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1.
Glycerol and glucose fermentation redox routes by Escherichia coli and their regulation by oxidizing and reducing reagents were investigated at different pHs. Cell growth was followed by decrease of pH and redox potential (E h ). During glycerol utilization at pH 7.5 ?pH, the difference between initial and end pH, was lower compared with glucose fermentation. After 8 h growth, during glycerol utilization E h dropped down to negative values (?150 mV) but during glucose fermentation it was positive (+50 mV). In case of glycerol H2 was evolved at the middle log phase while during glucose fermentation H2 was produced during early log phase. Furthermore, upon glycerol utilization, oxidizer potassium ferricyanide (1 mM) inhibited both cell growth and H2 formation. Reducing reagents dl-dithiothreitol (3 mM) and dithionite (1 mM) inhibited growth but stimulated H2 production. The findings point out the importance of reductive conditions for glycerol fermentation and H2 production by E. coli. 相似文献
2.
Yeva B. Dalyan Samvel G. Haroutiunian Gayane V. Ananyan Volodya I. Vardanyan Dmitri Y. Lando Valeri N. Madakyan 《Journal of biomolecular structure & dynamics》2013,31(5):677-687
Abstract Interactions of meso-tetra-(4-N-oxyethylpyridyl) porphyrin (TOEPyP(4)), its 3-N analog (TOEPyP(3)) and their Co, Cu, Ni, Zn metallocomplexes with duplex DNA have been investigated by uv/visible absorbance and circular dichrosim spectroscopies. Results reveal the interactions of these complexes with duplex DNA are of two types. (1) External binding of duplex DNA by metalloporphyrins containing Zn and Co, and (2) Binding of duplex DNA both externally and internally (by intercalation) by porphyrins not containing metals, and metalloporphyrins containing Cu and Ni. Results indicate that (4N-oxyethylpyridyl) porphyrins intercalate more preferably in the structure of duplex DNA and have weaker external binding than 3N-porphyrins. 相似文献
3.
Dmitri Y. Lando Alexander S. Fridman Samvel G. Haroutiunian Albert S. Benight Philippe Collery 《Journal of biomolecular structure & dynamics》2013,31(4):697-711
Abstract A theoretical method is developed for calculation of melting curves of covalent complexes of DNA with antitumor drugs. The method takes into account all the types of chemical modifications of the double helix caused by platinum compounds and DNA alkylating agents: 1) monofunctional adducts bound to one nucleotide; 2) intrastrand cross-links which appear due to bidentate binding of a drug molecule to two nucleotides that are included into the same DNA strand; 3) interstrand cross-links caused by bidentate binding of a molecule to two nucleotides of different strands. The developed calculation method takes into account the following double helix alterations at sites of chemical modifications: 1) a change in stability of chemically modified base pairs and neighboring ones, that is caused by all the types of chemical modifications; 2) a change in the energy of boundaries between helical and melted regions at sites of chemical modification (local alteration of the factor of cooperativity of DNA melting), that is caused by all the types of chemical modifications, too; 3) a change in the loop entropy factor of melted regions that include interstrand cross-links; 4) the prohibition of divergence of DNA strands in completely melted DNA molecules, which is caused by interstrand cross-links only. General equations are derived, and three calculation methods are proposed to calculate DNA melting curves and the parameters that characterize the helix-coil transition. 相似文献
4.
Alexander S. Fridman Viktor Brabec Samvel G. Haroutiunian Roger M. Wartell Dmitri Y. Lando 《Journal of biomolecular structure & dynamics》2013,31(4):533-545
Abstract DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links. 相似文献
5.
Avagyan S Glouchkova L Choi J Snoeck HW 《Journal of immunology (Baltimore, Md. : 1950)》2008,181(9):5904-5911
The hematopoietic stem and progenitor cell (HSPC) compartment is subject to extensive quantitative genetic variation. We have previously shown that TGF-beta2 at low concentrations enhances flt3 ligand-induced growth of HSPCs, while it is potently antiproliferative at higher concentrations. This in vitro enhancing effect was subject to quantitative genetic variation, for which a quantitative trait locus (QTL) was tentatively mapped to chromosome 4 (chr.4). Tgfb2(+/-) mice have a smaller and more slowly cycling HSPC compartment, which has a decreased serial repopulation capacity, and are less susceptible to the lethal effect of high doses of 5-fluorouracil. To unequivocally demonstrate that these phenotypes can be attributed to the enhancing effect of TGF-beta2 on HSPC proliferation observed in vitro and are therefore subject to mouse strain-dependent variation as well, we generated congenic mice where the telomeric region of chr.4 was introgressed from DBA/2 into C57BL/6 mice. In these mice, the enhancing effect of TGF-beta2 on flt3 signaling, but not the generic antiproliferative effect of high concentrations of TGF-beta2, was abrogated, confirming the location of this QTL, which we named tb2r1, on chr.4. These mice shared a smaller and more slowly cycling HSPC compartment and increased 5-fluorouracil resistance but not a decreased serial repopulation capacity with Tgfb2(+/-) mice. The concordance of phenotypes between Tgfb2(+/-) and congenic mice indicates that HSPC frequency and cycling are regulated by tb2r1, while an additional QTL in the telomeric region of chr.4 may regulate the serial repopulation capacity of hematopoietic stem cells. 相似文献
6.
Cyril Montoya Adrian Balasescu Sébastien Joannin Vincent Ollivier Jérémie Liagre Samvel Nahapetyan Ruben Ghukasyan David Colonge Boris Gasparyan Christine Chataigner 《Journal of human evolution》2013
The open-air site of Kalavan 1 is located in the Aregunyats mountain chain (at 1640 m above sea level) on the northern bank of Lake Sevan. It is the first Upper Palaeolithic site excavated in Armenia. Led by an Armenian-French team, several excavations (2005–2009) have revealed a well preserved palaeosoil, dated to around 14,000 BP (years before present), containing fauna, lithic artefacts, as well as several hearths and activity areas that structure the settlement. The initial studies enable placement of the site in its environment and justify palaeoethnological analysis of the Epigravettian human groups of the Lesser Caucasus. 相似文献
7.
Hydrostatic pressure has a vital role in the biological adaptation of the piezophiles, organisms that live under high hydrostatic pressure. However, the mechanisms by which piezophiles are able to adapt their proteins to high hydrostatic pressure is not well understood. One proposed hypothesis is that the volume changes of unfolding (ΔVTot) for proteins from piezophiles is distinct from those of nonpiezophilic organisms. Since ΔVTot defines pressure dependence of stability, we performed a comprehensive computational analysis of this property for proteins from piezophilic and nonpiezophilic organisms. In addition, we experimentally measured the ΔVTot of acylphosphatases and thioredoxins belonging to piezophilic and nonpiezophilic organisms. Based on this analysis we concluded that there is no difference in ΔVTot for proteins from piezophilic and nonpiezophilic organisms. Finally, we put forward the hypothesis that increased concentrations of osmolytes can provide a systemic increase in pressure stability of proteins from piezophilic organisms and provide experimental thermodynamic evidence in support of this hypothesis. 相似文献
8.
Arakelyan VB Haroutiunian SG Arakelyan HV Haroutiunian TS 《Journal of biomolecular structure & dynamics》2002,20(1):135-139
In the present work fluctuations of number of ligands adsorbed on macromolecule are investigated. We have taken into account the adsorption and desorption of ligands under the circumstance of some adsorption centers fluctuations affected by medium fluctuation. The correlation function and spectral density of number of ligands adsorbed on macromolecule are calculated. The properties of these fluctuations which allow identifying a noisemaker are determined. It has been shown, thatfas andsluggis adsorption can be distinguished by properties of dispersion and spectral density. It has been also shown, that comparison of experimental and theoretical correlation functions (or spectral densities) allows to calculate constants of ligand - adsorption center binding and unbinding. 相似文献
9.
A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats 总被引:3,自引:0,他引:3
Beloglazova N Brown G Zimmerman MD Proudfoot M Makarova KS Kudritska M Kochinyan S Wang S Chruszcz M Minor W Koonin EV Edwards AM Savchenko A Yakunin AF 《The Journal of biological chemistry》2008,283(29):20361-20371
Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs. 相似文献
10.
Greg Brown Alexander Singer Vladimir V. Lunin Michael Proudfoot Tatiana Skarina Robert Flick Samvel Kochinyan Ruslan Sanishvili Andrzej Joachimiak Aled M. Edwards Alexei Savchenko Alexander F. Yakunin 《The Journal of biological chemistry》2009,284(6):3784-3792
Gluconeogenesis is an important metabolic pathway, which produces glucose
from noncarbohydrate precursors such as organic acids, fatty acids, amino
acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of
gluconeogenesis, is found in all organisms, and five different classes of
these enzymes have been identified. Here we demonstrate that Escherichia
coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which
show different catalytic properties. We present the first crystal structure of
a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and
in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor
(phosphate). The crystal structure of the ligand-free GlpX revealed a compact,
globular shape with two α/β-sandwich domains. The core fold of GlpX
is structurally similar to that of Li+-sensitive phosphatases
implying that they have a common evolutionary origin and catalytic mechanism.
The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that
the active site is located between two domains and accommodates several
conserved residues coordinating two metal ions and the substrate. The third
metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate
strongly inhibited activity of both GlpX and YggF, and the crystal structure
of the GlpX complex with phosphate demonstrated that the inhibitor molecule
binds to the active site. Alanine replacement mutagenesis of GlpX identified
12 conserved residues important for activity and suggested that
Thr90 is the primary catalytic residue. Our data provide insight
into the molecular mechanisms of the substrate specificity and catalysis of
GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase
(FBPase,2 EC
3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of
fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It
is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis,
and the product, fructose 6-phosphate, is an important precursor in various
biosynthetic pathways (1). In
all organisms, gluconeogenesis is an important metabolic pathway that allows
the cells to synthesize glucose from noncarbohydrate precursors, such as
organic acids, amino acids, and glycerol. FBPases are members of the large
superfamily of lithium-sensitive phosphatases, which includes three families
of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167
sequences, Pfam data base). These enzymes show metal-dependent and
lithium-sensitive phosphomonoesterase activity and include inositol
polyphosphate 1-phosphatases, inositol monophosphatases (IMPases),
3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting
on both inositol 1,4-bisphosphate and PAP (PIPases)
(2). They possess a common
structural core with the active site lying between α+β and
α/β domains (3).
Li+-sensitive phosphatases are putative targets for lithium therapy
in the treatment of manic depressive patients
(4), whereas FBPases are
targets for the development of drugs for the treatment of noninsulin-dependent
diabetes (5,
6). In addition, FBPase is
required for virulence in Mycobacterium tuberculosis and
Leishmania major and plays an important role in the production of
lysine and glutamate by Corynebacterium glutamicum
(7,
8).Presently, five different classes of FBPases have been proposed based on
their amino acid sequences (FBPases I to V)
(9–11).
Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in
various prokaryotes. Types I, II, and III are primarily in bacteria, type IV
in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in
thermophilic prokaryotes from both domains
(11). Many organisms have more
than one FBPase, mostly the combination of types I + II or II + III, but no
bacterial genome has a combination of types I and III FBPases
(9). The type I FBPase is the
most widely distributed among living organisms and is the primary FBPase in
Escherichia coli, most bacteria, a few archaea, and all
eukaryotes (9,
11–15).
The type II FBPases are represented by the E. coli GlpX and FBPase
F-I from Synechocystis PCC6803
(9,
16); type III is represented
by the Bacillus subtilis FBPase
(17); type IV is represented
by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus
furiosus (18), MJ0109
from Methanococcus jannaschii
(19), and AF2372 from
Archaeoglobus fulgidus
(20); and type V is
represented by the FBPases TK2164 from Pyrococcus
(Thermococcus) kodakaraensis and ST0318 from Sulfolobus
tokodai (10,
21).Three-dimensional structures of the type I (from pig kidney, spinach
chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V
(ST0318) FBPases have been solved
(10,
11,
19,
20,
22,
23). FBPases I and IV and
inositol monophosphatases share a common sugar phosphatase fold organized in
five layered interleaved α-helices and β-sheets
(α-β-α-β-α)
(2,
19,
24). ST0318 (an FBPase V
enzyme) is composed of one domain with a completely different four-layer
α-β-β-α fold
(10). The FBPases from these
three classes (I, IV, and V) require divalent cations for activity
(Mg2+, Mn2+, or Zn2+), and their structures
have revealed the presence of three or four metal ions in the active site.E. coli has five Li+-sensitive phosphatases as follows:
CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase
II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a
3′-phosphoadenosine 5′-phosphatase involved in the cysteine
biosynthesis pathway (25,
26), whereas SuhB is an
inositol monophosphatase (IMPase) that is also known as a suppressor of
temperature-sensitive growth phenotypes in E. coli
(27,
28). Fbp is required for
growth on gluconeogenic substrates and probably represents the main
gluconeogenic FBPase (12).
This enzyme has been characterized both biochemically and structurally and
shown to be inhibited by low concentrations of AMP (IC50 15
μm) (11,
29,
30). The E. coli
GlpX, a class II enzyme FBPase, has been shown to possess a
Mn2+-dependent FBPase activity
(9). The increased expression
of glpX from a multicopy plasmid complemented the Fbp-
phenotype; however, the glpX knock-out strain grew normally on
gluconeogenic substrates (succinate or glycerol)
(9).In this study, we present the first structure of a class II FBPase, the
E. coli GlpX, in a free state and in the complex with FBP + metals or
phosphate. We have demonstrated that the fold of GlpX is similar to that of
the lithium-sensitive phosphatases. We have identified the GlpX residues
important for activity and proposed a catalytic mechanism. We have also showed
that YggF is a third FBPase in E. coli, which has distinct catalytic
properties and is more sensitive than GlpX to the inhibition by lithium or
phosphate. 相似文献