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1.
    
The X‐ray structure of ketose 3‐epimerase from Arthrobacter globiformis M30, which was previously reported to be a d ‐allulose 3‐epimerase (AgD‐AE), was determined at 1.96 Å resolution. The crystal belonged to the hexagonal space group P6522, with unit‐cell parameters a = b = 103.98, c = 256.53 Å. The structure was solved by molecular replacement using the structure of Mesorhizobium lotil ‐ribulose 3‐epimerase (MlL‐RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD‐AE formed a homotetramer with twofold symmetry. The overall structure of AgD‐AE was more similar to that of MlL‐RE than to the known structures of d ‐psicose (alternative name d ‐allulose) 3‐epimerases (d ‐PEs or d ‐AEs), although AgD‐AE and MlL‐RE have different substrate specificities. Both AgD‐AE and MlL‐RE have long helices in the C‐terminal region that would contribute to the stability of the homotetramer. AgD‐AE showed higher enzymatic activity for l ‐ribulose than d ‐allulose; however, AgD‐AE is stable and is a unique useful enzyme for the production of d ‐allulose from d ‐fructose.  相似文献   

2.
    
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3.
    
A pair of l ‐leucine (l ‐Leu) and d ‐leucine (d ‐Leu) was incorporated into α‐aminoisobutyric acid (Aib) peptide segments. The dominant conformations of four hexapeptides, Boc‐l ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1a), Boc‐d ‐Leu‐Aib‐Aib‐Aib‐Aib‐l ‐Leu‐OMe (1b), Boc‐Aib‐Aib‐l ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2a), and Boc‐Aib‐Aib‐d ‐Leu‐l ‐Leu‐Aib‐Aib‐OMe (2b), were investigated by IR, 1H NMR, CD spectra, and X‐ray crystallographic analysis. All peptides 1a,b and 2a,b formed 310‐helical structures in solution. X‐ray crystallographic analysis revealed that right‐handed (P) 310‐helices were present in 1a and 1b and a mixture of right‐handed (P) and left‐handed (M) 310‐helices was present in 2b in their crystalline states. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

4.
    
Two versions of the functional core of the rabbit voltage‐dependent calcium channel β2a subunit were expressed in Escherichia coli. These proteins were purified to homogeneity and screened for crystallization. Crystallization conditions were refined using the hanging‐drop vapour‐diffusion method and two crystal forms were pursued. Crystal form I is represented by thick rods with tetragonal symmetry, unit‐cell parameters a = b = 75, c = 165 Å and a diffraction limit of 3.4 Å which were obtained using ammonium sulfate as a precipitant. Crystal form II gives rise to plates with orthorhombic symmetry, unit‐cell parameters a = 35, b = 75, c = 165 Å and a diffraction limit of 2.3 Å which were grown using polyethylene glycol 20K as a precipitant.  相似文献   

5.
    
α‐Dioxygenases (α‐DOX) are heme‐containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α‐DOX oxygenate a variety of 14–20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro‐R hydrogen from the α‐carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H2O2). We determined the X‐ray crystal structures of wild type α‐DOX from Oryza sativa, the wild type enzyme in complex with H2O2, and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of α‐DOX such that the carboxylate forms ionic interactions with His‐311 and Arg‐559. Thr‐316 aids in the positioning of carbon‐2 for hydrogen abstraction. Twenty‐five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H2O2 structures provides insight into enzyme activation. The binding of H2O2 at the distal face of the heme displaces residues His‐157, Asp‐158, and Trp‐159 ～2.5 Å from their positions in the wild type structure. As a result, the Oδ2 atom of Asp‐158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp‐159 and Trp‐213 reorient, and the guanidinium group of Arg‐559 is repositioned near Tyr‐379, poised to interact with the carboxylate group of the substrate.  相似文献   

6.
    
Phytases are phosphatases that hydrolyze phytates to less phosphorylated myo‐inositol derivatives and inorganic phosphate. β‐Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH‐DI, a β‐propeller phytase from Bacillus sp. HJB17, was found to act synergistically with other single‐domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine‐substituted PhyH‐DI were obtained using the vapour‐diffusion method in a condition consisting of 0.2 M sodium chloride, 0.1 M Tris pH 8.5, 25%(w /v ) PEG 3350 at 289 K. X‐ray diffraction data were collected to 3.00 and 2.70 Å resolution, respectively, at 100 K. Native PhyH‐DI crystals belonged to space group C 121, with unit‐cell parameters a  = 156.84, b  = 45.54, c  = 97.64 Å, α = 90.00, β = 125.86, γ = 90.00°. The asymmetric unit contained two molecules of PhyH‐DI, with a corresponding Matthews coefficient of 2.17 Å3 Da−1 and a solvent content of 43.26%. Crystals of selenomethionine‐substituted PhyH‐DI belonged to space group C 2221, with unit‐cell parameters a  = 94.71, b = 97.03, c = 69.16 Å, α = β = γ = 90.00°. The asymmetric unit contained one molecule of the protein, with a corresponding Matthews coefficient of 2.44 Å3 Da−1 and a solvent content of 49.64%. Initial phases for PhyH‐DI were obtained from SeMet SAD data sets. These data will be useful for further studies of the structure–function relationship of PhyH‐DI.  相似文献   

7.
    
A single chiral cyclic α,α‐disubstituted amino acid with side‐chain methoxymethyl (MOM) protecting groups, (3S,4S)−1‐amino‐(3,4‐dimethoxymethoxy)cyclopentanecarboxylic acid [(S, S)‐Ac5cdOMOM], or side‐chain hydroxy groups, (3S,4S)−1‐amino‐(3,4‐dihydroxy)cyclopentanecarboxylic acid [(S, S)‐Ac5cdOH], was attached to the N‐terminal or C‐terminal position of α‐aminoisobutyric acid (Aib) tetrapeptide segments; i.e., we designed and synthesized four pentapeptides, Cbz‐[(S, S)‐Ac5cdOMOM]‐(Aib)4‐OEt ( 1 ), Cbz‐[(S, S)‐Ac5cdOH]‐(Aib)4‐OEt ( 2 ), Cbz‐(Aib)4‐[(S, S)‐Ac5cdOMOM]‐OMe ( 3 ), and Cbz‐(Aib)4‐[(S, S)‐Ac5cdOH]‐OMe ( 4 ). We then analyzed the peptides’ structures in the crystalline state. The four peptides all folded into 310‐helical structures; 1 formed a left‐handed (M) 310‐helix, 2 formed a mixture of right‐handed (P) and (M) 310‐helices, 3 formed a mixture of (P) and (M) 310‐helices, and 4 formed a (P) 310‐helix, respectively. In packing mode, the molecules of peptides 1 and 3 , which both possessed an Ac5cdOMOM residue, were connected by intermolecular hydrogen bonds along the peptide backbone (N H···O type). On the other hand, the packing of peptides 2 and 4 , which both contained an Ac5cdOH residue, was based on intermolecular hydrogen bonds derived from both the peptide backbone and the side‐chain hydroxy groups of the amino acid Ac5cdOH (O H···O type). © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 757–768, 2016.  相似文献   

8.
Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.  相似文献   

9.
    
The first bacterial member of the lipocalin protein family, Blc, was identified in Escherichia coli as an outer membrane lipoprotein that is expressed under conditions of environmental stress. Previous crystallographic studies in space group P212121 with two molecules per asymmetric unit, supported by static light‐scattering experiments in solution, indicated that Blc may form a functional homodimer with lysophospholipid binding activity. Here, a new crystal structure of recombinant Blc in space group I4122 with one molecule in the asymmetric unit is described. The crystal packing differs considerably from that observed previously, which was determined using an N‐terminally extended version of Blc dubbed `Blc‐X'. In particular, the characteristic large interface region that was previously described as being responsible for stable dimer formation is absent in the I4122 crystal structure. Thus, the dimerization behaviour of Blc‐X was most likely to be caused by the additional N‐terminal peptide segment resulting from the cloning strategy employed. In contrast, we used a native‐like N‐terminus for Blc with just the lipid‐anchored first Cys residue replaced by Ala. The fully monomeric status of this recombinant version of Blc in solution was confirmed by size‐exclusion chromatography as well as analytical ultracentrifugation. Consequently, these data shed new light on the previously postulated lipid‐binding mechanism and biological role of Blc. Beyond this, our findings illustrate that cloning artefacts, which frequently result from recombinant protein production for structural studies, must be considered with special caution when interpreting oligomerization and/or conformational effects.  相似文献   

10.
    
The number of new X‐ray crystallography‐based submissions to the Protein Data Bank appears to be at the beginning of a decline, perhaps signalling an end to the era of the dominance of X‐ray crystallography within structural biology. This letter, from the viewpoint of a young structural biologist, applies the Copernican method to the life expectancy of crystallography and asks whether the technique is still the mainstay of structural biology. A study of the rate of Protein Data Bank depositions allows a more nuanced analysis of the fortunes of macromolecular X‐ray crystallography and shows that cryo‐electron microscopy might now be outcompeting crystallography for new labour and talent, perhaps heralding a change in the landscape of the field.  相似文献   

11.
    
The preferred helical screw senses of chiral α‐amino acids with a Cα‐tetrasubstituted α‐carbon atom, as determined in the crystal state by X‐ray diffraction analyses on derivatives and peptides, are reviewed. This survey covers Cα‐methylated and Cα‐ethylated α‐amino acids, as well as α‐amino acids cyclized on the α‐carbon, including those characterized by the combination of lack of chirality at the α‐carbon with either side‐chain or axial chirality. Although, in general, chiral Cα‐tetrasubstituted α‐amino acids show a less pronounced bias toward a single helical screw sense than their proteinogenic (Cα‐trisubstituted) counterparts, our analysis highlights significant differences in terms of magnitude and direction of such a bias among the various sub‐families of residues, and between individual amino acids within each sub‐family as well. The experimental findings can be rationalized, at least in part, on the basis of steric considerations. © 2014 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 104: 46–64, 2015.  相似文献   

12.
    
Proteins of the Omp85 family chaperone the membrane insertion of β‐barrel‐shaped outer membrane proteins in bacteria, mitochondria, and probably chloroplasts and facilitate the transfer of nuclear‐encoded cytosolically synthesized preproteins across the outer envelope of chloroplasts. This protein family is characterized by N‐terminal polypeptide transport‐associated (POTRA) domains and a C‐terminal membrane‐embedded β‐barrel. We have investigated a recently identified Omp85 family member of Arabidopsis thaliana annotated as P39. We show by in vitro and in vivo experiments that P39 is localized in chloroplasts. The electrophysiological properties of P39 are consistent with those of other Omp85 family members confirming the sequence based assignment of P39 to this family. Bioinformatic analysis showed that P39 lacks any POTRA domain, while a complete 16 stranded β‐barrel including the highly conserved L6 loop is proposed. The electrophysiological properties are most comparable to Toc75‐V, which is consistent with the phylogenetic clustering of P39 in the Toc75‐V rather than the Toc75‐III branch of the Omp85 family tree. Taken together P39 forms a pore with Omp85 family protein characteristics. The bioinformatic comparison of the pore region of Toc75‐III, Toc75‐V, and P39 shows distinctions of the barrel region most likely related to function. Proteins 2017; 85:1391–1401. © 2014 Wiley Periodicals, Inc.  相似文献   

13.
    
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP‐μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP‐μNS(448–605) baculovirus formed rod‐shaped microcrystals (5–15 µm in length) which were reconstituted in high‐viscosity media (LCP and agarose) and investigated by serial femtosecond X‐ray diffraction using viscous jets at an X‐ray free‐electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit‐cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron‐density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), the current experimental strategy, which couples high‐viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.  相似文献   

14.
    
A single chiral cyclic α,α‐disubstituted amino acid, (3S,4S)‐1‐amino‐(3,4‐dimethoxy)cyclopentanecarboxylic acid [(S,S)‐Ac5cdOM], was placed at the N‐terminal or C‐terminal positions of achiral α‐aminoisobutyric acid (Aib) peptide segments. The IR and 1H NMR spectra indicated that the dominant conformations of two peptides Cbz‐[(S,S)‐Ac5cdOM]‐(Aib)4‐OEt ( 1) and Cbz‐(Aib)4‐[(S,S)‐Ac5cdOM]‐OMe (2) in solution were helical structures. X‐ray crystallographic analysis of 1 and 2 revealed that a left‐handed (M) 310‐helical structure was present in 1 and that a right‐handed (P) 310‐helical structure was present in 2 in their crystalline states. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.  相似文献   

15.
    
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16.
An α/β barrel is predicted for the three-dimensional (3D) structure of Bacillus subtilis ferrochelatase. To arrive at this structure, the THREADER program was used to find possible homologous 3D structures and to predict the secondary structure for the ferrochelatase sequence. The secondary structure was fit by hand to the selected homologous 3D structure then the MODELLER program was used to predict the fold of ferrochelatase. Molecular biological information about the conserved residues of ferrochelatase was used as the criteria to help select the homologous 3D structure used to predict the fold of ferrochelatase. Based on the predicted structure possible, ligands binding to the iron and protoporphyrin IX are discussed. The structure has been deposited in the Brookhaven database as ID 1FJI. © 1997 Wiley-Liss Inc.  相似文献   

17.
    
The crystal structure of an archaeal‐type phosphoenolpyruvate carboxylase from Clostridium perfringens has been determined based on X‐ray data extending to 3 Å. The asymmetric unit of the structure includes two tetramers (each a dimer‐of‐dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of phosphoenolpyruvate carboxylase and a malonate molecule is seen in the active‐site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose‐6‐phosphate (an activator) observed in the Escherichia coli and Zea mays phosphoenolpyruvate carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg++. phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal‐type phosphoenolpyruvate carboxylases with differing sensitivity to inhibition by aspartate. Proteins 2011; © 2011 Wiley‐Liss, Inc.  相似文献   

18.
    
Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P42212 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (VM) of 2.57 Å3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)8 core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)8 barrel. © 1995 Wiley-Liss, Inc.  相似文献   

19.
    
The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long‐lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty‐acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β‐oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosisl ‐3‐hydroxyacyl‐CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand‐free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann‐fold motif in the N‐terminal domain, while the helical C‐terminal domain mediates dimer formation. Comparison with the CoA‐ and NAD+‐bound human orthologue mitochondrial hydroxyacyl‐CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi‐catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self‐association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA–FadB complex as it lacks the N‐terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl‐CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein‐network database.  相似文献   

20.
    
To investigate early intermediates of β2‐microglobulin (β2m) amyloidogenesis, we solved the structure of β2m containing the amyloidogenic Pro32Gly mutation by X‐ray crystallography. One nanobody (Nb24) that efficiently blocks fibril elongation was used as a chaperone to co‐crystallize the Pro32Gly β2m monomer under physiological conditions. The complex of P32G β2m with Nb24 reveals a trans peptide bond at position 32 of this amyloidogenic variant, whereas Pro32 adopts the cis conformation in the wild‐type monomer, indicating that the cis to trans isomerization at Pro32 plays a critical role in the early onset of β2m amyloid formation.  相似文献   

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