Swimming Behavior of Pseudomonas aeruginosa Studied by Holographic 3D Tracking

Holographic 3D tracking was applied to record and analyze the swimming behavior of Pseudomonas aeruginosa. The obtained trajectories allow to qualitatively and quantitatively analyze the free swimming behavior of the bacterium. This can be classified into five distinct swimming patterns. In addition to the previously reported smooth and oscillatory swimming motions, three additional patterns are distinguished. We show that Pseudomonas aeruginosa performs helical movements which were so far only described for larger microorganisms. Occurrence of the swimming patterns was determined and transitions between the patterns were analyzed.     

Heat processing of soybean kernel and its effect on lysine availability and protein solubility

Soybean kernels of cultivars Bosa and ZPS 015 were used in the experiment. The contents of available lysine as well as water and salt soluble proteins, were analysed in fresh soybean kernels, soybean products made after the processes of dry extrusion, micronisation, microwave toasting and autoclaving. Utilizing a technological procedure of processing, kernels were exposed to temperatures from 57 to 150°C. The duration of exposure of the soybean kernels to the increased temperatures, ranged from 25-30 seconds in dry extrusion to 30 minutes in autoclaving. All treatments were subjected to different sources of heat, causing different thermodynamic processes to take place in kernels and change their chemical composition; i.e. nutritive quality. The content of water and salt soluble proteins decreased under the influence of higher temperatures in the course of all treatments of processing. The drop of solubility already was drastically effected by temperatures of 100°C in dry extrusion, while there was a gradual decrease in other treatments. The content of available lysine was determined by the modified Carpenter methods with DNFB. The processes of micronisation and microwave toasting showed the greatest effect on the reduction of lysine availability. Dry extrusion and autoclaving, performed within closed systems — in which the increased moisture content has a special effect — resulted in significantly smaller changes of the available lysine content.     

Exploration of the Drosophila acetylcholinesterase substrate activation site using a reversible inhibitor (Triton X-100) and mutated enzymes

Cholinesterases are activated at low substrate concentration, and this is followed by inhibition as the level of substrate increases. However, one of these two components is sometimes lacking. In Drosophila acetylcholinesterase, the two phases are present, allowing both phenomena to be studied. Several kinetic schemes can explain this complex kinetic behavior. Among them, one model assumes that activation results from the binding of a substrate molecule to a non-productive site affecting the entrance of a substrate molecule into the active site. To test this hypothesis, we looked for an inhibitor competitive for activation and we found Triton X-100. Using organophosphates or carbamates as hemisubstrates, we showed that Triton X-100 inhibits or increases phosphorylation or carbamoylation of the enzyme. In vitro mutagenesis of the residues lining the active site gorge allowed us to locate the Triton X-100 binding site at the rim of the gorge with glutamate 107 playing the major role. These results led to the hypothesis that substrate binding at this site affects the entrance of another substrate molecule into the active site cleft.     

Acceleration of Drosophila melanogaster acetylcholinesterase methanesulfonylation: peripheral ligand D-tubocurarine enhances the affinity for small methanesulfonylfluoride

D-Tubocurarine, a reversible peripheral inhibitor of cholinesterases accelerates methanesulfonylation of Drosophila melanogaster wild type and W359L mutant. The kinetic evaluation of the process was performed in a step-by-step analysis. The second order overall sulfonylation rate constants, determined from classical residual activity measurements, were used in the subsequent analysis of progress curves. The latter were obtained by measuring the hydrolysis of acetylthiocholine in a complex reaction system of enzyme, substrate, irreversible and reversible inhibitor. The underlying kinetic mechanisms, from such a complex data, could only be untangled by targeted inspection and successive incorporation of reaction steps for which experimental evidence existed. The study showed that the peripheral ligand D-tubocurarine, by binding at the entrance into the active site of the two investigated enzymes (Golicnik et al., Biochemistry 40 (2001) 1214), enhances the affinity for small methanesulfonylfluoride, rather to speeding up the formation of a stable covalent enzyme-inhibitor complex. The specific arrangements at the rim of the active site of each individual enzyme dictate the actual events which can be detected by kinetic means.     

Evidence for subdomain flexibility in Drosophila melanogaster acetylcholinesterase

The catalytic domain of the acetylcholinesterases is composed of a single polypeptide chain, the folding of which determines two subdomains. We have linked these two subdomains by mutating two residues, I327 and D375, to cysteines, to form a disulfide bridge. As a consequence, the hydrodynamic radius of the protein was reduced, suggesting that there is some flexibility in the subdomain connection. In addition to the smaller size, the mutated protein is more stable than the wild-type protein. Therefore, the flexibility between the two domains is a weak point in terms of protein stability. As expected from the location of the disulfide bond at the rim of the active site, the kinetic studies show that it affects interactions with peripheral ligands and the entrance of some of the bulkier substrates, like o-nitrophenyl acetate. In addition, the mutations affect the catalytic step for o-nitrophenyl acetate and phosphorylation by organophosphates, suggesting that this movement between the two subdomains is connected with the cooperativity between the peripheral and catalytic sites.     

Towards the first inhibitors of trihydroxynaphthalene reductase from Curvularia lunata: synthesis of artificial substrate, homology modelling and initial screening

Trihydroxynaphthalene reductase (3HNR) is an essential enzyme in the biosynthesis of fungal melanin and it represents an emerging target for the development of new fungicides and antimicotics. To promote the discovery of new inhibitors, an improved chemical synthesis of the artificial substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) was developed. A series of compounds were screened on 3HNR from Curvularia lunata, a known plant pathogen and an opportunistic human pathogen, and several structurally diverse hits were obtained. Homology modelling of 3HNR from C. lunata can explain their binding modes and will enable further structure-based design of new and improved inhibitors.     

Inhibition of Drosophila melanogaster acetylcholinesterase by high concentrations of substrate.

Acetylcholine hydrolysis by acetylcholinesterase is inhibited at high substrate concentrations. To determine the residues involved in this phenomenon, we have mutated most of the residues lining the active-site gorge but mutating these did not completely eliminate hydrolysis. Thus, we analyzed the effect of a nonhydrolysable substrate analogue on substrate hydrolysis and on reactivation of an analogue of the acetylenzyme. Analyses of various models led us to propose the following sequence of events: the substrate initially binds at the rim of the active-site gorge and then slides down to the bottom of the gorge where it is hydrolyzed. Another substrate molecule can bind to the peripheral site: (a) when the choline is still inside the gorge - it will thereby hinder its exit; (b) after choline has dissociated but before deacetylation occurs - binding at the peripheral site increases deacetylation rate but (c) if a substrate molecule bound to the peripheral site slides down to the bottom of the active-site before the catalytic serine is deacetylated, its new position will prevent the approach of water, thus blocking deacetylation.     

Rational polynomial equation as an unbiased approach for the kinetic studies of Drosophila melanogaster acetylcholinesterase reaction mechanism

The hydrolysis of substrates by cholinesterases does not follow the Michaelis–Menten reaction mechanism. The well-known inhibition by excess substrate is often accompanied by an unexpectedly high activity at low substrate concentrations. It appears that these peculiarities are the consequence of an unusual architecture of the active site, which conducts the substrate molecule over many stages before it is cleaved and released. Structural and kinetic data also suggest that two substrate molecules can attach at the same time to the free, as well as to the acetylated, enzyme. We present a procedure which provides an unbiased framework for mathematical modelling of such complex reaction mechanisms. It is based on regression analysis of a rational polynomial using classical initial rate data. The determination of polynomial degree reveals the number of independent parameters that can be evaluated from the available information. Once determined, these parameters can substantially facilitate the construction and evaluation of a kinetic model reflecting the expected molecular events in an enzymic reaction. We also present practical suggestions for testing the postulated kinetic model, using an original thermodynamic approach and an isolated effect in a specifically mutated enzyme.     

Transient Kinetic Approach to the Study of Acetylcholinesterase Reversible Inhibition by Eseroline

The kinetic rate constants for interaction of (?)-eseroline-(3aS-cis)-1,2,3,3a,8,8a-hexahydro-1,3a,8-trimethylpyrrolo-[2,3-b]indol-5-ol with electric eel acetylcholinesterase (EC 3.1.1.7, acetylcholine acetylhydrolase) were measured at a low substrate concentration according to a transient kinetic approach by using a rapid experimental technique. The measurements were carried out on a stopped-flow apparatus where pre-incubated samples of enzyme with various inhibitor concentrations were diluted with a buffer solution containing the substrate. The experimental data in the form of sigmoid-shaped progress curves were analysed by applying an explicit progress curve equation that described the time dependence of product released during the reaction. The kinetic parameters were evaluated by non-linear regression treatment and the values of the corresponding constants showed approximately the equal affinities of eseroline and eserine (cf. Stojan, J. and Zorko, M. (1997) Biochim. Biophys. Acta, 1337, 75-84.) for binding into the active centre of the enzyme. On the other hand, the kinetic rates for association and dissociation of eseroline were two grades of magnitude higher than those of eserine. The explanation appears to be a substantionally impaired gliding of eserine into the active site gorge by the great mobility of the carbamoyl tail as well as by its numerous possible interactions with the residues lining the gorge. Additionally, a study of the dependence of the transition phase information on the inhibitor concentration was carried out using our experimental data.     

Effect of substrate on the diiron(III) site in stearoyl acyl carrier protein delta 9-desaturase as disclosed by cryoreduction electron paramagnetic resonance/electron nuclear double resonance spectroscopy

The diiron center in stearoyl-acyl carrier protein (ACP) desaturase (DS) from castor plant Ricinus communis catalyzes the dioxygen- and NADPH-dependent introduction of a cis double bond between C9 and C10 of stearoyl-ACP. Radiolytic reduction of diferric DS at 77 K produces an electron paramagnetic resonance (EPR)-detectable mixed-valence center (or [DS(ox)](mv)) that is trapped in the conformation of the diferric precursor and thus provides a sensitive EPR/electron nuclear double resonance (ENDOR) probe of the structure of the diamagnetic diiron(III) state. The cryoreduced DS shows two distinct EPR signals, suggesting the presence of two diiron(III) states: the mu-oxo (major)- and mu-hydroxo (minor)-bridged diiron centers. ENDOR studies show that in the dominant oxo-bridged diferric state each iron(III) coordinates a histidine and a water along with other ligands. Samples containing stoichiometric amounts of stearoyl-ACP show pronounced changes in the EPR and (1)H ENDOR spectra of cryoreduced DS. EPR spectra of the cryoreduced DS-substrate complex reveal two distinct substates of the parent. EPR and ENDOR studies show that both major conformers of the diferric cluster have a mu-oxo bridge. ENDOR shows that the major conformer has a histidine and a water bound to both Fe ions. In the minor conformer, one of the irons has lost the terminal water ligand. The structure of the trapped [DS(ox)](mv) state relaxes upon annealing to 170 K: the mu-oxo bridge in the major cryoreduced DS species protonates on annealing to 170 K; this does not occur for the major DS-substrate complex, even upon annealing to 230 K. The relaxed Fe(II)Fe(III) center in cryoreduced DS and DS-substrate show much less intense and resolved (14)N ENDOR spectra than those of the structurally similar cryoreduced diiron center in ribonucleotide reductase (RNR) protein R2. This difference may reflect some differences in His-Fe bonds. The alterations in the diferric site of DS induced by substrate are suggested to be mediated by conformational changes in the polypeptide chain produced by substrate binding. These structural alterations may provide DS with an additional mechanism for tuning the redox potential of the diferric site. The mixed-valence states of DS are unstable at temperatures above 230 K.