Pulmonary granuloma caused by Pseudomonas andersonii sp nov.

Pulmonary granuloma is a common lesion for which gram-negative bacteria are rarely implicated as a cause. Hence, most physicians are unaware of this etiology. We isolated a gram-negative bacterium from a surgically resected pulmonary granuloma in a 42-year-old, nonimmunocompromised woman. Within the necrotizing granuloma, numerous organisms also were demonstrated by Gram stain, suggesting a cause-disease relationship. Characterization of the bacterium by sequence analysis of the 16S ribosomal gene, cellular fatty acid profiling, and microbiologic studies revealed a novel bacterium with a close relationship to Pseudomonas. We propose a new species for the bacterium, Pseudomonas andersonii. These results suggest that the differential diagnosis of a lung granuloma also should include this gram-negative bacterium as a potential causative agent, in addition to the more common infections caused by acid-fast bacilli and fungi. This bacterium was shown to be susceptible to most antibiotics that are active against gram-negative bacteria.

Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.

Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged.

Persistent binding of MgADP to the E187A mutant of Escherichia coli phosphofructokinase in the absence of allosteric effects.

MgADP binding to the allosteric site enhances the affinity of Escherichia coli phosphofructokinase (PFK) for fructose 6-phosphate (Fru-6-P). X-ray crystallographic data indicate that MgADP interacts with the conserved glutamate at position 187 within the allosteric site through an octahedrally coordinated Mg(2+) ion [Shirakihara, Y., and Evans, P. R. (1988) J. Mol. Biol. 204, 973-994]. Lau and Fersht reported that substituting an alanine for this glutamate within the allosteric site of PFK (i.e., mutant E187A) causes MgADP to lose its allosteric effect upon Fru-6-P binding [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. However, these authors later reported that MgADP inhibits Fru-6-P binding in the E187A mutant. The inhibition presumably occurs by preferential binding to the inactive (T) state complex of the Monod-Wyman-Changeux two-state model [Lau, F. T.-K., and Fersht, A. R. (1989) Biochemistry 28, 6841-6847]. The present study provides an alternative explanation of the role of MgADP in the E187A mutant. Using enzyme kinetics, steady-state fluorescence emission, and anisotropy, we performed a systematic linkage analysis of the three-ligand interaction between MgADP, Fru-6-P, and MgATP. We found that MgADP at low concentrations did not enhance or inhibit substrate binding. Anisotropy shows that MgADP binding at the allosteric site occurred even when MgADP produced no allosteric effect. However, as in the wild-type enzyme, the binding of MgADP to the active site in the mutant competitively inhibited MgATP binding and noncompetitively inhibited Fru-6-P binding. These results clarified the mechanism of a three-ligand interaction and offered a nontraditional perspective on allosteric mechanism.

MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase.

Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models.