Taxadiene synthase structure and evolution of modular architecture in terpene biosynthesis

With more than 55,000 members identified so far in all forms of life, the family of terpene or terpenoid natural products represents the epitome of molecular biodiversity. A well-known and important member of this family is the polycyclic diterpenoid Taxol (paclitaxel), which promotes tubulin polymerization and shows remarkable efficacy in cancer chemotherapy. The first committed step of Taxol biosynthesis in the Pacific yew (Taxus brevifolia) is the cyclization of the linear isoprenoid substrate geranylgeranyl diphosphate (GGPP) to form taxa-4(5),11(12)diene, which is catalysed by taxadiene synthase. The full-length form of this diterpene cyclase contains 862 residues, but a roughly 80-residue amino-terminal transit sequence is cleaved on maturation in plastids. We now report the X-ray crystal structure of a truncation variant lacking the transit sequence and an additional 27 residues at the N terminus, hereafter designated TXS. Specifically, we have determined structures of TXS complexed with 13-aza-13,14-dihydrocopalyl diphosphate (1.82?? resolution) and 2-fluorogeranylgeranyl diphosphate (2.25?? resolution). The TXS structure reveals a modular assembly of three α-helical domains. The carboxy-terminal catalytic domain is a class?I terpenoid cyclase, which binds and activates substrate GGPP with a three-metal ion cluster. The N-terminal domain and a third 'insertion' domain together adopt the fold of a vestigial class?II terpenoid cyclase. A class?II cyclase activates the isoprenoid substrate by protonation instead of ionization, and the TXS structure reveals a definitive connection between the two distinct cyclase classes in the evolution of terpenoid biosynthesis.     

Quantum gates and memory using microwave-dressed states

Trapped atomic ions have been used successfully to demonstrate basic elements of universal quantum information processing. Nevertheless, scaling up such methods to achieve large-scale, universal quantum information processing (or more specialized quantum simulations) remains challenging. The use of easily controllable and stable microwave sources, rather than complex laser systems, could remove obstacles to scalability. However, the microwave approach has drawbacks: it involves the use of magnetic-field-sensitive states, which shorten coherence times considerably, and requires large, stable magnetic field gradients. Here we show how to overcome both problems by using stationary atomic quantum states as qubits that are induced by microwave fields (that is, by dressing magnetic-field-sensitive states with microwave fields). This permits fast quantum logic, even in the presence of a small (effective) Lamb-Dicke parameter (and, therefore, moderate magnetic field gradients). We experimentally demonstrate the basic building blocks of this scheme, showing that the dressed states are long lived and that coherence times are increased by more than two orders of magnitude relative to those of bare magnetic-field-sensitive states. This improves the prospects of microwave-driven ion trap quantum information processing, and offers a route to extending coherence times in all systems that suffer from magnetic noise, such as neutral atoms, nitrogen-vacancy centres, quantum dots or circuit quantum electrodynamic systems.     

Microwave quantum logic gates for trapped ions

Control over physical systems at the quantum level is important in fields as diverse as metrology, information processing, simulation and chemistry. For trapped atomic ions, the quantized motional and internal degrees of freedom can be coherently manipulated with laser light. Similar control is difficult to achieve with radio-frequency or microwave radiation: the essential coupling between internal degrees of freedom and motion requires significant field changes over the extent of the atoms' motion, but such changes are negligible at these frequencies for freely propagating fields. An exception is in the near field of microwave currents in structures smaller than the free-space wavelength, where stronger gradients can be generated. Here we first manipulate coherently (on timescales of 20 nanoseconds) the internal quantum states of ions held in a microfabricated trap. The controlling magnetic fields are generated by microwave currents in electrodes that are integrated into the trap structure. We also generate entanglement between the internal degrees of freedom of two atoms with a gate operation suitable for general quantum computation; the entangled state has a fidelity of 0.76(3), where the uncertainty denotes standard error of the mean. Our approach, which involves integrating the quantum control mechanism into the trapping device in a scalable manner, could be applied to quantum information processing, simulation and spectroscopy.     

Detection of prokaryotic mRNA signifies microbial viability and promotes immunity

Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen-associated molecular patterns (PAMPs), which are detected by the immune system, are present in both live and killed vaccines, indicating that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the mammalian innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA as a vita-PAMP present only in viable bacteria, the recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even non-pathogenic bacteria in sterile tissues can trigger similar responses, provided that they are alive. Thus, the immune system actively gauges the infectious risk by searching PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers a state of alert not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.     

Oxysterols direct immune cell migration via EBI2

Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is a G-protein-coupled receptor that is required for humoral immune responses; polymorphisms in the receptor have been associated with inflammatory autoimmune diseases. The natural ligand for EBI2 has been unknown. Here we describe the identification of 7α,25-dihydroxycholesterol (also called 7α,25-OHC or 5-cholesten-3β,7α,25-triol) as a potent and selective agonist of EBI2. Functional activation of human EBI2 by 7α,25-OHC and closely related oxysterols was verified by monitoring second messenger readouts and saturable, high-affinity radioligand binding. Furthermore, we find that 7α,25-OHC and closely related oxysterols act as chemoattractants for immune cells expressing EBI2 by directing cell migration in vitro and in vivo. A critical enzyme required for the generation of 7α,25-OHC is cholesterol 25-hydroxylase (CH25H). Similar to EBI2 receptor knockout mice, mice deficient in CH25H fail to position activated B cells within the spleen to the outer follicle and mount a reduced plasma cell response after an immune challenge. This demonstrates that CH25H generates EBI2 biological activity in vivo and indicates that the EBI2-oxysterol signalling pathway has an important role in the adaptive immune response.     

Carbon loss from an unprecedented Arctic tundra wildfire

Arctic tundra soils store large amounts of carbon (C) in organic soil layers hundreds to thousands of years old that insulate, and in some cases maintain, permafrost soils. Fire has been largely absent from most of this biome since the early Holocene epoch, but its frequency and extent are increasing, probably in response to climate warming. The effect of fires on the C balance of tundra landscapes, however, remains largely unknown. The Anaktuvuk River fire in 2007 burned 1,039 square kilometres of Alaska's Arctic slope, making it the largest fire on record for the tundra biome and doubling the cumulative area burned since 1950 (ref. 5). Here we report that tundra ecosystems lost 2,016?±?435?g?C?m(-2) in the fire, an amount two orders of magnitude larger than annual net C exchange in undisturbed tundra. Sixty per cent of this C loss was from soil organic matter, and radiocarbon dating of residual soil layers revealed that the maximum age of soil C lost was 50 years. Scaled to the entire burned area, the fire released approximately 2.1?teragrams of C to the atmosphere, an amount similar in magnitude to the annual net C sink for the entire Arctic tundra biome averaged over the last quarter of the twentieth century. The magnitude of ecosystem C lost by fire, relative to both ecosystem and biome-scale fluxes, demonstrates that a climate-driven increase in tundra fire disturbance may represent a positive feedback, potentially offsetting Arctic greening and influencing the net C balance of the tundra biome.     

S-nitrosylation of NADPH oxidase regulates cell death in plant immunity

Changes in redox status are a conspicuous feature of immune responses in a variety of eukaryotes, but the associated signalling mechanisms are not well understood. In plants, attempted microbial infection triggers the rapid synthesis of nitric oxide and a parallel accumulation of reactive oxygen intermediates, the latter generated by NADPH oxidases related to those responsible for the pathogen-activated respiratory burst in phagocytes. Both nitric oxide and reactive oxygen intermediates have been implicated in controlling the hypersensitive response, a programmed execution of plant cells at sites of attempted infection. However, the molecular mechanisms that underpin their function and coordinate their synthesis are unknown. Here we show genetic evidence that increases in cysteine thiols modified using nitric oxide, termed S-nitrosothiols, facilitate the hypersensitive response in the absence of the cell death agonist salicylic acid and the synthesis of reactive oxygen intermediates. Surprisingly, when concentrations of S-nitrosothiols were high, nitric oxide function also governed a negative feedback loop limiting the hypersensitive response, mediated by S-nitrosylation of the NADPH oxidase, AtRBOHD, at Cys 890, abolishing its ability to synthesize reactive oxygen intermediates. Accordingly, mutation of Cys 890 compromised S-nitrosothiol-mediated control of AtRBOHD activity, perturbing the magnitude of cell death development. This cysteine is evolutionarily conserved and specifically S-nitrosylated in both human and fly NADPH oxidase, suggesting that this mechanism may govern immune responses in both plants and animals.