Response regulator ArcA of Salmonella enterica serovar Typhimurium downregulates expression of OmpD, a porin facilitating uptake of hydrogen peroxide.

Here we demonstrate that OmpD, the most abundant porin in Salmonella enterica serovar Typhimurium, facilitates uptake of hydrogen peroxide (H2O2) and that its expression is negatively regulated by ArcA upon peroxide exposure. When exposed to sublethal concentrations of H2O2, a S. Typhimurium ompD mutant showed decreased peroxide levels compared to those observed in the wild type strain, suggesting that H2O2 could be channeled inside the cell through OmpD. Further evidence came from in vitro studies using OmpD-containing reconstituted proteoliposomes, which showed enhanced H2O2 uptake compared to control liposomes with no porins. RT-PCR and western blot analyses were consistent with a negative regulation mechanism of ompD expression in wild type S. Typhimurium exposed to H2O2. In silico analysis aimed at detecting putative transcriptional regulator binding regions led to identification of an ArcA global regulator motif in the ompD promoter region. The interaction of ArcA with its putative binding site was confirmed in vitro by electrophoretic mobility shift assays. In addition, RT-PCR and western blot experiments demonstrated that the ompD downregulation, observed when the wild type strain was grown in the presence of H2O2, was not retained in arcA mutants, suggesting that ArcA could act as an ompD transcriptional repressor.

The ompW (porin) gene mediates methyl viologen (paraquat) efflux in Salmonella enterica serovar typhimurium.

Porins are channels that enable passive diffusion of hydrophilic solutes, nutrients and toxins through the outer bacterial membrane. This explains in part the ability of Gram-negative microorganisms to grow in several different environments, as well as their drug resistance. OmpD is an outer membrane channel that works with the inner membrane pump YddG to expel methyl viologen (MV) from Salmonella enterica serovar Typhimurium; this occurs independently of SmvA, also involved in MV resistance. On the other hand, DeltatolC strains show increased MV resistance when compared to wild-type cells, suggesting that there may be other porin(s) that could function with SmvA to pump MV out of S. typhimurium. A strong candidate is OmpW. Here we show that DeltaompW strains of S. typhimurium are 2.5-fold more sensitive to MV than the wild-type strain. Transcriptional fusions replacing ompW by lacZ show that ompW is induced at least 2-fold in the presence of MV. This result was observed both at the mRNA and protein levels, suggesting that ompW participates in MV resistance. In addition, DeltasmvADeltaompW strains are not fully complemented by smvA, suggesting that OmpW may function through an independent pathway different from that used by SmvA to move MV outside the cell.

Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is infrequent and suggests a low pK(a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK(a) of Lys(213). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.

Nucleotide specificity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase Kinetics, fluorescence spectroscopy, and molecular simulation studies.

Phosphoenolpyruvate carboxykinases, depending on the enzyme origin, preferentially use adenine or guanine nucleotides as substrates. In this work, analyses of the substrate specificity of the Saccharomyces cerevisiae ATP-dependent enzyme have been carried out. Kinetics studies gave relative values of k(cat)/K(m) for the nucleoside triphosphate complexes in the order ATP>>GTP>ITP>UTP>CTP. For the nucleoside diphosphate complexes the order is ADP>>GDP>IDP congruent withUDP>CDP. This shows that the enzyme has a strong preference for ADP (or ATP) over other nucleotides, being this preference about an order of magnitude higher for the diphosphorylated than for the triphosphorylated nucleosides. The calculated binding free energies (kcalmol(-1)) at 25 degrees C are 7.39 and 6.51 for ATP and ADP, respectively. These values decrease with the nucleotide structure in the same order than the kinetic specificity. The binding energy for any triphosphorylated nucleoside is more favourable than for the corresponding diphosphorylated compound, showing the relevance of the P(gamma) for nucleotide binding. Homology models of the adenine and guanine nucleotides in complex with the enzyme show that the base adopts a similar conformation in the diphosphorylated nucleosides while in the triphosphorylated nucleosides the sugar-base torsion angle is 61 degrees for ATP and -53 degrees for GTP. Differences are also noted in the distance between P(beta) and Mn2+ at site 1. This distance is almost the same in the ATP, GTP, and UTP complexes, however in the ADP, GDP and UDP complexes it is 2.9, 5.1, and 7A, respectively. Experimental data obtained with a Thr463Ala mutant enzyme agree with molecular simulation predictions. The results here presented are discussed in terms of the proposed interactions of the nucleotides with the protein.