Mechanism of Diol Dehydration by a Promiscuous Radical‐SAM Enzyme Homologue of the Antiviral Enzyme Viperin (RSAD2)

3´‐deoxy nucleotides are an important class of drugs because they interfere with metabolism of nucleotides and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds have largely been produced via multistep chemical synthesis and an enzyme with the ability to catalyse removal of 3´‐deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, high‐resolution mass spectrometry, and NMR spectroscopy we demonstrate that a thermostable fungal radical S‐adenosylmethionine (SAM) enzyme with similarity to the vertebrate antiviral enzyme viperin (RSAD2) can catalyze transformation of CTP, UTP, and 5‐bromo‐UTP to their 3ʹ‐deoxy‐3′,4ʹ‐didehydro analogues. We show that unlike the fungal enzyme human viperin can only catalyse transformation of CTP. Using electron paramagnetic resonance (EPR) spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical‐SAM enzymes. We predict that our work will help in utilizing a new class of radical‐SAM enzymes for biocatalytic synthesis of 3ʹ‐deoxy nucleotide/nucleoside analogues

An Introduction to Pyrolysis and Catalytic Pyrolysis: Versatile Techniques for Biomass Conversion

A significant proportion of renewable feedstocks research has been devoted to the production of bio-fuels and bio-chemicals from biomass, primarily via thermochemical conversion processes. This chapter presents an overview of alternative pyrolysis methodologies, which can convert biomass directly into solid (char), liquid (bio-oil), and gaseous products. In this report, the influence of the pyrolysis conditions employed, the design of reactor, and the nature of the biomass feedstock used upon the chemical composition of the product fractions are surveyed. A summary of the mechanisms by which the pyrolysis of the principle biomass constituents occurs is given. This is accompanied by an indication of the complications that arise as a result of the complex and heterogeneous nature of biomass, including the potential roles of the various salts and minerals naturally present in such feeds. From a commercial standpoint, the multi-component nature, different forms and structures of biomass, all pose significant challenges for the utilization of biomass in the manufacture of fuels and chemicals. Consequently, the in and ex situ use of a host of additives and catalysts (including molecular sieves, metal oxides, and transition metal-modified oxides) is reviewed, which have been incorporated in order to provide selectivity in pyrolytic biomass upgrading

Viperin, through its radical‐SAM activity, depletes cellular nucleotide pools and interferes with mitochondrial metabolism to inhibit viral replication

Viperin (RSAD2) is an antiviral radical S‐adenosylmethionine (SAM) enzyme highly expressed in different cell types upon viral infection. Recently, it has been reported that the radical‐SAM activity of viperin transforms cytidine triphosphate (CTP) to its analogue 3ʹ‐deoxy‐3′,4ʹ‐didehydro‐CTP (ddhCTP). Based on biochemical studies and cell biological experiments, it was concluded that ddhCTP and its nucleoside form ddhC do not affect the cellular concentration of nucleotide triphosphates (NTPs) but act as replication chain terminators. However, our re‐evaluation of the reported data and our data indicate that ddhCTP is not an effective viral chain terminator but depletes cellular nucleotide pools and interferes with mitochondrial activity to inhibit viral replication. Our analysis is consistent with a unifying view of the antiviral and radical‐SAM activities of viperin

Solution-state behaviour of algal mono-uronates evaluated by pure shift and compressive sampling NMR techniques

Sodium salts of the algal uronic-acids, d-mannuronic acid (HManA) and l-guluronic acid (HGulA) have been isolated and characterised in solution by nuclear magnetic resonance (NMR) spectroscopy. A suite of recently-described NMR experiments (including pure shift and compressive sampling techniques) were used to provide confident assignments of the pyranose forms of the two uronic acids at various pD values (from 7.5 to 1.4). The resulting high resolution spectra were used to determine several previously unknown parameters for the two acids, including their pKa values, the position of their isomeric equilibria, and their propensity to form furanurono-6,3-lactones. For each of the three parameters, comparisons are drawn with the behaviour of the related D-glucuronic (HGlcA) and D-galacturonic acids (HGalA), which have been previously studied extensively. This paper demonstrates how these new NMR spectroscopic techniques can be applied to better understand the properties of polyuronides and uronide-rich macroalgal biomass

Rapid, Heterogeneous Biocatalytic Hydrogenation and Deuteration in a Continuous Flow Reactor

The high selectivity of biocatalysis offers a valuable method for greener, more efficient production of enantiopure molecules. Operating immobilised enzymes in flow reactors can improve the productivity and handling of biocatalysts, and using H2 gas to drive redox enzymes bridges the gap to more traditional metal‐catalysed hydrogenation chemistry. Herein, we describe examples of H2‐driven heterogeneous biocatalysis in flow employing enzymes immobilised on a carbon nanotube column, achieving near‐quantitative conversion in <5 min residence time. Cofactor recycling is carried out in‐situ using H2 gas as a clean reductant, in a completely atom‐efficient process. The flow system is demonstrated for cofactor conversion, reductive amination and ketone reduction, and then extended to biocatalytic deuteration for the selective production of isotopically labelled chemicals

The crystalline state as a dynamic system: IR microspectroscopy under electrochemical control for a [NiFe] hydrogenase

Controlled formation of catalytically-relevant states within crystals of complex metalloenzymes represents a significant challenge to structure-function studies. Here we show how electrochemical control over single crystals of [NiFe] hydrogenase 1 (Hyd1) fromEscherichia colimakes it possible to navigate through the full array of active site states previously observed in solution. Electrochemical control is combined with synchrotron infrared microspectroscopy, which enables us to measure high signal-to-noise IR spectrain situfrom a small area of crystal. The output reports on active site speciationviathe vibrational stretching band positions of the endogenous CO and CN−ligands at the hydrogenase active site. Variation of pH further demonstrates how equilibria between catalytically-relevant protonation states can be deliberately perturbed in the crystals, generating a map of electrochemical potential and pH conditions which lead to enrichment of specific states. Comparison of in crystallo redox titrations with measurements in solution or of electrode-immobilised Hyd1 confirms the integrity of the proton transfer and redox environment around the active site of the enzyme in crystals. Slowed proton-transfer equilibria in the hydrogenase in crystallo reveals transitions which are only usually observable by ultrafast methods in solution. This study therefore demonstrates the possibilities of electrochemical control over single metalloenzyme crystals in stabilising specific states for further study, and extends mechanistic understanding of proton transfer during the [NiFe] hydrogenase catalytic cycle