p38α Mitogen-activated Protein Kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical genetic approaches and co-expression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif that allows auto-phosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Potentially, such intervention could circumvent the drawbacks seen when pharmacological inhibitors of p38 catalytic activity are used clinically.

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition.

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The aliphatic amidase from Pseudomonas aeruginosa belongs to the nitrilase superfamily, and Cys(166) is the nucleophile of the catalytic mechanism. A model of amidase was built by comparative modelling using the crystal structure of the worm nitrilase-fragile histidine triad fusion protein (NitFhit; Protein Data Bank accession number 1EMS) as a template. The amidase model predicted a catalytic triad (Cys-Glu-Lys) situated at the bottom of a pocket and identical with the presumptive catalytic triad of NitFhit. Three-dimensional models for other amidases belonging to the nitrilase superfamily also predicted Cys-Glu-Lys catalytic triads. Support for the structure for the P. aeruginosa amidase came from site-direct mutagenesis and from the locations of amino acid residues that altered substrate specificity or binding when mutated.

In a recent Nature leading article the possibility of the occurrence of resonance absorption at microwave frequencies in DNA was discussed in terms of the latest available theoretical and experimental evidence, and its implications were assessed. Because of the far-reaching nature of these implications it is important that the existence or otherwise of such absorption be firmly established. Here we report on a concerted effort in two independent laboratories which has involved the measurement of the dielectric properties of aqueous solutions of circular DNA molecules in the frequency range 1-10 GHz. No resonance absorption or any form of enhanced absorption was demonstrated.

The gene PA4866 from Pseudomonas aeruginosa is documented in the Pseudomonas genome database as encoding a 172 amino acid hypothetical acetyltransferase. We and others have described the 3D structure of this protein (termed pita) [Davies et al. (2005) Proteins: Struct., Funct., Bioinf. 61, 677-679; Nocek et al., unpublished results], and structures have also been reported for homologues from Agrobacterium tumefaciens (Rajashankar et al., unpublished results) and Bacillus subtilis [Badger et al. (2005) Proteins: Struct., Funct., Bioinf. 60, 787-796]. Pita homologues are found in a large number of bacterial genomes, and while the majority of these have been assigned putative phosphinothricin acetyltransferase activity, their true function is unknown. In this paper we report that pita has no activity toward phosphinothricin. Instead, we demonstrate that pita acts as an acetyltransferase using the glutamate analogues l-methionine sulfoximine and l-methionine sulfone as substrates, with Km(app) values of 1.3 +/- 0.21 and 1.3 +/- 0.13 mM and kcat(app) values of 505 +/- 43 and 610 +/- 23 s-1 for l-methionine sulfoximine and l-methionine sulfone, respectively. A high-resolution (1.55 A) crystal structure of pita in complex with one of these substrates (l-methionine sulfoximine) has been solved, revealing the mode of its interaction with the enzyme. Comparison with the apoenzyme structure has also revealed how certain active site residues undergo a conformational change upon substrate binding. To investigate the role of pita in P. aeruginosa, a mutant strain, Depp4, in which pita was inactivated through an in-frame deletion, was constructed by allelic exchange. Growth of strain Depp4 in the absence of glutamine was inhibited by l-methionine sulfoximine, suggesting a role for pita in protecting glutamine synthetase from inhibition.

Wild-type and site-specific mutants C166S and C166A (Cys-166-->Ser and Cys-166-->Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5'-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a=b=c=84 A; alpha=beta=gamma=75 degrees) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.