scholarly journals Transketolase from Leishmania mexicana has a dual subcellular localization

2004 ◽  
Vol 382 (2) ◽  
pp. 759-767 ◽  
Author(s):  
Nicola J. VEITCH ◽  
Dante A. MAUGERI ◽  
Juan Jose CAZZULO ◽  
Ylva LINDQVIST ◽  
Michael P. BARRETT
Keyword(s):  
Subcellular Localization ◽  
Three Dimensional ◽  
Total Activity ◽  
Pentose Phosphate ◽  
Dimensional Structure ◽  
Leishmania Mexicana ◽  
Gene Encoding ◽  
X Ray Crystallography ◽  
C Terminus ◽  
Targeting Signal

Transketolase has been characterized in Leishmania mexicana. A gene encoding this enzyme was identified and cloned. The gene was expressed in Escherichia coli and the protein was purified and characterized. An apparent Km of 2.75 mM for ribose 5-phosphate was determined. X-ray crystallography was used to determine the three-dimensional structure of the enzyme to a resolution of 2.2 Å (1 Å≡0.1 nm). The C-terminus of the protein contains a type-1 peroxisome-targeting signal, suggestive of a possible glycosomal subcellular localization. Subcellular localization experiments performed with promastigote forms of the parasite revealed that the protein was predominantly cytosolic, although a significant component of the total activity was associated with the glycosomes. Transketolase is thus the first enzyme of the nonoxidative branch of the pentose phosphate pathway whose presence has been demonstrated in a peroxisome-like organelle.

Crystal structure of the hinge domain of Smchd1 reveals its dimerization mode and nucleic acid–binding residues

2020 ◽  
Vol 13 (636) ◽  
pp. eaaz5599 ◽  
Author(s):  
Kelan Chen ◽  
Richard W. Birkinshaw ◽  
Alexandra D. Gurzau ◽  
Iromi Wanigasuriya ◽  
Ruoyun Wang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Muscular Dystrophy ◽  
Three Dimensional ◽  
Underlying Mechanism ◽  
Structural Features ◽  
Facioscapulohumeral Muscular Dystrophy ◽  
Dimensional Structure ◽  
Nucleic Acid Binding ◽  
X Ray Crystallography ◽  
Hinge Domain

Structural maintenance of chromosomes flexible hinge domain containing 1 (SMCHD1) is an epigenetic regulator in which polymorphisms cause the human developmental disorder, Bosma arhinia micropthalmia syndrome, and the degenerative disease, facioscapulohumeral muscular dystrophy. SMCHD1 is considered a noncanonical SMC family member because its hinge domain is C-terminal, because it homodimerizes rather than heterodimerizes, and because SMCHD1 contains a GHKL-type, rather than an ABC-type ATPase domain at its N terminus. The hinge domain has been previously implicated in chromatin association; however, the underlying mechanism involved and the basis for SMCHD1 homodimerization are unclear. Here, we used x-ray crystallography to solve the three-dimensional structure of the Smchd1 hinge domain. Together with structure-guided mutagenesis, we defined structural features of the hinge domain that participated in homodimerization and nucleic acid binding, and we identified a functional hotspot required for chromatin localization in cells. This structure provides a template for interpreting the mechanism by which patient polymorphisms within the SMCHD1 hinge domain could compromise function and lead to facioscapulohumeral muscular dystrophy.

scholarly journals Mechanisms of Congenital Myasthenia Caused by Three Mutations in the COLQ Gene

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaona Luo ◽  
Chunmei Wang ◽  
Longlong Lin ◽  
Fang Yuan ◽  
Simei Wang ◽  
...  
Keyword(s):  
Missense Mutation ◽  
Neuromuscular Junctions ◽  
Three Dimensional ◽  
Missense Variant ◽  
Homozygous Deletion ◽  
Dimensional Structure ◽  
Compound Heterozygous ◽  
Splicing Mutation ◽  
Gene Encoding ◽  
Congenital Myasthenia

The gene encoding collagen like tail subunit of asymmetric acetylcholinesterase (COLQ) is responsible for the transcription of three strands of collagen of acetylcholinesterase, which is attached to the endplate of neuromuscular junctions. Mutations in the COLQ gene are inherited in an autosomal-recessive manner and can lead to type V congenital myasthenia syndrome (CMS), which manifests as decreased muscle strength at birth or shortly after birth, respiratory failure, restricted eye movements, drooping of eyelids, and difficulty swallowing. Here we reported three variants within COLQ in two unrelated children with CMS. An intronic variant (c.393+1G>A) and a novel missense variant (p.Q381P) were identified as compound heterozygous in a 13-month-old boy, with the parents being carriers of each. An intragenic deletion including exons 14 and 15 was found in a homozygous state in a 12-year-old boy. We studied the relative expression of the COLQ and AChE gene in the probands' families, performed three-dimensional protein structural analysis, and analyzed the conservation of the missense mutation c.1142A>C (p.Q381P). The splicing mutation c.393+1G>A was found to affect the normal splicing of COLQ exon 5, resulting in a 27-bp deletion. The missense mutation c.1142A>C (p.Q381P) was located in a conserved position in different species. We found that homozygous deletion of COLQ exons 14–15 resulted in a 241-bp deletion, which decreased the number of amino acids and caused a frameshift translation. COLQ expression was significantly lower in the probands than in the probands' parents and siblings, while AChE expression was significantly higher. Moreover, the mutations were found to cause significant differences in the predicted three-dimensional structure of the protein. The splicing mutation c.393+1G>A, missense mutation c.1A>C (p.Q381P), and COLQ exon 14–15 deletion could cause CMS.

MRPC (Missing Regions in Polypeptide Chains): a knowledgebase

2019 ◽  
Vol 52 (6) ◽  
pp. 1422-1426
Author(s):  
Rajendran Santhosh ◽  
Namrata Bankoti ◽  
Adgonda Malgonnavar Padmashri ◽  
Daliah Michael ◽  
Jeyaraman Jeyakanthan ◽  
...  
Keyword(s):  
Protein Structures ◽  
Three Dimensional ◽  
Protein Molecule ◽  
Data Bank ◽  
Protein Crystal ◽  
Dimensional Structure ◽  
Protein Structure Analysis ◽  
Three Dimensional Structure ◽  
X Ray Crystallography ◽  
Polypeptide Chains

Missing regions in protein crystal structures are those regions that cannot be resolved, mainly owing to poor electron density (if the three-dimensional structure was solved using X-ray crystallography). These missing regions are known to have high B factors and could represent loops with a possibility of being part of an active site of the protein molecule. Thus, they are likely to provide valuable information and play a crucial role in the design of inhibitors and drugs and in protein structure analysis. In view of this, an online database, Missing Regions in Polypeptide Chains (MRPC), has been developed which provides information about the missing regions in protein structures available in the Protein Data Bank. In addition, the new database has an option for users to obtain the above data for non-homologous protein structures (25 and 90%). A user-friendly graphical interface with various options has been incorporated, with a provision to view the three-dimensional structure of the protein along with the missing regions using JSmol. The MRPC database is updated regularly (currently once every three months) and can be accessed freely at the URL http://cluster.physics.iisc.ac.in/mrpc.

scholarly journals The Prodigal Compound: Return of Ribosyl 1,5-Bisphosphate as an Important Player in Metabolism

2018 ◽  
Vol 83 (1) ◽  
Author(s):  
Bjarne Hove-Jensen ◽  
Ditlev E. Brodersen ◽  
M. Cemre Manav
Keyword(s):  
Three Dimensional ◽  
Bioinformatic Analysis ◽  
Pentose Phosphate ◽  
Dimensional Structure ◽  
Big Brother ◽  
Important Player ◽  
Domains Of Life ◽  
Important Intermediate ◽  
Metabolic Activities ◽  
Taxonomic Groups

SUMMARYRibosyl 1,5-bisphosphate (PRibP) was discovered 65 years ago and was believed to be an important intermediate in ribonucleotide metabolism, a role immediately taken over by its “big brother” phosphoribosyldiphosphate. Only recently has PRibP come back into focus as an important player in the metabolism of ribonucleotides with the discovery of the pentose bisphosphate pathway that comprises, among others, the intermediates PRibP and ribulose 1,5-bisphosphate (cf. ribose 5-phosphate and ribulose 5-phosphate of the pentose phosphate pathway). Enzymes of several pathways produce and utilize PRibP not only in ribonucleotide metabolism but also in the catabolism of phosphonates, i.e., compounds containing a carbon-phosphorus bond. Pathways for PRibP metabolism are found in all three domains of life, most prominently among organisms of the archaeal domain, where they have been identified either experimentally or by bioinformatic analysis within all of the four main taxonomic groups,Euryarchaeota, TACK, DPANN, and Asgard. Advances in molecular genetics of archaea have greatly improved the understanding of the physiology of PRibP metabolism, and reconciliation of molecular enzymology and three-dimensional structure analysis of enzymes producing or utilizing PRibP emphasize the versatility of the compound. Finally, PRibP is also an effector of several metabolic activities in many organisms, including higher organisms such as mammals. In the present review, we describe all aspects of PRibP metabolism, with emphasis on the biochemical, genetic, and physiological aspects of the enzymes that produce or utilize PRibP. The inclusion of high-resolution structures of relevant enzymes that bind PRibP provides evidence for the flexibility and importance of the compound in metabolism.

Structure and conformation of the hydrochloride of pseudoisocytidine, an antileukemic C-nucleoside

1980 ◽  
Vol 58 (16) ◽  
pp. 1633-1638 ◽  
Author(s):  
George I. Birnabaum ◽  
Kyoichi A. Watanabe ◽  
Jack J. Fox
Keyword(s):  
Heavy Atom ◽  
Three Dimensional ◽  
Direct Methods ◽  
Dimensional Structure ◽  
Side Chain ◽  
Hydrogen Atoms ◽  
Triclinic Space Group ◽  
X Ray Crystallography ◽  
Cell Dimensions ◽  
Sugar Ring

The three-dimensional structure of pseudoisocytidine hydrochloride was determined by X-ray crystallography. The crystals belong to the triclinic space group P1 and the cell dimensions are a = 6.623(2), b = 8.053(2), c = 6.201(2) Å, α = 108.35(2), β = 101.36(2), γ = 93.54(2) °. Intensity data were measured with a diffractometer and the structure was solved by a combination of heavy-atom and direct methods. Least-squares refinement, which included hydrogen atoms, converged at R = 0.040. The conformation about the glycosyl bond is anti (χCC = 21.6°), the pucker of the furanose ring is C(1′)exo, and the conformation of the —CH2OH side chain is gauche–trans (t). An examination of bond lengths indicates that of the three main resonance forms of the isocytosine cation the fully conjugated one contributes more to the structure than the cross-conjugated one. Bond angles in the sugar ring reflect its rare conformation.

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.

scholarly journals Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

2018 ◽  
Vol 19 (11) ◽  
pp. 3401 ◽  
Author(s):  
Ashutosh Srivastava ◽  
Tetsuro Nagai ◽  
Arpita Srivastava ◽  
Osamu Miyashita ◽  
Florence Tama
Keyword(s):  
Protein Dynamics ◽  
Computational Methods ◽  
Protein Structures ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

Three dimensional structure of the leishmania amastigote as revealed by computer-aided reconstruction from serial sections

Parasitology ◽  
1986 ◽  
Vol 92 (1) ◽  
pp. 13-23 ◽  
Author(s):  
G. H. Coombs ◽  
L. Tetley ◽  
V. A. Moss ◽  
K. Vickerman
Keyword(s):  
Cell Volume ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Leishmania Mexicana ◽  
Cell Organelles ◽  
3 Dimensional ◽  
Computer Aided ◽  
Total Cell Volume

SUMMARYComputer-aided reconstruction from serial sections has been used to analyse the 3-dimensional structure of entire amastigotes of Leishmania mexicana mexicana and to determine the number, arrangement and volume of each organelle. In two reconstructions, the lysosome-like ‘megasomes’ were the most numerous organelle, there being 34 in one amastigote, and they comprised as much as 15% of the total cell volume. In contrast, as few as 9 glycosomes were present, accounting for less than 1% of the cell volume. The unitary nature of the mitochondrion was confirmed and its complex basket-like structure was revealed. The spatial arrangement of the cell organelles is here displayed in stereo-pairs.

scholarly journals Structure, function and regulation of pyruvate carboxylase

1999 ◽  
Vol 340 (1) ◽  
pp. 1-16 ◽  
Author(s):  
Sarawut JITRAPAKDEE ◽  
John C. WALLACE
Keyword(s):  
Pyruvate Carboxylase ◽  
Limited Proteolysis ◽  
Three Dimensional ◽  
Point Mutations ◽  
Dimensional Structure ◽  
Molecular Defects ◽  
X Ray Crystallography ◽  
Neurotransmitter Substances ◽  
Translational Mechanisms ◽  
Structural Region

Pyruvate carboxylase (PC; EC 6.4.1.1), a member of the biotin-dependent enzyme family, catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. PC has been found in a wide variety of prokaryotes and eukaryotes. In mammals, PC plays a crucial role in gluconeogenesis and lipogenesis, in the biosynthesis of neurotransmitter substances, and in glucose-induced insulin secretion by pancreatic islets. The reaction catalysed by PC and the physical properties of the enzyme have been studied extensively. Although no high-resolution three-dimensional structure has yet been determined by X-ray crystallography, structural studies of PC have been conducted by electron microscopy, by limited proteolysis, and by cloning and sequencing of genes and cDNA encoding the enzyme. Most well characterized forms of active PC consist of four identical subunits arranged in a tetrahedron-like structure. Each subunit contains three functional domains: the biotin carboxylation domain, the transcarboxylation domain and the biotin carboxyl carrier domain. Different physiological conditions, including diabetes, hyperthyroidism, genetic obesity and postnatal development, increase the level of PC expression through transcriptional and translational mechanisms, whereas insulin inhibits PC expression. Glucocorticoids, glucagon and catecholamines cause an increase in PC activity or in the rate of pyruvate carboxylation in the short term. Molecular defects of PC in humans have recently been associated with four point mutations within the structural region of the PC gene, namely Val145 → Ala, Arg451 → Cys, Ala610 → Thr and Met743 → Thr.

scholarly journals Topography Prediction of Helical Transmembrane Proteins by a New Modification of the Sliding Window Method

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Maria N. Simakova ◽  
Nikolai N. Simakov
Keyword(s):  
Membrane Proteins ◽  
Transmembrane Domain ◽  
Domain Boundary ◽  
Three Dimensional ◽  
Transmembrane Proteins ◽  
Sliding Window ◽  
Dimensional Structure ◽  
X Ray ◽  
X Ray Crystallography ◽  
Window Method

Protein functions are specified by its three-dimensional structure, which is usually obtained by X-ray crystallography. Due to difficulty of handling membrane proteins experimentally to date the structure has only been determined for a very limited part of membrane proteins (<4%). Nevertheless, investigation of structure and functions of membrane proteins is important for medicine and pharmacology and, therefore, is of significant interest. Methods of computer modeling based on the data on the primary protein structure or the symbolic amino acid sequence have become an actual alternative to the experimental method of X-ray crystallography for investigating the structure of membrane proteins. Here we presented the results of the study of 35 transmembrane proteins, mainly GPCRs, using the novel method of cascade averaging of hydrophobicity function within the limits of a sliding window. The proposed method allowed revealing 139 transmembrane domains out of 140 (or 99.3%) identified by other methods. Also 236 transmembrane domain boundary positions out of 280 (or 84%) were predicted correctly by the proposed method with deviation from the predictions made by other methods that does not exceed the detection error of this method.

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