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1.
Nature ; 574(7780): 722-725, 2019 10.
Article in English | MEDLINE | ID: mdl-31645759

ABSTRACT

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.


Subject(s)
Chlorophyll/biosynthesis , Oxidoreductases Acting on CH-CH Group Donors/chemistry , Oxidoreductases Acting on CH-CH Group Donors/metabolism , Synechococcus/enzymology , Synechocystis/enzymology , Catalysis , Chlorophyll/chemistry , Molecular Structure , Photochemistry , Protochlorophyllide/metabolism , Structure-Activity Relationship , Substrate Specificity
2.
J Biol Chem ; 299(9): 105086, 2023 09.
Article in English | MEDLINE | ID: mdl-37495113

ABSTRACT

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.


Subject(s)
Hydrolases , NADP , Rhodobacteraceae , Escherichia coli/genetics , NADP/metabolism , Oxygen/chemistry , Vitamin B 12/metabolism , Phenols/chemistry , Phenols/metabolism , Electron Spin Resonance Spectroscopy , Hydrolases/chemistry , Hydrolases/genetics , Hydrolases/isolation & purification , Hydrolases/metabolism , Rhodobacteraceae/enzymology , Rhodobacteraceae/genetics , Protein Structure, Tertiary , Models, Molecular , Maltose-Binding Proteins/genetics , Maltose-Binding Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Coenzymes/metabolism
3.
J Am Chem Soc ; 146(17): 11726-11739, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38636166

ABSTRACT

Lysine dioxygenase (KDO) is an important enzyme in human physiology involved in bioprocesses that trigger collagen cross-linking and blood pressure control. There are several KDOs in nature; however, little is known about the factors that govern the regio- and stereoselectivity of these enzymes. To understand how KDOs can selectively hydroxylate their substrate, we did a comprehensive computational study into the mechanisms and features of 4-lysine dioxygenase. In particular, we selected a snapshot from the MD simulation on KDO5 and created large QM cluster models (A, B, and C) containing 297, 312, and 407 atoms, respectively. The largest model predicts regioselectivity that matches experimental observation with rate-determining hydrogen atom abstraction from the C4-H position, followed by fast OH rebound to form 4-hydroxylysine products. The calculations show that in model C, the dipole moment is positioned along the C4-H bond of the substrate and, therefore, the electrostatic and electric field perturbations of the protein assist the enzyme in creating C4-H hydroxylation selectivity. Furthermore, an active site Tyr233 residue is identified that reacts through proton-coupled electron transfer akin to the axial Trp residue in cytochrome c peroxidase. Thus, upon formation of the iron(IV)-oxo species in the catalytic cycle, the Tyr233 phenol loses a proton to the nearby Asp179 residue, while at the same time, an electron is transferred to the iron to create an iron(III)-oxo active species. This charged tyrosyl residue directs the dipole moment along the C4-H bond of the substrate and guides the selectivity to the C4-hydroxylation of the substrate.


Subject(s)
Catalytic Domain , Lysine , Protons , Hydroxylation , Lysine/metabolism , Lysine/chemistry , Electron Transport , Tyrosine/chemistry , Tyrosine/metabolism , Molecular Dynamics Simulation , Stereoisomerism , Mixed Function Oxygenases/chemistry , Mixed Function Oxygenases/metabolism , Humans , Iron/chemistry , Iron/metabolism
4.
Chembiochem ; : e202400672, 2024 Oct 14.
Article in English | MEDLINE | ID: mdl-39400489

ABSTRACT

Rubrobacter radiotolerans nerolidol synthase (NerS) and trans-α-bergamotene synthase (BerS) are among the first terpene synthases (TSs) discovered from thermotolerant bacteria, and, despite sharing the same substrate, make terpenoid products with different carbon scaffolds. Here, the potential thermostability of NerS and BerS was investigated, and NerS was found to retain activity up to 55 °C. A library of 22 NerS and BerS variants was designed to probe the differing reaction mechanisms of NerS and BerS, including residues putatively involved in substrate sequestration, cation-π stabilisation of reactive intermediates, and shaping of the active site contour. Two BerS variants showed improved in vivo titres vs the WT enzyme, and also yielded different ratios of the related sesquiterpenoids (E)-ß-farnesene and trans-α-bergamotene. BerS-L86F was proposed to encourage substrate isomerisation by cation-π stabilisation of the first cationic intermediate, resulting in a greater proportion of trans-α-bergamotene. By contrast, BerS-S82L significantly preferred (E)-ß-farnesene formation, attributed to steric blocking of the isomerisation step, consistent with what has been observed in several plant TSs. Our work highlights the importance of isomerisation as a key determinant of product outcome in TSs, and shows how a combined computational and experimental approach can characterise TSs and variants with improved and altered functionality.

5.
Chemistry ; : e202402604, 2024 Aug 27.
Article in English | MEDLINE | ID: mdl-39190221

ABSTRACT

The nonheme iron dioxygenase capreomycin C (CmnC) hydroxylates a free L-arginine amino acid regio- and stereospecifically at the C3-position as part of the capreomycin antibiotics biosynthesis. Little is known on its structure, catalytic cycle and substrate specificity and, therefore, a comprehensive computational study was performed. A large QM cluster model of CmnC was created of 297 atoms and the mechanisms for C3-H, C4-H and C5-H hydroxylation and C3-C4 desaturation were investigated. All low-energy pathways correspond to radical reaction mechanisms with an initial hydrogen atom abstraction followed by OH rebound to form alcohol product complexes. The work is compared to alternative L-Arg hydroxylating nonheme iron dioxygenases and the differences in active site polarity are compared. We show that a tight hydrogen bonding network in the substrate binding pocket positions the substrate in an ideal orientation for C3-H activation, whereby the polar groups in the substrate binding pocket induce an electric field effect that guides the selectivity.

6.
J Am Chem Soc ; 145(42): 22859-22865, 2023 Oct 25.
Article in English | MEDLINE | ID: mdl-37839071

ABSTRACT

To carry out reliable and comprehensive structural investigations, the exploitation of different complementary techniques is required. Here, we report that dual triplet-spin/fluorescent labels enable the first parallel distance measurements by electron spin resonance (ESR) and Förster resonance energy transfer (FRET) on exactly the same molecules with orthogonal chromophores, allowing for direct comparison. An improved light-induced triplet-triplet electron resonance method with 2-color excitation is used, improving the signal-to-noise ratio of the data and yielding a distance distribution that provides greater insight than the single distance resulting from FRET.

7.
Chemistry ; 29(42): e202300271, 2023 Jul 26.
Article in English | MEDLINE | ID: mdl-37159057

ABSTRACT

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.

8.
Biochemistry ; 61(17): 1735-1742, 2022 09 06.
Article in English | MEDLINE | ID: mdl-35979922

ABSTRACT

Calmodulin (CaM) is a highly dynamic Ca2+-binding protein that exhibits large conformational changes upon binding Ca2+ and target proteins. Although it is accepted that CaM exists in an equilibrium of conformational states in the absence of target protein, the physiological relevance of an elongated helical linker region in the Ca2+-replete form has been highly debated. In this study, we use PELDOR (pulsed electron-electron double resonance) EPR measurements of a doubly spin-labeled CaM variant to assess the conformational states of CaM in the apo-, Ca2+-bound, and Ca2+ plus target peptide-bound states. Our findings are consistent with a three-state conformational model of CaM, showing a semi-open apo-state, a highly extended Ca2+-replete state, and a compact target protein-bound state. Molecular dynamics simulations suggest that the presence of glycerol, and potentially other molecular crowding agents, has a profound effect on the relative stability of the different conformational states. Differing experimental conditions may explain the discrepancies in the literature regarding the observed conformational state(s) of CaM, and our PELDOR measurements show good evidence for an extended conformation of Ca2+-replete CaM similar to the one observed in early X-ray crystal structures.


Subject(s)
Calmodulin , Molecular Dynamics Simulation , Calcium/metabolism , Calmodulin/chemistry , Electrons , Protein Binding , Protein Conformation , Spin Labels
9.
Chembiochem ; 23(5): e202100688, 2022 03 04.
Article in English | MEDLINE | ID: mdl-35005823

ABSTRACT

Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α-terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation-π interactions.


Subject(s)
Eucalyptol , Catalytic Domain , Cyclization
10.
Nat Chem Biol ; 16(11): 1255-1260, 2020 11.
Article in English | MEDLINE | ID: mdl-32719558

ABSTRACT

The direct C-H carboxylation of aromatic compounds is an attractive route to the corresponding carboxylic acids, but remains challenging under mild conditions. It has been proposed that the first step in anaerobic microbial degradation of recalcitrant aromatic compounds is a UbiD-mediated carboxylation. In this study, we use the UbiD enzyme ferulic acid decarboxylase (Fdc) in combination with a carboxylic acid reductase to create aromatic degradation-inspired cascade reactions, leading to efficient functionalization of styrene through CO2 fixation. We reveal that rational structure-guided laboratory evolution can expand the substrate scope of Fdc, resulting in activity on a range of mono- and bicyclic aromatic compounds through a single mutation. Selected variants demonstrated 150-fold improvement in the conversion of coumarillic acid to benzofuran + CO2 and unlocked reactivity towards naphthoic acid. Our data demonstrate that UbiD-mediated C-H activation is a versatile tool for the transformation of aryl/alkene compounds and CO2 into commodity chemicals.


Subject(s)
Carbon Dioxide/chemistry , Carboxy-Lyases/metabolism , Hydrocarbons, Aromatic/metabolism , Oxidoreductases/metabolism , Amino Acid Sequence , Benzofurans/chemistry , Biocatalysis , Biodegradation, Environmental , Carboxy-Lyases/genetics , Carboxylic Acids/chemistry , Decarboxylation , Drug Evaluation, Preclinical , Enzyme Activation , Genomic Library , Hydrocarbons, Aromatic/chemistry , Models, Molecular , Molecular Structure , Mutation , Naphthalenes/chemistry , Oxidoreductases/genetics , Structure-Activity Relationship , Styrene/chemistry
11.
Nature ; 539(7630): 593-597, 2016 11 24.
Article in English | MEDLINE | ID: mdl-27851736

ABSTRACT

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.


Subject(s)
Oxidoreductases, N-Demethylating/chemistry , Oxidoreductases, N-Demethylating/metabolism , Pseudomonas mendocina/enzymology , Binding Sites , Coenzymes/metabolism , Crystallography, X-Ray , Dimethylamines/metabolism , Flavin Mononucleotide/metabolism , Heme/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Models, Molecular , NADP/metabolism , Oxidation-Reduction , Oxygen/metabolism , Protein Domains , Protein Subunits/chemistry , Protein Subunits/metabolism , Tetrahydrofolates/metabolism
12.
Angew Chem Int Ed Engl ; 61(50): e202212158, 2022 12 12.
Article in English | MEDLINE | ID: mdl-36250805

ABSTRACT

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.


Subject(s)
Methionine , Riboflavin , Amino Acids , Methionine/chemistry , Peptides/chemistry , Racemethionine , Vitamins , Catalysis
13.
Proteins ; 2021 Feb 25.
Article in English | MEDLINE | ID: mdl-33629765

ABSTRACT

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.

14.
Nature ; 517(7535): 513-516, 2015 Jan 22.
Article in English | MEDLINE | ID: mdl-25327251

ABSTRACT

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.


Subject(s)
Halogenation , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Phyllobacteriaceae/enzymology , Vitamin B 12/metabolism , Biocatalysis , Cobalt/chemistry , Cobalt/metabolism , Crystallography, X-Ray , Electron Spin Resonance Spectroscopy , Models, Molecular , Oxidation-Reduction , Oxygen/metabolism , Phenols/chemistry , Phenols/metabolism , Protein Conformation , Solubility , Vitamin B 12/chemistry
15.
Nature ; 522(7557): 502-6, 2015 Jun 25.
Article in English | MEDLINE | ID: mdl-26083743

ABSTRACT

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.


Subject(s)
Biocatalysis , Carboxy-Lyases/metabolism , Dimethylallyltranstransferase/metabolism , Flavins/metabolism , Pseudomonas aeruginosa/enzymology , Pseudomonas aeruginosa/metabolism , Ubiquinone/biosynthesis , Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/metabolism , Aspergillus niger/enzymology , Aspergillus niger/genetics , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Catalytic Domain , Crystallography, X-Ray , Cycloaddition Reaction , Decarboxylation , Dimethylallyltranstransferase/chemistry , Dimethylallyltranstransferase/genetics , Electron Transport , Flavin Mononucleotide/metabolism , Flavins/biosynthesis , Flavins/chemistry , Models, Molecular , Pseudomonas aeruginosa/genetics
16.
Nature ; 522(7557): 497-501, 2015 Jun 25.
Article in English | MEDLINE | ID: mdl-26083754

ABSTRACT

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.


Subject(s)
Biocatalysis , Carboxy-Lyases/metabolism , Cycloaddition Reaction , Alkenes/chemistry , Alkenes/metabolism , Aspergillus niger/enzymology , Aspergillus niger/genetics , Carboxy-Lyases/chemistry , Carboxy-Lyases/genetics , Crystallography, X-Ray , Decarboxylation , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Flavins/biosynthesis , Flavins/chemistry , Flavins/metabolism , Isomerism , Ligands , Models, Molecular , Ubiquinone/biosynthesis
17.
Chembiochem ; 21(7): 985-990, 2020 04 01.
Article in English | MEDLINE | ID: mdl-31682055

ABSTRACT

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. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.


Subject(s)
Intramolecular Lyases/metabolism , Monoterpenes/metabolism , Binding Sites , Catalytic Domain , Cyclization , Cyclohexane Monoterpenes/chemistry , Cyclohexane Monoterpenes/metabolism , Eucalyptol/chemistry , Eucalyptol/metabolism , Hydroxylation , Intramolecular Lyases/genetics , Molecular Dynamics Simulation , Monoterpenes/chemistry , Mutagenesis, Site-Directed , Stereoisomerism , Streptomyces/enzymology , Water/chemistry , Water/metabolism
18.
Chemistry ; 26(65): 14817-14822, 2020 Nov 20.
Article in English | MEDLINE | ID: mdl-32476171

ABSTRACT

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.


Subject(s)
Photons , Energy Transfer , Enzyme Activation , Fluorescent Dyes , Luminescence , Oxidation-Reduction , Substrate Specificity
19.
Faraday Discuss ; 221: 367-378, 2019 12 16.
Article in English | MEDLINE | ID: mdl-31544181

ABSTRACT

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.


Subject(s)
Biocatalysis , Enzymes/metabolism , Hydrogen/chemistry , Hydrogen/metabolism , Kinetics , Quantum Theory , Thermodynamics
20.
Nucleic Acids Res ; 44(3): e21, 2016 Feb 18.
Article in English | MEDLINE | ID: mdl-26405200

ABSTRACT

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems.


Subject(s)
Protein Biosynthesis , Transcription, Genetic , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Promoter Regions, Genetic , Riboswitch , Viral Proteins/genetics
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