Structure of a double CACHE chemoreceptor ligand-binding domain from Pseudomonas syringae provides insights into the basis of proline recognition.

Chemotaxis is the process of sensing chemical gradients and navigating towards favourable conditions. Bacterial chemotaxis is mediated by arrays of trans-membrane chemoreceptor proteins. The most common class of chemoreceptors have periplasmic ligand-binding domains (LBDs) that detect extracellular chemical signs and transduce these signals to the downstream chemotaxis machinery. The repertoire of chemoreceptor proteins in a bacterium determines the range of environmental signals to which it can respond. Pseudomonas syringae pv. actinidiae (Psa) is a plant pathogen which causes bacterial canker of kiwifruit (Actinidia sp.). Compared to many other bacteria, Psa has a large number of chemoreceptors encoded in its genome (43) and most of these remain uncharacterized. A previous study identified PscC as a potential chemoreceptor for l-proline and other amino acid ligands. Here, we have characterized the interaction of PscC-LBD with l-proline using a combination of isothermal titration calorimetry (ITC) and X-ray crystallography. ITC confirmed direct binding of l-proline to PscC-LBD with KD value of 5.0 µM. We determined the structure of PscC-LBD in complex with l-proline. Our structural analysis showed that PscC-LBD adopts similar double-CACHE fold to several other amino acid chemoreceptors. A comparison of the PscC-LDB to other dCACHE structures highlights residues in the binding cavity which contribute to its ligand specificity.

Impact of Goal-Directed Therapy on Delayed Ischemia After Aneurysmal Subarachnoid Hemorrhage: Randomized Controlled Trial.

BACKGROUND AND PURPOSE: Delayed cerebral ischemia (DCI) is the most important cause for a poor clinical outcome after a subarachnoid hemorrhage. The aim of this study was to assess whether goal-directed hemodynamic therapy (GDHT), as compared to standard clinical care, reduces the rate of DCI after subarachnoid hemorrhage. METHODS: We conducted a prospective randomized controlled trial. Patients >18 years of age with an aneurysmal subarachnoid hemorrhage were enrolled and randomly assigned to standard therapy or GDHT. Advanced hemodynamic monitoring and predefined GDHT algorithms were applied in the GDHT group. The primary end point was the occurrence of DCI. Functional outcome was assessed using the Glasgow Outcome Scale (GOS) 3 months after discharge. RESULTS: In total, 108 patients were randomized to the control (n=54) or GDHT group (n=54). The primary outcome (DCI) occurred in 13% of the GDHT group and in 32% of the control group patients (odds ratio, 0.324 [95% CI, 0.11-0.86]; P=0.021). Even after adjustment for confounding parameters, GDHT was found to be superior to standard therapy (hazard ratio, 2.84 [95% CI, 1.18-6.86]; P=0.02). The GOS was assessed 3 months after discharge in 107 patients; it showed more patients with a low disability (GOS 5, minor or no deficits) than patients with higher deficits (GOS 1-4) in the GDHT group compared with the control group (GOS 5, 66% versus 44%; GOS 1-4, 34% versus 56%; P=0.025). There was no significant difference in mortality between the groups. CONCLUSIONS: GDHT reduced the rate of DCI after subarachnoid hemorrhage with a better functional outcome (GOS=5) 3 months after discharge. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01832389.

Human CYP27A1 catalyzes hydroxylation of ß-sitosterol and ergosterol.

ß-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners adrenodoxin (Adx) and adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. ß-Sitosterol was found to be converted into 26-hydroxy-ß-sitosterol and 29-hydroxy-ß-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

Screening Chemoreceptor-Ligand Interactions by High-Throughput Thermal-Shift Assays.

Identifying the ligands sensed by chemoreceptors remains challenging, in part because current screening methods are low-throughput, costly, and/or time-consuming. In contrast, fluorescence thermal shift (FTS) assays provide a fast and inexpensive approach to chemoreceptor-ligand screening. In FTS assays, the temperature at which a protein denatures is measured by monitoring the fluorescence of a dye with affinity for hydrophobic regions of the protein, which are exposed as the protein unfolds. A detectable increase (or "shift") in the melting temperature (T m ) of the protein in the presence of a potential ligand indicates binding. Here, we present our protocol for using FTS assays for the screening of chemoreceptor ligands in a high-throughput, 96-well plate format. We have also included details on the use of Biolog Phenotype Microarray plates as a convenient ligand library, although the methods described should be generally applicable to other library formats as well.

Expression of human CYP27A1 in B. megaterium for the efficient hydroxylation of cholesterol, vitamin D3 and 7-dehydrocholesterol.

In the current work the ability of Bacillus megaterium to take up hydrophobic substrates and efficiently express eukaryotic membrane proteins was utilized for establishing a CYP27A1-based biocatalyst. The human mitochondrial cytochrome P450CYP27A1 was co-expressed with its redox partners adrenodoxin reductase (Adr) and adrenodoxin (Adx). CYP27A1 could be localized at the cell's polyhydroxybutyrate (PHB) granules, carbon storage serving organelle-like vesicles that can take up cholesterol, resulting in bioreactor-like structures in B. megaterium . The resulting whole cell system allowed the efficient biotechnological conversion of the CYP27A1 substrates cholesterol, 7-dehydrocholesterol (7-DHC) and vitamin D3. After 48 h, nearly 100% of cholesterol was metabolized producing a final concentration of 113.14 mg/l 27-hydroxycholesterol (27-HC). Moreover, 70% of vitamin D3 was converted into 25-hydroxyvitamin D3 (25-OH-D3) with a final concentration of 80.81 mg/l. Also more than 97% of 7-DHC were found to be metabolized into two products, corresponding to 26/27-hydroxy-7-dehydrocholesterol (P1) and 25-hydroxy-7-dehydrocholesterol (P2). To our knowledge this is the first CYP27A1-based whole-cell system, allowing the efficient and low-cost production of pharmaceutically interesting metabolites of this enzyme from relatively cheap substrates.