Selected publications

Selected publications – 2014/15

Conversion of alcohols to enantiopure amines through dual-enzyme hydroge-borrowing cascades
Mutti, Francesco G; Knaus, Tanya; Scrutton, Nigel S; Breuer, Michael; Turner, Nicholas J.
Science. 2015 Sept 25; 349(6255): 1525-1529; doi: 10.1126/science.aac9283″

Abstract

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale. We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines. The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product.
New cofactor supports α,β-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition.
Payne KA, White MD, Fisher K, Khara B, Bailey SS, Parker D, Rattray NJ, Trivedi DK, Goodacre R, Beveridge R, Barran P, Rigby SE, Scrutton NS, Hay S, Leys D.
Nature. 2015 Jun 25;522(7557):497-501. doi: 10.1038/nature14560

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.
UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis.
White MD, Payne KA, Fisher K, Marshall SA, Parker D, Rattray NJ, Trivedi DK, Goodacre R, Rigby SE, Scrutton NS, Hay S, Leys D.
Nature. 2015 Jun 25;522(7557):502-6. doi: 10.1038/nature14559

Abstract

Poly(ADP-ribosyl)ation is a reversible post-translational protein modification involved in the regulation of a number of cellular processes including DNA repair, chromatin structure, mitosis, transcription, checkpoint activation, apoptosis and asexual development. The reversion of poly(ADP-ribosyl)ation is catalysed by poly(ADP-ribose) (PAR) glycohydrolase (PARG), which specifically targets the unique PAR (1′′-2′) ribose–ribose bonds. Here we report the structure and mechanism of the first canonical PARG from the protozoan Tetrahymena thermophila. In addition, we reveal the structure of T. thermophila PARG in a complex with a novel rhodanine-containing mammalian PARG inhibitor RBPI-3. Our data demonstrate that the protozoan PARG represents a good model for human PARG and is therefore likely to prove useful in guiding structure-based discovery of new classes of PARG inhibitors.

Catalytic Mechanism of Cofactor-Free Dioxygenases and How They Circumvent Spin-Forbidden Oxygenation of Their Substrates.
Hernández-Ortega A, Quesne MG, Bui S, Heyes DJ, Steiner RA, Scrutton NS, de Visser SP.
J Am Chem Soc. 2015 Jun 17;137(23):7474-87. doi: 10.1021/jacs.5b03836

Abstract

Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the singlet spin state occurs. As revealed by steady- and transient-state kinetics the oxygen-dependent steps are rate-limiting, whereas Trp160 and His251 are essential residues for catalysis and contribute to substrate positioning and activation, respectively. Computational modeling further confirms the experimental observations and rationalizes the electron transfer pathways, and the effect of substrate and substrate binding pocket residues. Finally, we make a direct comparison with iron-based dioxygenases and explain the mechanistic and electronic differences with cofactor-free dioxygenases. Our multidisciplinary study confirms that the oxygenation reaction can take place in absence of any cofactor by a unique mechanism in which the specially designed fit-for-purpose active-site architecture modulates substrate reactivity toward oxygen.
Distinguishing Loss of Structure from Subunit Dissociation for Protein Complexes with Variable Temperature Ion Mobility Mass Spectrometry.
Pacholarz KJ, Barran PE.
Anal Chem. 2015 Jun 16 Paper
Synthesis of D- and L-Phenylalanine Derivatives by Phenylalanine Ammonia Lyases: A Multienzymatic Cascade Process.
Parmeggiani F, Lovelock SL, Weise NJ, Ahmed ST, Turner NJ.
Angew Chem Int Ed Engl. 2015 Volume 54, Issue 15, pages 4608–4611 doi: 10.1002/anie.201410670

Abstract

The synthesis of substituted D-phenylalanines in high yield and excellent optical purity, starting from inexpensive cinnamic acids, has been achieved with a novel one-pot approach by coupling phenylalanine ammonia lyase (PAL) amination with a chemoenzymatic deracemization (based on stereoselective oxidation and nonselective reduction). A simple high-throughput solid-phase screening method has also been developed to identify PALs with higher rates of formation of non-natural D-phenylalanines. The best variants were exploited in the chemoenzymatic cascade, thus increasing the yield and ee value of the D-configured product. Furthermore, the system was extended to the preparation of those L-phenylalanines which are obtained with a low ee value using PAL amination.
Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently.
Currin A, Swainston N, Day PJ, Kell DB.
Chem Soc Rev 2015 Mar 7;44(5):1172-239. doi: 10.1039/c4cs00351a

Abstract

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (Kd) and catalytic (kcat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving kcat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust.
Towards the free energy landscape for catalysis in mammalian nitric oxide synthases.
Leferink NG, Hay S, Rigby SE, Scrutton NS.
FEBS J. 2014 Dec 9. doi: 10.1111/febs.13171.

Abstract

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.
Excited-state charge separation in the photochemical mechanism of the light-driven enzyme protochlorophyllide oxidoreductase.
Heyes DJ, Hardman SJ, Hedison TM, Hoeven R, Greetham GM, Towrie M, Scrutton NS.
Angew Chem Int Ed Engl. 2015 Jan 26;54(5):1512-5 doi: 10.1002/anie.201409881

Abstract

The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond-microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called “reactive” intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.
Differences and Comparisons of the Properties and Reactivities of Iron(III)-hydroperoxo Complexes with Saturated Coordination Sphere.
Faponle AS, Quesne MG, Sastri CV, Banse F, de Visser SP.
Chemistry. 2014 Nov 14 doi: 10.1002/chem.201404918

Abstract

Heme and nonheme monoxygenases and dioxygenases catalyze important oxygen atom transfer reactions to substrates in the body. It is now well established that the cytochrome P450 enzymes react through the formation of a high-valent iron(IV)–oxo heme cation radical. Its precursor in the catalytic cycle, the iron(III)–hydroperoxo complex, was tested for catalytic activity and found to be a sluggish oxidant of hydroxylation, epoxidation and sulfoxidation reactions. In a recent twist of events, evidence has emerged of several nonheme iron(III)–hydroperoxo complexes that appear to react with substrates via oxygen atom transfer processes. Although it was not clear from these studies whether the iron(III)–hydroperoxo reacted directly with substrates or that an initial O O bond cleavage preceded the reaction. Clearly, the catalytic activity of heme and nonheme iron(III)–hydroperoxo complexes is substantially different, but the origins of this are still poorly understood and warrant a detailed analysis. In this work, an extensive computational analysis of aromatic hydroxylation by biomimetic nonheme and heme iron systems is presented, starting from an iron(III)–hydroperoxo complex with pentadentate ligand system (L52). Direct C O bond formation by an iron(III)–hydroperoxo complex is investigated, as well as the initial heterolytic and homolytic bond cleavage of the hydroperoxo group. The calculations show that [(L52)FeIII(OOH)]2+ should be able to initiate an aromatic hydroxylation process, although a low-energy homolytic cleavage pathway is only slightly higher in energy. A detailed valence bond and thermochemical analysis rationalizes the differences in chemical reactivity of heme and nonheme iron(III)–hydroperoxo and show that the main reason for this particular nonheme complex to be reactive comes from the fact that they homolytically split the O O bond, whereas a heterolytic O O bond breaking in heme iron(III)–hydroperoxo is found.
Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.
Payne KA, Quezada CP, Fisher K, Dunstan MS, Collins FA, Sjuts H, Levy C, Hay S, Rigby SE, Leys D.
Nature. 2015 Jan 22;517(7535):513-6 doi: 10.1038/nature13901

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.
Natural products: tools and more special issue.
Takano E, Breitling R.
ACS Synth Biol. 2013 Jul 19;2(7):352-3 doi: 10.1021/sb400063n
Accuracy and tractability of a kriging model of intramolecular polarizable multipolar electrostatics and its application to histidine.
Kandathil SM, Fletcher TL, Yuan Y, Knowles J, Popelier PL
J Comput Chem. 2013 May 29, 34, 1850–1861 doi: 10.1002/jcc.23333

Abstract

We propose a generic method to model polarization in the context of high-rank multipolar electrostatics. This method involves the machine learning technique kriging, here used to capture the response of an atomic multipole moment of a given atom to a change in the positions of the atoms surrounding this atom. The atoms are malleable boxes with sharp boundaries, they do not overlap and exhaust space. The method is applied to histidine where it is able to predict atomic multipole moments (up to hexadecapole) for unseen configurations, after training on 600 geometries distorted using normal modes of each of its 24 local energy minima at B3LYP/apc-1 level. The quality of the predictions is assessed by calculating the Coulomb energy between an atom for which the moments have been predicted and the surrounding atoms (having exact moments). Only interactions between atoms separated by three or more bonds (“1, 4 and higher” interactions) are included in this energy error. This energy is compared with that of a central atom with exact multipole moments interacting with the same environment. The resulting energy discrepancies are summed for 328 atom–atom interactions, for each of the 29 atoms of histidine being a central atom in turn. For 80% of the 539 test configurations (outside the training set), this summed energy deviates by less than 1 kcal mol.
Structural basis of kynurenine 3-monooxygenase inhibition.
Amaral M, Levy C, Heyes DJ, Lafite P, Outeiro TF, Giorgini F, Leys D, Scrutton NS.
Nature (2013) 496, 382–385 doi:10.1038/nature12039

Abstract

Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington’s-disease-relevant phenotypes in yeast, fruitfly and mouse models1, 2, 3, 4, 5, as well as in a mouse model of Alzheimer’s disease3. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders6, as well as cancer7, 8 and several peripheral inflammatory conditions9. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO–UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood–brain barrier in targeted therapies against neurodegenerative diseases such as Huntington’s, Alzheimer’s and Parkinson’s diseases.
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