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The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H‐transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme–substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water‐mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme–substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme. [ABSTRACT FROM AUTHOR]

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations. The authors previously showed that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita Baylis-Hillman (MBH) reaction. Here, they report another efficient MBHase that employs a non-canonical Nδ-methylhistidine nucleophile paired with a catalytic glutamate, providing an alternative mechanistic solution for MBH catalysis. [ABSTRACT FROM AUTHOR]

Time-resolved infrared spectroscopy reveals the flow of electron density through coenzyme B12 in the light-activated, bacterial transcriptional regulator, CarH. The protein stabilises a series of charge transfer states that result in a photoresponse that avoids reactive, and potentially damaging, radical photoproducts. [ABSTRACT FROM AUTHOR]

CarH is a coenzyme B12-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B12 Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B12-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation. Unexpectedly, in the absence of oxygen, Co-C bond cleavage is followed by reorientation of the corrin ring and a switch from a lower to upper histidine-Co ligation, corresponding to a pentacoordinate state. Under aerobic conditions, rapid flash-cooling of crystals prior to deterioration upon illumination confirm a similar B12-ligand switch occurs. Removal of the upper His-ligating residue prevents monomer formation upon illumination. Combined with detailed solution spectroscopy and computational studies, these data demonstrate the CarH photoresponse integrates B12 photo- and redox-chemistry to drive large-scale conformational changes through stepwise Co-ligation changes. CarH is a bacterial B12-binding photoreceptor involved in transcriptional regulation. Here, the authors provide insights into B12 dynamics and associated cobalt redox changes following light activation. These demonstrate the CarH response integrates light and oxygen sensing. [ABSTRACT FROM AUTHOR]

High‐valent metal‐oxo species play critical roles in enzymatic catalysis yet their properties are still poorly understood. In this work we report a combined experimental and computational study into biomimetic iron(IV)‐oxo and iron(III)‐oxo complexes with tight second‐coordination sphere environments that restrict substrate access. The work shows that the second‐coordination sphere slows the hydrogen atom abstraction step from toluene dramatically and the kinetics is zeroth order in substrate. However, the iron(II)‐hydroxo that is formed has a low reduction potential and hence cannot do OH rebound favorably. The tolyl radical in solution then reacts further with alternative reaction partners. By contrast, the iron(IV)‐oxo species reacts predominantly through OH rebound to form alcohol products. Our studies show that the oxidation state of the metal influences reactivities and selectivities with substrate dramatically and that enzymes will likely need an iron(IV) center to catalyze C−H hydroxylation reactions. [ABSTRACT FROM AUTHOR]

Access to new non‐canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored—despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non‐natural methionines through the development of a photochemical method for the radical α‐C−H functionalization of sulfides with alkenes, in water, using inexpensive and commercially‐available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two‐step synthesis of novel methionine analogues—by radical addition to unsaturated amino acid derivatives—and the chemoselective modification of peptide side‐chains to yield non‐natural methionine residues within small peptides. The mechanism of the bio‐inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies. [ABSTRACT FROM AUTHOR]

Access to new non‐canonical amino acid residues is crucial for medicinal chemistry and chemical biology. Analogues of the amino acid methionine have been far less explored—despite their use in biochemistry, pharmacology and peptide bioconjugation. This is largely due to limited synthetic access. Herein, we exploit a new disconnection to access non‐natural methionines through the development of a photochemical method for the radical α‐C−H functionalization of sulfides with alkenes, in water, using inexpensive and commercially‐available riboflavin (vitamin B2) as a photocatalyst. Our photochemical conditions allow the two‐step synthesis of novel methionine analogues—by radical addition to unsaturated amino acid derivatives—and the chemoselective modification of peptide side‐chains to yield non‐natural methionine residues within small peptides. The mechanism of the bio‐inspired flavin photocatalysis has been probed by experimental, DFT and TDDFT studies. [ABSTRACT FROM AUTHOR]

Isobutene is a high value gaseous alkene used as fuel additive and a chemical building block. As an alternative to fossil fuel derived isobutene, we here develop a modified mevalonate pathway for the production of isobutene from glucose in vivo. The final step in the pathway consists of the decarboxylation of 3-methylcrotonic acid, catalysed by an evolved ferulic acid decarboxylase (Fdc) enzyme. Fdc belongs to the prFMN-dependent UbiD enzyme family that catalyses reversible decarboxylation of (hetero)aromatic acids or acrylic acids with extended conjugation. Following a screen of an Fdc library for inherent 3-methylcrotonic acid decarboxylase activity, directed evolution yields variants with up to an 80-fold increase in activity. Crystal structures of the evolved variants reveal that changes in the substrate binding pocket are responsible for increased selectivity. Solution and computational studies suggest that isobutene cycloelimination is rate limiting and strictly dependent on presence of the 3-methyl group. Isobutene is a high value gaseous alkene that is widely used as fuel additive and a chemical building block. Here, the authors report an alternative pathway for isobutene bioproduction by directed evolution of prenylated FMN-dependent ferulic acid decarboxylase. [ABSTRACT FROM AUTHOR]

The widespread UbiD enzyme family utilises the prFMN cofactor to achieve reversible decarboxylation of acrylic and (hetero)aromatic compounds. The reaction with acrylic compounds based on reversible 1,3-dipolar cycloaddition between substrate and prFMN occurs within the confines of the active site. In contrast, during aromatic acid decarboxylation, substantial rearrangement of the substrate aromatic moiety associated with covalent catalysis presents a molecular dynamic challenge. Here we determine the crystal structures of the multi-subunit vanillic acid decarboxylase VdcCD. We demonstrate that the small VdcD subunit acts as an allosteric activator of the UbiD-like VdcC. Comparison of distinct VdcCD structures reveals domain motion of the prFMN-binding domain directly affects active site architecture. Docking of substrate and prFMN-adduct species reveals active site reorganisation coupled to domain motion supports rearrangement of the substrate aromatic moiety. Together with kinetic solvent viscosity effects, this establishes prFMN covalent catalysis of aromatic (de)carboxylation is afforded by UbiD dynamics. Understanding the structure and dynamics of enzymes is important for a number of applications. Here, the authors report on the crystal structure of vanillic acid decarboxylase, and show how the dynamics of the UbiD superfamily enzymes relate to the covalent catalysis of aromatic (de)carboxylation. [ABSTRACT FROM AUTHOR]

Molecular dynamics (MD) simulations are a popular method of studying protein structure and function, but are unable to reliably sample all relevant conformational space in reasonable computational timescales. A range of enhanced sampling methods are available that can improve conformational sampling, but these do not offer a complete solution. We present here a proof-of-principle method of combining MD simulation with machine learning to explore protein conformational space. An autoencoder is used to map snapshots from MD simulations onto a user-defined conformational landscape defined by principal components analysis or specific structural features, and we show that we can predict, with useful accuracy, conformations that are not present in the training data. This method offers a new approach to the prediction of new low energy/physically realistic structures of conformationally dynamic proteins and allows an alternative approach to enhanced sampling of MD simulations. [ABSTRACT FROM AUTHOR]

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light‐dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR–Pchlide–NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo‐electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation – a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development. [ABSTRACT FROM AUTHOR]

Upconverting phosphors (UCPs) convert multiple low energy photons into higher energy emission via the process of photon upconversion and offer an attractive alternative to organic fluorophores for use as luminescent probes. Here, UCPs were capped with functionalized silica in order to provide a surface to covalently conjugate proteins with surface‐accessible cysteines. Variants of green fluorescent protein (GFP) and the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) were then attached via maleimide‐thiol coupling in order to allow energy transfer from the UCP to the GFP or flavin cofactor of PETNR, respectively. PETNR retains its activity when coupled to the UCPs, which allows reversible detection of enzyme substrates via ratiometric sensing of the enzyme redox state. [ABSTRACT FROM AUTHOR]

While it is well established that thermally-activated quantum mechanical tunnelling of light particles (electrons and light atoms, typically hydrogen) plays a role in many enzyme-catalysed reactions, there are few definitive experimental signatures of atomic tunnelling and no clear methods of directly estimating the relative tunnelling contribution from typical experimental data. As most enzyme reactions involve the binding/capture of freely diffusing substrate(s), reactions are typically initiated by mixing and experimental conditions must then be compatible with liquid water (the solvent). This precludes the classic test of tunnelling: the observation of temperature-independent rate constants at cryogenic temperatures. Instead, H-tunnelling is usually inferred from kinetic isotope effects that are larger than the semiclassical limit. Often, the temperature dependence of the reaction is also measured over the experimentally accessible range (∼278–313 K for mesophilic enzymes), with resulting data analysed and interpreted using variations of Arrhenius, Eyring or Marcus theory. The apparent Arrhenius and Eyring activation parameters allow some quantitative comparison of different reactions, but do not directly provide any information about tunnelling, while the validity of parameters derived from non-adiabatic models such as Marcus theory are questionable due to the partially adiabatic nature of these reactions. Here, we use the correlation found between apparent activation enthalpy and entropy across several series of enzyme variants and tunnelling contributions determined using computational chemistry in an attempt to question and define new signatures of hydrogen tunnelling, which can be used to interpret typical experimental kinetic data measured for enzyme-catalysed reactions. [ABSTRACT FROM AUTHOR]

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth1–3. POR, which is one of three known light-dependent enzymes4,5, catalyses reduction of the photosensitizer and substrate protochlorophyllide to form the pigment chlorophyllide. Despite its biological importance, the structural basis for POR photocatalysis has remained unknown. Here we report crystal structures of cyanobacterial PORs from Thermosynechococcus elongatus and Synechocystis sp. in their free forms, and in complex with the nicotinamide coenzyme. Our structural models and simulations of the ternary protochlorophyllide–NADPH–POR complex identify multiple interactions in the POR active site that are important for protochlorophyllide binding, photosensitization and photochemical conversion to chlorophyllide. We demonstrate the importance of active-site architecture and protochlorophyllide structure in driving POR photochemistry in experiments using POR variants and protochlorophyllide analogues. These studies reveal how the POR active site facilitates light-driven reduction of protochlorophyllide by localized hydride transfer from NADPH and long-range proton transfer along structurally defined proton-transfer pathways. Crystal structures of cyanobacterial protochlorophyllide oxidoreductases reveal the basis of the photocatalytic activities of this enzyme, through the role of its active site in enabling the light-driven reduction of protochlorophyllide. [ABSTRACT FROM AUTHOR]

The development of graphene–polymer nanocomposite materials has been hindered by issues such as poor colloidal stability of graphene in liquid media, weak interactions between graphene and the host polymers as well as the lack of scalable and economical graphene synthesis routes. Chlorosulfonic acid (CSA) can spontaneously disperse graphene without the need for mechanical agitation, chemical functionalisation or surfactant stabilisation,1 however is incompatible with most polymers and organic materials. Here, we demonstrate how poly(p-phenylene terephthalamide) (PPTA) – the polymer which constitutes Kevlar – can be co-processed with graphene in CSA and wet-spun into nanocomposite fibres with minimal aggregation of graphene. [ABSTRACT FROM AUTHOR]

Most ene‐reductases belong to the Old Yellow Enzyme (OYE) family of flavin‐dependent oxidoreductases. OYEs use nicotinamide coenzymes as hydride donors to catalyze the reduction of alkenes that contain an electron‐withdrawing group. There have been many investigations of the structures and catalytic mechanisms of OYEs. However, the origin of coenzyme specificity in the OYE family is unknown. Structural NMR and X‐ray crystallographic data were used to rationally design variants of two OYEs, pentaerythritol tetranitrate reductase (PETNR) and morphinone reductase (MR), to discover the basis of coenzyme selectivity. PETNR has dual‐specificity and reacts with NADH and NADPH; MR accepts only NADH as hydride donor. Variants of a β‐hairpin motif in an active site loop of both these enzymes were studied using stopped‐flow spectroscopy. Specific attention was placed on the potential role of arginine residues within the β‐hairpin motif. Mutagenesis demonstrated that Arg130 governs the preference of PETNR for NADPH, and that Arg142 interacts with the coenzyme pyrophosphate group. These observations were used to switch coenzyme specificity in MR by replacing either Glu134 or Leu146 with arginine residues. These variants had increased (~15‐fold) affinity for NADH. Mutagenesis enabled MR to accept NADPH as a hydride donor, with E134R MR showing a significant (55‐fold) increase in efficiency in the reductive half‐reaction, when compared to the essentially unreactive wild‐type enzyme. Insight into the question of coenzyme selectivity in OYEs has therefore been addressed through rational redesign. This should enable coenzyme selectivity to be improved and switched in other OYEs. [ABSTRACT FROM AUTHOR]

mime-subtype="jpeg" mimetype="image" xlink:href="S2159685919000351_figAb" xmlns:xlink="http://www.w3.org/1999/xlink" /> Synthetic biology has a huge potential to produce the next generation of advanced materials by accessing previously unreachable (bio)chemical space. In this prospective review, we take a snapshot of current activity in this rapidly developing area, focusing on prominent examples for high-performance applications such as those required for protective materials and the aerospace sector. The continued growth of this emerging field will be facilitated by the convergence of expertise from a range of diverse disciplines, including molecular biology, polymer chemistry, materials science, and process engineering. This review highlights the most significant recent advances and addresses the cross-disciplinary challenges currently being faced. [ABSTRACT FROM AUTHOR]

The utilization of gaseous carbon dioxide instead of bicarbonate would greatly facilitate process development for enzyme catalyzed carboxylations on a large scale. As a proof-of-concept, 1,3-dihydroxybenzene (resorcinol) was carboxylated in the ortho-position using pressurized CO2 (∼30–40 bar) catalyzed by ortho-benzoic acid decarboxylases with up to 68% conversion. Optimization studies revealed tight pH-control and enzyme stability as the most important determinants. [ABSTRACT FROM AUTHOR]

Pentaerythritol tetranitrate reductase (PETNR) is a flavoenzyme possessing a broad substrate specificity and is a member of the Old Yellow Enzyme family of oxidoreductases. As well as having high potential as an industrial biocatalyst, PETNR is an excellent model system for studying hydrogen transfer reactions. Mechanistic studies performed with PETNR using stopped-flow methods have shown that tunneling contributes towards hydride transfer from the NAD(P)H coenzyme to the flavin mononucleotide (FMN) cofactor and fast protein dynamics have been inferred to facilitate this catalytic step. Herein, we report the near-complete 1H, 15N and 13C backbone resonance assignments of PETNR in a stoichiometric complex with the FMN cofactor in its native oxidized form, which were obtained using heteronuclear multidimensional NMR spectroscopy. A total of 97% of all backbone resonances were assigned, with 333 out of a possible 344 residues assigned in the 1H-15N TROSY spectrum. This is the first report of an NMR structural study of a flavoenzyme from the Old Yellow Enzyme family and it lays the foundation for future investigations of functional dynamics in hydride transfer catalytic mechanism. [ABSTRACT FROM AUTHOR]

Upconversion (UC) is a powerful mulitphoton mechanism that converts low-energy photons into higher energy emission. One of the most investigated UC systems is upconverting phosphors (UCPs). Here, a new, one-pot synthetic procedure was used to prepare water dispersible, visibly emissive, rare-earth doped UCPs that were capped with the functional groups oleic acid (OA), 6-aminohexanoic acid (AHA), and 6-maleimidohexanoic acid (MHA). These synthesized UCPs were characterized by UC luminescence, dynamic light scattering (DLS), transmission electron microscopy (TEM), and powder X-ray diffraction (pXRD). This study expands upon our previous proof-of-principle work in demonstrating the use of UCPs (both synthesized and commercial) to detect on the level and function of biological analytes, from enzymes to key disease biomarkers (PETNR, glucose oxidase, vitamin B12, and cytochrome c). By tailoring the absorption profile of the biomolecule cofactors to the UCP emission, a wide-range of analytes can be utilized. We also demonstrate the ability of our system to reversibly monitor the addition of enzyme substrates via repeat oxidation and reduction of pentaerythritol tetranitrate reductase (PETNR). [ABSTRACT FROM AUTHOR]

The haem-containing mono-oxygenase cytochrome P450 3A4 ( CYP3A4) and its redox partner NADPH-dependent cytochrome P450 oxidoreductase ( CPR) are among the most important enzymes in human liver for metabolizing drugs and xenobiotic compounds. They are membrane-bound in the endoplasmic reticulum ( ER). How ER colocalization and the complex ER phospholipid composition influence enzyme activity are not well understood. CPR and CYP3A4 were incorporated into phospholipid bilayer nanodiscs, both singly, and together in a 1 : 1 ratio, to investigate the significance of membrane insertion and the influence of varying membrane composition on steady-state reaction kinetics. Reaction kinetics were analysed using a fluorimetric assay with 7-benzyloxyquinoline as substrate for CYP3A4. Full activity of the mono-oxygenase system, with electron transfer from NADPH via CPR, could only be reconstituted when CPR and CYP3A4 were colocalized within the same nanodiscs. No activity was observed when CPR and CYP3A4 were each incorporated separately into nanodiscs then mixed together, or when soluble forms of CPR were mixed with preassembled CYP3A4-nanodiscs. Membrane integration and colocalization are therefore essential for electron transfer. Liver microsomal lipid had an enhancing effect compared with phosphatidylcholine on the activity of CPR alone in nanodiscs, and a greater enhancing effect on the activity of CPR- CYP3A4 nanodisc complexes, which was not matched by a phospholipid mixture designed to mimic the ER composition. Furthermore, liver lipid enhanced redox coupling within the system. Thus, natural ER lipids possess properties or include components important for enhanced catalysis by CPR- CYP3A4 nanodisc complexes. Our findings demonstrate the importance of using natural lipid preparations for the detailed analysis of membrane protein activity. [ABSTRACT FROM AUTHOR]

Catechol O-methyltransferase (COMT) is an enzyme that plays a major role in catechol neurotransmitter deactivation. Inhibition of COMT can increase neurotransmitter levels, which provides a means of treatment for Parkinson's disease, schizophrenia and depression. COMT exists as two isozymes: a soluble cytoplasmic form (S-COMT), expressed in the liver and kidneys and a membrane-bound form (MB-COMT), found mostly in the brain. Here we report the backbone H, N and C chemical shift assignments of S-COMT in complex with S-adenosyl- l-methionine, 3,5-dinitrocatechol and Mg. Assignments were obtained by heteronuclear multidimensional NMR spectroscopy. In total, 97 % of all backbone resonances were assigned in the complex, with 205 out of a possible 215 residues assigned in the H-N TROSY spectrum. Prediction of solution secondary structure from a chemical shift analysis using the TALOS+ webserver is in good agreement with published X-ray crystal structures. [ABSTRACT FROM AUTHOR]

Distinct mechanisms have been proposed for the biological dehalogenation catalyzed by cobalamin-dependent enzymes, with two recent crystallographic studies suggesting different mechanisms based on the observed interaction between the organohalide substrate and cobalamin. In one case, involving an aromatic dibromide substrate in NpRdhA, a novel CoII–Br interaction was observed using EPR, suggesting a mechanism involving a [Co…X…R] adduct. However, in the case of trichloroethylene in PceA, a significantly longer Co–Cl distance was observed in X-ray crystal structures, suggesting a dissociative electron transfer mechanism. Subsequent DFT models of these reactions have not reproduced these differences in binding modes. Here, we have performed molecular docking and DFT calculations to investigate and compare the interaction between different organohalides and cobalamin in both NpRdhA and PceA. In each case, despite differences in binding in the CoII state, the reaction likely proceeds via formation of a [Co…X…R] adduct in the CoI state that weakens the breaking carbon–halide bond, suggesting this could be a general mechanism for cobalamin-dependent dehalogenation. [ABSTRACT FROM AUTHOR]

The universal Per-ARNT-Sim (PAS) domain functions as a signal transduction module involved in sensing diverse stimuli such as small molecules, light, redox state and gases. The highly evolvable PAS scaffold can bind a broad range of ligands, including haem, flavins and metal ions. However, although these ligands can support catalytic activity, to our knowledge no enzymatic PAS domain has been found. Here we report characterization of the first PAS enzyme: a haem-dependent oxidative N-demethylase. Unrelated to other amine oxidases, this enzyme contains haem, flavin mononucleotide, 2Fe-2S and tetrahydrofolic acid cofactors, and specifically catalyses the NADPH-dependent oxidation of dimethylamine. The structure of the α subunit reveals that it is a haem-binding PAS domain, similar in structure to PAS gas sensors. The dimethylamine substrate forms part of a highly polarized oxygen-binding site, and directly assists oxygen activation by acting as both an electron and proton donor. Our data reveal that the ubiquitous PAS domain can make the transition from sensor to enzyme, suggesting that the PAS scaffold can support the development of artificial enzymes. [ABSTRACT FROM AUTHOR]

Enzyme-catalysed H-transfer reactions are ubiquitous, yet fundamental details of these reactions remain unresolved. In this perspective, we discuss the roles of nuclear quantum tunnelling and (compressive) dynamics during these reactions. Evidence for the coupling of specific substrate and/or protein vibrations to the chemical coordinate is considered and a case is made for the combination of multiple experimental and computational/theoretical approaches when studying these reactions. [ABSTRACT FROM AUTHOR]

Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins. [ABSTRACT FROM AUTHOR]

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases ( NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes. [ABSTRACT FROM AUTHOR]

The temperature dependence of kinetic isotope effects ( KIEs) has emerged as the main experimental probe of enzymatic H-transfer by quantum tunnelling. Implicit in the interpretation is a presumed role for dynamic coupling of H-transfer chemistry to the protein environment, the so-called 'promoting motions/vibrations hypothesis'. This idea remains contentious, and others have questioned the importance and/or existence of promoting motions/vibrations. New experimental methods of addressing this problem are emerging, including use of mass-modulated enzymes and time-resolved spectroscopy. The pressure dependence of KIEs has been considered as a potential probe of quantum tunnelling reactions, because semi-classical KIEs, which are defined by differences in zero-point vibrational energy, are relatively insensitive to kbar changes in pressure. Reported combined pressure and temperature ( p- T) dependence studies of H-transfer reactions are, however, limited. Here, we extend and review the available p- T studies that have utilized well-defined experimental systems in which quantum mechanical tunnelling is established. These include flavoproteins, quinoproteins, light-activated enzymes and chemical model systems. We show that there is no clear general trend between the p- T dependencies of the KIEs in these systems. Given the complex nature of p- T studies, we conclude that computational simulations using determined (e.g. X-ray) structures are also needed alongside experimental measurements of reaction rates/ KIEs to guide the interpretation of p- T effects. In providing new insight into H-transfer/environmental coupling, combined approaches that unite both atomistic understanding with experimental rate measurements will require careful evaluation on a case-by-case basis. Although individually informative, we conclude that p- T studies do not provide the more generalized insight that has come from studies of the temperature dependence of KIEs. [ABSTRACT FROM AUTHOR]

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1′ bond breakage following FMN reduction, leading to formation of the N5-C1′ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3′-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires. [ABSTRACT FROM AUTHOR]

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation. [ABSTRACT FROM AUTHOR]

Coenzyme B12-dependent enzymes such as ethanolamine ammonia lyase have remarkable catalytic power and some unique properties that enable detailed analysis of the reaction chemistry and associated dynamics. By selectively deuterating the substrate (ethanolamine) and/or the β-carbon of the 5′-deoxyadenosyl moiety of the intrinsic coenzyme B12, it was possible to experimentally probe both the forward and reverse hydrogen atom transfers between the 5′-deoxyadenosyl radical and substrate during single-turnover stopped-flow measurements. These data are interpreted within the context of a kinetic model where the 5′-deoxyadenosyl radical intermediate may be quasi-stable and rearrangement of the substrate radical is essentially irreversible. Global fitting of these data allows estimation of the intrinsic rate constants associated with CoC homolysis and initial H-abstraction steps. In contrast to previous stopped-flow studies, the apparent kinetic isotope effects are found to be relatively small. [ABSTRACT FROM AUTHOR]

Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis. [ABSTRACT FROM AUTHOR]

Fluorescent dyes that bind DNA have been demonstrated as a useful alternative to radionucleotides for the quantification of DNA and the in vitro measurement of the activity of DNA polymerases and nucleases. However, this approach is generally used in a semi-quantitative way to determine relative rates of reaction. In this report, we demonstrate a method for the simultaneous quantification of DNA in both its single-strand and double-strand forms using the dye Pico Green. This approach is used in a steady-state assay of DNA polymerase Klenow fragment exo−, where we determine kcat and Km values for the DNA polymerase that are in excellent agreement with literature values. [ABSTRACT FROM AUTHOR]

Gd4O2S:Yb:Tm rare-earth upconversion phosphors have been utilised to monitor the redox behaviour of flavin mononucleotide and report on the turnover of a flavo-protein, (pentaerythritol tetranitrate reductase). The presence of two bands separated by over 300 nm in the UCP emission spectra allows ratiometric signalling of these processes with high sensitivity. [ABSTRACT FROM AUTHOR]

A combination of the temperature- and pressure-dependencies of the kinetic isotope effect on the proton coupled electron transfer during ascorbate oxidation by ferricyanide suggests that this reference reaction may exploit vibrationally assisted quantum tunnelling of the transferred proton. [ABSTRACT FROM AUTHOR]

The ability of hemoproteins to discriminate between diatomic molecules, and the subsequent affinity for their chosen ligand, is fundamental to the existence of life. These processes are often controlled by precise structural arrangements in proteins, with heme pocket residues driving reactivity and specificity. One such protein is cytochrome c', which has the ability to bind nitric oxide ( NO) and carbon monoxide ( CO) on opposite faces of the heme, a property that is shared with soluble guanylate cycle. Like soluble guanylate cyclase, cytochrome c' also excludes O2 completely from the binding pocket. Previous studies have shown that the NO binding mechanism is regulated by a proximal arginine residue ( R124) and a distal leucine residue ( L16). Here, we have investigated the roles of these residues in maintaining the affinity for NO in the heme binding environment by using various time-resolved spectroscopy techniques that span the entire femtosecond-second temporal range in the UV-vis spectrum, and the femtosecond-nanosecond range by IR spectroscopy. Our findings indicate that the tightly regulated NO rebinding events following excitation in wild-type cytochrome c' are affected in the R124 A variant. In the R124 A variant, vibrational and electronic changes extend continuously across all time scales (from fs-s), in contrast to wild-type cytochrome c' and the L16 A variant. Based on these findings, we propose a NO (re)binding mechanism for the R124 A variant of cytochrome c' that is distinct from that in wild-type cytochrome c'. In the wider context, these findings emphasize the importance of heme pocket architecture in maintaining the reactivity of hemoproteins towards their chosen ligand, and demonstrate the power of spectroscopic probes spanning a wide temporal range. [ABSTRACT FROM AUTHOR]