Biocatalytic reductive amination as a route to isotopically labelled amino acids suitable for analysis of large proteins by NMR.

We demonstrate an atom-efficient and easy to use H2-driven biocatalytic platform for the enantioselective incorporation of 2H-atoms into amino acids. By combining the biocatalytic deuteration catalyst with amino acid dehydrogenase enzymes capable of reductive amination, we synthesised a library of multiply isotopically labelled amino acids from low-cost isotopic precursors, such as 2H2O and 15NH4+. The chosen approach avoids the use of pre-labeled 2H-reducing agents, and therefore vastly simplifies product cleanup. Notably, this strategy enables 2H, 15N, and an asymmetric centre to be introduced at a molecular site in a single step, with full selectivity, under benign conditions, and with near 100% atom economy. The method facilitates the preparation of amino acid isotopologues on a half-gram scale. These amino acids have wide applicability in the analytical life sciences, and in particular for NMR spectroscopic analysis of proteins. To demonstrate the benefits of the approach for enabling the workflow of protein NMR chemists, we prepared l-[α-2H,15N, ß-13C]-alanine and integrated it into a large (>400 kDa) heat-shock protein oligomer, which was subsequently analysable by methyl-TROSY techniques, revealing new structural information.

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) from Escherichia coli makes 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 spectra in situ from a small area of crystal. The output reports on active site speciation via the 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.

Opening the Egg Box: NMR spectroscopic analysis of the interactions between s-block cations and kelp monosaccharides.

The best-known theory accounting for metal-alginate complexation is the so-called "Egg Box" model. In order to gain greater insight into the metal-saccharide interactions that underpin this model, the coordination chemistry of the corresponding monomeric units of alginate, L-guluronate (GulA) and D-mannuronate (ManA) have been studied herein. GulA and ManA were exposed to solutions of different s-block cations and then analysed by 1H and 13C NMR spectroscopy. It was found that the α/ß ratio of the pyranose anomeric equilibria of GulA showed large pertubations from the starting value (α/ß = 0.21 ± 0.01) upon contact with 1.0 M Ca2+, Sr2+, and Ba2+ (α/ß = 1.50 ± 0.03, 1.20 ± 0.02, and 0.58 ± 0.02, respectively) at pD 7.9, but remained almost constant in the presence of Na+, K+, and Mg2+ (α/ß = 0.24 ± 0.01, 0.19 ± 0.01, and 0.26 ± 0.01, respectively). By comparison, no significant changes were observed in the α/ß ratios of ManA and related mono-uronates D-glucuronate (GlcA) and D-galacturonate (GalA) in the presence of all of the metal ions surveyed. Analysis of the 1H and 13C coordination chemical shift patterns indicate that the affinity of α-GulA for larger divalent cations is a consequence of the unique ax-eq-ax arrangement of hydroxyl groups found for this uronate anomer.

Hybrid Chemo-, Bio-, and Electrocatalysis for Atom-Efficient Deuteration of Cofactors in Heavy Water.

Deuterium-labeled nicotinamide cofactors such as [4-2H]-NADH can be used as mechanistic probes in biological redox processes and offer a route to the synthesis of selectively [2H] labeled chemicals via biocatalytic reductive deuteration. Atom-efficient routes to the formation and recycling of [4-2H]-NADH are therefore highly desirable but require careful design in order to alleviate the requirement for [2H]-labeled reducing agents. In this work, we explore a suite of electrode or hydrogen gas driven catalyst systems for the generation of [4-2H]-NADH and consider their use for driving reductive deuteration reactions. Catalysts are evaluated for their chemoselectivity, stereoselectivity, and isotopic selectivity, and it is shown that inclusion of an electronically coupled NAD+-reducing enzyme delivers considerable advantages over purely metal based systems, yielding exclusively [4S-2H]-NADH. We further demonstrate the applicability of these types of [4S-2H]-NADH recycling systems for driving reductive deuteration reactions, regardless of the facioselectivity of the coupled enzyme.

Synthesis of [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ cofactors through heterogeneous biocatalysis in heavy water.

This practitioner protocol describes the synthesis of a family of deuterated nicotinamide cofactors: [4S-2 H]NADH, [4R-2 H]NADH, [4-2 H2 ]NADH and [4-2 H]NAD+ . The application of a recently developed H2 -driven heterogeneous biocatalyst enables the cofactors to be prepared with high (>90%) 2 H-incorporation with 2 H2 O as the only isotope source.

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.

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 3'-deoxy-3',4'-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 and that ddhCTP acts as replication chain terminator. However, our re-evaluation of the reported data and new results 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.

Biocatalytic hydrogenations on carbon supports.

We describe the use of carbon as a versatile support for H2-driven redox biocatalysis for NADH-dependent CX bond reductions in batch and flow reactions. In each case, carbon is providing an electronic link between enzymes for H2 oxidation and reduction of the biological cofactor NAD+, as well as a support for a multi-enzyme biocatalysis system. Carbon nanopowders offer high surface areas for enzyme immobilization and good dispersion in aqueous solution for heterogeneous batch reactions. Difficulties in handling multi-wall carbon nanotubes in aqueous solution are overcome by growing them on quartz tubes to form carbon nanotube column reactors, and we show that these facilitate simple translation of H2-driven biocatalysis into flow processes. Using this flow reactor design, high conversions (90%) and total enzyme turnover numbers up to 54,000 could be achieved. Use of an entirely heterogeneous biocatalysis system simplifies recovery and re-use of the enzymes; combined with highly atom-efficient cofactor recycling, this means that high product purity can be achieved. We demonstrate these methods as platform approaches for overcoming challenges with NADH-dependent biocatalysis.

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

3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance 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 using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues.