Orchestration of enzymatic processing by thiazole/oxazole-modified microcin dehydrogenases.

Thiazole/oxazole-modified microcins (TOMMs) comprise a structurally diverse family of natural products with varied bioactivities linked by the presence of posttranslationally installed thiazol(in)e and oxazol(in)e heterocycles. The detailed investigation of the TOMM biosynthetic enzymes from Bacillus sp. Al Hakam (Balh) has provided significant insight into heterocycle biosynthesis. Thiazoles and oxazoles are installed by the successive action of an ATP-dependent cyclodehydratase (C- and D-protein) and a FMN-dependent dehydrogenase (B-protein), which are responsible for azoline formation and azoline oxidation, respectively. Although several studies have focused on the mechanism of azoline formation, many details regarding the role of the dehydrogenase (B-protein) in overall substrate processing remain unknown. In this work, we evaluated the involvement of the dehydrogenase in determining the order of ring formation as well as the promiscuity of the Balh and microcin B17 cyclodehydratases to accept a panel of noncognate dehydrogenases. In support of the observed promiscuity, a fluorescence polarization assay was utilized to measure binding of the dehydrogenase to the cyclodehydratase using the intrinsic fluorescence of the FMN cofactor. Ultimately, the noncognate dehydrogenases were shown to possess cyclodehydratase-independent activity. A previous study identified a conserved Lys-Tyr motif to be important for dehydrogenase activity. Using the tools developed in this study, the Lys-Tyr motif was shown neither to alter complex formation with the cyclodehydratase nor the reduction potential. Taken together with the known crystal structure of a homologue, our data suggest that the Lys-Tyr motif is of catalytic importance. Overall, this study provides a greater level of insight into the complex orchestration of enzymatic activity during TOMM biosynthesis.

Adrenalectomy amplifies aldosterone induced injury in cardiovascular tissue: an effect attenuated by adrenally derived steroids.

Aldosterone induces fibrotic changes in cardiovascular tissues but its effects have usually been demonstrated in models of pre-existing renal injury and/or hypertension. This study tests the hypothesis that aldosterone can directly induce vascular fibrotic changes in the absence of prior renal injury or hypertension. Experiments were conducted in intact or adrenalectomized (ADX) mice. Mice were divided into groups and treated for 1 week with vehicle or aldosterone (8 µg/kg/day)± inhibitor (800 µg/kg/day): CONTROLS, mice treated with aldosterone, ADX-CONTROLS, ADX+corticosterone (CORT 8 µg/kg/day), ADX with aldosterone, ADX with aldosterone plus the mineralocorticoid receptor (MR) antagonist RU-318, ADX with aldosterone+CORT (CORT inhibitor dose), and ADX with aldosterone+11-dehydro-CORT. Aortic smooth muscle to collagen ratio, aorta intimal thickness (µm), heart weight/body weight ratio (mg/gm), and left ventricular collagen (%) were measured. Prior to sacrifice, blood pressures were normal in all animals. Lower dose CORT alone had no effect on any of the variables examined. Aldosterone exposure was associated with extra-cellular matrix accumulation in cardiovascular tissues in intact mice and adrenalectomy exacerbated these effects. RU-318, CORT (inhibitor dose), and 11-deydro-CORT each attenuated the early fibrotic changes induced by aldosterone. In the heart, aldosterone exposure affected all the parameters measured and caused intimal hypercellularity with monocytes adhering to endothelial cells lining coronary vessels. Cultured endothelial cells exposed to aldosterone (10nM) released E-selectin, produced collagen, and promoted monocyte adhesion. These effects were inhibited by RU-318 and 11-deydro-CORT but not by CORT. Thus, adrenalectomy enhances aldosterone induced early fibrotic changes in heart and aorta. Aldosterone initially targets vascular endothelial cells. MR antagonists and 11-dehydro-CORT, an 11ß-HSD dehydrogenase end-product, directly attenuate these effects.

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.

The lipocalins are secreted proteins that bind small organic molecules. Scn-Ngal (also known as neutrophil gelatinase associated lipocalin, siderocalin, lipocalin 2) sequesters bacterial iron chelators, called siderophores, and consequently blocks bacterial growth. However, Scn-Ngal is also prominently expressed in aseptic diseases, implying that it binds additional ligands and serves additional functions. Using chemical screens, crystallography and fluorescence methods, we report that Scn-Ngal binds iron together with a small metabolic product called catechol. The formation of the complex blocked the reactivity of iron and permitted its transport once introduced into circulation in vivo. Scn-Ngal then recycled its iron in endosomes by a pH-sensitive mechanism. As catechols derive from bacterial and mammalian metabolism of dietary compounds, the Scn-Ngal-catechol-Fe(III) complex represents an unforeseen microbial-host interaction, which mimics Scn-Ngal-siderophore interactions but instead traffics iron in aseptic tissues. These results identify an endogenous siderophore, which may link the disparate roles of Scn-Ngal in different diseases.

Discovery of potent non-urea inhibitors of soluble epoxide hydrolase.

Soluble epoxide hydrolase (sEH) is a novel target for the treatment of hypertension and vascular inflammation. A new class of potent non-urea sEH inhibitors was identified via high throughput screening (HTS) and chemical modification. IC(50)s of the most potent compounds range from micromolar to low nanomolar.