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1.
Daniel Fernández Ester Boix Irantzu Pallarès Francesc X. Avilés Josep Vendrell 《Biopolymers》2010,93(2):178-185
A new triclinic crystal structure form of porcine pancreatic procarboxypeptidase B (PCPB) was obtained at higher resolution than the previously known tetragonal crystal structure. This new crystal polymorph has allowed for a corrected, accurate assignment of residues along the polypeptide chain based on the currently available gene sequence information and crystallographic data. The present structure shows unbound PCPB in a distinct molecular packing as compared to the previous benzamidine complexed form. Its catalytically important Tyr248 residue is oriented and hydrogen‐bonded to solvent water molecules, and locates the furthest away from the catalytic zinc ion as compared to previous structures. A relatively long stretch of residues flanking Tyr248 and guarding the access to the catalytic zinc ion was found to be sequentially unique to the M14 family of peptidases. Predictions from a normal mode analysis indicated that this stretch of residues belongs to a rigid subdomain in the protein structure. The specific presence of a tyrosyl residue at the most exposed position in this region would allow for a delicate balance between extreme hydrophobicity and hydrophilicity, and affect substrate binding and the kinetic efficiency of the enzyme. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 178–185, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
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Hwajung Choi Hee Jung Kim Atsushi Matsuura Bunzo Mikami Hye‐Jin Yoon Hyung Ho Lee 《Acta Crystallographica. Section D, Structural Biology》2015,71(10):2054-2065
The selection of correct metal ions with high fidelity against competing cellular cations is crucial for the function of many metalloenzymes; however, the understanding of the principles that govern metal selectivity is still incomplete. In this study, the crystal structure of the Tm1162 protein from Thermotoga maritima, a metallo‐β‐lactamase, is reported. Several crystal structures of wild‐type Tm1162 and its mutants were solved. Homologues of Tm1162 are widely distributed in bacteria and archaea, including several human pathogens. The monomer possesses an αβ/βα fold, with the core β‐strands having the β‐sheet sandwich structure common to the metallo‐β‐lactamase superfamily. Tm1162 exists as a trimer in the crystal and this trimeric unit is likely to be present in solution. In the trimer, three active sites reside at the interface between subunits, suggesting that the oligomeric assembly is crucial for catalysis. A new type of structurally encoded heterodinuclear site has been identified by confirming the identity of nickel‐containing heteronuclear sites in Tm1162 via X‐ray absorption spectroscopy and anomalous difference Fourier maps. The second coordination sphere, including His8 and Glu73, maintains the side‐chain orientations of histidines and stabilizes the metal‐binding site. Nickel coordination was crucial for the oligomerization of Tm1162. The nickel‐dependent and manganese‐dependent β‐lactamase and phosphodiesterase activities of Tm1162 have also been characterized. 相似文献
3.
Manoj Bhosale 《Biochemical and biophysical research communications》2010,395(1):76-81
Escherichia coli encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in ΔpepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of ΔpepN during nutritional downshift and high temperature stress. Purified PepA and PepB display broad substrate specificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in ΔpepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms. 相似文献
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The X-ray crystallographic structure of a thioredoxin from Thermus thermophilus was solved to 1.8 A resolution by molecular replacement. The crystals' space group was C2 with cell dimensions of a = 40.91, b = 95.44, c = 56.68 A, beta =91.41 degrees, with two molecules in the asymmetric unit. Unlike the reported thioredoxin structures, the biological unit of T. thermophilus thioredoxin is a dimer both in solution and in the crystal. The fold conforms to the \"thioredoxin fold\" that is common over a class of nine protein families including thioredoxin; however, the folded portion of this protein is much more compact than other thioredoxins previously solved by X-ray crystallography being reduced by one alpha-helix and one beta-strand. As with other thioredoxins, the active site is highly conserved even though the variation in sequence can be quite large. The T. thermophilus thioredoxin has some variability at the active site, especially compared with previously solved structures from bacterial sources. 相似文献
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Cryo‐electron microscopy (cryo‐EM) is a structural biological method that is used to determine the 3D structures of biomacromolecules. After years of development, cryo‐EM has made great achievements, which has led to a revolution in structural biology. In this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐EM are systematically reviewed. In addition, the new development direction of cryo‐EM—cryo‐electron tomography (cryo‐ET), is discussed in detail. Also, cryo‐EM is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐EM and drug development. This review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐EM, and to bringing them new insights. 相似文献
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Ravi Raghav Sonani Mahima Sharma Gagan Deep Gupta Vinay Kumar Datta Madamwar 《Acta Crystallographica. Section F, Structural Biology Communications》2015,71(8):998-1004
The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting‐drop vapour‐diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus‐2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit‐cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, β = 82.50, γ = 60.34°. The molecular‐replacement solution confirmed the presence of 12 αβ monomers in the P1 cell. The Phormidium PE elutes as an (αβ)3 trimer of αβ monomers from a molecular‐sieve column and exists as [(αβ)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher‐order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly. 相似文献
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Maxim Polikarpov Gleb Bourenkov Irina Snigireva Anatoly Snigirev Sophie Zimmermann Krisztian Csanko Sandor Brockhauser Thomas R. Schneider 《Acta Crystallographica. Section D, Structural Biology》2019,75(11):947-958
For the extraction of the best possible X‐ray diffraction data from macromolecular crystals, accurate positioning of the crystals with respect to the X‐ray beam is crucial. In addition, information about the shape and internal defects of crystals allows the optimization of data‐collection strategies. Here, it is demonstrated that the X‐ray beam available on the macromolecular crystallography beamline P14 at the high‐brilliance synchrotron‐radiation source PETRA III at DESY, Hamburg, Germany can be used for high‐energy phase‐contrast microtomography of protein crystals mounted in an optically opaque lipidic cubic phase matrix. Three‐dimensional tomograms have been obtained at X‐ray doses that are substantially smaller and on time scales that are substantially shorter than those used for diffraction‐scanning approaches that display protein crystals at micrometre resolution. Adding a compound refractive lens as an objective to the imaging setup, two‐dimensional imaging at sub‐micrometre resolution has been achieved. All experiments were performed on a standard macromolecular crystallography beamline and are compatible with standard diffraction data‐collection workflows and apparatus. Phase‐contrast X‐ray imaging of macromolecular crystals could find wide application at existing and upcoming low‐emittance synchrotron‐radiation sources. 相似文献
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Hsin‐Yi Wang Ying‐Ya Hsu Rong Chen Ting‐Shan Chan Hao Ming Chen Bin Liu 《Liver Transplantation》2015,5(10)
Efficient and earth abundant electrocatalysts for high‐performance oxygen evolution reaction (OER) are essential for the development of sustainable energy conversion technologies. Here, a new hierarchical Ni–Co oxide nanostructure, composed of small secondary nanosheets grown on primary nanosheet arrays, is synthesized via a topotactic transformation of Ni–Co layered double hydroxide. The Ni3+‐rich surface benefits the formation of NiOOH, which is the main redox site as revealed via in situ X‐ray absorption near edge structure and extended X‐ray absorption fine structure spectroscopy. The Ni–Co oxide hierarchical nanosheets (NCO–HNSs) deliver a stable current density of 10 mA cm?2 at an overpotential of ≈0.34 V for OER with a Tafel slope of as low as 51 mV dec?1 in alkaline media. The improvement in the OER activity can be ascribed to the synergy of large surface area offered by the 3D hierarchical nanostructure and the facile formation of NiOOH as the main active sites on the surface of NCO–HNSs to decrease the overpotential and facilitate the catalytic reaction. 相似文献
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Tomić A Abramić M Spoljarić J Agić D Smith DM Tomić S 《Journal of molecular recognition : JMR》2011,24(5):804-814
Human dipeptidyl peptidase III (DPP III) is a zinc-exopeptidase with implied roles in protein catabolism, pain modulation, and defense against oxidative stress. To understand the mode of ligand binding into its active site, we performed molecular modeling, site-directed mutagenesis, and biochemical analyses. Using the recently determined crystal structure of the human DPP III we built complexes between both, the wild-type (WT) protein and its mutant H568N with the preferred substrate Arg-Arg-2-naphthylamide (RRNA) and a competitive inhibitor Tyr-Phe-hydroxamate (Tyr-Phe-NHOH). The mutation of the conserved His568, structurally equivalent to catalytically important His231 in thermolysin, to Asn, resulted in a 1300-fold decrease of k(cat) for RRNA hydrolysis and in significantly lowered affinity for the inhibitor. Molecular dynamics simulations revealed the key protein-ligand interactions as well as the ligand-induced reorganization of the binding site and its partial closure. Simultaneously, the non-catalytic domain was observed to stretch and the opening at the wide side of the inter-domain cleft became enhanced. The driving force for these changes was the formation of the hydrogen bond between Asp372 and the bound ligand. The structural and dynamical differences, found for the ligand binding to the WT enzyme and the H568N mutant, and the calculated binding free energies, agree well with the measured affinities. On the basis of the obtained results we suggest a possible reaction mechanism. In addition, this work provides a foundation for further site-directed mutagenesis experiments, as well as for modeling the reaction itself. 相似文献
14.
Min-Guan Lin Meng-Chun Chi Vankadari Naveen Yi-Ching Li Long-Liu Lin Chwan-Deng Hsiao 《Acta Crystallographica. Section D, Structural Biology》2016,72(1):59-70
Trehalose‐6‐phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6‐phosphate (T6P) to yield glucose and glucose 6‐phosphate. The products of this reaction can be further metabolized by the energy‐generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA (BlTreA) and its R201Q mutant complexed with p‐nitrophenyl‐α‐d ‐glucopyranoside (R201Q–pPNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of BlTreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of BlTreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo‐1,6‐glucosidase (BcOgl) and Erwinia rhapontici isomaltulose synthase (NX‐5), the surface potential of the BlTreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of BlTreA. Strikingly, the 281HHLK284 motif and Lys292 play critical roles in substrate discrimination by BlTreA. 相似文献
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Sugadev Ragumani J. Michael Sauder Stephen K. Burley Subramanyam Swaminathan 《Proteins》2010,78(2):487-491
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Philip‐Kunio Naito Yu Ogawa Daisuke Sawada Yoshiharu Nishiyama Tadahisa Iwata Masahisa Wada 《Biopolymers》2016,105(7):361-368
We determined the crystal structure of anhydrous chitosan at atomic resolution, using X‐ray fiber diffraction data extending to 1.17 Å resolution. The unit cell [a = 8.129(7) Å, b = 8.347(6) Å, c = 10.311(7) Å, space group P212121] of anhydrous chitosan contains two chains having one glucosamine residue in the asymmetric unit with the primary hydroxyl group in the gt conformation, that could be directly located in the Fourier omit map. The molecular arrangement of chitosan is very similar to the corner chains of cellulose II implying similar intermolecular hydrogen bonding between O6 and the amine nitrogen atom, and an intramolecular bifurcated hydrogen bond from O3 to O5 and O6. In addition to the classical hydrogen bonds, all the aliphatic hydrogens were involved in one or two weak hydrogen bonds, mostly helping to stabilize cohesion between antiparallel chains. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 361–368, 2016. 相似文献
17.
Operando X‐ray diffraction (XRD) and X‐ray absorption spectroscopy (XAS) studies of Ge anodes are carried out to understand the effect of cycling rate on Ge phase transformation during charge/discharge process and to relate that effect to capacity. It is discovered that the formation of crystalline Li15Ge4 (c‐Li15Ge4) during lithiation is suppressed beyond a certain cycling rate. A very stable and reversible high capacity of ≈1800 mAh g?1 can be attained up to 100 cycles at a slow C‐rate of C/21 when there is complete conversion of Ge anode into c‐Li15Ge4. When the C‐rate is increased to ≈C/10, the lithiation reaction is more heterogeneous and a relatively high capacity of ≈1000 mAh g?1 is achieved with poorer electrochemical reversibility. An increase in C‐rate to C/5 and higher reduces the capacity (≈500 mAh g?1) due to an impeded transformation from amorphous LixGe to c‐Li15Ge4, and yet improves the electrochemical reversibility. A proposed mechanism is presented to explain the C‐rate dependent phase transformations and the relationship of these to capacity fading. The operando XRD and XAS results provide new insights into the relationship between structural changes in Ge and battery capacity, which are important for guiding better design of high‐capacity anodes. 相似文献
18.
Frithjof C. Küpper Catherine Leblanc Wolfram Meyer‐Klaucke Philippe Potin Martin C. Feiters 《Journal of phycology》2014,50(4):652-664
Members of various algal lineages are known to be strong producers of atmospherically relevant halogen emissions, that is a consequence of their capability to store and metabolize halogens. This study uses a noninvasive, synchrotron‐based technique, X‐ray absorption spectroscopy, for addressing in vivo bromine speciation in the brown algae Ectocarpus siliculosus, Ascophyllum nodosum, and Fucus serratus, the red algae Gracilaria dura, G. gracilis, Chondrus crispus, Osmundea pinnatifida, Asparagopsis armata, Polysiphonia elongata, and Corallina officinalis, the diatom Thalassiosira rotula, the dinoflagellate Lingulodinium polyedrum and a natural phytoplankton sample. The results highlight a diversity of fundamentally different bromine storage modes: while most of the stramenopile representatives and the dinoflagellate store mostly bromide, there is evidence for Br incorporated in nonaromatic hydrocarbons in Thalassiosira. Red algae operate various organic bromine stores – including a possible precursor (by the haloform reaction) for bromoform in Asparagopsis and aromatically bound Br in Polysiphonia and Corallina. Large fractions of the bromine in the red algae G. dura and C. crispus and the brown alga F. serratus are present as Br? defects in solid KCl, similar to what was reported earlier for Laminaria parts. These results are discussed according to different defensive strategies that are used within algal taxa to cope with biotic or abiotic stresses. 相似文献
19.
Taisuke Nomura Hisamu Iwase Naoki Saka Nobuyuki Takahashi Bunzo Mikami Kimihiko Mizutani 《Acta Crystallographica. Section D, Structural Biology》2019,75(4):426-436
Although endogenous animal cellulases have great potential for industrial applications such as bioethanol production, few investigations have focused on these enzymes. In this study, the glycoside hydrolase family 45 (GH45) subfamily B endoglucanase EG27II from the snail Ampullaria crossean was expressed using a Pichia pastoris expression system and the crystal structure of the apo form was determined at 1.00 Å resolution; this is the highest resolution structure of an animal endoglucanase. The results showed that EG27II has a double‐ψ six‐stranded β‐barrel and that the structure of EG27II more closely resembles those of subfamily C enzymes than those of subfamily A enzymes. The structure of EG27II complexed with cellobiose was also determined under cryoconditions and at room temperature at three pH values, pH 4.0, 5.5 and 8.0, and no structural changes were found to be associated with the change in pH. The structural comparison and catalytic activity measurements showed that Asp137 and Asn112 function as the catalytic acid and base, respectively, and that Asp27 is also an important residue for catalysis. These high‐resolution structures of EG27II provide a large amount of information for structure‐based enzyme modification and cell‐surface engineering, which will advance biofuel production using animal‐derived cellulases. 相似文献
20.
We determined the three-dimensional (3D) crystal structure of protein TM841, a protein product from a hypothetical open-reading frame in the genome of the hyperthermophile bacterium Thermotoga maritima, to 2.0 A resolution. The protein belongs to a large protein family, DegV or COG1307 of unknown function. The 35 kDa protein consists of two separate domains, with low-level structural resemblance to domains from other proteins with known 3D structures. These structural homologies, however, provided no clues for the function of TM841. But the electron density maps revealed clear density for a bound fatty-acid molecule in a pocket between the two protein domains. The structure indicates that TM841 has the molecular function of fatty-acid binding and may play a role in the cellular functions of fatty acid transport or metabolism. 相似文献