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International audience; Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.

International audience; Listeria monocytogenes is a human pathogen that employs a wide variety of virulence factors in order to adhere to, invade, and replicate within target cells. Internalins play key roles in processes ranging from adhesion to receptor recognition and are thus essential for infection. Recently, InlK, a surface-associated internalin, was shown to be involved in Listeria's ability to escape from autophagy by recruitment of the major vault protein (MVP) to the bacterial surface. Here, we report the structure of InlK, which harbors four domains arranged in the shape of a "bent arm". The structure supports a role for the "elbow" of InlK in partner recognition, as well as of two Ig-like pedestals intercalated by hinge regions in the projection of InlK away from the surface of the bacterium. The unusual fold and flexibility of InlK could be essential for MVP binding and concealment from recognition by molecules involved in the autophagic process.

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.

The development of high level β-lactam resistance in the pneumococcus requires the expression of an altered form of PBP1a, in addition to modified forms of PBP2b and PBP2x, which are necessary for the appearance of low levels of resistance. Here, we present the crystal structure of a soluble form of PBP1a from the highly resistant Streptococcus pneumoniae strain 5204 (minimal inhibitory concentration of cefotaxime is 12 mg·liter-1). Mutations T371A, which is adjacent to the catalytic nucleophile Ser370, and TSQF(574–577)NTGY, which lie in a loop bordering the active site cleft, were investigated by site-directed mutagenesis. The consequences of these substitutions on reaction kinetics with β-lactams were probed in vitro, and their effect on resistance was measured in vivo. The results are interpreted in the framework of the crystal structure, which displays a narrower, discontinuous active site cavity, compared with that of PBP1a from the β-lactam susceptible strain R6, as well as a reorientation of the catalytic Ser370.

The active site of flavoprotein oxidases frequently contains two active-site tyrosines, e.g., Y223 and Y238 in d-amino acid oxidases (DAAO) from Rhodotorula gracilis. In the past, these residues were individually mutated to phenylalanine and serine. To further study their role we undertake an interpretation of Y223F–Y238F double mutation. The spectral properties of the double mutant are similar to those of wild-type DAAO, suggesting a minimally altered active site, but its catalytic activity is lower. A first unexpected observation is that for a number of properties the double mutant is more similar to the wild-type DAAO than to the corresponding single-point mutants, e.g., similar kinetic mechanism, substrate specificity, and redox properties. The Y223F–Y238F DAAO also exhibits appreciably weaker binding of the competitive inhibitors’ carboxylic acids (synergistic effects), while the Km values for the substrate d-amino acids are only slightly changed, thus suggesting that the presence of an amino group in the ligand is important for binding to the double mutant. Synergistic effects of the substitutions are also evident on the rate constant of flavin reduction indicating anti-cooperative interaction of the two residues in the hydride transfer process. Our results indicate that Tyr223-OH and Tyr238-OH contribute to the high catalytic efficiency of DAAO allowing the precise alignment required for optimal catalysis.

Glycine oxidase (GO) is a homotetrameric flavoenzyme that contains one molecule of non-covalently bound flavin adenine dinucleotide per 47 kDa protein monomer. GO is active on various amines (sarcosine, N-ethylglycine, glycine) andd-amino acids (d-alanine,d-proline). The products of GO reaction with various substrates have been determined, and it has been clearly shown that GO catalyzes the oxidative deamination of primary and secondary amines, a reaction similar to that of d-amino acid oxidase, although its sequence homology is higher with enzymes such as sarcosine oxidase and N-methyltryptophane oxidase. GO shows properties that are characteristic of the oxidase class of flavoproteins: it stabilizes the anionic flavin semiquinone and forms a reversible covalent flavin-sulfite complex. The ∼300 mV separation between the two FAD redox potentials is in accordance with the high amount of the anionic semiquinone formed on photoreduction. GO can be distinguished from d-amino acid oxidase by its low catalytic efficiency and high apparent K m value ford-alanine. A number of active site ligands have been identified; the tightest binding is observed with glycolate, which acts as a competitive inhibitor with respect to sarcosine. The presence of a carboxylic group and an amino group on the substrate molecule is not mandatory for binding and catalysis.

Arg285, one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg285 result in an increase (≈300-fold) ofK m for the d-amino acid and in a large decrease (≈500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k 2), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg285 in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg285 DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg285 in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme.