Publications by authors named "Jennifer A Littlechild"

54 Publications

Structural Insights into a Novel Esterase from the East Pacific Rise and Its Improved Thermostability by a Semirational Design.

J Agric Food Chem 2021 Jan 14;69(3):1079-1090. Epub 2021 Jan 14.

Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Science, Fujian Normal University, Fuzhou 350117, China.

Lipolytic enzymes are essential biocatalysts in food processing as well as pharmaceutical and pesticide industries, catalyzing the cleavage of ester bonds in a variety of acyl chain substrates. Here, we report the crystal structure of an esterase from the deep-sea hydrothermal vent of the East Pacific Rise (EprEst). The X-ray structure of EprEst in complex with the ligand, acetate, has been determined at 2.03 Å resolution. The structure reveals a unique spatial arrangement and orientation of the helix cap domain and α/β hydrolase domain, which form a substrate pocket with preference for short-chain acyl groups. Molecular docking analysis further demonstrated that the active site pocket could accommodate -nitrophenyl (NP) carboxyl ligands of varying lengths (≤6 C atoms), with NP-butyrate ester predicted to have the highest binding affinity. Additionally, the semirational design was conducted to improve the thermostability of EprEst by enzyme engineering based on the established structure and multiple sequence alignment. A mutation, K114P, introduced in the hinge region of the esterase, which displayed increased thermostability and enzyme activity. Collectively, the structural and functional data obtained herein could be used as basis for further protein engineering to ultimately expand the scope of industrial applications of marine-derived lipolytic enzymes.
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http://dx.doi.org/10.1021/acs.jafc.0c06338DOI Listing
January 2021

A 'Split-Gene' Transketolase From the Hyper-Thermophilic Bacterium : Structure and Biochemical Characterization.

Front Microbiol 2020 30;11:592353. Epub 2020 Oct 30.

Henry Wellcome Building for Biocatalysis, Biosciences, University of Exeter, Exeter, United Kingdom.

A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a 'split-gene' identified in the genome of the hyperthermophilic bacterium, . The reconstituted active αβ tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted αβ tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β protein has been determined to 1.9 Å resolution. The structure of the active reconstituted αβ enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted 'split-gene' transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.
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http://dx.doi.org/10.3389/fmicb.2020.592353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7661550PMC
October 2020

Structural insights into the NAD-dependent formate dehydrogenase mechanism revealed from the NADH complex and the formate NAD ternary complex of the Chaetomium thermophilum enzyme.

J Struct Biol 2020 12 24;212(3):107657. Epub 2020 Oct 24.

Henry Wellcome Centre for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Electronic address:

The removal of carbon dioxide from the waste streams of industrial processes is a major challenge for creation of a sustainable circular economy. This makes the synthesis of formate from CO by NAD dependent formate dehydrogenases (FDHs) an attractive process for this purpose. The efficiency of this reaction is however low and to achieve a viable industrial process an optimised engineered enzyme needs to be developed. In order to understand the detailed enzymatic mechanism of catalysis structures of different cofactor and substrate complexes of the FDH from the thermophilic filamentous fungus, Chaetomium thermophilum have been determined to 1.2-1.3 Å resolution. The substrate formate is shown to be held by four hydrogen bonds in the FDH catalytic site within the ternary complex with substrate and NADand a secondary formate binding site is observed in crystals soaked with substrate. Water molecules are excluded from the FDH catalytic site when the substrate is bound. The angle between the plane of the NAD cofactor pyridine ring and the plane of the formate molecule is around 27°. Additionally, structures of a FDH mutant enzyme, N120C, in complex with the reduced form of the cofactor have also been determined both in the presence and absence of formate bound at the secondary site. These structures provide further understanding of the catalytic mechanism of this fungal enzyme.
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http://dx.doi.org/10.1016/j.jsb.2020.107657DOI Listing
December 2020

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family.

Plant J 2020 08 13;103(3):1215-1232. Epub 2020 Jun 13.

Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Science, Fujian Normal University, Fuzhou, 350117, P. R. China.

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium-dependent membrane-binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5-Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca -binding region in C2A domain is longer than classical C2 domains and a novel Ca binding site in the vWA domain. The structure of BON1 bound to Mn is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid-binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca . These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.
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http://dx.doi.org/10.1111/tpj.14797DOI Listing
August 2020

Using enzyme cascades in biocatalysis: Highlight on transaminases and carboxylic acid reductases.

Biochim Biophys Acta Proteins Proteom 2020 02 16;1868(2):140322. Epub 2019 Nov 16.

Living Systems Institute, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.. Electronic address:

Biocatalysis, the use of enzymes in chemical transformations, is an important green chemistry tool. Cascade reactions combine different enzyme activities in a sequential set of reactions. Cascades can occur within a living (usually bacterial) cell; in vitro in 'one pot' systems where the desired enzymes are mixed together to carry out the multi-enzyme reaction; or using microfluidic systems. Microfluidics offers particular advantages when the product of the reaction inhibits the enzyme(s). In vitro systems allow variation of different enzyme concentrations to optimise the metabolic 'flux', and the addition of enzyme cofactors as required. Cascades including cofactor recycling systems and modelling approaches are being developed to optimise cascades for wider industrial scale use. Two industrially important enzymes, transaminases and carboxylic acid reductases are used as examples regarding their applications in cascade reactions with other enzyme classes to obtain important synthons of pharmaceutical interest.
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http://dx.doi.org/10.1016/j.bbapap.2019.140322DOI Listing
February 2020

Engineering a Seven Enzyme Biotransformation using Mathematical Modelling and Characterized Enzyme Parts.

ChemCatChem 2019 Aug 4;11(15):3474-3489. Epub 2019 Jul 4.

Department of Biosciences University of Exeter Henry Wellcome Building for Biocatalysis Stocker Road Exeter EX4 4QD UK.

Multi-step enzyme reactions offer considerable cost and productivity benefits. Process models offer a route to understanding the complexity of these reactions, and allow for their optimization. Despite the increasing prevalence of multi-step biotransformations, there are few examples of process models for enzyme reactions. From a toolbox of characterized enzyme parts, we demonstrate the construction of a process model for a seven enzyme, three step biotransformation using isolated enzymes. Enzymes for cofactor regeneration were employed to make this in vitro reaction economical. Good modelling practice was critical in evaluating the impact of approximations and experimental error. We show that the use and validation of process models was instrumental in realizing and removing process bottlenecks, identifying divergent behavior, and for the optimization of the entire reaction using a genetic algorithm. We validated the optimized reaction to demonstrate that complex multi-step reactions with cofactor recycling involving at least seven enzymes can be reliably modelled and optimized.
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http://dx.doi.org/10.1002/cctc.201900646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774274PMC
August 2019

Structural basis for the Target DNA recognition and binding by the MYB domain of phosphate starvation response 1.

FEBS J 2019 07 24;286(14):2809-2821. Epub 2019 Apr 24.

Provincial University Key Laboratory of Cellular Stress Response and Metabolic Regulation, College of Life Science, Fujian Normal University, Fuzhou, China.

The phosphate starvation response 1 (PHR1) protein has a central role in mediating the response to phosphate starvation in plants. PHR1 is composed of a number of domains including a MYB domain involved with DNA binding and a coiled-coil domain proposed to be involved with dimer formation. PHR1 binds to the promoter of phosphate starvation-induced genes to control the levels of phosphate required for nutrition. Previous studies have shown that both the MYB domain and the coiled-coil domain of PHR1 are required for binding the target DNA. Here, we describe the crystal structure of the PHR1 MYB domain and two structures of its complex with the PHR1-binding DNA sequence (P1BS). Structural and isothermal titration calorimetry has been carried out showing that the MYB domain of PHR1 alone is sufficient for target DNA recognition and binding. Two copies of the PHR1 MYB domain bind to the same major groove of the P1BS DNA with few direct interactions between the individual MYB domains. In addition, the PHR1 MYB-P1BS DNA complex structures reveal amino acid residues involved in DNA recognition and binding. Mutagenesis of these residues results in lost or impaired ability of PHR1 MYB to bind to its target DNA. The results presented reveal the structural basis for DNA recognition by the PHR1 MYB domain and demonstrate that two PHR1 MYB domains attach to their P1BS DNA targeting sequence. DATABASE: Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 6J4K (PHR1 MYB), 6J4R (PHR1 MYB-R-P1BS), 6J5B (MYB-CC-R2-P1BS).
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http://dx.doi.org/10.1111/febs.14846DOI Listing
July 2019

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea and : Biochemical and Structural Characterization.

Front Bioeng Biotechnol 2019 24;7. Epub 2019 Jan 24.

Henry Wellcome Building for Biocatalysis, Biosciences, University of Exeter, Exeter, United Kingdom.

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, , and . These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to ()-methylbenzylamine but no activity to ()-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the enzyme and 2.0 Å for the enzyme. Also the enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another () selective class IV transaminase from the fungus which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.
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http://dx.doi.org/10.3389/fbioe.2019.00007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6353796PMC
January 2019

X-ray structure of Fasciola hepatica Sigma class glutathione transferase 1 reveals a disulfide bond to support stability in gastro-intestinal environment.

Sci Rep 2019 01 29;9(1):902. Epub 2019 Jan 29.

Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.

Sigma class GST (Prostaglandin D synthase), FhGST-S1, is present in the excretory-secretory products (ES) of the liver fluke parasite Fasciola hepatica as cargo of extracellular vesicles (EVs) released by the parasite. FhGST-S1 has a well characterised role in the modulation of the immune response; a key fluke intercession that allows for establishment and development within their hosts. We have resolved the three-dimensional structure of FhGST-S1 in complex with its co-factor glutathione, in complex with a glutathione-cysteine adduct, and in a glutathione disulfide complex in order to initiate a research pipeline to mechanistically understand how FhGST-S1 functions within the host environment and to rationally design selective inhibitors. The overall fold of FhGST-S1 shows high structural similarity to other Sigma class GSTs. However, a unique interdomain disulfide bond was found in the FhGST-S1 which could stabilise the structure within the host gastro-intestinal environment. The position of the two domains of the protein with respect to each other is seen to be crucial in the formation of the active site cleft of the enzyme. The interdomain disulfide bond raises the possibility of oxidative regulation of the active site of this GST protein.
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http://dx.doi.org/10.1038/s41598-018-37531-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6351632PMC
January 2019

New Thermophilic α/β Class Epoxide Hydrolases Found in Metagenomes From Hot Environments.

Front Bioeng Biotechnol 2018 16;6:144. Epub 2018 Oct 16.

Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milan, Italy.

Two novel epoxide hydrolases (EHs), Sibe-EH and CH65-EH, were identified in the metagenomes of samples collected in hot springs in Russia and China, respectively. The two α/β hydrolase superfamily fold enzymes were cloned, over-expressed in , purified and characterized. The new EHs were active toward a broad range of substrates, and in particular, Sibe-EH was excellent in the desymmetrization of -2,3-epoxybutane producing the (2,3)-diol product with exceeding 99%. Interestingly these enzymes also hydrolyse (4)-limonene-1,2-epoxide with Sibe-EH being specific for the isomer. The Sibe-EH is a monomer in solution whereas the CH65-EH is a dimer. Both enzymes showed high melting temperatures with the CH65-EH being the highest at 85°C retaining 80% of its initial activity after 3 h thermal treatment at 70°C making it the most thermal tolerant wild type epoxide hydrolase described. The Sibe-EH and CH65-EH have been crystallized and their structures determined to high resolution, 1.6 and 1.4 Å, respectively. The CH65-EH enzyme forms a dimer via its cap domains with different relative orientation of the monomers compared to previously described EHs. The entrance to the active site cavity is located in a different position in CH65-EH and Sibe-EH in relation to other known bacterial and mammalian EHs.
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http://dx.doi.org/10.3389/fbioe.2018.00144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198070PMC
October 2018

Structural characterization of geranylgeranyl pyrophosphate synthase GACE1337 from the hyperthermophilic archaeon Geoglobus acetivorans.

Extremophiles 2018 Nov 30;22(6):877-888. Epub 2018 Jul 30.

Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky Ave. 33, Bld. 2, Moscow, 119071, Russian Federation.

A novel type 1 geranylgeranyl pyrophosphate synthase GACE1337 has been identified within the genome of a newly identified hyperthermophilic archaeon Geoglobus acetivorans. The enzyme has been cloned and over-expressed in Escherichia coli. The recombinant enzyme has been biochemically and structurally characterized. It is able to catalyze the synthesis of geranylgeranyl pyrophosphate as a major product and of farnesyl pyrophosphate in smaller amounts, as measured by gas chromatography-mass spectrometry at an elevated temperature of 60 °C. Its ability to produce two products is consistent with the fact that GACE1337 is the only short-chain isoprenyl diphosphate synthase in G. acetivorans. Attempts to crystallize the enzyme were successful only at 37 °C. The three-dimensional structure of GACE1337 was determined by X-ray diffraction to 2.5 Å resolution. A comparison of its structure with those of related enzymes revealed that the Geoglobus enzyme has the features of both type I and type III geranylgeranyl pyrophosphate synthases, which allow it to regulate the product length. The active enzyme is a dimer and has three aromatic amino acids, two Phe, and a Tyr, located in the hydrophobic cleft between the two subunits. It is proposed that these bulky residues play a major role in the synthetic reaction by controlling the product elongation.
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http://dx.doi.org/10.1007/s00792-018-1044-5DOI Listing
November 2018

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase. Corrigendum.

Acta Crystallogr D Struct Biol 2018 04 6;74(Pt 4):379. Epub 2018 Apr 6.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, England.

A statement is amended in the article by Isupov et al. [(2015). Acta Cryst. D71, 2344-2353].
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http://dx.doi.org/10.1107/S205979831800150XDOI Listing
April 2018

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells.

Free Radic Biol Med 2018 05 17;120:246-254. Epub 2018 Mar 17.

University of Exeter Medical School, St Luke's Campus, Magdalen Road, Exeter EX1 2LU, UK. Electronic address:

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00-1.34) and 0.97 (0.00-1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.03.026DOI Listing
May 2018

Comments to Article by Willetts A. et al., Microorganisms 2016, 4, 38.

Microorganisms 2017 Sep 6;5(3). Epub 2017 Sep 6.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.

We would like to comment on recent work published in your journal in October 2016 by Willetts A. et al. [1].[...].
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http://dx.doi.org/10.3390/microorganisms5030054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5620645PMC
September 2017

Characterization of Carboxylic Acid Reductases as Enzymes in the Toolbox for Synthetic Chemistry.

ChemCatChem 2017 03 14;9(6):1005-1017. Epub 2017 Feb 14.

Biosciences, College of Life and Environmental Sciences University of Exeter, Stocker Road Devon Exeter EX4 4QD UK.

Carboxylic acid reductase enzymes (CARs) meet the demand in synthetic chemistry for a green and regiospecific route to aldehydes from their respective carboxylic acids. However, relatively few of these enzymes have been characterized. A sequence alignment with members of the ANL (Acyl-CoA synthetase/ NRPS adenylation domain/Luciferase) superfamily of enzymes shed light on CAR functional dynamics. Four unstudied enzymes were selected by using a phylogenetic analysis of known and hypothetical CARs, and for the first time, a thorough biochemical characterization was performed. Kinetic analysis of these enzymes with various substrates shows that they have a broad but similar substrate specificity. Electron-rich acids are favored, which suggests that the first step in the proposed reaction mechanism, attack by the carboxylate on the α-phosphate of adenosine triphosphate (ATP), is the step that determines the substrate specificity and reaction kinetics. The effects of pH and temperature provide a clear operational window for the use of these CARs, whereas an investigation of product inhibition by NADP, adenosine monophosphate, and pyrophosphate indicates that the binding of substrates at the adenylation domain is ordered with ATP binding first. This study consolidates CARs as important and exciting enzymes in the toolbox for sustainable chemistry and provides specifications for their use as a biocatalyst.
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http://dx.doi.org/10.1002/cctc.201601249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396282PMC
March 2017

Structural and biochemical characterisation of Archaeoglobus fulgidus esterase reveals a bound CoA molecule in the vicinity of the active site.

Sci Rep 2016 05 10;6:25542. Epub 2016 May 10.

The Henry Wellcome Building for Biocatalysis, Biosciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.

A new carboxyl esterase, AF-Est2, from the hyperthermophilic archaeon Archaeoglobus fulgidus has been cloned, over-expressed in Escherichia coli and biochemically and structurally characterized. The enzyme has high activity towards short- to medium-chain p-nitrophenyl carboxylic esters with optimal activity towards the valerate ester. The AF-Est2 has good solvent and pH stability and is very thermostable, showing no loss of activity after incubation for 30 min at 80 °C. The 1.4 Å resolution crystal structure of AF-Est2 reveals Coenzyme A (CoA) bound in the vicinity of the active site. Despite the presence of CoA bound to the AF-Est2 this enzyme has no CoA thioesterase activity. The pantetheine group of CoA partially obstructs the active site alcohol pocket suggesting that this ligand has a role in regulation of the enzyme activity. A comparison with closely related α/β hydrolase fold enzyme structures shows that the AF-Est2 has unique structural features that allow CoA binding. A comparison of the structure of AF-Est2 with the human carboxyl esterase 1, which has CoA thioesterase activity, reveals that CoA is bound to different parts of the core domain in these two enzymes and approaches the active site from opposite directions.
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http://dx.doi.org/10.1038/srep25542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4861933PMC
May 2016

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate.

PLoS One 2016 7;11(1):e0146454. Epub 2016 Jan 7.

Laboratory of Biotechnology, School of Chemical Engineering, National Technical University of Athens, Athens, Greece.

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146454PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4704807PMC
July 2016

The Structure of a Novel Thermophilic Esterase from the Planctomycetes Species, Thermogutta terrifontis Reveals an Open Active Site Due to a Minimal 'Cap' Domain.

Front Microbiol 2015 23;6:1294. Epub 2015 Nov 23.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter Exeter, UK.

A carboxyl esterase (TtEst2) has been identified in a novel thermophilic bacterium, Thermogutta terrifontis from the phylum Planctomycetes and has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity toward small p-nitrophenyl (pNP) carboxylic esters with optimal activity for pNP-acetate. The enzyme shows moderate thermostability retaining 75% activity after incubation for 30 min at 70°C. The crystal structures have been determined for the native TtEst2 and its complexes with the carboxylic acid products propionate, butyrate, and valerate. TtEst2 differs from most enzymes of the α/β-hydrolase family 3 as it lacks the majority of the 'cap' domain and its active site cavity is exposed to the solvent. The bound ligands have allowed the identification of the carboxyl pocket in the enzyme active site. Comparison of TtEst2 with structurally related enzymes has given insight into how differences in their substrate preference can be rationalized based upon the properties of their active site pockets.
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http://dx.doi.org/10.3389/fmicb.2015.01294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655241PMC
December 2015

Enzymes from Extreme Environments and Their Industrial Applications.

Front Bioeng Biotechnol 2015 13;3:161. Epub 2015 Oct 13.

Exeter Biocatalysis Centre, Biosciences, College of Life and Environmental Sciences, University of Exeter , Exeter , UK.

This article will discuss the importance of specific extremophilic enzymes for applications in industrial biotechnology. It will specifically address those enzymes that have applications in the area of biocatalysis. Such enzymes now play an important role in catalyzing a variety of chemical conversions that were previously carried out by traditional chemistry. The biocatalytic process is carried out under mild conditions and with greater specificity. The enzyme process does not result in the toxic waste that is usually produced in a chemical process that would require careful disposal. In this sense, the biocatalytic process is referred to as carrying out "green chemistry" which is considered to be environmentally friendly. Some of the extremophilic enzymes to be discussed have already been developed for industrial processes such as an l-aminoacylase and a γ-lactamase. The industrial applications of other extremophilic enzymes, including transaminases, carbonic anhydrases, dehalogenases, specific esterases, and epoxide hydrolases, are currently being assessed. Specific examples of these industrially important enzymes that have been studied in the authors group will be presented in this review.
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http://dx.doi.org/10.3389/fbioe.2015.00161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4602302PMC
November 2015

The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer-Villiger monooxygenase.

Acta Crystallogr D Biol Crystallogr 2015 Nov 31;71(Pt 11):2344-53. Epub 2015 Oct 31.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, England.

The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer-Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.
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http://dx.doi.org/10.1107/S1399004715017939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631483PMC
November 2015

Archaeal Enzymes and Applications in Industrial Biocatalysts.

Archaea 2015 30;2015:147671. Epub 2015 Sep 30.

Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, Stocker Road, Exeter EX4 4QD, UK.

Archaeal enzymes are playing an important role in industrial biotechnology. Many representatives of organisms living in "extreme" conditions, the so-called Extremophiles, belong to the archaeal kingdom of life. This paper will review studies carried by the Exeter group and others regarding archaeal enzymes that have important applications in commercial biocatalysis. Some of these biocatalysts are already being used in large scale industrial processes for the production of optically pure drug intermediates and amino acids and their analogues. Other enzymes have been characterised at laboratory scale regarding their substrate specificity and properties for potential industrial application. The increasing availability of DNA sequences from new archaeal species and metagenomes will provide a continuing resource to identify new enzymes of commercial interest using both bioinformatics and screening approaches.
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http://dx.doi.org/10.1155/2015/147671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606452PMC
November 2015

Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries.

FEBS J 2015 Aug 16;282(15):2879-94. Epub 2015 Jun 16.

Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Milano, Italy.

The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2-diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene-1,2-epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk-LEH and CH55-LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re-LEH) in terms of stereoselectivity. Tomsk-LEH and CH55-LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re-LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk-LEH and poly(ethylene glycol) for CH55-LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.
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http://dx.doi.org/10.1111/febs.13328DOI Listing
August 2015

Structural studies of a thermophilic esterase from a new Planctomycetes species, Thermogutta terrifontis.

FEBS J 2015 Aug 17;282(15):2846-57. Epub 2015 Jun 17.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, UK.

Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over-expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p-nitrophenyl (pNP) carboxylic esters, with optimal activity for pNP-propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue D-malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP-esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP-butyrate.
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http://dx.doi.org/10.1111/febs.13326DOI Listing
August 2015

The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability.

Acta Crystallogr D Biol Crystallogr 2014 Oct 27;70(Pt 10):2607-18. Epub 2014 Sep 27.

Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, England.

Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.
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http://dx.doi.org/10.1107/S1399004714016526DOI Listing
October 2014

Determination of protein-ligand interactions using differential scanning fluorimetry.

J Vis Exp 2014 Sep 13(91):51809. Epub 2014 Sep 13.

Department of Biosciences, University of Exeter;

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most of these require access to specialist equipment, and often require a degree of expertise to effectively establish reliable experiments and analyze data. Differential scanning fluorimetry (DSF) is being increasingly used as a robust method for initial screening of proteins for interacting small molecules, either for identifying physiological partners or for hit discovery. This technique has the advantage that it requires only a PCR machine suitable for quantitative PCR, and so suitable instrumentation is available in most institutions; an excellent range of protocols are already available; and there are strong precedents in the literature for multiple uses of the method. Past work has proposed several means of calculating dissociation constants from DSF data, but these are mathematically demanding. Here, we demonstrate a method for estimating dissociation constants from a moderate amount of DSF experimental data. These data can typically be collected and analyzed within a single day. We demonstrate how different models can be used to fit data collected from simple binding events, and where cooperative binding or independent binding sites are present. Finally, we present an example of data analysis in a case where standard models do not apply. These methods are illustrated with data collected on commercially available control proteins, and two proteins from our research program. Overall, our method provides a straightforward way for researchers to rapidly gain further insight into protein-ligand interactions using DSF.
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http://dx.doi.org/10.3791/51809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4692391PMC
September 2014

The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca.

FEBS J 2014 May 7;281(9):2240-53. Epub 2014 Apr 7.

Henry Wellcome Building for Biocatalysis, College of Life and Environmental Sciences, University of Exeter, EX4 4QD, UK.

Unlabelled: During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)-enantiospecificity. Transaminases with (R)-specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)-specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)-selective deamination of several substrates including (R)-α-methylbenzylamine, as well as production of (R)-amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)-selective amine : pyruvate transaminases.

Database: The atomic coordinates and structure factors for the Nectria TAm in holoenzyme and gabaculine-bound forms have been deposited in the PDB as entries 4cmd and 4cmf respectively.
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http://dx.doi.org/10.1111/febs.12778DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4255305PMC
May 2014

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae.

FEBS Lett 2014 May 5;588(9):1616-22. Epub 2014 Mar 5.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. Electronic address:

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.
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http://dx.doi.org/10.1016/j.febslet.2014.02.056DOI Listing
May 2014

Functional and structural characterisation of a viral cytochrome b5.

FEBS Lett 2013 Nov 4;587(22):3633-9. Epub 2013 Oct 4.

Plymouth Marine Laboratory, Prospect Place, The Hoe, Plymouth PL1 3DH, UK; Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK.

Cytochrome b5 is a ubiquitous electron transport protein. The sequenced viral OtV-2 genome, which infects Ostreococcus tauri, was predicted to encode a putative cytochrome b5 enzyme. Using purified OtV-2 cytochrome b5 we confirm this protein has identical spectral properties to purified human cytochrome b5 and additionally that the viral enzyme can substitute for yeast cytochrome b5 in yeast cytochrome P450 51 mediated sterol 14α-demethylation. The crystal structure of the OtV-2 cytochrome b5 enzyme reveals a single domain, comprising four β sheets, four α helices and a haem moiety, which is similar to that found in larger eukaryotic cytochrome proteins. As a product of a horizontal gene transfer event involving a subdomain of the host fumarate reductase-like protein, OtV-2 cytochrome b5 appears to have diverged in function and is likely to have evolved an entirely new role for the virus during infection. Indeed, lacking a hydrophobic C-terminal anchor, OtV-2 encodes the first cytosolic cytochrome b5 characterised. The lack of requirement for membrane attachment (in contrast to all other microsomal cytochrome b5s) may be a reflection of the small size of the host cell, further emphasizes the unique nature of this virus gene product and draws attention to the potential importance of cytochrome b5 metabolic activity at the extremes of cellular scale.
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http://dx.doi.org/10.1016/j.febslet.2013.09.035DOI Listing
November 2013

Characterisation of an L-haloacid dehalogenase from the marine psychrophile Psychromonas ingrahamii with potential industrial application.

Mar Biotechnol (NY) 2013 Dec 16;15(6):695-705. Epub 2013 Aug 16.

The Henry Wellcome Building for Biocatalysis, Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.

The recombinant L-haloacid dehalogenase from the marine bacterium Psychromonas ingrahamii has been cloned and over-expressed in Escherichia coli. It shows activity towards monobromoacetic (100 %), monochloroacetic acid (62 %), S-chloropropionic acid (42 %), S-bromopropionic acid (31 %), dichloroacetic acid (28 %) and 2-chlorobutyric acid (10 %), respectively. The L-haloacid dehalogenase has highest activity towards substrates with shorter carbon chain lengths (≤ C3), without preference towards a chlorine or bromine at the α-carbon position. Despite being isolated from a psychrophilic bacterium, the enzyme has mesophilic properties with an optimal temperature for activity of 45 °C. It retains above 70 % of its activity after being incubated at 65 °C for 90 min before being assayed at 25 °C. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. The V max and K m were calculated to be 0.6 μM min(-1) mg(-1) and 1.36 mM with monobromoacetic acid, respectively. This solvent-resistant and stable L-haloacid dehalogenase from P. ingrahamii has potential to be used as a biocatalyst in industrial processes.
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http://dx.doi.org/10.1007/s10126-013-9522-3DOI Listing
December 2013