Publications by authors named "Helen S Toogood"

32 Publications

Thermal, electrochemical and photochemical reactions involving catalytically versatile ene reductase enzymes.

Enzymes 2020 18;47:491-515. Epub 2020 Jul 18.

EPSRC/BBSRC Future Biomanufacturing Research Hub, BBSRC/EPSRC Synthetic Biology Research Centre SYNBIOCHEM Manchester Institute of Biotechnology and Department of Chemistry, School of Natural Sciences, The University of Manchester, Manchester, United Kingdom. Electronic address:

Successful exploitation of biocatalytic processes employing flavoproteins requires the implementation of cost-effective solutions to circumvent the need to supply costly nicotinamide coenzymes as reducing equivalents. Chemical syntheses harnessing the power of the flavoprotein ene reductases will likely increase the range and/or optical purity of available fine chemicals and pharmaceuticals due to their ability to catalyze asymmetric bioreductions. This review will outline current progress in the design of alternative routes to ene reductase flavin activation, most notably within the Old Yellow Enzyme family. A variety of chemical, enzymatic, electrochemical and photocatalytic routes have been employed, designed to eliminate the need for nicotinamide coenzymes or provide cost-effective alternatives to efficient recycling. Photochemical approaches have also enabled novel mechanistic routes of ene reductases to become available, opening up the possibility of accessing a wider range of non-natural chemical diversity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/bs.enz.2020.05.012DOI Listing
December 2020

Renewable and tuneable bio-LPG blends derived from amino acids.

Biotechnol Biofuels 2020 14;13:125. Epub 2020 Jul 14.

EPSRC/BBSRC Future Biomanufacturing Research Hub, BBSRC/EPSRC, Synthetic Biology Research Centre SYNBIOCHEM Manchester Institute of Biotechnology and School of Chemistry, The University of Manchester, Manchester, M1 7DN UK.

Background: Microbial biorefinery approaches are beginning to define renewable and sustainable routes to clean-burning and non-fossil fuel-derived gaseous alkanes (known as 'bio-LPG'). The most promising strategies have used a terminal fatty acid photodecarboxylase, enabling light-driven propane production from externally fed waste butyric acid. Use of (a robust extremophile microbial chassis) with these pathways has enabled bio-LPG production under non-sterile conditions and using waste biomass as the carbon source. Here, we describe new engineering approaches to produce next-generation pathways that use amino acids as fuel precursors for bio-LPG production (propane, butane and isobutane blends).

Results: Multiple pathways from the amino acids valine, leucine and isoleucine were designed in for the production of propane, isobutane and butane, respectively. A branched-chain keto acid decarboxylase-dependent pathway utilising fatty acid photodecarboxylase was the most effective route, generating higher alkane gas titres over alternative routes requiring coenzyme A and/or aldehyde deformylating oxygenase. Isobutane was the major gas produced in standard (mixed amino acid) medium, however valine supplementation led to primarily propane production. Transitioning pathways into strain TQ10 enabled fermentative production of mixed alkane gases under non-sterile conditions on simple carbon sources. Chromosomal integration of inducible (~ 180 mg/g cells/day) and constitutive (~ 30 mg/g cells/day) pathways into generated production strains shown to be stable for up to 7 days.

Conclusions: This study highlights new microbial pathways for the production of clean-burning bio-LPG fuels from amino acids. The use of stable production strains could lead to gas production in the field under non-sterile conditions following process optimisation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13068-020-01766-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362463PMC
July 2020

Discovery, Characterisation, Engineering and Applications of Ene Reductases for Industrial Biocatalysis.

ACS Catal 2019 May 20;8(4):3532-3549. Epub 2018 Mar 20.

School of Chemistry, Faculty of Science and Engineering, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.

Recent studies of multiple enzyme families collectively referred to as ene-reductases (ERs) have highlighted potential industrial application of these biocatalysts in the production of fine and speciality chemicals. Processes have been developed whereby ERs contribute to synthetic routes as isolated enzymes, components of multi-enzyme cascades, and more recently in metabolic engineering and synthetic biology programmes using microbial cell factories to support chemicals production. The discovery of ERs from previously untapped sources and the expansion of directed evolution screening programmes, coupled to deeper mechanistic understanding of ER reactions, have driven their use in natural product and chemicals synthesis. Here we review developments, challenges and opportunities for the use of ERs in fine and speciality chemicals manufacture. The ER research field is rapidly expanding and the focus of this review is on developments that have emerged predominantly over the last 4 years.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.8b00624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542678PMC
May 2019

C3 and C6 Modification-Specific OYE Biotransformations of Synthetic Carvones and Sequential BVMO Chemoenzymatic Synthesis of Chiral Caprolactones.

Chemistry 2019 Feb 15;25(12):2983-2988. Epub 2019 Jan 15.

Manchester Institute of Biotechnology and the School of Chemistry, The University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.

The scope for biocatalytic modification of non-native carvone derivatives for speciality intermediates has hitherto been limited. Additionally, caprolactones are important feedstocks with diverse applications in the polymer industry and new non-native terpenone-derived biocatalytic caprolactone syntheses are thus of potential value for industrial biocatalytic materials applications. Biocatalytic reduction of synthetic analogues of R-(-)-carvone with additional substituents at C3 or C6, or both C3 and C6, using three types of OYEs (OYE2, PETNR and OYE3) shows significant impact of both regio-substitution and the substrate diastereomer. Bioreduction of (-)-carvone derivatives substituted with a Me and/or OH group at C6 is highly dependent on the diastereomer of the substrate. Derivatives bearing C6 substituents larger than methyl moieties are not substrates. Computer docking studies of PETNR with both (6S)-Me and (6R)-Me substituted (-)-carvone provides a model consistent with the outcomes of bioconversion. The products of bioreduction were efficiently biotransformed by the Baeyer-Villiger monooxygenase (BVase) CHMO_Phi1 to afford novel trisubstituted lactones with complete regioselectivity to provide a new biocatalytic entry to these chiral caprolactones. This provides both new non-native polymerization feedstock chemicals, but also with enhanced efficiency and selectivity over native (+)-dihydrocarvone Baeyer-Villigerase expansion. Optimum enzymatic reactions were scaled up to 60-100 mg, demonstrating the utility for preparative biocatalytic synthesis of both new synthetic scaffold-modified dihydrocarvones and efficient biocatalytic entry to new chiral caprolactones, which are potential single-isomer chiral polymer feedstocks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/chem.201805219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468273PMC
February 2019

From Bugs to Bioplastics: Total (+)-Dihydrocarvide Biosynthesis by Engineered Escherichia coli.

Chembiochem 2019 03 21;20(6):785-792. Epub 2019 Jan 21.

School of Chemistry, Faculty of Science and Engineering, University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK.

The monoterpenoid lactone derivative (+)-dihydrocarvide ((+)-DHCD) can be polymerised to form shape-memory polymers. Synthetic biology routes from simple, inexpensive carbon sources are an attractive, alternative route over chemical synthesis from (R)-carvone. We have demonstrated a proof-of-principle in vivo approach for the complete biosynthesis of (+)-DHCD from glucose in Escherichia coli (6.6 mg L ). The pathway is based on the Mentha spicata route to (R)-carvone, with the addition of an 'ene'-reductase and Baeyer-Villiger cyclohexanone monooxygenase. Co-expression with a limonene synthesis pathway enzyme enables complete biocatalytic production within one microbial chassis. (+)-DHCD was successfully produced by screening multiple homologues of the pathway genes, combined with expression optimisation by selective promoter and/or ribosomal binding-site screening. This study demonstrates the potential application of synthetic biology approaches in the development of truly sustainable and renewable bioplastic monomers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201800606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6850611PMC
March 2019

Chemoenzymatic Synthesis of the Intermediates in the Peppermint Monoterpenoid Biosynthetic Pathway.

J Nat Prod 2018 07 6;81(7):1546-1552. Epub 2018 Jul 6.

BBSRC/EPSRC Manchester Synthetic Biology Research Centre for Fine and Speciality Chemicals (SYNBIOCHEM), Manchester Institute of Biotechnology and School of Chemistry , The University of Manchester , 131 Princess Street , Manchester M1 7DN , U.K.

A chemoenzymatic approach providing access to all four intermediates in the peppermint biosynthetic pathway between limonene and menthone/isomenthone, including noncommercially available intermediates (-)- trans-isopiperitenol (2), (-)-isopiperitenone (3), and (+)- cis-isopulegone (4), is described. Oxidation of (+)-isopulegol (13) followed by enolate selenation and oxidative elimination steps provides (-)-isopiperitenone (3). A chemical reduction and separation route from (3) provides both native (-)- trans-isopiperitenol (2) and isomer (-)- cis-isopiperitenol (18), while enzymatic conjugate reduction of (-)-isopiperitenone (3) with IPR [(-)-isopiperitenone reductase)] provides (+)- cis-isopulegone (4). This undergoes facile base-mediated chemical epimerization to (+)-pulegone (5), which is subsequently shown to be a substrate for NtDBR ( Nicotiana tabacum double-bond reductase) to afford (-)-menthone (7) and (+)-isomenthone (8).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jnatprod.7b01026DOI Listing
July 2018

Engineering the "Missing Link" in Biosynthetic (-)-Menthol Production: Bacterial Isopulegone Isomerase.

ACS Catal 2018 Mar 24;8(3):2012-2020. Epub 2018 Jan 24.

Manchester Centre for Fine and Speciality Chemicals (SYNBIOCHEM) and School of Chemistry, Manchester Institute of Biotechnology, University of Manchester, Manchester M1 7DN, United Kingdom.

The realization of a synthetic biology approach to microbial (1,2,5)-()-menthol () production relies on the identification of a gene encoding an isopulegone isomerase (IPGI), the only enzyme in the biosynthetic pathway as yet unidentified. We demonstrate that Δ5-3-ketosteroid isomerase (KSI) from can act as an IPGI, producing ()-(+)-pulegone (()-) from (+)--isopulegone (). Using a robotics-driven semirational design strategy, we identified a key KSI variant encoding four active site mutations, which confer a 4.3-fold increase in activity over the wild-type enzyme. This was assisted by the generation of crystal structures of four KSI variants, combined with molecular modeling of binding to identify key active site residue targets. The KSI variant was demonstrated to function efficiently within cascade biocatalytic reactions with downstream enzymes pulegone reductase and (-)-menthone:(-)-menthol reductase to generate from . This study introduces the use of a recombinant IPGI, engineered to function efficiently within a biosynthetic pathway for the production of in microorganisms.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acscatal.7b04115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5937688PMC
March 2018

Biocatalytic Routes to Lactone Monomers for Polymer Production.

Biochemistry 2018 04 22;57(13):1997-2008. Epub 2018 Mar 22.

Centre for Computational Chemistry, School of Chemistry , University of Bristol , Cantock's Close , Bristol BS8 1TS , U.K.

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMO) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMO. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMO for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.8b00169DOI Listing
April 2018

Retooling microorganisms for the fermentative production of alcohols.

Curr Opin Biotechnol 2018 04 6;50:1-10. Epub 2017 Sep 6.

Manchester Institute of Biotechnology, Department of Chemistry, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. Electronic address:

Bioengineering and synthetic biology approaches have revolutionised the field of biotechnology, enabling the introduction of non-native and de novo pathways for biofuels production. This 'retooling' of microorganisms is also applied to the utilisation of mixed carbon components derived from lignocellulosic biomass, a major technical barrier for the development of economically viable fermentations. This review will discuss recent advances in microorganism engineering for efficient production of alcohols from waste biomass. These advances span the introduction of new pathways to alcohols, host modifications for more cost-effective utilisation of lignocellulosic waste and modifications of existing pathways for generating new fuel additives.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.copbio.2017.08.010DOI Listing
April 2018

Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

Angew Chem Weinheim Bergstr Ger 2016 Aug 13;128(33):9748-9752. Epub 2016 Jul 13.

Manchester Institute of Biotechnology University of Manchester Manchester M1 7DN UK.

Three enzymes of the essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ange.201603785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988499PMC
August 2016

Pinpointing a Mechanistic Switch Between Ketoreduction and "Ene" Reduction in Short-Chain Dehydrogenases/Reductases.

Angew Chem Int Ed Engl 2016 08 13;55(33):9596-600. Epub 2016 Jul 13.

Manchester Institute of Biotechnology, University of Manchester, Manchester, M1 7DN, UK.

Three enzymes of the Mentha essential oil biosynthetic pathway are highly homologous, namely the ketoreductases (-)-menthone:(-)-menthol reductase and (-)-menthone:(+)-neomenthol reductase, and the "ene" reductase isopiperitenone reductase. We identified a rare catalytic residue substitution in the last two, and performed comparative crystal structure analyses and residue-swapping mutagenesis to investigate whether this determines the reaction outcome. The result was a complete loss of native activity and a switch between ene reduction and ketoreduction. This suggests the importance of a catalytic glutamate vs. tyrosine residue in determining the outcome of the reduction of α,β-unsaturated alkenes, due to the substrate occupying different binding conformations, and possibly also to the relative acidities of the two residues. This simple switch in mechanism by a single amino acid substitution could potentially generate a large number of de novo ene reductases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201603785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988501PMC
August 2016

A 'Plug and Play' Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffolds in .

ChemistrySelect 2016 Jun;1(9):1893-1896

SYNBIOCHEM, Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, Manchester M1 7DN (UK).

The terpenoids constitute one of the largest and most diverse classes of natural compounds with applications as pharmaceuticals, flavorings and fragrances, pesticides and biofuels. Synthetic biology is ideally placed to create new routes to this chemical diversity and facilitation of new compound discovery. The C10 monoterpenoids display a huge structural diversity produced from a single substrate, geranyl diphosphate, by a family of monoterpene cyclases and synthases (mTC/S). Here we employ a library of mTC/S in a single 'plug and play' platform system for the production of over 30 different monoterpenoids in Escherichia coli by fermentation on glucose. These products include several compounds never before produced in engineered microbes demonstrating the power of this approach to rapidly create routes to structural diversity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/slct.201600563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947754PMC
June 2016

Light-driven biocatalytic reduction of α,β-unsaturated compounds by ene reductases employing transition metal complexes as photosensitizers.

Catal Sci Technol 2016 Jan 26;6(1):169-177. Epub 2015 Oct 26.

Manchester Institute of Biotechnology , Faculty of Life Sciences , University of Manchester , 131 Princess Street , Manchester , M1 7DN , UK . Email:

Efficient and cost effective nicotinamide cofactor regeneration is essential for industrial-scale bio-hydrogenations employing flavin-containing biocatalysts such as the Old Yellow Enzymes. A direct flavin regeneration system using visible light to initiate a photoredox cycle and drive biocatalysis is described, and shown to be effective in driving biocatalytic activated alkene reduction. Using Ru(ii) or Ir(iii) complexes as photosensitizers, coupled with an electron transfer mediator (methyl viologen) and sacrificial electron donor (triethanolamine) drives catalytic turnover of two Old Yellow Enzymes with multiple oxidative substrates. Therefore, there is great potential in the development of light-driven biocatalytic systems, providing an alternative to the reliance on enzyme-based cofactor regeneration systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5cy01642hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4786955PMC
January 2016

Enzymatic Menthol Production: One-Pot Approach Using Engineered Escherichia coli.

ACS Synth Biol 2015 Oct 5;4(10):1112-23. Epub 2015 Jun 5.

Medicines Research Centre, GlaxoSmithKline , Gunnel's Wood Road, Stevenage, Herts SG1 2NY, U.K.

Menthol isomers are high-value monoterpenoid commodity chemicals, produced naturally by mint plants, Mentha spp. Alternative clean biosynthetic routes to these compounds are commercially attractive. Optimization strategies for biocatalytic terpenoid production are mainly focused on metabolic engineering of the biosynthesis pathway within an expression host. We circumvent this bottleneck by combining pathway assembly techniques with classical biocatalysis methods to engineer and optimize cell-free one-pot biotransformation systems and apply this strategy to the mint biosynthesis pathway. Our approach allows optimization of each pathway enzyme and avoidance of monoterpenoid toxicity issues to the host cell. We have developed a one-pot (bio)synthesis of (1R,2S,5R)-(-)-menthol and (1S,2S,5R)-(+)-neomenthol from pulegone, using recombinant Escherichia coli extracts containing the biosynthetic genes for an "ene"-reductase (NtDBR from Nicotiana tabacum) and two menthone dehydrogenases (MMR and MNMR from Mentha piperita). Our modular engineering strategy allowed each step to be optimized to improve the final production level. Moderate to highly pure menthol (79.1%) and neomenthol (89.9%) were obtained when E. coli strains coexpressed NtDBR with only MMR or MNMR, respectively. This one-pot biocatalytic method allows easier optimization of each enzymatic step and easier modular combination of reactions to ultimately generate libraries of pure compounds for use in high-throughput screening. It will be, therefore, a valuable addition to the arsenal of biocatalysis strategies, especially when applied for (semi)-toxic chemical compounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssynbio.5b00092DOI Listing
October 2015

New developments in 'ene'-reductase catalysed biological hydrogenations.

Curr Opin Chem Biol 2014 Apr 6;19:107-15. Epub 2014 Mar 6.

Manchester Institute of Biotechnology, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK. Electronic address:

Asymmetric biocatalytic hydrogenations are important reactions performed primarily by members of the Old Yellow Enzyme family. These reactions have great potential in the chemosynthesis of a variety of industrially useful synthons due to the generation of up to two stereogenic centres. In this review, additional enzyme classes capable of asymmetric hydrogenations will be discussed, as will examples of multienzyme cascading reactions. New and improved technology that enhances the commercial viability of biotransformations are included, such as the nicotinamide coenzyme-independent reactions. This review will focus on progress in this field within the last two years, with emphasis on industrial applications of this technology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cbpa.2014.01.019DOI Listing
April 2014

Nanofibrillar Peptide hydrogels for the immobilization of biocatalysts for chemical transformations.

Macromol Rapid Commun 2014 May 7;35(9):868-74. Epub 2014 Mar 7.

School of Chemical Engineering and Analytical Science, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester, M1, 7DN, UK.

Enzymes are attractive, "green" alternatives to chemical catalysts within the industrial sector, but their robustness to environmental conditions needs optimizing. Here, an enzyme is tagged chemically and recombinantly with a self-assembling peptide that allows the conjugate to spontaneously assemble with pure peptide to form β-sheet-rich nanofibers decorated with tethered enzyme. Above a critical concentration, these fibers entangle and form a 3D hydrogel. The immobilized enzyme catalyzes chemical transformations and critically its stability is increased significantly where it retains activity after exposure to high temperatures (90 °C) and long storage times (up to 12 months).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/marc.201400027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4316184PMC
May 2014

Biocatalytic Asymmetric Alkene Reduction: Crystal Structure and Characterization of a Double Bond Reductase from .

ACS Catal 2013 Mar 21;3(3):370-379. Epub 2013 Jan 21.

Manchester Institute of Biotechnology, School of Chemistry, and Faculty of Life Sciences, University of Manchester , Manchester, U.K.

The application of biocatalysis for the asymmetric reduction of activated C=C is a powerful tool for the manufacture of high-value chemical commodities. The biocatalytic potential of "-ene" reductases from the Old Yellow Enzyme (OYE) family of oxidoreductases is well-known; however, the specificity of these enzymes toward mainly small molecule substrates has highlighted the need to discover "-ene" reductases from different enzymatic classes to broaden industrial applicability. Here, we describe the characterization of a flavin-free double bond reductase from (NtDBR), which belongs to the leukotriene B dehydrogenase (LTD) subfamily of the zinc-independent, medium chain dehydrogenase/reductase superfamily of enzymes. Using steady-state kinetics and biotransformation reactions, we have demonstrated the regio- and stereospecificity of NtDBR against a variety of α,β-unsaturated activated alkenes. In addition to catalyzing the reduction of typical LTD substrates and several classical OYE-like substrates, NtDBR also exhibited complementary activity by reducing non-OYE substrates (i.e., reducing the exocyclic C=C double bond of ()-pulegone) and in some cases showing an opposite stereopreference in comparison with the OYE family member pentaerythritol tetranitrate (PETN) reductase. This serves to augment classical OYE "-ene" reductase activity and, coupled with its aerobic stability, emphasizes the potential industrial value of NtDBR. Furthermore, we also report the X-ray crystal structures of the holo-, binary NADP(H)-bound, and ternary [NADP and 4-hydroxy-3-methoxycinnamaldehyde ()-bound] NtDBR complexes. These will underpin structure-driven site-saturated mutagenesis studies aimed at enhancing the reactivity, stereochemistry, and specificity of this enzyme.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cs300709mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4990313PMC
March 2013

A surprising observation that oxygen can affect the product enantiopurity of an enzyme-catalysed reaction.

FEBS J 2012 Nov 12;279(22):4160-71. Epub 2012 Oct 12.

Manchester Interdisciplinary Biocentre and School of Chemistry, University of Manchester, UK.

Enzymes are natural catalysts, controlling reactions with typically high stereospecificity and enantiospecificity in substrate selection and/or product formation. This makes them useful in the synthesis of industrially relevant compounds, particularly where highly enantiopure products are required. The flavoprotein pentaerythritol tetranitrate (PETN) reductase is a member of the Old Yellow Enzyme family, and catalyses the asymmetric reduction of β-alkyl-β-arylnitroalkenes. Under aerobic conditions, it additionally undergoes futile cycles of NAD(P)H reduction of flavin, followed by reoxidation by oxygen, which generates the reactive oxygen species (ROS) hydrogen peroxide and superoxide. Prior studies have shown that not all reactions catalysed by PETN reductase yield enantiopure products, such as the reduction of (E)-2-phenyl-1-nitroprop-1-ene (PNE) to produce (S)-2-phenyl-1-nitropropane (PNA) with variable enantiomeric excess (ee). Recent independent studies of (E)-PNE reduction by PETN reductase showed that the major product formed could be switched to (R)-PNA, depending on the reaction conditions. We investigated this phenomenon, and found that the presence of oxygen and ROS influenced the overall product enantiopurity. Anaerobic reactions produced consistently higher nitroalkane (S)-PNA product yields than aerobic reactions (64% versus 28%). The presence of oxygen dramatically increased the preference for (R)-PNA formation (up to 52% ee). Conversely, the presence of the ROS superoxide and hydrogen peroxide switched the preference to (S)-PNA product formation. Given that oxygen has no role in the natural catalytic cycle, these findings demonstrate a remarkable ability to manipulate product enantiopurity of this enzyme-catalysed reaction by simple manipulation of reaction conditions. Potential mechanisms of this unusual behaviour are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.12008DOI Listing
November 2012

ELDOR spectroscopy reveals that energy landscapes in human methionine synthase reductase are extensively remodelled following ligand and partner protein binding.

Chembiochem 2011 Apr 25;12(6):863-7. Epub 2011 Feb 25.

Faculty of Life Sciences and Manchester Interdisciplinary Biocentre, University of Manchester, Manchester, UK.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201000630DOI Listing
April 2011

A site-saturated mutagenesis study of pentaerythritol tetranitrate reductase reveals that residues 181 and 184 influence ligand binding, stereochemistry and reactivity.

Chembiochem 2011 Mar 4;12(5):738-49. Epub 2011 Mar 4.

Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

We have conducted a site-specific saturation mutagenesis study of H181 and H184 of flavoprotein pentaerythritol tetranitrate reductase (PETN reductase) to probe the role of these residues in substrate binding and catalysis with a variety of α,β-unsaturated alkenes. Single mutations at these residues were sufficient to dramatically increase the enantiopurity of products formed by reduction of 2-phenyl-1-nitropropene. In addition, many mutants exhibited a switch in reactivity to predominantly catalyse nitro reduction, as opposed to CC reduction. These mutants showed an enhancement in a minor side reaction and formed 2-phenylpropanal oxime from 2-phenyl-1-nitropropene. The multiple binding conformations of hydroxy substituted nitro-olefins in PETN reductase were examined by using both structural and catalytic techniques. These compounds were found to bind in both active and inhibitory complexes; this highlights the plasticity of the active site and the ability of the H181/H184 couple to coordinate with multiple functional groups. These properties demonstrate the potential to use PETN reductase as a scaffold in the development of industrially useful biocatalysts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201000662DOI Listing
March 2011

Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries.

Chembiochem 2010 Nov;11(17):2433-47

Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201000527DOI Listing
November 2010

Biocatalysis with thermostable enzymes: structure and properties of a thermophilic 'ene'-reductase related to old yellow enzyme.

Chembiochem 2010 Jan;11(2):197-207

Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.

We report the crystal structure of a thermophilic "ene" reductase (TOYE) isolated from Thermoanaerobacter pseudethanolicus E39. The crystal structure reveals a tetrameric enzyme and an active site that is relatively large compared to most other structurally determined and related Old Yellow Enzymes. The enzyme adopts higher order oligomeric states (octamers and dodecamers) in solution, as revealed by sedimentation velocity and multiangle laser light scattering. Bead modelling indicates that the solution structure is consistent with the basic tetrameric structure observed in crystallographic studies and electron microscopy. TOYE is stable at high temperatures (T(m)>70 degrees C) and shows increased resistance to denaturation in water-miscible organic solvents compared to the mesophilic Old Yellow Enzyme family member, pentaerythritol tetranitrate reductase. TOYE has typical ene-reductase properties of the Old Yellow Enzyme family. There is currently major interest in using Old Yellow Enzyme family members in the preparative biocatalysis of a number of activated alkenes. The increased stability of TOYE in organic solvents is advantageous for biotransformations in which water-miscible organic solvents and biphasic reaction conditions are required to both deliver novel substrates and minimize product racemisation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.200900570DOI Listing
January 2010

Reduction of aliphatic nitroesters and N-nitramines by Enterobacter cloacae PB2 pentaerythritol tetranitrate reductase: quantitative structure-activity relationships.

FEBS J 2008 Dec 5;275(24):6192-203. Epub 2008 Nov 5.

Institute of Biochemistry, Vilnius, Lithuania.

Enterobacter cloacae PB2 NADPH:pentaerythritol tetranitrate reductase (PETNR) performs the biodegradation of explosive organic nitrate esters via their reductive denitration. In order to understand the enzyme substrate specificity, we have examined the reactions of PETNR with organic nitrates (n = 15) and their nitrogen analogues, N-nitramines (n = 4). The reactions of these compounds with PETNR were accompanied by the release of 1-2 mol of nitrite per mole of compound, but were not accompanied by their redox cycling and superoxide formation. The reduction rate constants (k(cat)/K(m)) of inositol hexanitrate, diglycerol tetranitrate, erythritol tetranitrate, mannitol hexanitrate and xylitol pentanitrate were similar to those of the established PETNR substrates, PETN and glycerol trinitrate, whereas the reactivities of hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine were three orders of magnitude lower. The log k(cat)/K(m) value of the compounds increased with a decrease in the enthalpy of formation of the hydride adducts [DeltaH(f)(R-O-N(OH)O(-)) or DeltaH(f)(R(1),R(2) > N-N(OH)O(-))], and with an increase in their lipophilicity (octanol/water partition coefficient, log P(ow)), and did not depend on their van der Waals' volumes. Hydrophobic organic nitroesters and hydrophilic N-nitramines compete for the same binding site in the reduced enzyme form. The role of the hydrophobic interaction of PETNR with glycerol trinitrate was supported by the positive dependence of glycerol trinitrate reactivity on the solution ionic strength. The discrimination of nitroesters and N-nitramines according to their log P(ow) values seems to be a specific feature of the Old Yellow Enzyme family of flavoenzymes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2008.06744.xDOI Listing
December 2008

Structure-Based Insight into the Asymmetric Bioreduction of the C=C Double Bond of alpha,beta-Unsaturated Nitroalkenes by Pentaerythritol Tetranitrate Reductase.

Adv Synth Catal 2008 Nov;350(17):2789-2803

Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, U.K.

Biocatalytic reduction of alpha- or beta-alkyl-beta-arylnitroalkenes provides a convenient and efficient method to prepare chiral substituted nitroalkanes. Pentaerythritol tetranitrate reductase (PETN reductase) from Enterobacter cloacae st. PB2 catalyses the reduction of nitroolefins such as 1-nitrocyclohexene (1) with steady state and rapid reaction kinetics comparable to other old yellow enzyme homologues. Furthermore, it reduces 2-aryl-1-nitropropenes (4a-d) to their equivalent (S)-nitropropanes 9a-d. The enzyme shows a preference for the (Z)-isomer of substrates 4a-d, providing almost pure enantiomeric products 9a-d (ees up to > 99%) in quantitative yield, whereas the respective (E)-isomers are reduced with lower enantioselectivity (63-89% ee) and lower product yields. 1-Aryl-2-nitropropenes (5a, b) are also reduced efficiently, but the products (R)-10 have lower optical purities. The structure of the enzyme complex with 1-nitrocyclohexene (1) was determined by X-ray crystallography, revealing two substrate-binding modes, with only one compatible with hydride transfer. Models of nitropropenes 4 and 5 in the active site of PETN reductase predicted that the enantioselectivity of the reaction was dependent on the orientation of binding of the (E)- and (Z)-substrates. This work provides a structural basis for understanding the mechanism of asymmetric bioreduction of nitroalkenes by PETN reductase.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/adsc.200800561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2854801PMC
November 2008

Novel haem co-ordination variants of flavocytochrome P450BM3.

Biochem J 2009 Jan;417(1):65-76

Manchester Interdisciplinary Biocentre, University of Manchester, Faculty of Life Sciences, 131 Princess Street, Manchester M17DN, UK.

Bacillus megaterium flavocytochrome P450 BM3 is a catalytically self-sufficient fatty acid hydroxylase formed by fusion of soluble NADPH-cytochrome P450 reductase and P450 domains. Selected mutations at residue 264 in the haem (P450) domain of the enzyme lead to novel amino acid sixth (distal) co-ordination ligands to the haem iron. The catalytic, spectroscopic and thermodynamic properties of the A264M, A264Q and A264C variants were determined in both the intact flavocytochromes and haem domains of P450 BM3. Crystal structures of the mutant haem domains demonstrate axial ligation of P450 haem iron by methionine and glutamine ligands trans to the cysteine thiolate, creating novel haem iron ligand sets in the A264M/Q variants. In contrast, the crystal structure of the A264C variant reveals no direct interaction between the introduced cysteine side chain and the haem, although EPR data indicate Cys(264) interactions with haem iron in solution. The A264M haem potential is elevated by comparison with wild-type haem domain, and substrate binding to the A264Q haem domain results in a approximately 360 mV increase in potential. All mutant haem domains occupy the conformation adopted by the substrate-bound form of wild-type BM3, despite the absence of added substrate. The A264M mutant (which has higher dodecanoate affinity than wild-type BM3) co-purifies with a structurally resolved lipid. These data demonstrate that a single mutation at Ala(264) is enough to perturb the conformational equilibrium between substrate-free and substrate-bound P450 BM3, and provide firm structural and spectroscopic data for novel haem iron ligand sets unprecedented in nature.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BJ20081133DOI Listing
January 2009

Dynamics driving function: new insights from electron transferring flavoproteins and partner complexes.

FEBS J 2007 Nov 17;274(21):5481-504. Epub 2007 Oct 17.

Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester, UK.

Electron transferring flavoproteins (ETFs) are soluble heterodimeric FAD-containing proteins that function primarily as soluble electron carriers between various flavoprotein dehydrogenases. ETF is positioned at a key metabolic branch point, responsible for transferring electrons from up to 10 primary dehydrogenases to the membrane-bound respiratory chain. Clinical mutations of ETF result in the often fatal disease glutaric aciduria type II. Structural and biophysical studies of ETF in complex with partner proteins have shown that ETF partitions the functions of partner binding and electron transfer between (a) a 'recognition loop', which acts as a static anchor at the ETF-partner interface, and (b) a highly mobile redox-active FAD domain. Together, this enables the FAD domain of ETF to sample a range of conformations, some compatible with fast interprotein electron transfer. This 'conformational sampling' enables ETF to recognize structurally distinct partners, whilst also maintaining a degree of specificity. Complex formation triggers mobility of the FAD domain, an 'induced disorder' mechanism contrasting with the more generally accepted models of protein-protein interaction by induced fit mechanisms. We discuss the implications of the highly dynamic nature of ETFs in biological interprotein electron transfer. ETF complexes point to mechanisms of electron transfer in which 'dynamics drive function', a feature that is probably widespread in biology given the modular assembly and flexible nature of biological electron transfer systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2007.06107.xDOI Listing
November 2007

Mechanism of coenzyme binding to human methionine synthase reductase revealed through the crystal structure of the FNR-like module and isothermal titration calorimetry.

Biochemistry 2007 Oct 25;46(42):11833-44. Epub 2007 Sep 25.

Faculty of Life Sciences, University of Manchester, Manchester Interdisciplinary Biocentre, 131 Princess Street, Manchester M1 7DN, United Kingdom.

Human methionine synthase reductase (MSR) is a 78 kDa flavoprotein that regenerates the active form of cobalamin-dependent methionine synthase (MS). MSR contains one FAD and one FMN cofactor per polypeptide and functions in the sequential transfer of reducing equivalents from NADPH to MS via its flavin centers. We report the 1.9 A crystal structure of the NADP+-bound FNR-like module of MSR that spans the NADP(H)-binding domain, the FAD-binding domain, the connecting domain, and part of the extended hinge region, a feature unique to MSR. The overall fold of the protein is similar to that of the corresponding domains of the related diflavin reductase enzymes cytochrome P450 reductase and neuronal nitric oxide synthase (NOS). However, the extended hinge region of MSR, which is positioned between the NADP(H)/FAD- and FMN-binding domains, is in an unexpected orientation with potential implications for the mechanism of electron transfer. Compared with related flavoproteins, there is structural variation in the NADP(H)-binding site, in particular regarding those residues that interact with the 2'-phosphate and the pyrophosphate moiety of the coenzyme. The lack of a conserved binding determinant for the 2'-phosphate does not weaken the coenzyme specificity for NADP(H) over NAD(H), which is within the range expected for the diflavin oxidoreductase family of enzymes. Isothermal titration calorimetry reveals a binding constant of 37 and 2 microM for binding of NADP+ and 2',5'-ADP, respectively, for the ligand-protein complex formed with full-length MSR or the isolated FNR module. These values are consistent with Ki values (36 microM for NADP+ and 1.4 microM for 2',5'-ADP) obtained from steady-state inhibition studies. The relatively weaker binding of NADP+ to MSR compared with other members of the diflavin oxidoreductase family might arise from unique electrostatic repulsive forces near the 5'-pyrophosphate moiety and/or increased hydrophobic stacking between Trp697 and the re face of the FAD isoalloxazine ring. Small structural permutations within the NADP(H)-binding cleft have profound affects on coenzyme binding, which likely retards catalytic turnover of the enzyme in the cell. The biological implications of an attenuated mechanism of MS reactivation by MSR on methionine and folate metabolism are discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi701209pDOI Listing
October 2007

Crystal structure and solution characterization of the activation domain of human methionine synthase.

FEBS J 2007 Feb;274(3):738-50

Faculty of Life Sciences, University of Manchester, UK.

Human methionine synthase (hMS) is a multidomain cobalamin-dependent enzyme that catalyses the conversion of homocysteine to methionine by methyl group transfer. We report here the 1.6 A crystal structure of the C-terminal activation domain of hMS. The structure is C-shaped with the core comprising mixed alpha and beta regions, dominated by a twisted antiparallel beta sheet with a beta-meander region. These features, including the positions of the active-site residues, are similar to the activation domain of Escherichia coli cobalamin-dependent MS (MetH). Structural and solution studies suggest a small proportion of hMS activation domain exists in a dimeric form, which contrasts with the monomeric form of the E. coli homologue. Fluorescence studies show that human activation domain interacts with the FMN-binding domain of human methionine synthase reductase (hMSR). This interaction is enhanced in the presence of S-adenosyl-methionine. Binding of the D963E/K1071N mutant activation domain to the FMN domain of MSR is weaker than with wild-type activation domain. This suggests that one or both of the residues D963 and K1071 are important in partner binding. Key differences in the sequences and structures of hMS and MetH activation domains are recognized and include a major reorientation of an extended 3(10)-containing loop in the human protein. This structural alteration might reflect differences in their respective reactivation complexes and/or potential for dimer formation. The reported structure is a component of the multidomain hMS : MSR complex, and represents an important step in understanding the impact of clinical mutations and polymorphisms in this key electron transfer complex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1742-4658.2006.05618.xDOI Listing
February 2007

Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3.

J Biol Chem 2007 Jan 31;282(1):564-72. Epub 2006 Oct 31.

School of Chemical Engineering and Analytical Science, Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, UK.

Two novel P450 heme iron ligand sets were generated by directed mutagenesis of the flavocytochrome P450 BM3 heme domain. The A264H and A264K variants produce Cys-Fe-His and Cys-Fe-Lys axial ligand sets, which were validated structurally and characterized by spectroscopic analysis. EPR and magnetic circular dichroism (MCD) provided fingerprints defining these P450 ligand sets. Near IR MCD spectra identified ferric low spin charge-transfer bands diagnostic of the novel ligands. For the A264K mutant, this is the first report of a Cys-Fe-Lys near-IR MCD band. Crystal structure determination showed that substrate-free A264H and A264K proteins crystallize in distinct conformations, as observed previously in substrate-free and fatty acid-bound wild-type P450 forms, respectively. This, in turn, likely reflects the positioning of the I alpha helix section of the protein that is required for optimal configuration of the ligands to the heme iron. One of the monomers in the asymmetric unit of the A264H crystals was in a novel conformation with a more open substrate access route to the active site. The same species was isolated for the wildtype heme domain and represents a novel conformational state of BM3 (termed SF2). The "locking" of these distinct conformations is evident from the fact that the endogenous ligands cannot be displaced by substrate or exogenous ligands. The consequent reduction of heme domain conformational heterogeneity will be important in attempts to determine atomic structure of the full-length, multidomain flavocytochrome, and thus to understand in atomic detail interactions between its heme and reductase domains.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M607949200DOI Listing
January 2007

Stabilization of non-productive conformations underpins rapid electron transfer to electron-transferring flavoprotein.

J Biol Chem 2005 Aug 23;280(34):30361-6. Epub 2005 Jun 23.

Department of Biochemistry, University of Leicester, Henry Wellcome Building, Lancaster Road, LE1 7RH, Leicester United Kingdom.

Crystal structures of protein complexes with electron-transferring flavoprotein (ETF) have revealed a dual protein-protein interface with one region serving as anchor while the ETF FAD domain samples available space within the complex. We show that mutation of the conserved Glu-165beta in human ETF leads to drastically modulated rates of interprotein electron transfer with both medium chain acyl-CoA dehydrogenase and dimethylglycine dehydrogenase. The crystal structure of free E165betaA ETF is essentially identical to that of wild-type ETF, but the crystal structure of the E165betaA ETF.medium chain acyl-CoA dehydrogenase complex reveals clear electron density for the FAD domain in a position optimal for fast interprotein electron transfer. Based on our observations, we present a dynamic multistate model for conformational sampling that for the wild-type ETF. medium chain acyl-CoA dehydrogenase complex involves random motion between three distinct positions for the ETF FAD domain. ETF Glu-165beta plays a key role in stabilizing positions incompatible with fast interprotein electron transfer, thus ensuring high rates of complex dissociation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M505562200DOI Listing
August 2005