scholarly journals An Uncharacterized Member of the Ribokinase Family in Thermococcus kodakarensis Exhibits myo-Inositol Kinase Activity

2013 ◽  
Vol 288 (29) ◽  
pp. 20856-20867 ◽  
Author(s):  
Takaaki Sato ◽  
Masahiro Fujihashi ◽  
Yukika Miyamoto ◽  
Keiko Kuwata ◽  
Eriko Kusaka ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Three Dimensional ◽  
Fold Increase ◽  
Adenosine Kinase ◽  
Dimensional Structure ◽  
Uncharacterized Protein ◽  
Thermococcus Kodakarensis ◽  
Sugar Phosphates ◽  
Biochemical Analyses ◽  
Physiological Substrates

Here we performed structural and biochemical analyses on the TK2285 gene product, an uncharacterized protein annotated as a member of the ribokinase family, from the hyperthermophilic archaeon Thermococcus kodakarensis. The three-dimensional structure of the TK2285 protein resembled those of previously characterized members of the ribokinase family including ribokinase, adenosine kinase, and phosphofructokinase. Conserved residues characteristic of this protein family were located in a cleft of the TK2285 protein as in other members whose structures have been determined. We thus examined the kinase activity of the TK2285 protein toward various sugars recognized by well characterized ribokinase family members. Although activity with sugar phosphates and nucleosides was not detected, kinase activity was observed toward d-allose, d-lyxose, d-tagatose, d-talose, d-xylose, and d-xylulose. Kinetic analyses with the six sugar substrates revealed high Km values, suggesting that they were not the true physiological substrates. By examining activity toward amino sugars, sugar alcohols, and disaccharides, we found that the TK2285 protein exhibited prominent kinase activity toward myo-inositol. Kinetic analyses with myo-inositol revealed a greater kcat and much lower Km value than those obtained with the monosaccharides, resulting in over a 2,000-fold increase in kcat/Km values. TK2285 homologs are distributed among members of Thermococcales, and in most species, the gene is positioned close to a myo-inositol monophosphate synthase gene. Our results suggest the presence of a novel subfamily of the ribokinase family whose members are present in Archaea and recognize myo-inositol as a substrate.

scholarly journals The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

2020 ◽  
Vol 295 (32) ◽  
pp. 11364-11376 ◽  
Author(s):  
Andrew C. Hausrath ◽  
Nicholas A. Ramirez ◽  
Alan T. Ly ◽  
Megan M. McEvoy
Keyword(s):  
Metal Binding ◽  
Biochemical Characterization ◽  
Copper Ions ◽  
Three Dimensional ◽  
Copper Resistance ◽  
Dimensional Structure ◽  
Copper Binding ◽  
Uncharacterized Protein ◽  
X Ray Crystallography ◽  
Biochemical Analyses

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa. Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

Hemoglobin Biosynthesis in Vitreoscilla stercoraria DW: Cloning, Expression, and Characterization of a New Homolog of a Bacterial Globin Gene

1998 ◽  
Vol 64 (6) ◽  
pp. 2220-2228 ◽  
Author(s):  
Meenal Joshi ◽  
Shekhar Mande ◽  
Kanak L. Dikshit
Keyword(s):  
Globin Gene ◽  
Three Dimensional ◽  
Fold Increase ◽  
Binding Pocket ◽  
Growth Conditions ◽  
Dimensional Structure ◽  
Strictly Aerobic ◽  
Coding Region ◽  
Oxygen Control ◽  
Constitutive Production

ABSTRACT In the strictly aerobic, gram-negative bacteriumVitreoscilla strain C1, oxygen-limited growth conditions create a more than 50-fold increase in the expression of a homodimeric heme protein which was recognized as the first bacterial hemoglobin (Hb). The recently determined crystal structure ofVitreoscilla Hb has indicated that the heme pocket of microbial globins differs from that of eukaryotic Hbs. In an attempt to understand the diverse functions of Hb-like proteins in prokaryotes, we have cloned and characterized the gene (vgb) encoding an Hb-like protein from another strain of Vitreoscilla,V. stercoraria DW. Several silent changes were observed within the coding region of the V. stercoraria vgb gene. Apart from that, V. stercoraria Hb exhibited interesting differences between the A and E helices. Compared to its Hb counterpart from Vitreoscilla strain C1, the purified preparation ofV. stercoraria Hb displays a slower autooxidation rate. The differences between Vitreoscilla Hb and V. stercoraria Hb were mapped onto the three-dimensional structure of Vitreoscilla Hb, which indicated that the four changes, namely, Ile7Val, Ile9Thr, Ile10Ser, and Leu62Val, present within theV. stercoraria Hb fall in the region where the A and E helices contact each other. Therefore, alteration in the relative orientation of the A and E helices and the corresponding conformational change in the heme binding pocket of V. stercoraria Hb can be correlated to its slower autooxidation rate. In sharp contrast to the oxygen-regulated biosynthesis of Hb in Vitreoscillastrain C1, production of Hb in V. stercoraria has been found to be low and independent of oxygen control, which is supported by the absence of a fumarate and nitrate reductase regulator box within the V. stercoraria vgb promoter region. Thus, the regulation mechanisms of the Hb-encoding gene appear to be quite different in the two closely related species ofVitreoscilla. The relatively slower autooxidation rate ofV. stercoraria Hb, lack of oxygen sensitivity, and constitutive production of Hb suggest that it may have some other function(s) in the cellular physiology of V. stercorariaDW, together with facilitated oxygen transport, predicted for earlier reported Vitreoscilla Hb.

scholarly journals O2- and NO-Sensing Mechanism through the DevSR Two-Component System in Mycobacterium smegmatis

2008 ◽  
Vol 190 (20) ◽  
pp. 6795-6804 ◽  
Author(s):  
Jin-Mok Lee ◽  
Ha Yeon Cho ◽  
Hyo Je Cho ◽  
In-Jeong Ko ◽  
Sae Woong Park ◽  
...  
Keyword(s):  
Mycobacterium Smegmatis ◽  
Kinase Activity ◽  
Binding Capacity ◽  
Three Dimensional ◽  
Heme Iron ◽  
Dimensional Structure ◽  
Comparison Analysis ◽  
Two Component System ◽  
Transport Chain ◽  
Autokinase Activity

ABSTRACT The DevS histidine kinase of Mycobacterium smegmatis contains tandem GAF domains (GAF-A and GAF-B) in its N-terminal sensory domain. The heme iron of DevS is in the ferrous state when purified and is resistant to autooxidation from a ferrous to a ferric state in the presence of O2. The redox property of the heme and the results of sequence comparison analysis indicate that DevS of M. smegmatis is more closely related to DosT of Mycobacterium tuberculosis than DevS of M. tuberculosis. The binding of O2 to the deoxyferrous heme led to a decrease in the autokinase activity of DevS, whereas NO binding did not. The regulation of DevS autokinase activity in response to O2 and NO was not observed in the DevS derivatives lacking its heme, indicating that the ligand-binding state of the heme plays an important role in the regulation of DevS kinase activity. The redox state of the quinone/quinol pool of the respiratory electron transport chain appears not to be implicated in the regulation of DevS activity. Neither cyclic GMP (cGMP) nor cAMP affected DevS autokinase activity, excluding the possibility that the cyclic nucleotides serve as the effector molecules to modulate DevS kinase activity. The three-dimensional structure of the putative GAF-B domain revealed that it has a GAF folding structure without cyclic nucleotide binding capacity.

First model of dimeric LRRK2: the challenge of unrevealing the structure of a multidomain Parkinson's-associated protein

2016 ◽  
Vol 44 (6) ◽  
pp. 1635-1641 ◽  
Author(s):  
Giambattista Guaitoli ◽  
Bernd K. Gilsbach ◽  
Francesco Raimondi ◽  
Christian Johannes Gloeckner
Keyword(s):  
Kinase Activity ◽  
Structural Model ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Rational Drug Design ◽  
Dimensional Structure ◽  
Rational Drug ◽  
Its Gene ◽  
Lrrk2 Gene ◽  
Successful Approach

Mutations within the leucine-rich repeat kinase 2 (LRRK2) gene represent the most common cause of Mendelian forms of Parkinson's disease, among autosomal dominant cases. Its gene product, LRRK2, is a large multidomain protein that belongs to the Roco protein family exhibiting GTPase and kinase activity, with the latter activity increased by pathogenic mutations. To allow rational drug design against LRRK2 and to understand the cross-regulation of the G- and the kinase domain at a molecular level, it is key to solve the three-dimensional structure of the protein. We review here our recent successful approach to build the first structural model of dimeric LRRK2 by an integrative modeling approach.

scholarly journals Improving the Thermostability and Activity of Transaminase From Aspergillus terreus by Charge-Charge Interaction

2021 ◽  
Vol 9 ◽  
Author(s):  
Jia-Ren Cao ◽  
Fang-Fang Fan ◽  
Chang-Jiang Lv ◽  
Hong-Peng Wang ◽  
Ye Li ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Aspergillus Terreus ◽  
Three Dimensional ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Wild Type ◽  
Enzyme Thermal Stability ◽  
Α Helix ◽  
Dynamics Simulations

Transaminases that promote the amination of ketones into amines are an emerging class of biocatalysts for preparing a series of drugs and their intermediates. One of the main limitations of (R)-selective amine transaminase from Aspergillus terreus (At-ATA) is its weak thermostability, with a half-life (t1/2) of only 6.9 min at 40°C. To improve its thermostability, four important residue sites (E133, D224, E253, and E262) located on the surface of At-ATA were identified using the enzyme thermal stability system (ETSS). Subsequently, 13 mutants (E133A, E133H, E133K, E133R, E133Q, D224A, D224H, D224K, D224R, E253A, E253H, E253K, and E262A) were constructed by site-directed mutagenesis according to the principle of turning the residues into opposite charged ones. Among them, three substitutions, E133Q, D224K, and E253A, displayed higher thermal stability than the wild-type enzyme. Molecular dynamics simulations indicated that these three mutations limited the random vibration amplitude in the two α-helix regions of 130–135 and 148–158, thereby increasing the rigidity of the protein. Compared to the wild-type, the best mutant, D224K, showed improved thermostability with a 4.23-fold increase in t1/2 at 40°C, and 6.08°C increase in T5010. Exploring the three-dimensional structure of D224K at the atomic level, three strong hydrogen bonds were added to form a special “claw structure” of the α-helix 8, and the residues located at 151–156 also stabilized the α-helix 9 by interacting with each other alternately.

scholarly journals A Phosphofructokinase Homolog fromPyrobaculum calidifontisDisplays Kinase Activity towards Pyrimidine Nucleosides and Ribose 1-Phosphate

2018 ◽  
Vol 200 (16) ◽  
Author(s):  
Iram Aziz ◽  
Tahira Bibi ◽  
Naeem Rashid ◽  
Riku Aono ◽  
Haruyuki Atomi ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Catalytic Efficiency ◽  
Content Type ◽  
Reading Frame ◽  
Thermococcus Kodakarensis ◽  
Pyrobaculum Calidifontis ◽  
Cell Extracts ◽  
Activity Measurements ◽  
Phosphate Donor ◽  
Sugar Phosphates

ABSTRACTThe genome of the hyperthermophilic archaeonPyrobaculum calidifontiscontains an open reading frame, Pcal_0041, annotated as encoding a PfkB family ribokinase, consisting of phosphofructokinase and pyrimidine kinase domains. Among the biochemically characterized enzymes, the Pcal_0041 protein was 37% identical to the phosphofructokinase (Ape_0012) fromAeropyrum pernix, which displayed kinase activity toward a broad spectrum of substrates, including sugars, sugar phosphates, and nucleosides, and 36% identical to a phosphofructokinase fromDesulfurococcus amylolyticus. To examine the biochemical function of the Pcal_0041 protein, we cloned and expressed the gene and purified the recombinant protein. Although the Pcal_0041 protein contained a putative phosphofructokinase domain, it exhibited only low levels of phosphofructokinase activity. The recombinant enzyme catalyzed the phosphorylation of nucleosides and, to a lower extent, sugars and sugar phosphates. Surprisingly, among the substrates tested, the highest activity was detected with ribose 1-phosphate (R1P), followed by cytidine and uridine. The catalytic efficiency (kcat/Km) toward R1P was 11.5 mM−1· s−1. ATP was the most preferred phosphate donor, followed by GTP. Activity measurements with cell extracts ofP. calidifontisindicated the presence of nucleoside phosphorylase activity, which would provide the means to generate R1P from nucleosides. The study suggests that, in addition to the recently identified ADP-dependent ribose 1-phosphate kinase (R1P kinase) inThermococcus kodakarensisthat functions in the pentose bisphosphate pathway, R1P kinase is also present in members of the Crenarchaeota.IMPORTANCEThe discovery of the pentose bisphosphate pathway inThermococcus kodakarensishas clarified how this archaeon can degrade nucleosides. Homologs of the enzymes of this pathway are present in many members of the Thermococcales, suggesting that this metabolism occurs in these organisms. However, this is not the case in other archaea, and degradation mechanisms for nucleosides or ribose 1-phosphate are still unknown. This study reveals an important first step in understanding nucleoside metabolism in Crenarchaeota and identifies an ATP-dependent ribose 1-phosphate kinase inPyrobaculum calidifontis. The enzyme is structurally distinct from previously characterized archaeal members of the ribokinase family and represents a group of proteins found in many crenarchaea.

scholarly journals How do I get the most out of my protein sequence using bioinformatics tools?

2021 ◽  
Vol 77 (9) ◽  
pp. 1116-1126 ◽  
Author(s):  
Joana Pereira ◽  
Vikram Alva
Keyword(s):  
Tertiary Structure ◽  
Three Dimensional ◽  
Coiled Coils ◽  
Dimensional Structure ◽  
Sequence Motifs ◽  
Uncharacterized Protein ◽  
Conserved Sequence ◽  
Transmembrane Segments ◽  
Combined Use ◽  
Evolutionary Relatedness

Biochemical and biophysical experiments are essential for uncovering the three-dimensional structure and biological role of a protein of interest. However, meaningful predictions can frequently also be made using bioinformatics resources that transfer knowledge from a well studied protein to an uncharacterized protein based on their evolutionary relatedness. These predictions are helpful in developing specific hypotheses to guide wet-laboratory experiments. Commonly used bioinformatics resources include methods to identify and predict conserved sequence motifs, protein domains, transmembrane segments, signal sequences, and secondary as well as tertiary structure. Here, several such methods available through the MPI Bioinformatics Toolkit (https://toolkit.tuebingen.mpg.de) are described and how their combined use can provide meaningful information on a protein of unknown function is demonstrated. In particular, the identification of homologs of known structure using HHpred, internal repeats using HHrepID, coiled coils using PCOILS and DeepCoil, and transmembrane segments using Quick2D are focused on.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

Three-Dimensional Reconstruction Using Stereo Pairs from Serial Thick Sections

1977 ◽  
Vol 35 ◽  
pp. 562-563
Author(s):  
Robert Glaeser ◽  
Thomas Bauer ◽  
David Grano
Keyword(s):  
Three Dimensional ◽  
Dimensional Structure ◽  
Serial Sections ◽  
Thin Sections ◽  
3 Dimensional ◽  
Transmission Electron ◽  
Freeze Dried ◽  
Dimensional Reconstruction ◽  
Stereo Imagery ◽  
Whole Mounts

In transmission electron microscopy, the 3-dimensional structure of an object is usually obtained in one of two ways. For objects which can be included in one specimen, as for example with elements included in freeze- dried whole mounts and examined with a high voltage microscope, stereo pairs can be obtained which exhibit the 3-D structure of the element. For objects which can not be included in one specimen, the 3-D shape is obtained by reconstruction from serial sections. However, without stereo imagery, only detail which remains constant within the thickness of the section can be used in the reconstruction; consequently, the choice is between a low resolution reconstruction using a few thick sections and a better resolution reconstruction using many thin sections, generally a tedious chore. This paper describes an approach to 3-D reconstruction which uses stereo images of serial thick sections to reconstruct an object including detail which changes within the depth of an individual thick section.

Electron Microscopy of Cytoplasmic Contractile Proteins

1978 ◽  
Vol 36 (3) ◽  
pp. 606-614 ◽  
Author(s):  
T.D. Pollard ◽  
P. Maupin
Keyword(s):  
Electron Microscopy ◽  
Actin Filaments ◽  
Three Dimensional ◽  
Uranyl Acetate ◽  
Contractile Proteins ◽  
Dimensional Structure ◽  
X Ray Diffraction ◽  
Cytoplasmic Matrix ◽  
Computer Image Processing ◽  
Nonmuscle Cells

In this paper we review some of the contributions that electron microscopy has made to the analysis of actin and myosin from nonmuscle cells. We place particular emphasis upon the limitations of the ultrastructural techniques used to study these cytoplasmic contractile proteins, because it is not widely recognized how difficult it is to preserve these elements of the cytoplasmic matrix for electron microscopy. The structure of actin filaments is well preserved for electron microscope observation by negative staining with uranyl acetate (Figure 1). In fact, to a resolution of about 3nm the three-dimensional structure of actin filaments determined by computer image processing of electron micrographs of negatively stained specimens (Moore et al., 1970) is indistinguishable from the structure revealed by X-ray diffraction of living muscle.

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