Antifungal activity of cinnamic acid derivatives involves inhibition of benzoate 4-hydroxylase (CYP53).

AIMS: CYP53A15, from the sorghum pathogen Cochliobolus lunatus, is involved in detoxification of benzoate, a key intermediate in aromatic compound metabolism in fungi. Because this enzyme is unique to fungi, it is a promising drug target in fungal pathogens of other eukaryotes. METHODS AND RESULTS: In our work, we showed high antifungal activity of seven cinnamic acid derivatives against C. lunatus and two other fungi, Aspergillus niger and Pleurotus ostreatus. To elucidate the mechanism of action of cinnamic acid derivatives with the most potent antifungal properties, we studied the interactions between these compounds and the active site of C. lunatus cytochrome P450, CYP53A15. CONCLUSION: We demonstrated that cinnamic acid and at least four of the 42 tested derivatives inhibit CYP53A15 enzymatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: By identifying selected derivatives of cinnamic acid as possible antifungal drugs, and CYP53 family enzymes as their targets, we revealed a potential inhibitor-target system for antifungal drug development.

Kinetic characterization of all steps of the interaction between acetylcholinesterase and eserine.

The three-step carbamylenzyme mechanism of the action of eserine on acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) has been known for a long time, but its complete kinetic characterization has never been done. Some of our investigations indicated that the determination of missing kinetic parameters should include the inspection of the enzyme-eserine interaction in a very wide range of eserine concentrations. Therefore, the activity of acetylcholinesterase as a function of time in the presence of low concentrations of eserine comparable to the enzyme concentration was followed. The reaction mechanism was analysed by fitting numerically integrated differential equations that describe the time dependences of all reactants and reaction intermediates to these data. Additionally, the progress curve measurements at higher eserine concentrations were carried out on a stopped-flow apparatus. The corresponding progress curve equations were derived and the kinetic parameters evaluated by non-linear regression treatment. The complex analysis confirmed the three-step mechanism. The values of the constants showed that the very high affinity of eserine for binding into the active centre of the enzyme is not so much a consequence of the fast initial complex formation but rather a consequence of its slow dissociation. The subsequent covalent bonding of eserine is also slow, but faster than the dissociation of the initial complex. In this manner, the decarbamoylation is the only process responsible for the reactivation of acetylcholinesterase after removal of eserine.

On the inhibition of cholinesterase by D-tubocurarine.

Some investigation in this laboratory pointed to an unexpectedly slow inhibition of cholinesterase by D-tubocurarine, occurring in addition to a typically instantaneous inhibition. In order to elucidate this phenomenon, the hydrolysis of butyrylthiocholine catalyzed by cholinesterase was recorded, in the absence and presence of D-tubocurarine, on a stopped-flow apparatus. Experimental results were analyzed by classical kinetic methods and by means of mathematical modeling. It was found that the inhibition is of a double character, consisting of an instantaneous phase and a slow one occurring in a minute time scale. It seems that the action of D-tubocurarine is a consequence of an instantaneous binding of D-tubocurarine to a peripheral site, followed by a relatively slow conformational transition in the enzyme.

Electron paramagnetic resonance reveals altered topography of the active center gorge of acetylcholinesterase after binding of fasciculin to the peripheral site.

Fasciculin, a peptidic toxin from snake venom, inhibits mammalian and fish acetylcholinesterases (AChE) by binding to the peripheral site of the enzyme. This site is located at the rim of a narrow, deep gorge which leads to the active center triad, located at its base. The proposed mechanisms for AChE inhibition by fasciculin include allosteric events resulting in altered conformation of the AChE active center gorge. However, a fasciculin-induced altered topography of the active center gorge has not been directly demonstrated. Using electron paramagnetic resonance with the spin-labeled organophosphate 1-oxyl-2,2,6, 6-tetramethyl-4-piperidinylethylphosphorofluoridate (EtOSL) specifically bound to the catalytic serine of mouse AChE (mAChE), we show that bound fasciculin on mAChE slows down, but does not prevent phosphorylation of the active site serine by EtOSL and protects the gorge conformation against thermal denaturation. Most importantly, a restricted freedom of motion of the spin label bound to the fasciculin-associated mAChE, compared to mAChE, is evidenced. Molecular models of mAChE and fasciculin-associated mAChE with tethered EtOSL enantiomers indicate that this restricted motion is due to greater proximity of the S-EtOSL nitroxide radical to the W86 residue in the fasciculin-associated enzyme. Our results demonstrate a topographical alteration indicative of a restricted conformation of the active center gorge of mAChE with bound fasciculin at its rim.

A novel keratin 5 mutation (K5V186L) in a family with EBS-K: a conservative substitution can lead to development of different disease phenotypes.

Epidermolysis bullosa simplex is a hereditary skin blistering disorder caused by mutations in the KRT5 or KRT14 genes. More than 50 different mutations have been described so far. These, and reports of other keratin gene mutations, have highlighted the existence of mutation "hotspots" in keratin proteins at which sequence changes are most likely to be detrimental to protein function. Pathogenic mutations that occur outside these hotspots are usually associated with less severe disease phenotypes. We describe a novel K5 mutation (V186L) that produces a conservative amino acid change (valine to leucine) at position 18 of the 1A helix. The phenotype of this case is unexpectedly severe for the location of the mutation, which lies outside the consensus helix initiation motif mutation hotspot, and other mutations at this position have been associated in Weber--Cockayne (mild) epidermolysis bullosa simplex only. The mutation was confirmed by mismatch-allele-specific polymerase chain reaction and the entire KRT5 coding region was sequenced, but no other changes were identified. De novo K5/K14 (mutant and wild-type) filament assembly in cultured cells was studied to determine the effect of this mutation on filament polymerization and stability. A computer model of the 1A region of the K5/K14 coiled-coil was generated to visualize the structural impact of this mutation and to compare it with an analogous mutation causing mild disease. The results show a high level of concordance between genetic, cell culture and molecular modeling data, suggesting that even a conservative substitution can cause severe dysfunction in a structural protein, depending on the size and structure of the amino acid involved.

Autocatalytic processing of recombinant human procathepsin B is a bimolecular process.

Cathepsin B and other lysosomal cysteine proteinases are synthesized as inactive zymogens, which are converted to their mature forms by other proteases or by autocatalytic processing. Procathepsin B autoactivation was shown in vitro at pH 4.5 to be a bimolecular process with K(s) and k(cat) values of 2.1+/-0.9 microM and 0.12+/-0.02 s(-1)6.0. However, in the presence of 0.5 microg/ml of dextran sulfate, relatively rapid processing is observed even at pH 6.5 (t(1/2) approximately 90 min), suggesting that glycosaminoglycans are involved in in vivo processing of lysosomal cysteine proteases.

A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase.

Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 microM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 microM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.

Searching for the physiological function of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: studies of substrate specificity and expression analysis.

17beta-hydroxysteroid dehydrogenase from the filamentous fungus Cochliobolus lunatus (17beta-HSDcl) has recently been characterized. Since its function is still unclear, we performed substrate specificity studies to obtain some indications about its physiological function. Different steroids were studied as putative substrates of recombinant 17beta-HSDcl, androgens and estrogens, brassinosteroids, and the fungal steroid herbarulid. Among these androgens and estrogens were most efficiently converted. The following substrates in decreasing order were best reduced: 4-estrene-3,17-dione, 5alpha-androstane-3,17-dione, 4-androstene-3,17-dione and estrone. Two typical inhibitors were tested: carbenoxolone--a representative inhibitor of the SDR family and quercetin--a diagnostic inhibitor of carbonyl reductases. Among these two quercetin was more efficient. Expression studies revealed that 17beta-HSDcl is mainly expressed in the stationary phase of growth indicating its possible involvement in secondary metabolism.

Substrate recognition sites in 14alpha-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51.

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068-3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh's analysis [J. Biol. Chem. 267 (1992) 83-90]. While Gotoh's SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.

Effect of tetramethylammonium, choline and edrophonium on insect acetylcholinesterase: test of a kinetic model.

Cholinesterases display a non-Michaelian behaviour with respect to substrate concentration. With the insect enzyme, there is an activation at low substrate concentrations and an inhibition at high concentrations. Previous studies allow us to propose a kinetic model involving a secondary non-productive binding site for the substrate. Unexpectedly, this secondary site has a very high affinity for the substrate when the enzyme is free. On the contrary, when the catalytic site of the enzyme is occupied a strong decrease of this affinity was observed. Moreover, a substrate molecule bound to the peripheral site results in a global decrease of the acylation and/or the deacylation step. Kinetic studies with three reversible inhibitors, tetramethylammonium, edrophonium and choline supported the kinetic model and enable its further refinement.

Inhibition of Drosophila acetylcholinesterase by 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide.

The kinetic behaviour of Drosophila melanogaster acetylcholinesterase toward its substrate shows, in comparison with classic Michaelis-Menten kinetics, an apparent homotropic cooperative double activation-inhibition pattern. In order to construct an appropriate kinetic model and obtain further information on the mechanism of the catalytic action of this enzyme, the hydrolysis of acetylthiocholine in the absence and presence of different concentrations of synthetic quaternary methylphosphonate, 7-(methylethoxyphosphinyloxy)1-methyl-quinolinium iodide (MEPQ), was followed on a stopped-flow apparatus. The reaction at low substrate concentrations was followed until the change of the absorbance became negligible and at high concentrations only initial parts were recorded. A simultaneous analysis of the progress curves using numerical integration showed that the powerful organophosphonate inhibitor binds and compete with the substrate for the same binding sites. The results are also in accordance with the hypothesis that virtually every substrate or quasi-substrate molecule that enters into the gorge of active site is hydrolysed.

17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus: structural and functional aspects.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) activity has been described in all filamentous fungi tested, but until now only one 17beta-HSD from Cochliobolus lunatus (17beta-HSDcl) was sequenced. We examined the evolutionary relationship among 17beta-HSDcl, fungal reductases, versicolorin reductase (Ver1), trihydroxynaphthalene reductase (THNR), and other homologous proteins. In the phylogenetic tree 17beta-HSDcl formed a separate branch with Ver1, while THNRs reside in another branch, indicating that 17beta-HSDcl could have similar function as Ver1. The structural relationship was investigated by comparing a model structure of 17beta-HSDcl to several known crystal structures of the short chain dehydrogenase/reductase (SDR) family. A similarity was observed to structures of bacterial 7alpha-HSD and plant tropinone reductase (TR). Additionally, substrate specificity revealed that among the substrates tested the 17beta-HSDcl preferentially catalyzed reductions of steroid substrates with a 3-keto group, Delta(4) or 5alpha, such as: 4-estrene-3,17-dione and 5alpha-androstane-3,17-dione.

The influence of an organosulfonate on the reaction between Ellman's reagent and thiocholine.

The aim of this work was to elucidate the effect of methanesulfonyl fluoride on the detection reaction for the determination of cholinesterase activity. The effect of methanesulfonyl fluoride was studied by monitoring the time course of the appearance and disappearance of the detection reaction product using a stopped-flow technique. The obtained experimental data were analyzed by progress curve analysis. It was found that the effect of methanesulfonyl fluoride results from a reaction between methanesulfonyl fluoride and the detection reaction product with the following rate constant: k1 = 5.33 x 10(-2) M-1s-1. In evaluating the effect of methanesulfonyl fluoride, the decomposition of this agent in water was included in the analysis because the decomposition considerably affected the reaction between methanesulfonyl fluoride and the product. The corresponding rate constant was found to be k2 = 4.9 x 10(-5) M-1s-1.

Trihydroxynaphthalene reductase of Curvularia lunata--a target for flavonoid action?

Melanin protects dark-pigmented fungi from environmental stresses and serves as an important virulence factor in plant and human pathogenic fungi. The enzymes of melanin biosynthesis thus represent interesting targets for the development of fungicides and new selective antimycotics. In Curvularia lunata, a facultative plant and human pathogen, melanin is produced from 1,8-dihydroxynaphthalene via the pentaketide pathway. Recently, the melanin biosynthetic enzyme trihydroxynaphthalene reductase (3HNR) of C. lunata was cloned and expressed in Escherichia coli, enabling further inhibition studies. Here, we have examined structurally different flavonoids (flavones, flavonols, isoflavones and flavanones) as inhibitors of recombinant 3HNR by following the NADP(+)-dependant oxidation of a non-physiological substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one. At 40 microM substrate concentration the most potent inhibitors were five flavones that are hydroxylated at positions 5 and 7: apigenin (IC(50), 3.1 microM), acacetin (IC(50), 4.9 microM), diosmetin (IC(50), 5.7 microM), 5,7-dihydroxyflavone (IC(50), 5.8 microM) and luteolin (IC(50), 6.8 microM). Flavonol (kaempferol; IC(50), 7.9 microM), isoflavone (genistein; IC(50), >50 microM) and flavanone (naringenin; IC(50), 26 microM) derivates were less potent than their corresponding flavone analogue apigenin. Among the isoflavones and flavanones, biochanin A was the most active (IC(50), 12 microM). Kinetic studies confirmed that apigenin and biochanin A, the best inhibitors among the flavones and isoflavones, act as competitive inhibitors of 3HNR, with K(i) values of 1.2 microM and 6.5 microM, respectively. Docking of apigenin and biochanin A into the active site of C. lunata 3HNR revealed their possible binding modes, in which they are stacked between the phenol ring of Tyr208 and the coenzyme nicotinamide moiety, forming two H-bonds with Ser149 and Ser228, and Ser149 and Tyr163, respectively. In vivo inhibition study showed that apigenin and one of the less potent inhibitors, baicalein affect fungal pigmentation and growth. Knowing that the flavonoids are formed in plants in response to fungal attack, they can be considered as potential physiological inhibitors of 3HNR.

Homology built model of acetylcholinesterase from Drosophila melanogaster.

Acetylcholinesterases from Drosophila melanogaster and Torpedo marmorata possess 35% identical residues. We built a homology model of the Drosophila enzyme on the basis of the known three-dimensional structure of Torpedo acetylcholinesterase, which revealed an oval rim of the active site gorge with an additional hollow which could accept small charged ligands more firmly than the corresponding surface in the Torpedo enzyme. This difference at the peripheral site, together with the kinetics of W121A and W359L mutants, suggests coordinate action of important hydrophobic residues that form the active site gorge during the catalytic process. It may also account for the activation-inhibition kinetic pattern which is characteristic for the insect enzyme.

Equations for progress curves of some kinetic models of enzyme-single substrate-single slow binding modifier system.

A procedure is described by means of which the equations for progress curves for the kinetic models that include fast and slow reaction steps can be derived. It is based on combined assumptions of equilibrium and steady-state and uses Laplace transformation for solving the systems of differential equations. The progress curve equations and the significance of the corresponding parameters are given for some most frequently occurring models describing the influence of a slow binding modifier on a single substrate enzyme reaction.

Slow detection reaction can mimic initial inhibition of an enzymic reaction.

An analysis of sigmoid-shaped progress curves in the reaction between Electric Eel acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7, AChE) and its substrate acetylthiocholine in low concentrations at pH 7 is presented. In order to be able to explain an initial apparent inhibition of the enzyme-substrate reaction, the rate of detection reaction had to be taken into account. The theoretical curves obtained by the fitting of differential equations for the reaction mechanism to the data of six progress curves simultaneously, exactly reproduce the course of the experimental curves. The measurements performed with various concentrations of detection reagent confirm the proposed cause of sigmoidity.

Velocity of Ellman's reaction and its implication for kinetic studies in the millisecond time range.

A detailed study of the velocity of the reaction between Ellman's reagent and thiocholine was undertaken, in order to test the possibilities of this reaction as a detection method for the earlier stages of cholinesterases reactions. Experiments were carried out on a stopped-flow apparatus with a built-in spectrophotometer. The obtained experimental data were analyzed by fitting the data to theoretical kinetic equations derived for the reaction. In this way, a complete kinetic characterization of the reaction was obtained. An important practical result derived from our investigations is the finding that, under most experimental conditions, the Ellman's reaction is more than sufficiently rapid as a detection method. However, in the case of reactions in the time scale of 200 milliseconds or less, this being 5 times the half life of Ellman's reaction at standard conditions, one has to consider the interference of this reaction with the enzyme reaction itself.

Mechanism of eserine action on the hydrolysis of butyrylthiocholine by butyrylcholinesterase.

The mechanism of the interaction of eserine with butyrylcholinesterase has been proposed only on the basis of analogy with acetylcholinesterase. Here the interactions was studied in detail and the results analysed by classical kinetic methods and by means of mathematical modelling. An appropriate kinetic scheme was developed, an adequate equation derived and the corresponding kinetic parameters evaluated. The findings suggest that a fast but relatively weak binding of eserine to the enzyme's active site is followed by a slow acylation step and by an even slower rate limiting deacylation step so misrepresenting eserine as an irreversible inhibitor. The proposed kinetic scheme also suggests that the reaction of eserine with a peripheral substrate site is unlikely as seen with the substrate, butyrylthiocholine.

Interaction of Drosophila acetylcholinesterases with D-tubocurarine: an explanation of the activation by an inhibitor.

Homotropic cooperativity in Drosophila melanogaster acetylcholinesterase seems to be a consequence of an initial substrate binding to a high-affinity peripheral substrate binding site situated around the negative charge of D413 (G335, Torpedo numbering). An appropriate mutation which turns the peripheral binding site to a low-affinity spot abolishes apparent activation but improves the overall enzyme effectiveness. This contradiction can be explained as less effective inhibition due to a shorter occupation of such a peripheral site. A similar effect can be achieved by an appropriate peripheral inhibitor such as TC, which can in special cases, when less effective heterotropic inhibition prevails over homotropic, acts as an activator. At the highest substrate concentrations, however, these enzymes are always inhibited, although steric components may influence the strength of inhibition like in the F368G mutant (F290, Torpedo numbering). Cooperative effects thus may include a steric component, but covering of the entrance must affect influx and efflux to different extents.