SignatureFinder enables sequence mining to identify cobalamin-dependent photoreceptor proteins.

Photoreceptors control cellular processes in response to light. Most photoreceptors sense blue or red light, but the recent discovery of the cobalamin-dependent photoreceptor, CarH, has expanded the wavelength range of photoreception to other regions of the electromagnetic spectrum to include the green light region. Further identification of cobalamin-dependent green light-sensitive photoreceptors has been hampered owing to poor annotation of the light responsiveness of cobalamin-binding domains (CBDs) in public databases.

Photocobilins integrate B and bilin photochemistry for enzyme control.

Photoreceptor proteins utilise chromophores to sense light and trigger a biological response. The discovery that adenosylcobalamin (or coenzyme B) can act as a light-sensing chromophore heralded a new field of B-photobiology. Although microbial genome analysis indicates that photoactive B-binding domains form part of more complex protein architectures, regulating a range of molecular-cellular functions in response to light, experimental evidence is lacking.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

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 N-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned.

Mechanistic implications of the ternary complex structural models for the photoenzyme protochlorophyllide oxidoreductase.

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.

Decoding Catalysis by Terpene Synthases.

The review by Christianson, published in 2017 on the twentieth anniversary of the emergence of the field, summarizes the foundational discoveries and key advances in terpene synthase/cyclase (TS) biocatalysis (Christianson, D. W. , (17), 11570-11648.

Redox driven B-ligand switch drives CarH photoresponse.

CarH is a coenzyme B-dependent photoreceptor involved in regulating carotenoid biosynthesis. How light-triggered cleavage of the B Co-C bond culminates in CarH tetramer dissociation to initiate transcription remains unclear. Here, a series of crystal structures of the CarH B-binding domain after illumination suggest formation of unforeseen intermediate states prior to tetramer dissociation.

How Is Substrate Halogenation Triggered by the Vanadium Haloperoxidase from ?

Vanadium haloperoxidases (VHPOs) are unique enzymes in biology that catalyze a challenging halogen transfer reaction and convert a strong aromatic C-H bond into C-X (X = Cl, Br, I) with the use of a vanadium cofactor and HO. The VHPO catalytic cycle starts with the conversion of hydrogen peroxide and halide (X = Cl, Br, I) into hypohalide on the vanadate cofactor, and the hypohalide subsequently reacts with a substrate. However, it is unclear whether the hypohalide is released from the enzyme or otherwise trapped within the enzyme structure for the halogenation of organic substrates.

Photoinduced Electron Transfer from a 1,4,5,6-Tetrahydro Nicotinamide Adenine Dinucleotide (Phosphate) Analogue to Oxidized Flavin in an Ene-Reductase Flavoenzyme.

Recent reports have described the use of ene-reductase flavoenzymes to catalyze non-natural photochemical reactions. These studies have focused on using reduced flavoenzyme, yet oxidized flavins have superior light harvesting properties. In a binary complex of the oxidized ene-reductase pentaerythritol tetranitrate reductase with the nonreactive nicotinamide coenzyme analogs 1,4,5,6-tetrahydro NAD(P)H, visible photoexcitation of the flavin mononucleotide (FMN) leads to one-electron transfer from the NAD(P)H to FMN, generating a NAD(P)H cation radical and anionic FMN semiquinone.

Mechanism of Action of Flavin-Dependent Halogenases.

To rationally engineer the substrate scope and selectivity of flavin-dependent halogenases (FDHs), it is essential to first understand the reaction mechanism and substrate interactions in the active site. FDHs have long been known to achieve regioselectivity through an electrophilic aromatic substitution at C7 of the natural substrate Trp, but the precise role of a key active-site Lys residue remains ambiguous. Formation of hypochlorous acid (HOCl) at the cofactor-binding site is achieved by the direct reaction of molecular oxygen and a single chloride ion with reduced FAD and flavin hydroxide, respectively.

A Vitamin B-Photocatalysed Approach to Methionine Analogues.

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.

A Vitamin B -Photocatalysed Approach to Methionine Analogues.

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.

How Photoactivation Triggers Protochlorophyllide Reduction: Computational Evidence of a Stepwise Hydride Transfer during Chlorophyll Biosynthesis.

The photochemical reaction catalyzed by enzyme protochlorophyllide oxidoreductase (POR), a rare example of a photoactivated enzyme, is a crucial step during chlorophyll biosynthesis and involves the fastest known biological hydride transfer. Structures of the enzyme with bound substrate protochlorophyllide (PChlide) and coenzyme nicotinamide adenine dinucleotide phosphate (NADPH) have recently been published, opening up the possibility of using computational approaches to provide a comprehensive understanding of the excited state chemistry. Herein, we propose a complete mechanism for the photochemistry between PChlide and NADPH based on density functional theory (DFT) and time-dependent DFT calculations that is consistent with recent experimental data.

Chelator-Based Parameterization of the 12-6-4 Lennard-Jones Molecular Mechanics Potential for More Realistic Metal Ion-Protein Interactions.

Metal ions are associated with a variety of proteins and play critical roles in a wide range of biochemical processes. There are multiple ways to study and quantify protein-metal ion interactions, including molecular dynamics simulations. Recently, the AMBER molecular mechanics forcefield was modified to include a 12-6-4 Lennard-Jones potential, which allows for a better description of nonbonded terms through the additional pairwise coefficients.

Engineering an efficient and enantioselective enzyme for the Morita-Baylis-Hillman reaction.

The combination of computational design and directed evolution could offer a general strategy to create enzymes with new functions. So far, this approach has delivered enzymes for a handful of model reactions. Here we show that new catalytic mechanisms can be engineered into proteins to accelerate more challenging chemical transformations.

Structural basis of terephthalate recognition by solute binding protein TphC.

Biological degradation of Polyethylene terephthalate (PET) plastic and assimilation of the corresponding monomers ethylene glycol and terephthalate (TPA) into central metabolism offers an attractive route for bio-based molecular recycling and bioremediation applications. A key step is the cellular uptake of the non-permeable TPA into bacterial cells which has been shown to be dependent upon the presence of the key tphC gene. However, little is known from a biochemical and structural perspective about the encoded solute binding protein, TphC.

Interplay between chromophore binding and domain assembly by the B-dependent photoreceptor protein, CarH.

Organisms across the natural world respond to their environment through the action of photoreceptor proteins. The vitamin B-dependent photoreceptor, CarH, is a bacterial transcriptional regulator that controls the biosynthesis of carotenoids to protect against photo-oxidative stress. The binding of B to CarH monomers in the dark results in the formation of a homo-tetramer that complexes with DNA; B photochemistry results in tetramer dissociation, releasing DNA for transcription.

Photocatalysis as the 'master switch' of photomorphogenesis in early plant development.

Enzymatic photocatalysis is seldom used in biology. Photocatalysis by light-dependent protochlorophyllide oxidoreductase (LPOR)-one of only a few natural light-dependent enzymes-is an exception, and is responsible for the conversion of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis. Photocatalysis by LPOR not only regulates the biosynthesis of the most abundant pigment on Earth but it is also a 'master switch' in photomorphogenesis in early plant development.

Predicting new protein conformations from molecular dynamics simulation conformational landscapes and machine learning.

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.

Dual role of the active site 'lid' regions of protochlorophyllide oxidoreductase in photocatalysis and plant development.

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.

Characterization of the structure and interactions of P450 BM3 using hybrid mass spectrometry approaches.

The cytochrome P450 monooxygenase P450 BM3 (BM3) is a biotechnologically important and versatile enzyme capable of producing important compounds such as the medical drugs pravastatin and artemether, and the steroid hormone testosterone. BM3 is a natural fusion enzyme comprising two major domains: a cytochrome P450 (heme-binding) catalytic domain and a NADPH-cytochrome P450 reductase (CPR) domain containing FAD and FMN cofactors in distinct domains of the CPR. A crystal structure of full-length BM3 enzyme is not available in its monomeric or catalytically active dimeric state.

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation.

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled.

Structural basis for enzymatic photocatalysis in chlorophyll biosynthesis.

The enzyme protochlorophyllide oxidoreductase (POR) catalyses a light-dependent step in chlorophyll biosynthesis that is essential to photosynthesis and, ultimately, all life on Earth. POR, which is one of three known light-dependent enzymes, 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.

What are the signatures of tunnelling in enzyme-catalysed reactions?

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.

Equatorial Active Site Compaction and Electrostatic Reorganization in Catechol--methyltransferase.

Catechol--methyltransferase (COMT) is a model S-adenosyl-l-methionine (SAM) dependent methyl transferase, which catalyzes the methylation of catecholamine neurotransmitters such as dopamine in the primary pathway of neurotransmitter deactivation in animals. Despite extensive study, there is no consensus view of the physical basis of catalysis in COMT. Further progress requires experimental data that directly probes active site geometry, protein dynamics and electrostatics, ideally in a range of positions along the reaction coordinate.