous ancestor during the evolution of hydroxymethyl pyrimidine kinases.

In the family of ATP-dependent vitamin kinases, several bifunctional enzymes that phosphorylate hydroxymethyl pyrimidine (HMP) and pyridoxal (PL) have been described besides enzymes specific towards HMP. To determine how bifunctionality emerged, we reconstructed the sequence of three ancestors of HMP kinases, experimentally resurrected, and assayed the enzymatic activity of their last common ancestor. The latter has ∼ 8-fold higher specificity for HMP due to a glutamine residue (Gln44) that is a key determinant of the specificity towards HMP, although it is capable of phosphorylating both substrates.

Bifunctional ADP-dependent phosphofructokinase/glucokinase activity in the order Methanococcales--biochemical characterization of the mesophilic enzyme from Methanococcus maripaludis.

In some archaea, the phosphorylation of glucose and fructose 6-phosphate (fructose 6P) is carried out by enzymes that are specific for either substrate and that use ADP as phosphoryl donor. In the hyperthermophilic archaeon Methanocaldococcus jannaschii, a bifunctional enzyme able to phosphorylate glucose and fructose 6P has been described. To determine whether the ability to phosphorylate both glucose and fructose 6P is a common feature for all enzymes of the order Methanococcales, we expressed, purified and characterized the unique homologous protein of the mesophilic archaea Methanococcus maripaludis.

Crystal structure, SAXS and kinetic mechanism of hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis reveal a conserved mechanism for catalysis.

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Thermococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function.