Substrate binding in the allosteric site mimics homotropic cooperativity in the SIS-fold glucosamine-6-phosphate deaminases.

Glucosamine-6-phosphate (GlcN6P) deaminases from Escherichia coli (EcNagBI) and Shewanella denitrificans (SdNagBII) are special examples of what constitute nonhomologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography. ITC experiments revealed two different binding sites with distinctive thermodynamic signatures: a single binding site per monomer for the allosteric activator N-acetylglucosamine 6-phosphate (GlcNAc6P) and two binding sites per monomer for the transition-state analog 2-amino-2-deoxy-D-glucitol 6-phosphate (GlcNol6P). Crystallographic data demonstrated the existence of an unusual allosteric site that can bind both GlcNAc6P and GlcNol6P, implying that the homotropic activation of this enzyme arises from the occupation of the allosteric site by the substrate. In this work we describe the presence of this novel allosteric site in the SIS-fold deaminases, which is responsible for the homotropic and heterotropic activation of SdNagBII by GlcN6P and GlcNAc6P, respectively. This study unveils an original mechanism to generate a high degree of homotropic activation in SdNagBII, mimicking the allosteric and cooperative properties of hexameric EcNagBI but with a reduced number of subunits.

A native IgE in complex with profilin provides insights into allergen recognition and cross-reactivity.

Allergies have become a rising health problem, where plentiful substances can trigger IgE-mediated allergies in humans. While profilins are considered minor allergens, these ubiquitous proteins are primary molecules involved in cross-reactivity and pollen-food allergy syndrome. Here we report the first crystal structures of murine Fab/IgE, with its chains naturally paired, in complex with the allergen profilin from Hevea brasiliensis (Hev b 8). The crystallographic models revealed that the IgE's six complementarity-determining regions (CDRs) interact with the allergen, comprising a rigid paratope-epitope surface of 926 Å2, which includes an extensive network of interactions. Interestingly, we also observed previously unreported flexibility at Fab/IgE's elbow angle, which did not influence the shape of the paratope. The Fab/IgE exhibits a high affinity for Hev b 8, even when using 1 M NaCl in BLI experiments. Finally, based on the encouraging cross-reactivity assays using two mutants of the maize profilin (Zea m 12), this antibody could be a promising tool in IgE engineering for diagnosis and research applications.

The K+-Dependent and -Independent Pyruvate Kinases Acquire the Active Conformation by Different Mechanisms.

Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K+-dependent, whereas those with K117 are K+-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K+-independent pyruvate kinases using the K+-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K+-dependent and -independent mutants. K+-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K+-dependent enzymes. E117K mutant was K+-independent and the enzyme acquired the active conformation by a different mechanism. In the K+-independent apoenzyme of Mycobacterium tuberculosis, K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.

Energetic and structural effects of the Tanford transition on ligand recognition of bovine ß-lactoglobulin.

Bovine ß-lactoglobulin, an abundant protein in whey, is a promising nanocarrier for peroral administration of drug-like hydrophobic molecules, a process that involves transit through the different acidic conditions of the human digestive tract. Among the several pH-induced conformational rearrangements that this lipocalin undergoes, the Tanford transition is particularly relevant. This transition, which occurs with a midpoint around neutral pH, involves a conformational change of the E-F loop that regulates accessibility to the primary binding site. The effect of this transition on the ligand binding properties of this protein has scarcely been explored. In this study, we carried out an energetic and structural characterization of ß-lactoglobulin molecular recognition at pH values above and below the zone in which the Tanford transition occurs. The combined analysis of crystallographic, calorimetric, and molecular dynamics data sheds new light on the interplay between self-association, ligand binding, and the Tanford pre- and post-transition conformational states, revealing novel aspects underlying the molecular recognition mechanism of this enigmatic lipocalin.

The structure of a novel membrane-associated 6-phosphogluconate dehydrogenase from Gluconacetobacter diazotrophicus (Gd6PGD) reveals a subfamily of short-chain 6PGDs.

The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.

Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8.

The production of specific antibodies able to recognize allergens from different sources or block interactions between allergens and antibodies mediating allergic reactions is crucial for developing successful tools for diagnostics and therapeutics. Panallergens are highly conserved proteins present in widely different species, implicated in relevant cross-reactions. The panallergen latex profilin (Hev b 8) has been associated with the latex-food-pollen syndrome. We generated five monoclonal IgGs and one IgE from murine hybridomas against recombinant Hev b 8 and evaluated their interaction with this allergen using ELISA and biolayer interferometry (BLI). Affinity purified mAbs exhibited high binding affinities towards rHev b 8, with KD1 values ranging from 10-10 M to 10-11 M. Some of these antibodies also recognized the recombinant profilins from maize and tomato (Zea m 12 and Sola l 1), and the ash tree pollen (Fra e 2). Competition ELISA demonstrated that some mAb pairs could bind simultaneously to rHev b 8. Using BLI, we detected competitive, non-competitive, and partial-competition interactions between pairs of mAbs with rHev b 8, suggesting the existence of at least two non-overlapping epitopes on the surface of this allergen. Three-dimensional models of the Fv of 1B4 and 2D10 IgGs and docking simulations of these Fvs with rHev b 8 revealed these epitopes. Furthermore, these two mAbs inhibited the interaction of polyclonal IgE and IgG4 antibodies from profilin-allergic patients with rHev b 8, indicating that the mAbs and the antibodies present in sera from allergic patients bind to overlapping epitopes on the allergen. These mAbs can be useful tools for immune-localization studies, immunoassay development, or standardization of allergenic products.

The substrate of the glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa provides structural stability.

In general, eukaryotic glucose-6-phosphate dehydrogenases (G6PDHs) are structurally stabilized by NADP+. Here we show by spectrofluorometric analysis, thermal and urea denaturation, and trypsin proteolysis, that a different mechanism stabilizes the enzyme from Pseudomonas aeruginosa (PaG6PDH) (EC 1.1.1.363). The spectrofluorometric analysis of the emission of 8-anilino-1-naphthalenesulfonic acid (ANS) indicates that this stabilization is the result of a structural change in the enzyme caused by G6P. The similarity between the Kd values determined for the PaG6PDH-G6P complex (78.0 ±â€¯7.9 µM) and the K0.5 values determined for G6P (57.9 ±â€¯2.5 and 104.5 ±â€¯9.3 µM in the NADP+- and NAD+-dependent reactions, respectively) suggests that the structural changes are the result of G6P binding to the active site of PaG6PDH. Modeling of PaG6PDH indicated the residues that potentially bind the ligand. These results and a phylogenetic analysis of the amino acid sequences of forty-four G6PDHs, suggest that the stabilization observed for PaG6PDH could be a characteristic that distinguishes this and other G6PDHs that use NAD+ and NADP+ from those that use NADP+ only or preferentially, such as those found in eukaryotes. This characteristic could be related to the metabolic roles these enzymes play in the organisms to which they belong.

Amino and carboxy-terminal extensions of yeast mitochondrial DNA polymerase assemble both the polymerization and exonuclease active sites.

Human and yeast mitochondrial DNA polymerases (DNAPs), POLG and Mip1, are related by evolution to bacteriophage DNAPs. However, mitochondrial DNAPs contain unique amino and carboxyl-terminal extensions that physically interact. Here we describe that N-terminal deletions in Mip1 polymerases abolish polymerization and decrease exonucleolytic degradation, whereas moderate C-terminal deletions reduce polymerization. Similarly, to the N-terminal deletions, an extended C-terminal deletion of 298 amino acids is deficient in nucleotide addition and exonucleolytic degradation of double and single-stranded DNA. The latter observation suggests that the physical interaction between the amino and carboxyl-terminal regions of Mip1 may be related to the spread of pathogenic POLG mutant along its primary sequence.

A biophysical and structural study of two chitinases from Agave tequilana and their potential role as defense proteins.

Plant chitinases are enzymes that have several functions, including providing protection against pathogens. Agave tequilana is an economically important plant that is poorly studied. Here, we identified a chitinase from short reads of the A. tequilana transcriptome (AtChi1). A second chitinase, differing by only six residues from the first, was isolated from total RNA of plants infected with Fusarium oxysporum (AtChi2). Both enzymes were overexpressed in Escherichia coli and analysis of their sequences indicated that they belong to the class I glycoside hydrolase family19, whose members exhibit two domains: a carbohydrate-binding module and a catalytic domain, connected by a flexible linker. Activity assays and thermal shift experiments demonstrated that the recombinant Agave enzymes are highly thermostable acidic endochitinases with Tm values of 75 °C and 71 °C. Both exhibit a molecular mass close to 32 kDa, as determined by MALDI-TOF, and experimental pIs of 3.7 and 3.9. Coupling small-angle x-ray scattering information with homology modeling and docking simulations allowed us to structurally characterize both chitinases, which notably show different interactions in the binding groove. Even when the six different amino acids are all exposed to solvent in the loops located near the linker and opposite to the binding site, they confer distinct kinetic parameters against colloidal chitin and similar affinity for (GlnNAc)6, as shown by isothermal titration calorimetry. Interestingly, binding is more enthalpy-driven for AtChi2. Whereas the physiological role of these chitinases remains unknown, we demonstrate that they exhibit important antifungal activity against chitin-rich fungi such as Aspergillus sp. DATABASE: SAXS structural data are available in the SASBDB database with accession numbers SASDDE7 and SASDDA6. ENZYMES: Chitinases (EC3.2.1.14).