Structural modelling of substrate binding and inhibition in penicillin V acylase from Pectobacterium atrosepticum

Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation.     

Kinetically controlled acylation of 6-APA catalyzed by penicillin acylase from Streptomyces lavendulae: effect of reaction conditions in the enzymatic synthesis of penicillin V

Abstract

Enzymatic synthesis of penicillin V (penV) by acylation of 6-aminopenicillanic acid (6-APA) was carried out using methyl phenoxyacetate (MPOA) as activated acyl donor and soluble penicillin acylase from Streptomyces lavendulae (SlPVA) as biocatalyst. The effect of different reaction conditions on penV synthesis was investigated, such as enzyme concentration, pH, molar ratio of 6-APA to MPOA, as well as presence of DMSO as water-miscible co-solvent at different concentrations. Time-course profiles of all reactions followed the typical pattern of kinetically controlled synthesis (KCS) of β-lactam antibiotics: penV concentration reached a maximum (highest yield or Y_max) and then decreased gradually. Such maximum was higher at pH 7.0, observing that final penV concentration was abruptly reduced when basic pH values were employed in the reaction. Under the selected conditions (100?mM Tris/HCl buffer pH 7.0, 30?°C, 2.7% (v/v) DMSO, 20?mM MPOA, 0.3 UI/ml of SlPVA), Y_max was enhanced by increasing the substrate molar ratio (6-APA to MPOA) up to 5, reaching a maximum of 94.5% and a S/H value of 16.4 (ratio of synthetic activity to hydrolytic activity). As a consequence, the use of an excess of 6-APA as nucleophile has allowed us to obtain some of the highest Y_max and S/H values among those reported in literature for KCS of β-lactam antibiotics. Although many penicillin G acylases (PGAs) have been described in kinetically controlled acylations, SlPVA should be considered as a different enzyme in the biocatalytic tool-box for novel potential synthetic processes, mainly due to its different substrate specificity compared to PGAs.     

Controlling the hydration of covalently immobilised penicillin G amidase in low-water medium: properties and use of Celite R-640

The study describes how Celite R-640 adsorbs liquid water in toluene and vapour water from a gas phase. In toluene, Celite R-640 is able to maintain the water activity (a_w) constant within broad ranges of water concentrations. The a_w values are strongly related to the original hydration of the Celite batches, but prolonged drying confers comparable and reproducible properties to the different batches. The use of Celite R-640 in controlling the hydration and activity of covalently immobilised PGA in toluene is reported.     

Reducibility of cytochromes b in aerobic beef-heart mitochondria treated with antimycin

Under anaerobic conditions cytochrome b in beef-heart mitochondria is partially reduced in the presence of NADH, whereas other cytochromes are completely reduced. Addition of antimycin together with oxygen under these conditions causes an immediate reduction of cytochromes b-558, b-562 and b-566 and oxidation of cytochrome c. During the subsequent transient aerobic steady state cytochromes b-558 and b-566 are rapidly re-oxidized without changes in redox state of cytochrome c, but cytochrome b-562 remains reduced. When oxygen is consumed by the leak through or around the antimycin-inhibition site, cytochrome b-562 becomes oxidized with concomitant reduction of cytochrome c.

The cytochromes b in lyophilized beef-heart mitochondria are more readily accessible to electrons from NADH, and in the presence of antimycin and NADH a complete and stable reduction is obtained under both aerobic and anaerobic conditions. Gradual addition of rotenone under these conditions causes re-oxidation of cytochromes b in which oxidation of cytochromes b-558 and b-566 precedes that of cytochrome b-562.

It is concluded that (1) the effect of antimycin in the presence of oxygen involves all three cytochromes b, (2) the reducibility of the cytochromes b in the aerobic steady state of antimycin-treated mitochondria is dependent upon the potential of the substrate redox couple registered on the cytochromes, and (3) the midpoint potential of cytochrome b-562 in the presence of antimycin is higher than that of cytochrome b-558 or b-566.     

Oxidation of both termini of p- and m-xylene by Escherichia coli transformed with xylene monooxygenase gene

Xylene monooxygenase (XMO) from Pseudomonas putida mt-2 catalyzes oxidation of methyl group of toluene and xylenes. While it has been postulated that this enzyme oxidizes one methyl group of xylene, we observed that both methyl groups in p- and m-xylene were oxidized to alcohol and aldehyde when the relevant genes (xylM and xylA) were co-expressed in Escherichia coli C600 and MC4100. When p-xylene was used as a substrate, p-hydroxymethylbenzaldehyde and p-xylyleneglycol were identified, in addition to p-methylbenzylalcohol and p-tolualdehyde. When m-xylene was used as a substrate, m-hydroxymethylbenzaldehyde and m-xylyleneglycol were identified, in addition to m-methylbenzylalcohol and m-tolualdehyde. Ratio of the products varied significantly according to the reaction condition and host strain, presumably reflecting the relative activity of XMO and host-derived dehydrogenase(s). Using various oxidized compounds as substrates, it was indicated that dialcohol (p- or m-xylyleneglycol) was formed via p- or m-hydroxymethylbenzaldehyde, respectively, rather than directly from corresponding monoalcohol (p- or m-methybenzylalcohol).     

Steroid metabolism in the hormone dependent MCF-7 human breast carcinoma cell line and its two hormone resistant subpopulations MCF-7/LCC1 and MCF-7/LCC2

Androgen and estrogen metabolism was investigated in the hormone-dependent human breast cancer cell line MCF-7 and its two hormone-resistant sublines MCF-7/LCC1 and MCF-7/LCC2. Using the product isolation method, the activity of aromatase, 5-reductase, 3/β-hydroxysteroid oxidoreductase and 17β-hydroxysteroid oxidoreductase were investigated isolating the following steroids: estriol (E₃), estradiol (E₂), estrone (E₁), 3/β-androstanediol (A-diol), testosterone (T), dihydrotestosterone (DHT), androsterone (AND), androstenedion (4-AD) and androstanedione (A-dion). For all experiments, cells were preincubated with cortisol and subsequently incubated with [¹⁴C]T or [¹⁴C]4-AD as the substrate in medium without phenol red and with serum charcoal stripped of steroids. The results showed no aromatase activity in any of the cell lines under the experimental conditions used, and preincubation with cortisol had no effect on the enzyme activity. With [¹⁴C]T as the substrate, the metabolized level of DHT was very similar in the three cell lines, though MCF-7/LCC1 and MCF-7/LCC2 utilized the substrate to a much lesser extent. The amount of DHT and 4-AD produced were comparable in the two hormone-resistant cell lines, while the amount of 4-AD was significantly higher in MCF-7 cells. No differences in enzyme activity were found in the three cell lines when [¹⁴C]4-AD was used as the substrate. This study showed an altered androgen metabolism in the MCF-7/LCC1 and MCF-7/LCC2 sublines compared to the parent MCF-7. However, since treatment with DHT and T inhibited cell growth equally well in all three tumor cell lines, it is unlikely that the found differences in steroid metabolism was involved in the acquisition of the endocrine resistance of the two MCF-7 sublines.     

Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.     

Regioselective metabolism of phenanthrene in Atlantic cod (Gadus morhua): studies on the effects of monooxygenase inducers and role of cytochromes P-450

The polycyclic aromatic hydrocarbon phenanthrene was converted mainly (>90%) to the 1,2-dihydrodiol when metabolized in vivo by the marine teleost cod. This is also found in other bony fishes, but contrary to what is known from cartilaginous fish, crustaceans and mammals, where the K-region 9,10-dihydrodiol is the main metabolite. When liver microsomal preparations from differently pretreated cod were incubated with phenanthrene in vitro, the metabolic profile was dramatically different from the in vivo pattern, as shown by gas chromatography—mass spectrometry. The microsomes from untreated, phenanthrene, phenobarbital and pregnenolone-16-carbonitrile-treated cod converted phenanthrene mainly, but to a varying extent, to the 9,10-dihydrodiol. Treatment with β-naphthoflavone (BNF), however, resulted in a large increase in the oxidation at the 1,2-position, along with a four- to seven-fold increase in specific activity. The major cytochrome P-450 isozyme purified from BNF-treated cod liver (P-450c) showed highest activity with phenanthrene (a turnover of 0.18 nmol/min per nmol P-450), but with about equal selectivity for the 1,2- and 9,10-region of the substrate in a reconstituted system with phospholipid and NADPH-cytochrome P-450 reductase. The low regioselectivity was also observed as a lack of regioselective inhibition of microsomal phenanthrene metabolism with antiserum to cod P-450c. Two of the minor isozymes, cod cytochromes P-450b and d, showed a similar turnover to P-450c, but with a stronger selectivity for the 1,2-position (55–60%). The results indicate that other control systems, in addition to the content of individual P-450-forms in the regulatory systems, in addition to the content of individual P-450-forms in the endoplasmic reticulum, are involved in the in vivo transformation of phenanthrene by cod to the 1,2-dihydrodiol metabolite.     

Solanesyl diphosphate synthase reaction with artificial substrates. Formation of R and S-enantiomers of 4- and 8- methyl derivatives of geranylgeranyl diphosphate

(E)- and (Z-3-Methyl-3-pentenyl diphosphates acted as artificial substrates in the reaction with geranyl diphosphate catalyzed by solanesyl diphosphate synthase of Micrococcus luteus. The reactions of the E- and Z-isomers proceeded in the same stereochemical manner as that with the natural substrate but stopped at the stage of two steps of condensation, forming C₁₆- and C₂₂-prenyl diphosphates having extra one and two methyl groups at 4- and 4,8-positions, respectively.     

Probing conformations of the glycerol backbones of triacyglycerols in the active site of lipase by 1,2-cyclopentane-carbamates: The meso effect for the enzyme inhibition

The aim of this study is to probe the glycerol backbone conformation of the substrate (or inhibitor) in the active site of Pseudomonas species lipase by the 1,2-cyclopentandiol analogues of the ethylene glycerol carbamate inhibitors. Cyclopentane-carbamates, cis-1,2-di-N-n- butylcarbamyl-cyclopentane (1) and trans-1,2-di-N-n-butylcarbamyl-cyclopentane (2), are the conformationally constrained analogues of 1,2-di-N-n-butylcarbamyl ethane (3). All carbamates are synthesized and characterized as the pseudo-substrate inhibitors of the enzyme. Cis-cyclopentane-di-carbamate (1) is a more potent inhibitor than both ethane-di-carbamate (3) and trans-cyclopentane-di-carbamate (2) probably because the glycerol backbone conformations of cis-cyclopentane-di-carbamate (1) are constrained by the cyclopentane ring and cis-cyclopentane-di-cabamate (1) is a meso compound but trans-cyclopentane-di-carbamate (2) is a racemate.     

Effects of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase and glutathione S-transferase activities in rat brain

The effects of acute and therapeutic doses of phenobarbital and sodium salicylate on cytochrome P-450 mixed function oxygenase (EC 1.14.14.1) and glutathione S-transferase (EC 2.5.1.18) activities have been studied in rat brain and compared with those of rat liver. P-450 enzymic activity was assayed by N-demethylation of p-chloro-N-methylaniline and 1-chloro-2,4-dinitrobenzene was used as substrate for glutathione S-transferase activity. The acute effects of a single daily dose of phenobarbital (75 mg/kg/day;i.p.) and sodium salicylate (500 mg/kg/day;i.p.) for 3 days increased cytochrome P-450 as well as glutathione S-transferase in rat liver. But the same doses of both drugs decreased glutathione S-transferase levels in rat brain and increased cytochrome P-450 dependent N-demethylation of p-chloro-N-methylaniline. The therapeutic doses of sodium salicylate (50 mg/kg/day;i.p.) and phenobarbital (10 mg/kg/day;i.p.) daily for 21 days increased cytochrome P-450 in rat liver as well as in brain. The increase in brain glutathione S-transferase by prolonged treatment of phenobarbital was significant compared to the control values.     

Comparison of different approaches and computer programs for progress curve analysis of enzyme kinetics

For the analysis of enzyme kinetics, a variety of programs exists. These programs apply either algebraic or dynamic parameter estimation, requiring different approaches for data fitting. The choice of approach and computer program is usually subjective, and it is generally assumed that this choice has no influence on the obtained parameter estimates. However, this assumption has not yet been verified comprehensively. Therefore, in this study, five computer programs for progress curve analysis were compared with respect to accuracy and minimum data amount required to obtain accurate parameter estimates. While two of these five computer programs (MS‐Excel, Origin) use algebraic parameter estimation, three computer programs (Encora, ModelMaker, gPROMS) are able to perform dynamic parameter estimation. For this comparison, the industrially important enzyme penicillin amidase (EC 3.5.1.11) was studied, and both experimental and in silico data were used. It was shown that significant differences in the estimated parameter values arise by using different computer programs, especially if the number of data points is low. Therefore, deviations between parameter values reported in the literature could simply be caused by the use of different computer programs.     

Effect of organic solvents on cell-bound penicillin V acylase activity of Erwinia aroideae (DSMZ 30186): A permeabilization effect

Erwinia aroideae (DSMZ 30186) is a potential microbial culture to produce intracellular penicillin V acylase (PVA). The whole cell PVA activity was improved by permeabilization with various organic solvents. The cell-bound PVA activity showed an eightfold increase upon treatment with chloroform (5 μL/mg_{dry biomass}) for 10 min and diethyl ether (10 μL/mg_{dry biomass}) for 45 min. Hexane, toluene, ethyl acetate and dichloromethane enhanced the enzyme activity up to two-, six-, four- and two-fold, respectively; whereas, PVA activity declined drastically on permeabilization with acetone, pyridine and alcohols. The physicochemical properties of the organic solvents used for permeabilization were correlated with the change in activity. It was found that solvents with high hydrophobicity (log P > 0.68) and lower dielectric constant (<9) were relatively effective in increasing PVA activity. These results allow systematic selection of suitable solvent for best performance.     

Biotransformation of nitriles by Rhodococcus equi A4 immobilized in LentiKats

Whole cells of Rhodococcus equi A4, a producer of nitrile hydratase and amidase activities, were immobilized in lens-shaped hydrogel particles, LentiKats^®. The immobilized biocatalyst was applied to the biotransformation of benzonitrile, 3-cyanopyridine, (R,S)-3-hydroxy-2-methylenebutanenitrile and (R,S)-3-hydroxy-2-methylene-3-phenylpropanenitrile. The stability of the nitrile hydratase during the repeated use of the biocatalyst was dependent on the type of the substrate. The enzyme was most stable during the transformation of (R,S)-3-hydroxy-2-methylenebutanenitrile. No significant loss of the amidase activity was observed within the course of the biocatalytic reaction.     

Molecular orbital studies of oxygen activation and mechanisms of cytochromes P-450-mediated oxidative metabolism of xenobiotics

Molecular dimensions and molecular orbital calculations of the electronic structures of 56 substrates, inhibitors and inducers of the cytochromes P-448 and other families of the cytochromes P-450 are reported. Substrates of the cytochromes P-448 are shown to be planar molecules with relatively large values of area/depth², and to have electronic structures with relatively low values for ΔE, the difference in energy between the frontier orbitals (E(LEMO) − E(HOMO)). Substrates of other families of the cytochromes P-450 are globular molecules, with relatively low values of area/depth² and relatively high values of ΔE. Molecular orbital calculations of the active oxygen species, singlet oxygen and superoxy anion, have also been made. Singlet oxygen is a poor electron donor (low values of E(HOMO)) but a good electron acceptor (low values of E(LEMO)), whereas superoxy anion is a good electron donor and a poor electron acceptor. Cytochrome P-448 substrates, which are good electron donors, would preferentially accept singlet oxygen, a good electron acceptor; substrates of the other families of cytochrome P-450, which are less effective electron donors, would preferentially accept superoxy anion, a good electron donor, although substrates of both cytochromes P-448 and other P-450s may accept both species of active oxygen. Together with recent published evidence, these data provide a greater understanding of the mode of activation of oxygen by the various families of the cytochromes P-450, and to the insertion of active oxygen into the substrates. Mechanisms are proposed for the oxygenation of substrates, namely, epoxidation involving singlet oxygen and hydroxylation by superoxy anion. Finally, a detailed explanation of the cytochrome P-450 cycle is discussed, and mechanisms of the different types of oxidative metabolism are presented.     

Breast cancer inhibiting diastereomeric diacetato[1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) derivatives: synthesis and studies on the relationship between reactivity and antitumor activity

Antitumor active [1,2-bis(4-fluorophenyl)ethylenediamine]platinum(II) diastereoisomers containing acetic acid derivatives as ‘leaving groups’ (acetate: meso/rac-4F-Pt(Ac)₂; monochloroacetate: meso/rac-4F-Pt(ClAc)₂; dichloroacetate: meso/rac-4F-Pt(Cl₂Ac)₂; trichloroacetate: meso/rac-4F-Pt(Cl₃Ac)₂; glycolate: meso/rac-4F-Pt(OHAc)₂; phenylacetate: meso/rac-4F-Pt(PhAc)₂) were synthesized and characterized by IR and ¹H NMR spectroscopy. In all complexes except meso/rac-4F-Pt(PhAc)₂, which exist as [meso/rac-4F-PtPhAc]⁺PhAc⁻, both carboxylic acid residues are coordinated to platinum. Kinetic studies on the reaction behavior of the title compounds with nucleophiles were performed by using iodide as nucleophile. The studies show that the new complexes react with nucleophiles predominantly via the ‘solvent path’ (i.e. via the reactive intermediates = Pt(X)(OH₂)⁺ and =Pt(OH₂)₂²⁺. Therefore the rates of reactions in which the reactive species are formed affect the antitumor activity of the complexes as well as their inactivation by bionucleophiles during the transport to the tumor. The extent of accumulation in the tumor cell, too, influences the antitumor activity of a complex. The rate constants are discussed in view of the activities of the respective complexes on the human MCF-7 breast cancer cell line. From the title compounds the Cl₂Ac and Cl₃Ac derivatives do not come close to the standard cisplatin, neither in chemical reactivity nor in biological activity. Meso/rac-4F-Pt(Ac)₂ and meso/rac-4F-Pt(ClAc)₂, respectively, show similar hydrolysis rates but lower antitumor activities than cisplatin, presumably due to a reduced drug uptake by the tumor cell. Meso/rac-4F-Pt(PhAc)₂ compare well with their standard carboplatin in respect to both properties. Other than the remaining, poorly water soluble title compounds, meso/rac-4F-Pt(OHAc)₂ equal their standard cisplatin in terms of water solubility and antitumor activity rac-4F-Pt(OHAc)₂ > meso-4F--Pt(OHAc)₂). However, they are markedly faster hydrolyzed than cisplatin. By use of rac-4F-Pt(Ac)₂ as an example it was confirmed that, in contrast to the parent compound rac-4F-PtCl₂, the new complex type is also active under in vivo conditions owing to its markedly lower reactivity (mainly due to the lack of a direct substitution by strong nucleophiles), which entails a reduced inactivation of the drug on its way to the tumor. The in vitro testing on tumor cell lines combined with the evaluation of the water solubility and with kinetic studies on the reaction with nucleophiles is a useful method for the preselection of potent platinum complexes deserving further thorough in vitro and in vivo investigations.     

The cytochromes of Chlorobium thiosulfatophilum

Three c-type cytochromes (c-551, c-553, c-555) have been isolated and characterized from a strain of the green photosynthetic bacterium Chlorobium thiosulfatophilum. These cytochromes are atypical when compared to horse heart cytochrome c in many properties, among them: oxidation-reduction potential at pH 7.0 (c-551, 135 mV; c-553, 98 mV; c-555, 145 mV), molecular weight (c-551, 45000–60000; c-553, 50000; c-555, 10000) and isolelectric point (c-551, 6.0; c-553, 6.7). No protoheme was detected in whole cells or cell-free extracts.     

The fractionation of plant photoactive pigment-protein complexes I and II

The pigment-protein complexes enriched with Photosystem I (PPC-I) and Photosystem II (PPC-II) were obtained using sievorptive chromatography on DEAE-Sephadex column. Both types of complexes contain Chlorophyll a, β-carotene and minor quantities of Chl b. Red absorbance maxima are located at 676 nm and 673 nm for PPC-I and PPC-II, respectively. The degrees of reaction centre enrichment were measured by the method of differential spectrophotometry: PPC-I has one P-700 per 35 bulk Chl a molecules, PPC-II contains one P-680 per 18 bulk Chl a molecules. The yield of PPC-II is 7–10 times lower than that of PPC-I. After one chromatographic procedure the amount of P-680 in PPC-I preparation does not exceed 7% of that of P-700, the amount of P-700 in PPC-II preparation 2% of that of P-680. The product of PPC-II degradation was studied.     

Canavanine derivatives useful in peptide synthesis

Summary The objective of this work is to investigate the possibilities for introducing the currently used N-, N_G- and C-protective groups into the canavanine molecule and the preparation of canavanines selectively blocked at the guanidino function. These novel compounds will find application in the synthesis of canavanine derivatives expected to be amino acid antimetabolites and of canavanine modified biologically active peptides.