Crystal structure of full-length cytotoxic necrotizing factor CNFY reveals molecular building blocks for intoxication

Cytotoxic necrotizing factors (CNFs) are bacterial single-chain exotoxins that modulate cytokinetic/oncogenic and inflammatory processes through activation of host cell Rho GTPases. To achieve this, they are secreted, bind surface receptors to induce endocytosis and translocate a catalytic unit into the cytosol to intoxicate host cells. A three-dimensional structure that provides insight into the underlying mechanisms is still lacking. Here, we determined the crystal structure of full-length Yersinia pseudotuberculosis CNFY. CNFY consists of five domains (D1-D5), and by integrating structural and functional data we demonstrate that D1-3 act as export and translocation module for the catalytic unit (D4-5) or fused β-lactamase reporter proteins. We further found that domain D4, which possesses structural similarity to ADP-ribosyl transferases, but had no equivalent catalytic activity, changed its position to interact extensively with D5 in the crystal structure of the free D4-5 fragment. This liberates D5 from a semi-blocked conformation in full-length CNFY, leading to higher deamidation activity. Finally, sequence comparisons identified the CNF translocation module in many uncharacterized bacterial proteins, suggesting its usability as a universal drug delivery tool.

Crystal structure of the catalytic unit of GH 87-type α-1,3-glucanase Agl-KA from Bacillus circulans

Glycoside hydrolase (GH) 87-type α-1,3-glucanase hydrolyses the α-1,3-glucoside linkages of α-1,3-glucan, which is found in fungal cell walls and extracellular polysaccharides produced by oral Streptococci. In this study, we report on the molecular structure of the catalytic unit of GH 87-type α-1,3-glucanase, Agl-KA, from Bacillus circulans, as determined by x-ray crystallography at a resolution of 1.82 Å. The catalytic unit constitutes a complex structure of two tandemly connected domains—the N-terminal galactose-binding-like domain and the C-terminal right-handed β-helix domain. While the β-helix domain is widely found among polysaccharide-processing enzymes, complex formation with the galactose-binding-like domain was observed for the first time. Biochemical assays showed that Asp1067, Asp1090 and Asp1091 are important for catalysis, and these residues are indeed located at the putative substrate-binding cleft, which forms a closed end and explains the product specificity.

Aerobic catabolism of sterols by microorganisms: key enzymes that open the 3-ketosteroid nucleus

ABSTRACT Aerobic degradation of the sterol tetracyclic nucleus by microorganisms comprises the catabolism of A/B-rings, followed by that of C/D-rings. B-ring rupture at the C9,10-position is a key step involving 3-ketosteroid Δ1-dehydrogenase (KstD) and 3-ketosteroid 9α-hydroxylase (KstH). Their activities lead to the aromatization of C4,5-en-containing A-ring causing the rupture of B-ring. C4,5α-hydrogenated 3-ketosteroid could be produced by the growing microorganism containing a 5α-reductase. In this case, the microorganism synthesizes, in addition to KstD and KstH, a 3-ketosteroid Δ4-(5α)-dehydrogenase (Kst4D) in order to produce the A-ring aromatization, and consequently B-ring rupture. KstD and Kst4D are FAD-dependent oxidoreductases. KstH is composed of a reductase and a monooxygenase. This last component is the catalytic unit; it contains a Rieske-[2Fe-2S] center with a non-haem mononuclear iron in the active site. Published data regarding these enzymes are reviewed.

Early photophysical events of a ruthenium(ii) molecular dyad capable of performing photochemical water oxidation and of its model compounds

Early events in a water oxidation chromophore-catalyst species are studied; ultrafast energy transfer occurs from the chromophore to the catalytic unit.

Peptide-based development of PKA activators

Activation of the PKA catalytic unit by small peptide (SE1). Development of peptidomimetics.