Citrate Binding Research Articles

Functional interaction of region 4 of the extracytoplasmic function sigma factor FecI with the cytoplasmic portion of the FecR transmembrane protein of the Escherichia coli ferric citrate transport system.

Transcriptional regulation of the ferric citrate transport genes of Escherichia coli is initiated by the binding of ferric citrate to the outer membrane protein FecA. This binding elicits a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm, where the sigma factor FecI directs the RNA polymerase to the promoter upstream of the fecABCDE genes. An in vivo deletion analysis using a bacterial two-hybrid system assigned the interaction of the FecR and FecI proteins to the cytoplasmic portion of the FecR transmembrane protein and region 4 of FecI. Missense mutations randomly generated by PCR were localized to region 4 of FecI, and the mutants were impaired with regard to the interaction of FecR with FecI and fecB-lacZ transcription. The cloned region 4 of FecI interfered with fecB-lacZ transcription. Interaction of N-proximal regions of predicted FecR homologs with region 4 of predicted FecI homologs of Pseudomonas aeruginosa was demonstrated. The interaction was specific in that only cognate protein pairs interacted with each other; no interactions occurred between heterologous combinations of the P. aeruginosa proteins and between a P. aeruginosa FecI homolog and E. coli FecR. The results demonstrate that region 4 of FecI specifically binds FecR and that this binding is necessary for FecI to function as a sigma factor.

Kinetic and biochemical analyses on the reaction mechanism of a bacterial ATP-citrate lyase.

The prokaryotic ATP-citrate lyase is considered to be a key enzyme of the carbon dioxide-fixing reductive tricarboxylic acid (RTCA) cycle. Kinetic examination of the ATP-citrate lyase from the green sulfur bacterium Chlorobium limicola (Cl-ACL), an alpha(4)beta(4) heteromeric enzyme, revealed that the enzyme displayed typical Michaelis-Menten kinetics toward ATP with an apparent K(m) value of 0.21 +/- 0.04 mm. However, strong negative cooperativity was observed with respect to citrate binding, with a Hill coefficient (n(H)) of 0.45. Although the dissociation constant of the first citrate molecule was 0.057 +/- 0.008 mm, binding of the first citrate molecule to the enzyme drastically decreased the affinity of the enzyme for the second molecule by a factor of 23. ADP was a competitive inhibitor of ATP with a K(i) value of 0.037 +/- 0.006 mm. Together with previous findings that the enzyme catalyzed the reaction only in the direction of citrate cleavage, these kinetic features indicated that Cl-ACL can regulate both the direction and carbon flux of the RTCA cycle in C. limicola. Furthermore, in order to gain insight on the reaction mechanism, we performed biochemical analyses of Cl-ACL. His273 of the alpha subunit was indicated to be the phosphorylated residue in the catalytic center, as both catalytic activity and phosphorylation of the enzyme by ATP were abolished in an H273A mutant enzyme. We found that phosphorylation of the subunit was reversible. Nucleotide preference for activity was in good accordance with the preference for phosphorylation of the enzyme. Although residues interacting with nucleotides in the succinyl-CoA synthetase from Escherichia coli were conserved in AclB, AclA alone could be phoshorylated with the same nucleotide specificity observed in the holoenzyme. However, AclB was necessary for enzyme activity and contributed to enhance phosphorylation and stabilization of AclA.

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Structural basis of gating by the outer membrane transporter FecA.

Siderophore-mediated acquisition systems facilitate iron uptake. We present the crystallographic structure of the integral outer membrane receptor FecA from Escherichia coli with and without ferric citrate at 2.5 and 2.0 angstrom resolution. FecA is composed of three distinct domains: the barrel, plug, and NH2-terminal extension. Binding of ferric citrate triggers a conformational change of the extracellular loops that close the external pocket of FecA. Ligand-induced allosteric transitions are propagated through the outer membrane by the plug domain, signaling the occupancy of the receptor in the periplasm. These data establish the structural basis of gating for receptors dependent on the cytoplasmic membrane protein TonB. By compiling available data for this family of receptors, we propose a mechanism for the energy-dependent transport of siderophores.

Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis

The lutoid-body (bottom) fraction of latex from the rubber tree ( Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a β-1,3-glucanase. The content and properties of the latter enzyme differ between lutoid-body fractions from four different rubber clones (cultivars). While the enzyme from clone GT.1 is a glycoprotein with carbohydrate attached to two glycosylation sites, the enzymes from other clones contain little or no carbohydrate. Latex from clone GT.1 has a higher β-1,3-glucanase content than those from the other three clones, but with a significantly lower specific activity. The enzyme exhibits a pH optimum at 4.5, but there is a second one at 6.7. Peptides isolated from β-1,3-glucanase of clone GT.1 showed that the enzyme is heterogeneous at the C-terminus, probably as a result of removal of a vacuolar targeting sequence by an endopeptidase, followed by further removal of C-terminal residues by a carboxypeptidase-like activity. This incomplete digestion can be related to glycosylation at the extreme C-terminus of the mature enzyme. Non-glycosylated Hevea β-1,3-glucanases exhibit less C-terminal heterogeneity. A homologue of the antifungal protein osmotin was isolated from rubber clones which are less susceptible to fungal diseases. Another identified protein is identical to a citrate binding protein (CBP), already sequenced as cDNA, but with cleaved-off N-terminal signal and C-terminal vacuolar targeting peptides. Four C-terminal propeptides of vacuolar proteins in Hevea are positively identified, which is a valuable contribution to previously known examples of this type of processing.

The Application of Calcium Phosphate Precipitation Chemistry to Phosphorus Recovery: The Influence of Organic Ligands

This paper describes current knowledge of phosphate precipitation chemistry in the context of phosphorus recovery from wastewaters, and presents experimental results on the effect of organic species, as key potential inhibitors, to the precipitation of calcium phosphate. The supersaturation required for precipitation at 25°C, pH 7, 0.1 M ionic strength and near-stoichiometric (for hydroxylapatite) calcium to phosphate molar ratio was determined under spontaneous precipitation conditions. The experiments were carried out in air. The phase precipitating at the critical concentration was allowed to grow under constant supersaturation. The influence of organic ligands on the precipitation was investigated using two small molecular weight organic ligands, acetate and citrate, present at a concentration of 10−3 M. The precipitate was studied using X-ray diffraction and scanning electron microscopy. Good reproducibility of the experiments, which were carried out in triplicate, was observed. The study assessed the supersaturation necessary for spontaneous precipitation of hydroxylapatite to be 10.93, calculated using a solubility constant of log K=−57.74. The required supersaturation was not affected by the presence of acetate. However, citrate was found to increase the supersaturation required for precipitation to 11.73. It is likely that this increase is due to binding of citrate on the active growth sites of newly formed nuclei, thereby inhibiting precipitation. All experiments showed formation of a single phase: micro-crystalline hydroxylapatite.

Competition between citrate and heptapeptide DDSDEEN binding to DNA in presence of divalent cations.

The binding of citrate and acidic peptide DDSDEEN with DNA in the presence of divalent cations is compared. Citric acid shows a higher number of binding sites on the DNA compared to the peptide; this is probably due to the bigger sitric hindrance of the peptide compared to the citric acid for the binding in the DNA grooves. Moreover. DNA preincubated with saturating amounts of citric acid is not available for the binding with successively added peptide. Therefore the peptide and citrate binding sites to some extent overlap.

Metal triggered fluorescence sensing of citrate using a synthetic receptor

A metal containing fluorescent chemosensor was designed, synthesized, and studied for the quantification of citrate in common beverages. The sensor consists of Cu(II) bound by a 1,10-phenanthroline ligand which is attached to a bis(aminoimidazolium) receptor (5). Receptor 5 was designed such that binding of the metal creates an additional binding site for citrate. This additional binding interaction was found to increase the metal and citrate binding constants in a cooperative manner, yielding a minimum 2.0 fold increase in the citrate binding constant and a minimum 2.0 fold increase in the Cu(II) binding constant. Further, 5 was designed so that binding of Cu(II) quenches a photo-excited state of the 1,10-phenanthroline fluorophore. Thus, addition of citrate to 5–Cu(II) resulted in an increase of the fluorescence of the system. The nature of the fluorescence modulation upon citrate binding was probed using a model compound (6–Cu(II)). The data support an increase of electron density on the metal due to the donating ability of a carboxylate anion of citrate. In a sensing assay, the receptor is effective for measuring citrate concentrations in the micromolar range in highly competitive media. We believe this is the first demonstration of anion sensing in which the fluorescence emission is modulated due to a perturbation in the metal quenching effect upon analyte binding.

Control of the Ferric Citrate Transport System of Escherichia coli : Mutations in Region 2.1 of the FecI Extracytoplasmic-Function Sigma Factor Suppress Mutations in the FecR Transmembrane Regulatory Protein

Transcription of the ferric citrate transport genes is initiated by binding of ferric citrate to the FecA protein in the outer membrane of Escherichia coli K-12. Bound ferric citrate does not have to be transported but initiates a signal that is transmitted by FecA across the outer membrane and by FecR across the cytoplasmic membrane into the cytoplasm, where the FecI extracytoplasmic-function (ECF) sigma factor becomes active. In this study, we isolated transcription initiation-negative missense mutants in the cytoplasmic region of FecR that were located at four sites, L13Q, W19R, W39R, and W50R, which are highly conserved in FecR-like open reading frames of the Pseudomonas aeruginosa, Pseudomonas putida, Bordetella pertussis, Bordetella bronchiseptica, and Caulobacter crescentus genomes. The cytoplasmic portion of the FecR mutant proteins, FecR(1-85), did not interact with wild-type FecI, in contrast to wild-type FecR(1-85), which induced FecI-mediated fecB transport gene transcription. Two missense mutations in region 2.1 of FecI, S15A and H20E, partially restored induction of ferric citrate transport gene induction of the fecR mutants by ferric citrate. Region 2.1 of sigma(70) is thought to bind RNA polymerase core enzyme; the residual activity of mutated FecI in the absence of FecR, however, was not higher than that of wild-type FecI. In addition, missense mutations in the fecI promoter region resulted in a twofold increased transcription in fecR wild-type cells and a partial restoration of fec transport gene transcription in the fecR mutants. The mutations reduced binding of the Fe(2+) Fur repressor and as a consequence enhanced fecI transcription. The data reveal properties of the FecI ECF factor distinct from those of sigma(70) and further support the novel transcription initiation model in which the cytoplasmic portion of FecR is important for FecI activity.

Surface signaling in ferric citrate transport gene induction: interaction of the FecA, FecR, and FecI regulatory proteins.

In Escherichia coli, transcription of the ferric citrate transport genes fecABCDE is controlled by a novel signal transduction mechanism that starts at the cell surface. Binding of ferric citrate to the outer membrane protein FecA initiates a signal that is transmitted by FecR across the cytoplasmic membrane into the cytoplasm where FecI, the sigma factor, is activated. Interaction between the signaling proteins was demonstrated by utilizing two methods. In in vitro binding assays, FecR that was His tagged at the N terminus [(His)(10)-FecR] and bound to a Ni-nitrilotriacetic acid agarose column was able to retain FecA, and FecR that was His tagged at the C terminus [FecR-(His)(6)] retained FecI on the column. An N-terminally truncated, induction-negative but transport-active FecA protein did not bind to (His)(10)-FecR. The in vivo assay involved the determination of the FecA, FecR, and FecI interacting domains with the bacterial two-hybrid Lex-based system. FecA(1-79) interacts with FecR(101-317) and FecR(1-85) interacts with FecI(1-173). These data clearly support a model that proposes interaction of the periplasmic N terminus of FecA with the periplasmic C-terminal portion of FecR and interaction of the cytoplasmic N terminus of FecR with FecI, which results in FecI activation.

A citrate-binding site in calmodulin.

Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca(2+)-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein.

Characterisation of citrate and iron citrate uptake by cultured rat hepatocytes

Background/Aims: The endogenous low molecular weight iron chelator, citrate, is considered to be an important contributor to iron transport and the liver the main site of uptake of iron citrate in subjects suffering from diseases of iron overload. Moreover, the citrate-metabolising enzyme, aconitase, is implicated in the regulation of cellular iron metabolism. This study was undertaken to determine the role of citrate and ferric citrate in the uptake of iron by rat hepatocytes. Methods: Cultured rat hepatocytes were incubated (37°C, 15 min) with 100 μM [ 14C]-citrate in the presence or absence of 1.0 μM 55Fe. Membrane-bound and intracellular radiolabel were separated by incubation with the general protease, Pronase. Results: Our results suggest that ferric citrate uptake is mediated by a specific citrate binding site which exhibits a higher affinity for citrate in the presence of iron than in this absence. Citrate was internalised by hepatocytes with at least 70% being oxidised to CO 2 within 15 min. Citrate uptake was pH-dependent, did not require the presence of sodium and increased with increasing iron concentration. Metabolic energy, anion channels, the Na +, K +-ATPase and vesicle acidification do not appear to play a role in uptake of ferric citrate, but functional sulphydryl groups may be involved. Conclusion: The data suggest either that ferric citrate complexes with higher molar ratios of iron to citrate relative to the incubation medium are bound preferentially to the membrane, or that once citrate has delivered its iron to the membrane, the complex dissociates and the components are internalised separately.

Mechanisms of ferric citrate uptake by human hepatoma cells.

The mechanisms of uptake of non-transferrin-bound iron by human hepatoma cells (HuH7) were investigated using 59Fe-citrate and [14C]citrate. The amount of iron associated with the cells increased linearly with time, whereas citrate uptake reached a plateau after 45 min, resulting in a cellular accumulation of iron over citrate. The cells displayed high-affinity membrane binding sites for citrate with maximum binding of 118 +/- 17 pmol citrate/mg protein and a dissociation constant of 21 +/- 2 microM (n = 3). Iron uptake was saturable with a maximum uptake rate of 1.95 +/- 0.43 pmol . mg protein-1 . min-1 and an apparent Michaelis constant of 1.1 +/- 0.1 microM. Nonradioactive ferric citrate and citrate inhibited 59Fe uptake to a similar degree. This suggests that the uptake of citrate-bound iron is dependent on either binding to specific citrate binding sites or the concentration of unbound iron. The uptake of iron was inhibited by ferricyanide (>100 microM) and ferrous iron chelators but stimulated by ferrocyanide and ascorbate, suggesting that the iron is reduced from Fe3+ to Fe2+ and transported into the cell by an iron carrier-mediated step.

SigX of Bacillus subtilis replaces the ECF sigma factor fecI of Escherichia coli and is inhibited by RsiX.

Analysis of the Bacillus subtilis genome sequence revealed two open reading frames, designated sigX and ypuN (now termed rsiX), that are homologous to fecI and fecR, respectively, of Escherichia coli. fecI encodes a sigma 70-type factor that is necessary for transcription of the ferric citrate transport genes fecABCDE. fecR encodes a cytoplasmic transmembrane protein that is required for the induction of fec transport gene transcription by ferric citrate binding to the FecA outer membrane receptor protein. Investigation of the SigX and RsiX activities disclosed that they are not involved in ferric citrate utilization--since ferric citrate did not serve as an iron source for B. subtilis SG64--or in the regulation of any other ferric siderophore transport system tested. Strains deleted for sigX or rsiX displayed no phenotype under aerobic or anoxic conditions. However, cloned sigX complemented an E. coli fecI mutant, and the Fur box upstream of sigX responded to the E. coli iron regulatory protein Fur. The purified SigX protein was required for in vitro transcription of a sigX-containing DNA fragment by the E. coli RNA polymerase core enzyme. Autoregulation of sigX was also found in vivo using a sigX'-lacZ gene fusion. RsiX inhibited SigX activity in vivo and in vitro and stabilized the SigX protein. RsiX was localized in the membrane fraction. When RsiX is present, SigX is found in the membrane fraction; in the absence of RsiX, some SigX is detectable in the cytoplasm. We conclude that SigX is a sigma factor that belongs to the ECF (extracytoplasmic function) sigma 70-factor family. It is not known which promoters are recognized by SigX in B. subtilis. SigX may be involved in the regulation of iron metabolism, as evidenced by its activity in E. coli.

A Synthetic Receptor Selective for Citrate

Applied molecular recognition—strong and selective binding of citrate in highly competitive media such as water and orange juice is reported. Five steps are enough to synthesize receptor 1 that is complementary in charge and shape of citrate. Electrostatic interactions and a network of hydrogen bonds, preorganized by steric effects, are the design principles responsible for the binding of receptor and citrate [Eq. (a)].

Investigations of the human aluminium biokinetics with 26Al and AMS

Continuing the investigations on two healthy volunteers and on two patients with renal failure, the aluminium biokinetics in humans was studied by administering oral and intravenous doses of 26Al to three further healthy volunteers. Blood samples were drawn at times between 20 min and half a year after administration of the doses. The complete daily urine was collected during the first nine days, spot urine samples were taken at later times when blood samples were obtained. Creatinin renal clearances and haematocrit values were also obtained in the time period of the investigations. The 26Al concentrations of the samples were measured using the Munich Tandem accelerator. An open compartment model was developed to describe the time dependences of the measured 26Al concentrations in blood and urine and to establish the human Al biokinetics. The model comprises stomach and duodenum for oral administration, a central compartment consisting of blood plasma and interstitial fluid with transferrin and citrate binding and three peripheral compartments which are needed to describe the time dependence for the long observation period of up to three years. Excretion of Al was mainly described from plasma citrate via the kidneys into the urine and to a lesser extent from the plasma transferrin via the liver into the stool. Time constants between the compartments, fractional intestinal absorption factors and aluminium renal clearances were derived. It was found that the sizes of two peripheral compartments of the patients with renal failure were different to those of the healthy volunteers.

Transcription induction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton system and the electrochemical potential of the cytoplasmic membrane.

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12. Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport. Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment. The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor Fecl, which then directs the RNA polymerase to the fec promoter upstream of fecA.

The Tonoplast-associated Citrate Binding Protein (CBP) of Hevea brasiliensis: PHOTOAFFINITY LABELING, PURIFICATION, AND CLONING OF THE CORRESPONDING GENE

A detailed comparison of citrate uptake into the vacuole-like lutoids of rubber tree (Hevea brasiliensis Muell. Arg.) and of malate and citrate transport into barley (Hordeum vulgare L.) vacuoles revealed very similar transport specificities. In order to identify proteins mediating the transport, two photoreactive analogues (N'-(2-hydroxy-5-azido)-diazo-N-3,5-benzenedicarboxylic acid and 5-azidoisophthalic acid) of malate/citrate were synthesized and found to efficiently inhibit citrate uptake into barley vacuoles (Ki = 18 microM) and Hevea lutoid vesicles (Ki = 27 microM). In vacuoles from both plant species, these photoaffinity probes specifically labeled a single protein with a molecular mass of 23.6 kDa. This citrate binding protein (CBP) was purified to homogeneity from Hevea lutoids, and amino acid sequences were determined for NH2-terminal and tryptic peptides. Using degenerate oligonucleotides of the NH2-terminal sequence, a cDNA coding for the CBP protein of Hevea was isolated. The cDNA codes for a precursor protein of 238 amino acids, containing an NH2-terminal 31-amino acid signal sequence for endoplasmic reticulum targeting, a prerequisite for vacuolar localization. The mature CBP does not show significant sequence similarities to any known primary protein structure and thus represents a member of a novel class of proteins.

Scanning electron microscopy and molecular modeling of inhibition of calcium oxalate monohydrate crystal growth by citrate and phosphocitrate.

Binding of citrate and phosphocitrate to calcium oxalate monohydrate crystals has been studied using scanning electron microscopy (SEM) and molecular modeling. Phosphocitrate structure has been resolved using low temperature X-ray analysis and ab initio computational methods. The (-1 0 1) crystal surface of calcium oxalate monohydrate is involved in binding of citrate and phosphocitrate, as shown by SEM and molecular modeling. Citrate and phosphocitrate conformations and binding energies to (-1 0 1) faces have been obtained and compared to binding to another set of calcium-rich planes (0 1 0). Difference in inhibitory properties of these compounds has been attributed to better coordination of functional groups of phosphocitrate with calcium ions in (-1 0 1). Relevance of this study to design of new calcium oxalate monohydrate inhibitors is discussed.

Crystal Structures of Aconitase with Trans-aconitate and Nitrocitrate Bound

Crystal structures of mitochondrial aconitase with the inhibitors trans -aconitate and nitrocitrate bound to the [4Fe-4S] cluster have been solved and refined at 2·05 Å resolution with R-factors of 0·168 and 0·172, respectively. Crystallization of aconitase with the substrates citrate and cis-aconitate has not been possible because the enzyme turns over and selects enzyme with isocitrate bound into the crystal lattice. Therefore we have analyzed crystal structures of the enzyme complexed with inhibitor analogs of these two substrates. The structure with nitrocitrate bound provides a model for citrate binding. The structure with trans -aconitate bound provides a model for cis-aconitate binding in two ways: Fe4 of the [4Fe-4S] cluster is five-coordinate and the carbon at the Cβ position is trigonal. These results allow the model for the reaction mechanism to be extended to all three natural substrates of aconitase. The results support a model in which citrate and isocitrate form similar chelate structures related by 180° rotation about the Cα-Cβ bond while the intermediate cis -aconitate binds in either of two ways (citrate mode or isocitrate mode). In both inhibitor complexes a H2O molecule is also bound to Fe4. In the structure with nitrocitrate bound, partial occupancy of sulfate in the active site is observed accompanied by hydroxyl binding to Fe4. Comparison of the structures with isocitrate, trans-aconitate, nitrocitrate and sulfate bound reveals preferred orientations for the three types of oxygens ligated to Fe4 (carboxyl, hydroxyl and H2O) supporting the proposed roles for His101, Asp165 and His167 in the catalytic mechanism.