scholarly journals Crystallization and preliminary crystallographic analysis of human muscle phosphofructokinase, the main regulator of glycolysis

2014 ◽  
Vol 70 (5) ◽  
pp. 578-582 ◽  
Author(s):  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Torsten Schöneberg ◽  
Norbert Sträter
Keyword(s):  
Three Dimensional ◽  
Crystallographic Analysis ◽  
Human Muscle ◽  
Yeast Cells ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Diffusion Method ◽  
Rabbit Muscle ◽  
Main Regulator ◽  
Purification Protocol

Whereas the three-dimensional structure and the structural basis of the allosteric regulation of prokaryotic 6-phosphofructokinases (Pfks) have been studied in great detail, knowledge of the molecular basis of the allosteric behaviour of the far more complex mammalian Pfks is still very limited. The human muscle isozyme was expressed heterologously in yeast cells and purified using a five-step purification protocol. Protein crystals suitable for diffraction experiments were obtained by the vapour-diffusion method. The crystals belonged to space groupP6222 and diffracted to 6.0 Å resolution. The 3.2 Å resolution structure of rabbit muscle Pfk (rmPfk) was placed into the asymmetric unit and optimized by rigid-body and groupB-factor refinement. Interestingly, the tetrameric enzyme dissociated into a dimer, similar to the situation observed in the structure of rmPfk.

scholarly journals Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Santhosh Gatreddi ◽  
Sayanna Are ◽  
Insaf Ahmed Qureshi
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Protozoan Parasite ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Gene Encoding ◽  
Cell Parameters ◽  
Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

METHODS FOR INDICATION OF NONCULTURATED VIABLE MICROORGANISMS WHEN CONTROL OF CRITICAL POINTS OF LIVESTOCK, POULTRY AND FOOD PRODUCTION TECHNOLOGY

2021 ◽  
Vol 1 (4) ◽  
pp. 441-447
Author(s):  
Ekaterina M. Lenchenko ◽  
◽  
Damir I. Udavliev ◽  
Inna B. Pavlova ◽  
◽  
...  
Keyword(s):  
Cell Wall ◽  
Luminescence Spectrum ◽  
Three Dimensional ◽  
Yeast Cells ◽  
Dimensional Structure ◽  
Heterogeneous Structure ◽  
Bacterial Cells ◽  
Microscopic Fungi ◽  
Gram Positive Bacteria ◽  
Red Luminescence

The results of morphometric and densitometric parameters biofilms are presented, effective methods of detecting uncultivated viable microorganisms isolated from a representative sample of objects of veterinary and sanitary supervision are tested and selected. Optical, luminescent and scanning electron microscopy revealed the formation of a three-dimensional structure biofilms in the form a dense network consisting of gram-negative and gram-positive bacteria, yeast cells, hyphal and pseudohyphalic forms, surrounded by an intercellular polymer matrix. The presence hyphae of microscopic fungi causes an increase in the number of cells adhered to the substrate, microcolonies were formed from bacteria and yeast cells of microscopic fungi. The pathogenesis of the syndrome of overgrowth of microorganisms is provided by the presence of various dissociative variants, the dispersion of uncultivated bacterial cells, which gain advantages in the hyperagregation of the architectonics of heterogeneous biofilms. Multilayer membranes, vesicles, cells with a defective cell wall, spheroplasts, protoplasts, L-shapes, needle-like and giant structures, and revertant cells were identified. The dynamics of changes in the viable structures microorganisms was characterized by alternating periods of decrease and increase in the intensity of biofilm formation. When detecting the viability of microorganisms in the composition biofilms, viable and non-viable cells were differentiated – a green luminescence spectrum and a red luminescence spectrum, respectively. The dissociation of the population caused an increase in the concentration of R-dissociant cells with a higher growth rate, cell lysis was detected after 48–72 h of cultivation, a change in the ratio phenotypic forms was observed – the M-dissociant was predominant. The study of the heterogeneous structure of the population, without disturbing the natural architectonics of biofilms, revealed direct correlations (r = 0,89) between morphometric (≥90 % of the field of view) and densitometric parameters (OD). The efficiency of a nutrient medium containing pancreatic hydrolyzate, mannitol, L-asparagine and glycerol was established for the repair of the cell wall, the reversal of L-forms of microorganisms.

scholarly journals Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 114
Author(s):  
R. Elliot Murphy ◽  
Alexandra B. Samal ◽  
Gunnar Eastep ◽  
Ruba H. Ghanam ◽  
Peter E. Prevelige ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Late Phase ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Membrane Interactions ◽  
Env Protein ◽  
Gag Assembly ◽  
Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

scholarly journals Preliminary crystallographic analysis of Xyn52B2, a GH52 β-D-xylosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (12) ◽  
pp. 1675-1682 ◽  
Author(s):  
Roie Dann ◽  
Shifra Lansky ◽  
Noa Lavid ◽  
Arie Zehavi ◽  
Valery Belakhov ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Crystal Form ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Glycoside Hydrolase Family ◽  
Data Sets ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Average Unit

Geobacillus stearothermophilusT6 is a thermophilic bacterium that possesses an extensive hemicellulolytic system, including over 40 specific genes that are dedicated to this purpose. For the utilization of xylan, the bacterium uses an extracellular xylanase which degrades xylan to decorated xylo-oligomers that are imported into the cell. These oligomers are hydrolyzed by side-chain-cleaving enzymes such as arabinofuranosidases, acetylesterases and a glucuronidase, and finally by an intracellular xylanase and a number of β-xylosidases. One of these β-xylosidases is Xyn52B2, a GH52 enzyme that has already proved to be useful for various glycosynthesis applications. In addition to its demonstrated glycosynthase properties, interest in the structural aspects of Xyn52B2 stems from its special glycoside hydrolase family, GH52, the structures and mechanisms of which are only starting to be resolved. Here, the cloning, overexpression, purification and crystallization of Xyn52B2 are reported. The most suitable crystal form that has been obtained belonged to the orthorhombicP212121space group, with average unit-cell parametersa = 97.7,b= 119.1,c = 242.3 Å. Several X-ray diffraction data sets have been collected from flash-cooled crystals of this form, including the wild-type enzyme (3.70 Å resolution), the E335G catalytic mutant (2.95 Å resolution), a potential mercury derivative (2.15 Å resolution) and a selenomethionine derivative (3.90 Å resolution). These data are currently being used for detailed three-dimensional structure determination of the Xyn52B2 protein.

scholarly journals Overexpression, crystallization and preliminary X-ray crystallographic analysis of the ectoine hydroxylase fromSphingopyxis alaskensis

2014 ◽  
Vol 70 (4) ◽  
pp. 493-496 ◽  
Author(s):  
Astrid Hoeppner ◽  
Nils Widderich ◽  
Erhard Bremer ◽  
Sander H. J. Smits
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Oxidative Decarboxylation ◽  
Expression And Purification ◽  
Crystal Forms ◽  
X Ray ◽  
Mononuclear Iron ◽  
Vapour Diffusion ◽  
Purification Protocol

The ectoine hydroxylase (EctD) is a member of the non-haem-containing iron(II)- and 2-oxoglutarate-dependent dioxygenase superfamily. Its mononuclear iron centre is a prerequisite for the activity of this enzyme and promotes the O2-dependent oxidative decarboxylation of 2-oxoglutarate, which is coupled to a two-electron oxidation of the substrate ectoine to yield 5-hydroxyectoine. An expression and purification protocol for the EctD enzyme fromSphingopyxis alaskensiswas developed and the protein was crystallized using the sitting-drop vapour-diffusion method. This resulted in two different crystal forms, representing the apo and iron-bound forms of the enzyme.

scholarly journals Folding domains and intramolecular ionic interactions of lysine residues in glyceraldehyde 3-phosphate dehydrogenase

1977 ◽  
Vol 161 (1) ◽  
pp. 49-62 ◽  
Author(s):  
J M Lambert ◽  
R N Perham
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Three Dimensional ◽  
Ion Pair ◽  
Dimensional Structure ◽  
Ionic Interactions ◽  
Rabbit Muscle ◽  
Amino Groups ◽  
Muscle Enzyme ◽  
Lysine Residues

1. Treatment with methyl acetimidate was used to probe the topography of several tetrameric glyceraldehyde 3-phosphate dehydrogenases, in particular the holoenzymes from rabbit muscle and Bacillus stearothermophilus. During the course of the reaction with the rabbit muscle enzyme, the number of amino groups fell rapidly from the starting value of 27 per subunit to a value of approx. five per subunit. This number could be lowered further to values between one and two per subunit by a second treatment with methyl acetimidate. The enzyme remained tetrameric throughout and retained 50% of its initial catalytic activity at the end of the experiment. 2. Use of methyl [1-14C]acetimidate and small-scale methods of protein chemistry showed that only one amino group per subunit, that of lysine-306, was completely unavailable for reaction with imido ester in the native enzyme. This results is consistent with the structure of the highly homologous glyceraldehyde 3-phosphate dehydrogenase of lobster muscle deduced from X-ray-crystallographic analysis, since lysine-306 can be seen to form an intrachain ion-pair with aspartic acid-241 in the hydrophobic environment of a subunit-subunit interface. 3. Several other amino groups in the rabbit muscle enzyme that reacted only slowly with the reagent were also identified chemically. These were found to be located entirely in the C-terminal half of the polypeptides chain, which comprises a folding domain associated with catalytic activity and subunit contact in the three-dimensional structure. Slow reaction of these ‘surface’ amino groups with methyl acetimidate is attributed to intramolecular ionic interactions of the amino groups with neighbouring side-chain carboxyl groups, a conclusion that is compatible with the reported three-dimensional structure and with the dependence of the reaction of ionic stength. 4. Very similar results were obtained with the enzymes from B. stearothermophilus and from ox muscle and ox liver, supporting the view that the ion-pair involving lysine-306 and aspartic acid-241 will be a common structural feature in glyceraldehyde-3-phosphate dehydrogenases. The B. stearothermophilus enzyme was fully active after modification. 5. No differences could be detected between the enzymes from ox muscle and ox liver, in accord with other evidence that points to the identify of these enzymes. 6. The pattern of slowly reacting amino groups in the enzyme from B. stearothermophilus, although similar to that of the mammalian enzymes, indicated one or two additional intramolecular ionic interactions of lysine residues that might contribute to the thermal stability of this enzyme.

scholarly journals Three-dimensional Structure of Goose-type Lysozyme from the Egg White of the Australian Black Swan, Cygnus atratus

1985 ◽  
Vol 38 (1) ◽  
pp. 13 ◽  
Author(s):  
NW Isaacs ◽  
K JMachin ◽  
M Masakuni
Keyword(s):  
Three Dimensional ◽  
Egg White ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Hen Egg White Lysozyme ◽  
Amino Acid Sequence Homology ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Domestic Hen ◽  
Hen Egg

The egg white of C. atratus contains two forms of lysozyme, a 'chick-type' which is similar to that found in the egg white of the domestic hen, and a 'goose-type' similar to that found in the egg white of the Embden goose. The molecular structure of the goose-type lysozyme has been determined at a resolution of a 2' 8 A by X-ray crystallographic analysis. The structure consists of two domains linked by a long stretch of a-helix. In all, there are seven helical segments in the structure. While there is no amino acid sequence homology with either hen egg-white or bacteriophage T4lysozymes, there are portions of the structure where the folding of the main chain is similar to that found in portions of either hen egg-white lysozyme or T4 lysozyme or both. In particular, there is a consistency of structure in the arrangement of acid groups in the catalytic site.

scholarly journals Structural insights into the cause of human RSPH4A primary ciliary dyskinesia

2021 ◽  
pp. mbc.E20-12-0806
Author(s):  
Yanhe Zhao ◽  
Justine Pinskey ◽  
Jianfeng Lin ◽  
Weining Yin ◽  
Patrick R. Sears ◽  
...  
Keyword(s):  
Primary Ciliary Dyskinesia ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Radial Spoke ◽  
Radial Spokes ◽  
Cilia And Flagella ◽  
Tract Infections ◽  
Ciliary Dyskinesia

Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Protein 4 homolog A ( RSPH4A) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of RSPH4A-/- PCD remains elusive. Here, we present the native, three-dimensional structure of RSPH4A-/- human respiratory cilia using samples collected non-invasively from a PCD patient. Using cryo-electron tomography and subtomogram averaging, we compared the structures of control and RSPH4A-/- cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to RSPH1-/- cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in RSPH4A-/- cilia. However, RSPH4A-/- cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for RSPH4A-/- PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. [Media: see text]

scholarly journals Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

2016 ◽  
Vol 198 (10) ◽  
pp. 1499-1512 ◽  
Author(s):  
Sonali Dhindwal ◽  
Leticia Gomez-Gil ◽  
David B. Neau ◽  
Thi Thanh My Pham ◽  
Michel Sylvestre ◽  
...  
Keyword(s):  
Polychlorinated Biphenyl ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Degradation Pathway ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Biphenyl Dioxygenase ◽  
Pcb Congeners ◽  
Biphenyl Degradation ◽  
Insight Into

ABSTRACTBiphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400in which a seven-residue segment,335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356,333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400and BphAEB356. BphAEII9also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparentkcat/Kmof 2.2 ± 0.5 mM−1s−1, versus 0.9 ± 0.5 mM−1s−1for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes.IMPORTANCEBiphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.

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