Crystal structures of the C2B domain of Rabphilin-3A and the PP2C-like phosphatase tPphA of Thermosynechococcus elongatus BP-1

Die vorliegende Arbeit befaßt sich im ersten Teil mit der Röntgenkristallstruktur der C2B-Domäne von Rabphilin-3A , die auch einen Teil der linker-Region zwischen der C2A- und der C2B-Domäne des Rabphilin-3A enthält, und im zweiten Teil mit der Röntgenkristallstruktur der PP2C-ähnlichen Phosphatase tPphA von Thermosynechococcus elongatus BP-1. Beide Strukturen konnten nach Expression, Reinigung und Kristallisation des nativen und Se-Met Proteins mittels MAD (multiple-wavelength anomalous dispersion) gelöst werden. In beiden Fällen wurden Kristalle in zwei verschiedenen Raumgruppen erhalten, die jeweils miteinander verglichen wurden.C2-Domänen sind intrazelluläre, ca. 130 Aminosäuren umfassende Proteinmodule, welche als charakteristisches Strukturmerkmal ein achtsträngiges, antiparalleles β-Sandwich aufweisen und zumeist eine hohe Kalziumionen-Affinität zeigen. Sie sind sowohl an der Exocytose, als auch an der Regulierung der Neurotransmitterfreisetzung im synaptischen Vesikelzyklus beteiligt. Rabphilin-3A enthält sowohl eine Rab-Bindungsdomäne, als auch zwei C2-Domänen (C2A und C2B). Die NMR-Struktur der C2B-Domäne war bekannt. Da sie jedoch nicht die hohe Kalziumionen-Affinität erklären konnte, wurde in dieser Arbeit die Kristallstruktur der C2B-Domäne und ein Teil der linker-Region mittels Röntgenstrukturanalyse untersucht. Die Kristallstruktur weicht in der Kalziumbindungsstelle von der NMR-Struktur ab und erklärt durch ideale Koordination der Kalziumionen die hohe Kalzium-Affinität.Cyanobakterien sind in der Lage, sowohl Nitrat, Nitrit oder Ammonium als Stickstoffquelle zu nutzen (Stickstoff-Assimilation), als auch aus molekularem Stickstoff biologisch verwendbare Stickstoffverbindungen aufzubauen (Stickstoff-Fixierung). Sowohl Stickstoff-Assimilation als auch Stickstoff-Fixierung führen zur Bildung von Ammonium, welches als zentrales Ausgangsprodukt für den GS/GOGAT-Zyklus (Glutamin-Synthetase/ Glutamat-2-Oxoglutarat-Amidotransferase) dient. Dieser Zyklus wird über die Regulierung der Glutamin-Synthetase kontrolliert, welche von den PII-Proteinen übernommen wird. Die PII-Proteine werden dabei je nach Kohlenstoff-Stickstoffbilanz der Zelle entweder phosphoryliert oder dephosphoryliert, um in die Aktivität der Glutamin-Synthetase einzugreifen. Die Dephosphorylierung übernimmt eine PP2C-ähnliche Phosphatase (PII-Phosphatase): tPphA. Die Struktur der tPphA wurde in dieser Arbeit röntgenkristallographisch untersucht und mit anderen PP2C-ähnlichen Phosphatasen verglichen, um mögliche Rückschlüsse auf den katalytischen Mechanismus ziehen zu können. Die hohe Ähnlichkeit der tPphA mit der bakteriellen Phosphatase deutet auf einen vergleichbaren Mechanismus der beiden Phosphatasen hin, der jedoch in beiden Fällen ungeklärt ist

Comparison of Classical Serotyping and PremiTest Assay for Routine Identification of Common Salmonella enterica Serovars▿ †

The commercial PremiTest Salmonella kit uses a multiplexed DNA typing test aimed at identifying common serovars of Salmonella enterica. It was used in assays over a 9-month period in the Belgian reference laboratory that performs the routine identification of Salmonella strains of animal origin. A blind analysis of 754 strains was conducted in parallel by classical serotyping and the PremiTest assay. Full results were available for 685 strains (90.8%) by serotyping, while the remaining 69 strains were found to be nontypeable due to either a lack of surface antigen expression or autoagglutination properties. When the PremiTest assay (version 4.2) was performed with crude bacterial extracts, it identified 658 strains (87.3%), including most strains found to be nontypeable by serotyping. In contrast, it gave no, wrong, dual, or noninterpretable results for 96 strains, for which 23 were caused by assay failures. When purified DNA instead of crude extracts were tested, the number of strains successfully identified to the serovar level increased to 714 (94.7%), while all assay failures were cleared. Our conclusion is that, in its actual development stage, the application of the investigated kit to purified DNA samples offers a valuable alternative to classical serotyping for laboratories performing the routine identification of Salmonella strains belonging to commonly encountered serovars and isolated from a given geographical area, assuming that the system has been validated beforehand with a significant number of strains originating from that particular area

Carbonic anhydrase inhibitors. Inhibition of the β-class enzymes from the fungal pathogens Candida albicans and Cryptococcus neoformans with aliphatic and aromatic carboxylates

The inhibition of the beta-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic fungi Cryptococcus neoformans (Can2) and Candida albicans (Nce103) with carboxylates such as the C1-C5 aliphatic carboxylates, oxalate, malonate, maleate, malate, pyruvate, lactate, citrate and some benzoates has been investigated. The best Can2 inhibitors were acetate and maleate (K(I)s of 7.3-8.7 microM), whereas formate, acetate, valerate, oxalate, maleate, citrate and 2,3,5,6-tetrafluorobenzoate showed less effective inhibition, with K(I)s in the range of 42.8-88.6 microM. Propionate, butyrate, malonate, L-malate, pyruvate, L-lactate and benzoate, were weak Can2 inhibitors, with inhibition constants in the range of 225-1267 microM. Nce103 was more susceptible to inhibition with carboxylates compared to Can2, with the best inhibitors (maleate, benzoate, butyrate and malonate) showing K(I)s in the range of 8.6-26.9 microM. L-Malate and pyruvate together with valerate were the less efficient Nce103 inhibitors (K(I)s of 87.7-94.0 microM), while the remaining carboxylates showed a compact behavior of efficient inhibitors (K(I)s in the range of 35.1-61.6 microM). Notably the inhibition profiles of the two fungal beta-CAs was very different from that of the ubiquitous host enzyme hCA II (belonging to the alpha-CA family), with maleate showing selectivity ratios of 113.6 and 115 for Can2 and Nce103, respectively, over hCA II inhibition. Therefore, maleate is a promising starting lead molecule for the development of better, low nanomolar, selective beta-CA inhibitors

Carbonic anhydrase inhibitors. Inhibition and homology modeling studies of the fungal beta-carbonic anhydrase from Candida albicans with sulfonamides

The beta-carbonic anhydrase (CA, EC 4.2.1.1) from the fungal pathogen Candida albicans (Nce103) is involved in a CO(2) sensing pathway critical for the pathogen life cycle and amenable to drug design studies. Herein we report an inhibition study of Nce103 with a library of sulfonamides and one sulfamate, showing that Nce103, similarly to the related enzyme from Cryptococcus neoformans Can2, is inhibited by these compounds with K(I)s in the range of 132 nM-7.6 microM. The best Nce103 inhibitors were acetazolamide, methazolamide, bromosulfanilamide, and 4-hydroxymethylbenzenesulfonamide (K(I)

Structure and Inhibition of the CO2-Sensing Carbonic Anhydrase Can2 from the Pathogenic Fungus Cryptococcus neoformans

In the pathogenic fungus Cryptococcus neoformans, a CO2-sensing system is essential for survival in the natural environment (∼ 0.03% CO2) and mediates the switch to virulent growth in the human host (∼ 5% CO2). This system is composed of the carbonic anhydrase (CA) Can2, which catalyzes formation of bicarbonate, and the fungal, bicarbonate-stimulated adenylyl cyclase Cac1. The critical role of these enzymes for fungal metabolism and pathogenesis identifies them as targets for antifungal drugs. Here, we prove functional similarity of Can2 to the CA Nce103 from Candida albicans and describe its biochemical and structural characterization. The crystal structure of Can2 reveals that the enzyme belongs to the “plant-type” β-CAs but carries a unique N-terminal extension that can interact with the active-site entrance of the dimer. We further tested a panel of compounds, identifying nanomolar Can2 inhibitors, and present the structure of a Can2 complex with the inhibitor and product analog acetate, revealing insights into interactions with physiological ligands and inhibitors