| 1. |
- Alzari, Pedro M., et al.
(författare)
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Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE
- 2006
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62, s. 1103-1113
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Tidskriftsartikel (refereegranskat)abstract
- The implementation of high- throughput ( HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe ( SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non- ligation- based cloning techniques used, namely Gateway, ligation- indendent cloning of PCR products ( LIC- PCR) and In- Fusion, with LIC- PCR emerging as the most cost- effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library- based screening for soluble constructs and parallel small- scale high- density fermentation.
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| 2. |
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| 3. |
- Aricescu, A R, et al.
(författare)
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Eukaryotic expression: developments for structural proteomics.
- 2006
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Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 62, s. 1114-1124
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Tidskriftsartikel (refereegranskat)abstract
- The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.
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| 4. |
- Backmark, Anna, 1979, et al.
(författare)
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Affinity tags can reduce merohedral twinning of membrane protein crystals
- 2008
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; D64, s. 1183-1186
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Tidskriftsartikel (refereegranskat)abstract
- This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
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| 5. |
- Björkelid, Christofer, et al.
(författare)
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Structural and functional studies of mycobacterial IspD enzymes
- 2011
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 67, s. 403-414
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.
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| 6. |
- Björkelid, Christofer, et al.
(författare)
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Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
- 2012
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68, s. 134-143
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Tidskriftsartikel (refereegranskat)abstract
- A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.
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| 7. |
- Castell, Alina, et al.
(författare)
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Structural analysis of mycobacterial branched-chain aminotransferase : implications for inhibitor design
- 2010
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 66, s. 549-557
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Tidskriftsartikel (refereegranskat)abstract
- The branched-chain aminotransferase (BCAT) of Mycobacterium tuberculosis has been characterized as being essential to the survival of the bacterium. The enzyme is pyridoxal 5'-phosphate-dependent and belongs to the aminotransferase IIIa subfamily, to which the human BCATs also belong. The overall sequence similarity is high within the subfamily and the sequence identity among the active-site residues is high. In order to identify structurally unique features of M. tuberculosis BCAT, X-ray structural and functional analyses of the closely related BCAT from M. smegmatis were carried out. The crystal structures include the apo form at 2.2 angstrom resolution and a 1.9 angstrom structure of the holo form cocrystallized with the inhibitor O-benzylhydroxylamine (Obe). The analyses highlighted the active-site residues Tyr209 and Gly243 as being structurally unique characteristics of the mycobacterial BCATs relative to the human BCATs. The inhibitory activities of Obe and ammonium sulfate were verified in an inhibition assay. Modelling of the inhibitor Obe in the substrate pocket indicated potential for the design of a mycobacterial-specific inhibitor.
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| 8. |
- Charavgi, Maria-Despoina, et al.
(författare)
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The structure of a novel glucuronoyl esterase from Myceliophthora thermophila gives new insights into its role as a potential biocatalyst
- 2013
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 69:1, s. 63-73
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Tidskriftsartikel (refereegranskat)abstract
- The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-[beta]-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an [alpha]/[beta]-hydrolase fold with a three-layer [alpha][beta][alpha]-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.
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| 9. |
- Cheeseman, J. D., et al.
(författare)
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Structure of an aryl esterase from Pseudomonas fluorescens
- 2004
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 60:7, s. 1237-1243
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Tidskriftsartikel (refereegranskat)abstract
- The structure of PFE, an aryl esterase from Pseudomonas fluorescens, has been solved to a resolution of 1.8 Å by X-ray diffraction and shows a characteristic α/β-hydrolase fold. In addition to catalyzing the hydrolysis of esters in vitro, PFE also shows low bromoperoxidase activity. PFE shows highest structural similarity, including the active-site environment, to a family of non-heme bacterial haloperoxidases, with an r.m.s. deviation in 271 Cα atoms between PFE and its five closest structural neighbors averaging 0.8 Å. PFE has far less similarity (r.m.s. deviation in 218 Cα atoms of 5.0 Å) to P. fluorescens carboxyl esterase. PFE favors activated esters with small acyl groups, such as phenyl acetate. The X-ray structure of PFE reveals a significantly occluded active site. In addition, several residues, including Trp28 and Met95, limit the size of the acyl-binding pocket, explaining its preference for small acyl groups.
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| 10. |
- Chen, Yang, et al.
(författare)
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Structure of AadA from Salmonella enterica : a monomeric aminoglycoside (3'')(9) adenyltransferase
- 2015
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71, s. 2267-2277
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Tidskriftsartikel (refereegranskat)abstract
- Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.
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| 11. |
- Cohen, Serge X., et al.
(författare)
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Towards complete validated models in the next generation of ARP/wARP
- 2004
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 60:Pt 12 Pt 1, s. 2222-9
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Tidskriftsartikel (refereegranskat)abstract
- The design of a new versatile control system that will underlie future releases of the automated model-building package ARP/wARP is presented. A sophisticated expert system is under development that will transform ARP/wARP from a very useful model-building aid to a truly automated package capable of delivering complete, well refined and validated models comparable in quality to the result of intensive manual checking, rebuilding, hypothesis testing, refinement and validation cycles of an experienced crystallographer. In addition to the presentation of this control system, recent advances, ideas and future plans for improving the current model-building algorithms, especially for completing partially built models, are presented. Furthermore, a concept for integrating validation routines into the iterative model-building process is also presented.
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| 12. |
- Dimarogona, M, et al.
(författare)
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The structure of a GH10 xylanase from Fusarium oxysporum reveals the presence of an extended loop on top of the catalytic cleft
- 2012
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 68:7, s. 735-742
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Tidskriftsartikel (refereegranskat)abstract
- Xylanase enzymes have been the focus of considerable research in recent decades owing to their extensive use in a variety of biotechnological applications. Previous structural studies of a number of GH10 xylanases revealed that all GH10 family members have the (β/α)8-barrel fold and their catalytic site is conserved. The structure of a new GH10 xylanase from Fusarium oxysporum (FoXyn10a) was determined at 1.94 Å resolution from crystals belonging to the tetragonal space group P41212 with five molecules per asymmetric unit. Comparison of the structure of FoXyn10a with previously determined structures of GH10 family members indicated that most of the differences were located in the loop regions between the ordered secondary-structure elements of the barrel, as expected. However, alignment of FoXyn10a with sequence and structural homologues denoted an atypically long loop connecting strand β6b and helix 6 that was only present in one other GH10 xylanase, the structure of which is not known. This structural feature may be of functional importance, with potential implications in the catalytic efficiency of the enzyme.
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| 13. |
- Dobritzsch, Doreen, 1972-, et al.
(författare)
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Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri
- 2003
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 59:Pt 7, s. 1267-1269
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Tidskriftsartikel (refereegranskat)abstract
- In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.
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| 14. |
- Dobritzsch, Doreen, 1972-, et al.
(författare)
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Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase
- 2001
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 57:Pt 1, s. 153-155
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Tidskriftsartikel (refereegranskat)abstract
- Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.
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| 15. |
- Ekström, Fredrik, 1973-, et al.
(författare)
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Crystallization of the actin-binding domain of human alpha-actinin : analysis of microcrystals of SeMet-labelled protein
- 2003
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Ingår i: Acta Crystallographica Section D. - : Blackwell Munksgaard. - 0907-4449 .- 1399-0047. ; 59:Pt 4, s. 724-726
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Tidskriftsartikel (refereegranskat)abstract
- Alpha-actinin forms antiparallel homodimers that cross-link actin filaments from adjacent sarcomeres within the Z-discs of striated muscle. The N-terminal actin-binding domain (ABD) is composed of two calponin homology (CH) domains followed by four spectrin-like repeats and a calmodulin-like EF-hand domain at the C-terminus. The ABD of human alpha-actinin crystallizes in space group P2(1), with unit-cell parameters a = 101.9, b = 38.4, c = 154.9 A, beta = 109.2 degrees. A complete native data set from a native crystal was collected extending to 2.0 A resolution and a single-wavelength anomalous dispersion (SAD) data set to 2.9 A resolution was collected from a selenomethionine-labelled microcrystal using the microfocusing beamline ID-13 at the ESRF. Analysis of the anomalous contribution shows a rapid decrease in the sigma(normal)/sigma(anomal) ratio owing to radiation damage.
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| 16. |
- Espaillat, Akbar, et al.
(författare)
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Structural basis for the broad specificity of a new family of amino-acid racemases
- 2014
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Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell. - 0907-4449 .- 1399-0047. ; 70, s. 79-90
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Tidskriftsartikel (refereegranskat)abstract
- Broad-spectrum amino-acid racemases (Bsrs) enable bacteria to generate noncanonical D-amino acids, the roles of which in microbial physiology, including the modulation of cell-wall structure and the dissolution of biofilms, are just beginning to be appreciated. Here, extensive crystallographic, mutational, biochemical and bioinformatic studies were used to define the molecular features of the racemase BsrV that enable this enzyme to accommodate more diverse substrates than the related PLP-dependent alanine racemases. Conserved residues were identified that distinguish BsrV and a newly defined family of broad-spectrum racemases from alanine racemases, and these residues were found to be key mediators of the multispecificity of BrsV. Finally, the structural analysis of an additional Bsr that was identified in the bioinformatic analysis confirmed that the distinguishing features of BrsV are conserved among Bsr family members.
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| 17. |
- Fogg, M. J., et al.
(författare)
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Application of the use of high-throughput technologies to the determination of protein structures of bacterial and viral pathogens
- 2006
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 62:10, s. 1196-1207
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Tidskriftsartikel (refereegranskat)abstract
- The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
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| 18. |
- Frankaer, C. G., et al.
(författare)
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The structures of T6, T3R3 and R6 bovine insulin: combining X-ray diffraction and absorption spectroscopy
- 2012
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Ingår i: Acta Crystallographica Section D-Biological Crystallography. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 68, s. 1259-1271
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structures of three conformations, T6, T3R3 and R6, of bovine insulin were solved at 1.40, 1.30 and 1.80 angstrom resolution, respectively. All conformations crystallized in space group R3. In contrast to the T6 and T3R3 structures, different conformations of the N-terminal B-chain residue PheB1 were observed in the R6 insulin structure, resulting in an eightfold doubling of the unit-cell volume upon cooling. The zinc coordination in each conformation was studied by X-ray absorption spectroscopy (XAS), including both EXAFS and XANES. Zinc adopts a tetrahedral coordination in all R3 sites and an octahedral coordination in T3 sites. The coordination distances were refined from XAS with a standard deviation of <0.01 angstrom. In contrast to the distances determined from the medium-resolution crystal structures, the XAS results were in good agreement with similar coordination geometries found in small molecules, as well as in other high-resolution insulin structures. As the radiation dose for XRD experiments is two orders of magnitude higher compared with that of XAS experiments, the single crystals were exposed to a higher degree of radiation damage that affected the zinc coordination in the T3 sites in particular. Furthermore, XANES spectra for the zinc sites in T6 and R6 insulin were successfully calculated using finite difference methods and the bond distances and angles were optimized from a quantitative XANES analysis.
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| 19. |
- Friemann, Rosmarie, et al.
(författare)
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Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme system
- 2009
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Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 65, s. 24-33
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe-2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe-2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed.
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| 20. |
- Grahn, Elin, et al.
(författare)
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Crystallization and preliminary X-ray crystallographic studies of a lectin from the mushroom Marasmius oreades
- 2004
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Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 1399-0047 .- 0907-4449. ; 60:11, s. 2038-2039
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Tidskriftsartikel (refereegranskat)abstract
- The Marasmius oreades agglutinin (MOA) recognizes blood group B oligosaccharides. This mushroom lectin belongs to the ricin superfamily and is currently the only lectin known with exclusive specificity for Galα1,3Gal-structures, as occur in the subterminally fucosylated blood group B epitope Galα1,3(Fucα1,2)Galβ1,4GlcNAc (MOA's preferred ligand) or without fucosylation in the xenotransplantation epitope. MOA has been co-crystallized with the linear blood group B trisaccharide Galα1,3Galβ1,4GlcNAc using the hanging-drop vapour-diffusion technique at room temperature. MOA crystals were grown in the presence of ammonium formate and HEPES buffer. A 3.0 Å data set has been collected. Preliminary analysis of the X-ray data is consistent with space group P3 1 or P3 2 and unit-cell parameters a = b = 105, c = 113 Å, with two dimers per asymmetric unit. © 2004 International Union of Crystallography.
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| 21. |
- Haddad Momeni, Majid, et al.
(författare)
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Expression, crystal structure and cellulase activity of the thermostable cellobiohydrolase Cel7A from the fungus Humicola grisea var. thermoidea
- 2014
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Ingår i: Acta Crystallographica Section D: Biological Crystallography. - 0907-4449 .- 1399-0047. ; 70, s. 2356-2366
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Tidskriftsartikel (refereegranskat)abstract
- Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10 degrees C higher melting temperature (T-m of 72.5 degrees C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65 degrees C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 angstrom resolution; R-work and R-free of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.
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| 22. |
- Hasse, Dirk, et al.
(författare)
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Structure of Arabidopsis thaliana Rubisco activase
- 2015
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 71:Pt 4, s. 800-808
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Tidskriftsartikel (refereegranskat)abstract
- The CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is inactivated by the formation of dead-end complexes with inhibitory sugar phosphates. In plants and green algae, the ATP-dependent motor protein Rubisco activase restores catalytic competence by facilitating conformational changes in Rubisco that promote the release of the inhibitory compounds from the active site. Here, the crystal structure of Rubisco activase from Arabidopsis thaliana is presented at 2.9 Å resolution. The structure reveals an AAA+ two-domain structure. More than 100 residues in the protein were not visible in the electron-density map owing to conformational disorder, but were verified to be present in the crystal by mass spectrometry. Two sulfate ions were found in the structure. One was bound in the loop formed by the Walker A motif at the interface of the domains. A second sulfate ion was bound at the N-terminal end of the first helix of the C-terminal domain. The protein packs in a helical fashion in the crystal, as observed previously for Rubisco activase, but differences in the helical pitch indicate flexibility in the packing of the protein.
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| 23. |
- Hällberg, B. Martin, et al.
(författare)
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Crystallization and preliminary X-ray diffraction analysis of pyranose 2-oxidase from the white-rot fungus Trametes multicolor
- 2004
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Ingår i: Acta Crystallographica Section D. - : International Union of Crystallography (IUCr). - 0907-4449 .- 1399-0047. ; 60, s. 197-199
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetrameric flavoenzyme that catalyzes the oxidation of D-glucose to 2-keto-D-glucose. P2Ox participates in lignin degradation by white-rot fungi and a tentative role of the enzyme is the production of H2O2 for lignin peroxidases. Crystals of Trametes multicolor P2Ox were grown from monomethylether PEG 2000, sodium acetate, MgCl2 and Ta6Br12. They belong to space group P2(1), with unit-cell parameters a = 99.9, b = 101.7, c = 135.6 Angstrom, beta = 90.85degrees. X-ray diffraction data to 2.0 Angstrom resolution were collected using synchrotron radiation. Self-rotation function calculations suggest that the asymmetric unit contains one homotetramer with 222 point-group symmetry.
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| 24. |
- Ingvarsson, Henrik, et al.
(författare)
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Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1
- 2007
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Ingår i: Acta Crystallographica Section D. - 0907-4449 .- 1399-0047. ; 63:2, s. 249-259
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Tidskriftsartikel (refereegranskat)abstract
- Mycobacterium tuberculosis caseinolytic protease ClpP1 (Mt ClpP1) is a self-compartmentalized protease consisting of two heptameric rings stacked on top of each other, thus enclosing a catalytic chamber. Within the chamber, which can be reached through two axial pores, each of the 14 identical monomers possesses a serine protease active site. The unfolding and translocation of substrates into the chamber are mediated by associated hexameric ATPases covering the axial pores. Three crystal structures of Mt ClpP1, determined by molecular replacement, are presented in this study. Two of the models were refined to a resolution of 2.6 A and the third to 3.0 A. It was found that disorder in the handle domain affects the formation and configuration of the tetradecamer and results in condensed structures with larger equatorial pores when compared with ClpPs from other species. Additionally, this disorder accompanies conformational changes of the residues in the catalytic triad. The models also reveal structural differences within the N-terminal hairpin-loop domain, which possibly reflect the significant differences in amino-acid sequence between Mt ClpP1 and other ClpP homologues in this region.
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| 25. |
- Jacobson, Frida, 1975, et al.
(författare)
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High resolution X-ray structures of the oxidised and reduced forms of nitrite reductase from Rhodobacter sphaeroides 2.4.3
- 2005
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Ingår i: Acta Crystallography D. - 0907-4449 .- 1399-0047. ; 61, s. 1190-1198
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Tidskriftsartikel (refereegranskat)abstract
- Nitrite reductase is an enzyme operating in the denitrification pathway which catalyses the conversion of nitrite (NO2(-)) to gaseous nitric oxide (NO). Here, crystal structures of the oxidized and reduced forms of the copper-containing nitrite reductase from Rhodobacter sphaeroides 2.4.3 are presented at 1.74 and 1.85 A resolution, respectively. Whereas the structure of the enzyme is very similar to those of other copper-containing nitrite reductases, folding as a trimer and containing two copper sites per monomer, the structures reported here enable conformational differences between the oxidized and reduced forms of the enzyme to be identified. In the type 1 copper site, a rotational perturbation of the side chain of the copper ligand Met182 occurs upon reduction. At the type 2 copper site, a dual conformation of the catalytic residue His287 is observed in the oxidized structure but is lacking in the reduced structure, such that the interactions of the oxidized type 2 copper ion can be regarded as adopting octahedral geometry. These findings shed light on the structural mechanism of the reduction of a copper-bound nitrite to nitric oxide and water.
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