Crystal Structures of Apo and Liganded 4‑Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β‑Keto Acid

The enzymes in the catechol <i>meta</i>-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria <i>Pseudomonas putida</i> G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg²⁺ as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD

Structural and Kinetic Properties of the Aldehyde Dehydrogenase NahF, a Broad Substrate Specificity Enzyme for Aldehyde Oxidation

The salicylaldehyde dehydrogenase (NahF) catalyzes the oxidation of salicylaldehyde to salicylate using NAD⁺ as a cofactor, the last reaction of the upper degradation pathway of naphthalene in <i>Pseudomonas putida</i> G7. The naphthalene is an abundant and toxic compound in oil and has been used as a model for bioremediation studies. The steady-state kinetic parameters for oxidation of aliphatic or aromatic aldehydes catalyzed by 6xHis-NahF are presented. The 6xHis-NahF catalyzes the oxidation of aromatic aldehydes with large <i>k</i>_cat/<i>K</i>_m values close to 10⁶ M^–1 s^–1. The active site of NahF is highly hydrophobic, and the enzyme shows higher specificity for less polar substrates than for polar substrates, e.g., acetaldehyde. The enzyme shows α/β folding with three well-defined domains: the oligomerization domain, which is responsible for the interlacement between the two monomers; the Rossmann-like fold domain, essential for nucleotide binding; and the catalytic domain. A salicylaldehyde molecule was observed in a deep pocket in the crystal structure of NahF where the catalytic C284 and E250 are present. Moreover, the residues G150, R157, W96, F99, F274, F279, and Y446 were thought to be important for catalysis and specificity for aromatic aldehydes. Understanding the molecular features responsible for NahF activity allows for comparisons with other aldehyde dehydrogenases and, together with structural information, provides the information needed for future mutational studies aimed to enhance its stability and specificity and further its use in biotechnological processes

Cellular and humoral response induced in BALB/c mice by immunization with rLiHyp1 plus saponin.

<p>Single cells suspensions were obtained from the spleens of mice, four weeks after vaccination. Cells were non-stimulated (medium; background control) or stimulated with rLiHyp1 (20 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. IFN-γ, IL-12, GM-CSF, IL-4, and IL-10 levels were measured in culture supernatants by capture ELISA (<b>A</b>). Each bar represents the mean ± standard deviation (SD) of data from four individual mice per group. Statistically significant differences in the IFN-γ, IL-12 and GM-CSF levels between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>B</b>); and between IL-12/IL-10 and IL-12/IL-4 levels (<b>C</b>) are also showed. Statistically significant differences in the ratios between the rLiHyp1 plus saponin group and control groups were observed (*** <i>P</i><0.0001). The ratio between rLiHyp1-specific IgG1 and IgG2a antibodies was obtained for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and control groups was also observed (* <i>P</i><0.005) (<b>D</b>).</p

Analysis of the cellular and humoral response and of the involvement of IL-12, CD4 and CD8 T cells in the IFN-γ production after <i>L. infantum</i> challenge.

<p>Single cells suspensions were obtained from the spleens of mice, 10 weeks after infection. Cells were non-stimulated (medium; background control) or stimulated with <i>L. infantum</i> SLA (25 µg mL⁻¹) for 48 h at 37°C, 5% CO₂. Levels of IFN-γ, IL-12, GM-CSF, IL-4 and IL-10 were measured in culture supernatants by capture ELISA. Mean ± standard deviation (SD) of the cytokines levels determined in four individual mice per group is shown (<b>A</b>). Statistically significant differences between the rLiHyp1 plus saponin group and the control mice (saline and saponin groups) were observed (*** <i>P<0.0001</i>). The analysis of the involvement of IL-12 and CD4 and CD8 T cells in the IFN-γ production is showed (<b>B</b>). Levels of IFN-γ in the supernatants of spleen cells cultures stimulated with SLA, as explained above, in the absence (positive control) or in the presence of anti-IL-12, anti-CD4, or anti-CD8 monoclonal antibodies were measured. Statistically significant differences between non-treated control cells and cultures incubated with anti-CD4 and anti-IL-12 monoclonal antibodies were observed (*** <i>P</i><0.0001). The ratio between IFN-γ/IL-10 and IFN-γ/IL-4 levels (<b>C</b>), and between IL-12/IL-10 and IL-12/IL-4 levels (<b>D</b>), are also showed. Statistically significant differences between the rLiHyp1 plus saponin group and the control groups were observed (***<i>P</i><0.0001). The ratio between SLA-specific IgG1 and IgG2a antibodies levels were calculated for sera of each individual mouse within their respective vaccination group and statistically significant difference between the rLiHyp1 plus saponin group and the control groups was also observed (* <i>P</i><0.005) (<b>E</b>).</p

Protection of BALB/c mice vaccinated with rLiHyp1 plus saponin against <i>L. infantum</i>.

<p>Mice inoculated with saline, saponin, or rLiHyp1 plus saponin were subcutaneously infected with virulent 1×10⁷ stationary-phase promastigotes of <i>L. infantum</i>. The number of parasites in the liver (<b>A</b>), spleen (<b>B</b>), bone marrow (<b>C</b>), and paws' draining lymph nodes (<b>D</b>) was measured, 10 weeks after challenge by a limiting-dilution technique. Mean ± standard deviation (SD) of four mice in each group is shown. Statistically significant differences in the parasite load in all evaluated organs between the rLiHyp1 plus saponin group and control mice (saline and saponin groups) are showed (in numbers). Data shown in this study are representative of two independent experiments, which presented similar results.</p