Publications by authors named "Michail N Isupov"

58 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 basis for SARM1 inhibition and activation under energetic stress.

Elife 2020 11 13;9. Epub 2020 Nov 13.

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.

SARM1, an executor of axonal degeneration, displays NADase activity that depletes the key cellular metabolite, NAD+, in response to nerve injury. The basis of SARM1 inhibition and its activation under stress conditions are still unknown. Here, we present cryo-EM maps of SARM1 at 2.9 and 2.7 Å resolutions. These indicate that SARM1 homo-octamer avoids premature activation by assuming a packed conformation, with ordered inner and peripheral rings, that prevents dimerization and activation of the catalytic domains. This inactive conformation is stabilized by binding of SARM1's own substrate NAD+ in an allosteric location, away from the catalytic sites. This model was validated by mutagenesis of the allosteric site, which led to constitutively active SARM1. We propose that the reduction of cellular NAD+ concentration contributes to the disassembly of SARM1's peripheral ring, which allows formation of active NADase domain dimers, thereby further depleting NAD+ to cause an energetic catastrophe and cell death.
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http://dx.doi.org/10.7554/eLife.62021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688312PMC
November 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

The crystal structure of human microsomal triglyceride transfer protein.

Proc Natl Acad Sci U S A 2019 08 8;116(35):17251-17260. Epub 2019 Aug 8.

Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90220 Oulu, Finland;

Microsomal triglyceride transfer protein (MTP) plays an essential role in lipid metabolism, especially in the biogenesis of very low-density lipoproteins and chylomicrons via the transfer of neutral lipids and the assembly of apoB-containing lipoproteins. Our understanding of the molecular mechanisms of MTP has been hindered by a lack of structural information of this heterodimeric complex comprising an MTPα subunit and a protein disulfide isomerase (PDI) β-subunit. The structure of MTP presented here gives important insights into the potential mechanisms of action of this essential lipid transfer molecule, structure-based rationale for previously reported disease-causing mutations, and a means for rational drug design against cardiovascular disease and obesity. In contrast to the previously reported structure of lipovitellin, which has a funnel-like lipid-binding cavity, the lipid-binding site is encompassed in a β-sandwich formed by 2 β-sheets from the C-terminal domain of MTPα. The lipid-binding cavity of MTPα is large enough to accommodate a single lipid. PDI independently has a major role in oxidative protein folding in the endoplasmic reticulum. Comparison of the mechanism of MTPα binding by PDI with previously published structures gives insights into large protein substrate binding by PDI and suggests that the previous structures of human PDI represent the "substrate-bound" and "free" states rather than differences arising from redox state.
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http://dx.doi.org/10.1073/pnas.1903029116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6717300PMC
August 2019

Structural Evidence for an Octameric Ring Arrangement of SARM1.

J Mol Biol 2019 09 3;431(19):3591-3605. Epub 2019 Jul 3.

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel. Electronic address:

SARM1 induces axonal degeneration in response to various insults and is therefore considered an attractive drug target for the treatment of neuro-degenerative diseases as well as for brain and spinal cord injuries. SARM1 activity depends on the integrity of the protein's SAM domains, as well as on the enzymatic conversion of NAD+ to ADPR (ADP Ribose) products by the SARM1's TIR domain. Therefore, inhibition of either SAM or TIR functions may constitute an effective therapeutic strategy. However, there is currently no SARM1-directed therapeutic approach available because of an insufficient structural and mechanistic understanding of this protein. In this study, we found that SARM1 assembles into an octameric ring. This arrangement was not described before in other SAM proteins, but is reminiscent of the apoptosome and inflammasome-well-known apoptotic ring-like oligomers. We show that both SARM1 and the isolated tandem SAM domains form octamers in solution, and electron microscopy analysis reveals an octameric ring of SARM1. We determined the crystal structure of SAM and found that it also forms a closed octameric ring in the crystal lattice. The SAM ring interactions are mediated by complementing "lock and key" hydrophobic grooves and inserts and electrostatic charges between the neighboring protomers. We have mutated several interacting SAM interfaces and measured how these mutations affect SARM1 apoptotic activity in cultured cells, and in this way identified critical oligomerization sites that facilitate cell death. These results highlight the importance of oligomerization for SARM1 function and reveal critical epitopes for future targeted drug development.
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http://dx.doi.org/10.1016/j.jmb.2019.06.030DOI Listing
September 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

Structural Principles in Robo Activation and Auto-inhibition.

Cell 2019 04 7;177(2):272-285.e16. Epub 2019 Mar 7.

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Israel. Electronic address:

Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos' switch from "off" to "on" states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1-8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation.
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http://dx.doi.org/10.1016/j.cell.2019.02.004DOI Listing
April 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

An N-Terminal Extension to UBA5 Adenylation Domain Boosts UFM1 Activation: Isoform-Specific Differences in Ubiquitin-like Protein Activation.

J Mol Biol 2019 02 6;431(3):463-478. Epub 2018 Nov 6.

Department of Biochemistry and Molecular Biology, The Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. Electronic address:

Modification of proteins by the ubiquitin-like protein, UFM1, requires activation of UFM1 by the E1-activating enzyme, UBA5. In humans, UBA5 possesses two isoforms, each comprising an adenylation domain, but only one containing an N-terminal extension. Currently, the role of the N-terminal extension in UFM1 activation is not clear. Here we provide structural and biochemical data on UBA5 N-terminal extension to understand its contribution to UFM1 activation. The crystal structures of the UBA5 long isoform bound to ATP with and without UFM1 show that the N-terminus not only is directly involved in ATP binding but also affects how the adenylation domain interacts with ATP. Surprisingly, in the presence of the N-terminus, UBA5 no longer retains the 1:2 ratio of ATP to UBA5, but rather this becomes a 1:1 ratio. Accordingly, the N-terminus significantly increases the affinity of ATP to UBA5. Finally, the N-terminus, although not directly involved in the E2 binding, stimulates transfer of UFM1 from UBA5 to the E2, UFC1.
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http://dx.doi.org/10.1016/j.jmb.2018.10.007DOI Listing
February 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

The molecular basis of protein toxin HicA-dependent binding of the protein antitoxin HicB to DNA.

J Biol Chem 2018 12 18;293(50):19429-19440. Epub 2018 Oct 18.

From the School of Chemistry, University of Bristol Cantock's Close, Bristol BS8 1TS, United Kingdom,

Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex.
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http://dx.doi.org/10.1074/jbc.RA118.005173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302177PMC
December 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

Molecular mechanism for the subversion of the retromer coat by the effector RidL.

Proc Natl Acad Sci U S A 2017 12 11;114(52):E11151-E11160. Epub 2017 Dec 11.

Structural Biology Unit, Centro de Investigación Cooperativa en Biociencias, 48160 Derio, Spain;

Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
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http://dx.doi.org/10.1073/pnas.1715361115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5748213PMC
December 2017

The Crystal Structure and Conformations of an Unbranched Mixed Tri-Ubiquitin Chain Containing K48 and K63 Linkages.

J Mol Biol 2017 12 27;429(24):3801-3813. Epub 2017 Oct 27.

Department of Biochemistry and Molecular Biology, the Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel. Electronic address:

The ability of ubiquitin to function in a wide range of cellular processes is ascribed to its capacity to cause a diverse spectrum of modifications. While a target protein can be modified with monoubiquitin, it can also be modified with ubiquitin chains. The latter include seven types of homotypic chains as well as mixed ubiquitin chains. In a mixed chain, not all the isopeptide bonds are restricted to a specific lysine of ubiquitin, resulting in a chain possessing more than one type of linkage. While structural characterization of homotypic chains has been well elucidated, less is known about mixed chains. Here we present the crystal structure of a mixed tri-ubiquitin chain at 3.1-Å resolution. In the structure, the proximal ubiquitin is connected to the middle ubiquitin via K48 and these two ubiquitins adopt a compact structure as observed in K48 di-ubiquitin. The middle ubiquitin links to the distal ubiquitin via its K63 and these ubiquitins adopt two conformations, suggesting a flexible structure. Using small-angle X-ray scattering, we unexpectedly found differences between the conformational ensembles of the above tri-ubiquitin chains and chains possessing the same linkages but in the reverse order. In addition, cleavage of the K48 linkage by DUB is faster if this linkage is at the distal end. Taken together, our results suggest that in mixed chains, not only the type of the linkages but also their sequence determine the structural and functional properties of the chain.
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http://dx.doi.org/10.1016/j.jmb.2017.10.027DOI Listing
December 2017

Structural History of Human SRGAP2 Proteins.

Mol Biol Evol 2017 06;34(6):1463-1478

The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel.

In the development of the human brain, human-specific genes are considered to play key roles, conferring its unique advantages and vulnerabilities. At the time of Homo lineage divergence from Australopithecus, SRGAP2C gradually emerged through a process of serial duplications and mutagenesis from ancestral SRGAP2A (3.4-2.4 Ma). Remarkably, ectopic expression of SRGAP2C endows cultured mouse brain cells, with human-like characteristics, specifically, increased dendritic spine length and density. To understand the molecular mechanisms underlying this change in neuronal morphology, we determined the structure of SRGAP2A and studied the interplay between SRGAP2A and SRGAP2C. We found that: 1) SRGAP2A homo-dimerizes through a large interface that includes an F-BAR domain, a newly identified F-BAR extension (Fx), and RhoGAP-SH3 domains. 2) SRGAP2A has an unusual inverse geometry, enabling associations with lamellipodia and dendritic spine heads in vivo, and scaffolding of membrane protrusions in cell culture. 3) As a result of the initial partial duplication event (∼3.4 Ma), SRGAP2C carries a defective Fx-domain that severely compromises its solubility and membrane-scaffolding ability. Consistently, SRGAP2A:SRAGP2C hetero-dimers form, but are insoluble, inhibiting SRGAP2A activity. 4) Inactivation of SRGAP2A is sensitive to the level of hetero-dimerization with SRGAP2C. 5) The primal form of SRGAP2C (P-SRGAP2C, existing between ∼3.4 and 2.4 Ma) is less effective in hetero-dimerizing with SRGAP2A than the modern SRGAP2C, which carries several substitutions (from ∼2.4 Ma). Thus, the genetic mutagenesis phase contributed to modulation of SRGAP2A's inhibition of neuronal expansion, by introducing and improving the formation of inactive SRGAP2A:SRGAP2C hetero-dimers, indicating a stepwise involvement of SRGAP2C in human evolutionary history.
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http://dx.doi.org/10.1093/molbev/msx094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435084PMC
June 2017

Crystal structure of the type IV secretion system component CagX from Helicobacter pylori.

Acta Crystallogr F Struct Biol Commun 2017 03 28;73(Pt 3):167-173. Epub 2017 Feb 28.

State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Science, Fuzhou 350002, People's Republic of China.

Helicobacter pylori, a Gram-negative bacterial pathogen prevalent in the human population, is the causative agent of severe gastric diseases. An H. pylori type IV secretion (T4S) system encoded by the cytotoxin-associated gene pathogenicity island (cagPAI) is responsible for communication with host cells. As a component of the cagPAI T4S system core complex, CagX plays an important role in virulence-protein translocation into the host cells. In this work, the crystal structure of the C-terminal domain of CagX (CagXct), which is a homologue of the VirB9 protein from the VirB/D4 T4S system, is presented. CagXct is only the second three-dimensional structure to be elucidated of a VirB9-like protein. Another homologue, TraO, which is encoded on the Escherichia coli conjugative plasmid pKM101, shares only 19% sequence identity with CagXct; however, there is a remarkable similarity in tertiary structure between these two β-sandwich protein domains. Most of the residues that are conserved between CagXct and TraO are located within the protein core and appear to be responsible for the preservation of this domain fold. The studies presented here will contribute to our understanding of different bacterial T4S systems.
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http://dx.doi.org/10.1107/S2053230X17001376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5349311PMC
March 2017

Molecular symmetry-constrained systematic search approach to structure solution of the coiled-coil SRGAP2 F-BARx domain.

Acta Crystallogr D Struct Biol 2016 12 29;72(Pt 12):1241-1253. Epub 2016 Nov 29.

The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel.

SRGAP2 (Slit-Robo GTPase-activating protein 2) is a cytoplasmic protein found to be involved in neuronal branching, restriction of neuronal migration and restriction of the length and density of dendritic postsynaptic spines. The extended F-BAR (F-BARx) domain of SRGAP2 generates membrane protrusions when expressed in COS-7 cells, while most F-BARs induce the opposite effect: membrane invaginations. As a first step to understand this discrepancy, the F-BARx domain of SRGAP2 was isolated and crystallized after co-expression with the carboxy domains of the protein. Diffraction data were collected from two significantly non-isomorphous crystals in the same monoclinic C2 space group. A correct molecular-replacment solution was obtained by applying a molecular symmetry-constrained systematic search approach that took advantage of the conserved biological symmetry of the F-BAR domains. It is shown that similar approaches can solve other F-BAR structures that were previously determined by experimental phasing. Diffraction data were reprocessed with a high-resolution cutoff of 2.2 Å, chosen using less strict statistical criteria. This has improved the outcome of multi-crystal averaging and other density-modification procedures.
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http://dx.doi.org/10.1107/S2059798316016697DOI Listing
December 2016

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

Unraveling the B. pseudomallei Heptokinase WcbL: From Structure to Drug Discovery.

Chem Biol 2015 Dec;22(12):1622-32

Department of Biosciences, University of Exeter, Henry Wellcome Building, Stocker Road, Exeter EX4 4QD, UK. Electronic address:

Gram-negative bacteria utilize heptoses as part of their repertoire of extracellular polysaccharide virulence determinants. Disruption of heptose biosynthesis offers an attractive target for novel antimicrobials. A critical step in the synthesis of heptoses is their 1-O phosphorylation, mediated by kinases such as HldE or WcbL. Here, we present the structure of WcbL from Burkholderia pseudomallei. We report that WcbL operates through a sequential ordered Bi-Bi mechanism, loading the heptose first and then ATP. We show that dimeric WcbL binds ATP anti-cooperatively in the absence of heptose, and cooperatively in its presence. Modeling of WcbL suggests that heptose binding causes an elegant switch in the hydrogen-bonding network, facilitating the binding of a second ATP molecule. Finally, we screened a library of drug-like fragments, identifying hits that potently inhibit WcbL. Our results provide a novel mechanism for control of substrate binding and emphasize WcbL as an attractive anti-microbial target for Gram-negative bacteria.
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http://dx.doi.org/10.1016/j.chembiol.2015.10.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4691232PMC
December 2015

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

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

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