A fully reversible 25-hydroxy steroid kinase involved in oxygen-independent cholesterol side-chain oxidation.

The degradation of cholesterol and related steroids by microbes follows fundamentally different strategies in aerobic and anaerobic environments. In anaerobic bacteria, the primary C26 of the isoprenoid side chain is hydroxylated without oxygen via a three-step cascade: (i) water-dependent hydroxylation at the tertiary C25, (ii) ATP-dependent dehydration to form a subterminal alkene, and (iii) water-dependent hydroxylation at the primary C26 to form an allylic alcohol. However, the enzymes involved in the ATP-dependent dehydration have remained unknown. Here, we isolated an ATP-dependent 25-hydroxy-steroid kinase (25-HSK) from the anaerobic bacterium Sterolibacterium denitrificans. This highly active enzyme preferentially phosphorylated the tertiary C25 of steroid alcohols, including metabolites of cholesterol and sitosterol degradation or 25-OH-vitamin D3. Kinetic data were in agreement with a sequential mechanism via a ternary complex. Remarkably, 25-HSK readily catalyzed the formation of γ-(18O)2-ATP from ADP and the C25-(18O)2-phosphoester. The observed full reversibility of 25-HSK with an equilibrium constant below one can be rationalized by an unusual high phosphoryl transfer potential of tertiary steroid C25-phosphoesters, which is ≈20 kJ mol-1 higher than that of standard sugar phosphoesters and even slightly greater than the ß,γ-phosphoanhydride of ATP. In summary, 25-HSK plays an essential role in anaerobic bacterial degradation of zoo- and phytosterols and shows only little similarity to known phosphotransferases.

Thiocoumarin Caged Nucleotides: Synthetic Access and Their Photophysical Properties.

Photocages have been successfully applied in cellular signaling studies for the controlled release of metabolites with high spatio-temporal resolution. Commonly, coumarin photocages are activated by UV light and the quantum yields of uncaging are relatively low, which can limit their applications in vivo. Here, syntheses, the determination of the photophysical properties, and quantum chemical calculations of 7-diethylamino-4-hydroxymethyl-thiocoumarin (thio-DEACM) and caged adenine nucleotides are reported and compared to the widely used 7-diethylamino-4-hydroxymethyl-coumarin (DEACM) caging group. In this comparison, thio-DEACM stands out as a phosphate cage with improved photophysical properties, such as red-shifted absorption and significantly faster photolysis kinetics.

ATP-dependent hydroxylation of an unactivated primary carbon with water.

Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C-H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C-H activations beyond canonical oxygenase-dependent reactions.

Photolysis of Caged Inositol Pyrophosphate InsP8 Directly Modulates Intracellular Ca2+ Oscillations and Controls C2AB Domain Localization.

Inositol pyrophosphates constitute a family of hyperphosphorylated signaling molecules involved in the regulation of glucose uptake and insulin sensitivity. While our understanding of the biological roles of inositol heptaphosphates (PP-InsP5) has greatly improved, the functions of the inositol octaphosphates ((PP)2-InsP4) have remained unclear. Here we present the synthesis of two enantiomeric cell-permeant and photocaged (PP)2-InsP4 derivatives and apply them to study the functions in living ß-cells. Photorelease of the naturally occurring isomer 1,5-(PP)2-InsP4 led to an immediate and concentration-dependent reduction of intracellular calcium oscillations, while other caged inositol pyrophosphates (3,5-(PP)2-InsP4, 5-PP-InsP5, 1-PP-InsP5, 3-PP-InsP5) showed no immediate effect. Furthermore, uncaging of 1,5-(PP)2-InsP4 but not 3,5-(PP)2-InsP4 induced translocation of the C2AB domain of granuphilin from the plasma membrane to the cytosol. Granuphilin is involved in membrane docking of secretory vesicles. This suggests that 1,5-(PP)2-InsP4 impacts ß-cell activity by regulating granule localization and/or priming and calcium signaling in concert.

Multiple Light Control Mechanisms in ATP-Fueled Non-equilibrium DNA Systems.

Fuel-driven self-assemblies are gaining ground for creating autonomous systems and materials, whose temporal behavior is preprogrammed by a reaction network. However, up to now there has been a lack of simple external control mechanisms of the transient behavior, at best using remote and benign light control. Even more challenging is to use different wavelengths to modulate the reactivity of different components of the system, for example, as fuel or building blocks. Success would enable such systems to navigate along different trajectories in a wavelength-dependent fashion. Herein, we introduce the first examples of light control in ATP-fueled, dynamic covalent DNA polymerization systems organized in an enzymatic reaction network of concurrent ATP-powered ligation and restriction. We demonstrate concepts for light activation and modulation by introducing caged ATP derivatives and caged DNA building blocks, making it possible to realize light-activated fueling, self-sorting in structure and behavior, and transition across different wavelength-dependent dynamic steady states.

Cyclotriphosphate: A Brief History, Recent Developments, and Perspectives in Synthesis.

There has been a recent upsurge in the study and application of approaches utilizing cyclotriphosphate 1 (cyclo-TP, also known as trimetaphosphate, TMP) and/or proceeding through its analogues in synthetic chemistry to access modified oligo- and polyphosphates. This is especially useful in the area of chemical nucleotide synthesis, but by no means restricted to it. Enabled by new high yielding and easy-to-implement methodologies, these approaches promise to open up an area of research that has previously been underappreciated. Additionally, refinements of concepts of prebiotic phosphorylation chemistry have been disclosed that ultimately rely on cyclo-TP 1 as a precursor, placing it as a potentially central compound in the emergence of life. Given the importance of such concepts for our understanding of prebiotic chemistry in combination with the need to readily access modified polyphosphates for structural and biological studies, this paper will discuss selected recent developments in the field of cyclo-TP chemistry, briefly touch on ultraphosphate chemistry, and highlight areas in which further developments can be expected.

Investigating the Toxicity of the Aeruginosin Chlorosulfopeptides by Chemical Synthesis.

Harmful algal blooms are becoming more prevalent all over the world, and identification and mechanism-of-action studies of the responsible toxins serve to protect ecosystems, livestock, and humans alike. In this study, the chlorosulfopeptide aeruginosin 828A, which rivals the well-known toxin microcystin LR in terms of crustacean toxicity, has been synthesized for the first time. Furthermore, three congeners with different permutations of the chloride and sulfate groups were prepared, thereby enabling toxicity studies without the risk of contamination by other natural toxins. Toxicity assays with the sensitive crustacean Thamnocephalus platyurus demonstrated that the introduction of a sulfate group leads to pronounced toxicity, and NMR spectroscopic evidence suggests that the chloride substituent modulates the conformation, which in turn might influence protease inhibition.