Simultaneous Improvement in the Thermostability and Catalytic Activity of Epoxidase Lsd18 for the Synthesis of Lasalocid A.

Enzymes used in the synthesis of natural products are potent catalysts, capable of efficient and stereoselective chemical transformations. Lsd18 catalyzes two sequential epoxidations during the biosynthesis of lasalocid A, a polyether polyketide natural product. We performed protein engineering on Lsd18 to improve its thermostability and catalytic activity. Utilizing structure-guided methods of FoldX and Rosetta-ddG, we designed 15 mutants of Lsd18. Screening of these mutants using thermal shift assay identified stabilized variants Lsd18-T189M, Lsd18-S195M, and the double mutant Lsd18-T189M-S195M. Trypsin digestion, molecular dynamic simulation, circular dichroism (CD) spectroscopy, and X-ray crystallography provided insights into the molecular basis for the improved enzyme properties. Notably, enhanced hydrophobic interaction within the enzyme core and interaction of the protein with the FAD cofactor appear to be responsible for its better thermostability.

Environmental pro-oxidants induce altered envelope protein profiles in human keratinocytes.

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known downstream protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Determination of Eight Coccidiostats in Eggs by Liquid-Liquid Extraction-Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry.

A method for the simultaneous determination of robenidine, halofuginone, lasalocid, monensin, nigericin, salinomycin, narasin, and maduramicin residues in eggs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The sample preparation method used a combination of liquid-liquid extraction (LLE) and solid-phase extraction (SPE) technology to extract and purify these target compounds from eggs. The target compounds were separated by gradient elution using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography (UPLC). Tandem mass spectrometry was used to quantitatively and qualitatively analyze the target compounds via electrospray ionization (ESI+) and multiple reaction monitoring mode. The HPLC-MS/MS and UPLC-MS/MS methods were validated according to the requirements defined by the European Union and the Food and Drug Administration. The limits of detection and limits of quantification of the eight coccidiostats in eggs were 0.23-0.52 µg/kg and 0.82-1.73 µg/kg for HPLC-MS/MS, and 0.16-0.42 µg/kg and 0.81-1.25 µg/kg for UPLC-MS/MS, respectively. The eggs were spiked with four concentrations of the eight coccidiostats, and the HPLC-MS/MS and UPLC-MS/MS average recoveries were all higher than 71.69% and 72.26%, respectively. Compared with the HPLC-MS/MS method, utilizing UPLC-MS/MS had the advantages of low reagent consumption, a short detection time, and high recovery and precision. Finally, the HPLC-MS/MS and UPLC-MS/MS methods were successfully applied to detect eight coccidiostats in 40 eggs.

Coccidiostats in milk: development of a multi-residue method and transfer of salinomycin and lasalocid from contaminated feed.

A confirmatory multi-residue method was developed for the determination in milk of 19 coccidiostats (amprolium, arprinocid, clazuril, clopidol, decoquinate, diclazuril, ethopabate, halofuginone, lasalocid, maduramicin, monensin, narasin, nicarbazin, nequinate, robenidine, salinomycin, semduramicin, toltrazuril sulfone and toltrazuril sulfoxide). Sample preparation utilising extraction with organic solvent and clean up by SPE and freezing was found reliable and time-efficient. Optimised chromatography and MS conditions with positive and negative ESI achieved sufficient sensitivity and selectivity. Validation experiments has proven method usefulness for routine analysis of coccidiostats in milk samples. An on-farm study conducted on dairy cows fed with experimentally contaminated feed with salinomycin and lasalocid showed negligible transfer to milk. No residues of lasalocid were found in collected samples. Salinomycin was found only in 5 of 168 samples analysed, while the concentrations of salinomycin in those samples (0.119-0.179 µg kg-1) was significantly below the limit of salinomycin in milk set by European Union legislation. Such low concentrations of both coccidiostats cannot be explained by conjugation during dairy cows' metabolism, as shown by experiments with enzymatic hydrolysis.

Development of intramammary delivery systems containing lasalocid for the treatment of bovine mastitis: impact of solubility improvement on safety, efficacy, and milk distribution in dairy cattle.

BACKGROUND: Mastitis is a major disease of dairy cattle. Given the recent emergence of methicillin-resistant Staphylococcus aureus as a cause of bovine mastitis, new intramammary (IMA) treatments are urgently required. Lasalocid, a member of the polyether ionophore class of antimicrobial agents, has not been previously administered to cows by the IMA route and has favorable characteristics for development as a mastitis treatment. This study aimed to develop an IMA drug delivery system (IMDS) of lasalocid for the treatment of bovine mastitis. METHODS: Minimum inhibitory concentrations (MICs) were determined applying the procedures recommended by the Clinical and Laboratory Standards Institute. Solid dispersions (SDs) of lasalocid were prepared and characterized using differential scanning calorimetry and Fourier transform infrared spectroscopy. IMDSs containing lasalocid of micronized, nano-sized, or as SD form were tested for their IMA safety in cows. Therapeutic efficacy of lasalocid IMDSs was tested in a bovine model involving experimental IMA challenge with the mastitis pathogen Streptococcus uberis. RESULTS: Lasalocid demonstrated antimicrobial activity against the major Gram-positive mastitis pathogens including S. aureus (MIC range 0.5-8 µg/mL). The solubility test confirmed limited, ion-strength-dependent water solubility of lasalocid. A kinetic solubility study showed that SDs effectively enhanced water solubility of lasalocid (21-35-fold). Polyvinylpyrrolidone (PVP)-lasalocid SD caused minimum mammary irritation in treated cows and exhibited faster distribution in milk than either nano or microsized lasalocid. IMDSs with PVP-lasalocid SD provided effective treatment with a higher mastitis clinical and microbiological cure rate (66.7%) compared to cloxacillin (62.5%). CONCLUSION: Lasalocid SD IMDS provided high cure rates and effectiveness in treating bovine mastitis with acceptable safety in treated cows.

Epoxide hydrolase-lasalocid a structure provides mechanistic insight into polyether natural product biosynthesis.

Biosynthesis of some polyether natural products involves a kinetically disfavored epoxide-opening cyclic ether formation, a reaction termed anti-Baldwin cyclization. One such example is the biosynthesis of lasalocid A, an ionophore antibiotic polyether. During lasalocid A biosynthesis, an epoxide hydrolase, Lsd19, converts the bisepoxy polyketide intermediate into the tetrahydrofuranyl-tetrahydropyran product. We report the crystal structure of Lsd19 in complex with lasalocid A. The structure unambiguously shows that the C-terminal domain of Lsd19 catalyzes the intriguing anti-Baldwin cyclization. We propose a general mechanism for epoxide selection by ionophore polyether epoxide hydrolases.

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis.

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure-to our knowledge-of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Sorption and degradation in soils of veterinary ionophore antibiotics: monensin and lasalocid.

Monensin and lasalocid are polyether ionophores commonly used in the beef and poultry industries for the prevention of coccidial infections and promotion of growth. These ionophores can exhibit higher toxicity than many other antibiotics; thus, evaluating their fate in the environments associated with concentrated feed operations is important. Sorption of monensin and lasalocid was measured in eight soils of varying physiochemical composition. Organic carbon-normalized sorption coefficients (log Koc) ranged from 2.1 to 3.8 for monensin and from 2.9 to 4.2 for lasalocid and were inversely correlated to equilibrium soil-solution pH. Degradation of lasalocid and monensin in two contrasting soils with and without manure amendment was measured in moist soils at 23 degrees C and 0.03 MPa moisture potential. The half-life of both compounds in the fresh nonsterile soils was less than 4 d, for which monensin degraded slightly faster than lasalocid. Fresh liquid manure amendments did not significantly alter degradation of either compound. Based on parallel 60Co-sterilized soil experiments, some abiotic degradation of monensin was apparent, whereas lasalocid only degraded in the presence of microbes. Analysis of beef-derived lagoon effluent used for irrigation confirmed that monensin can be present at low-ppb to low-ppm concentrations in the aqueous and suspended solids fractions, respectively; however, subsequent analysis of drainage water in a nearby ditch suggested that attenuation by soil after land application will greatly reduce the amount entering surface waters.

7Li and 23Na NMR studies of transmembrane cation transport mediated by ionophore lasalocid A.

Ion transport across phospholipid vesicles was studied by 7Li and 23Na-NMR using an aqueous anionic paramagnetic shift reagent, dysprosium nitrilotriacetate [Dy(NTA)2](3-), mediated by ionophores, lasalocid A and A23187. The intra- and extracellular 7Li and 23Na-NMR signals were well separated (20 Hz) at mM concentration of the shift reagent. The observed data on the rate constant for lithium transport across DPPC vesicles at various concentrations of the ionophores indicated that lasalocid A is a more efficient carrier for lithium ion compared with the sodium ion transport by this ionophore, while A23187 was not specific to either of the ions (Li or Na).

Unusual behavior of ion transport mediated by polyene antibiotics. Activation energies for the exchange of Na+ ions through liposomal membranes studied by 23Na-NMR spectroscopy.

23Na-NMR spectroscopy has been applied to study the transport of Na+ ions across unilamellar vesicle membranes mediated by ionophores. The ionophores used were amphotericin B and nystatin. Also, monensin, lasalocid A and gramicidin D were included for the sake of comparison since the transport processes of these ionophores are well characterized as carrier-, carrier- and channel-types, respectively. The 23Na-NMR techniques employed were 2D-EXSY (exchange spectroscopy) and the ID time-course monitoring techniques, and the measurement exchange rates. These techniques were also applied at different temperatures, and the activation parameters were determined for the ionophore-mediated Na+ exchange. These activation parameters disclosed an unusual behavior of amphotericin B and nystatin. That is, cation transport through the membrane is decreased on increasing the temperature of measurement, resulting in negative values for the apparent enthalpy of activation. Such unusual behavior is related to the fluidity of the model membrane and to intermolecular interactions in the membrane.

Fitness of amines for pseudocyclic conformation of lasalocid A.

Ionophore lasalocid A selectively transports amines across lipid membranes. The structures of amine-lasalocid complexes were calculated by a molecular mechanics technique. Four typical catecholamines were considered: serotonin, dopamine, noradrenaline and adrenaline. The fitness energy was introduced consisting of tension energies of amine and ionophore and the interaction energy between them. Four fitted complexes of the first three amines and two of the last one were found. They are characterized by interaction between lasalocid ligand oxygens and amine ammonium groups, and also between their aromatic rings. The fitness energy increase correlates well with the decrease of the transportation rate in the considered sequence of amines.

Binding of radiolabeled monensin and lasalocid to ruminal microorganisms and feed.

Gram-negative, ionophore-resistant ruminal bacteria and Gram-positive, ionophore-sensitive species bound similar amounts of [14C]lasalocid, but neither group bound large amounts of [14C]monensin. Membrane vesicles also bound more lasalocid than monensin (P < .05). The binding was first-order at low cell or vesicle concentrations and saturable at high cell or vesicle densities. Streptococcus bovis was inhibited by both monensin and lasalocid (5 microM), but cells that were re-incubated in medium lacking ionophore grew rapidly. Lasalocid-treated cells grew very slowly when they were resuspended in fresh medium. Based on these results, it seemed that lasalocid had a higher affinity for bacterial membranes than monensin. Mixed bacteria, however, bound nearly equal amounts of [14C]monensin and [14C]lasalocid (P > .05). Monensin binding was greatly reduced when the mixed ruminal bacteria were pretreated with Tris+EDTA (P < .05), but Tris+EDTA did not affect the binding of lasalocid. Mixed ruminal protozoa always took up more lasalocid than monensin (P < .05), but feed particles bound equal amounts of [14C]lasalocid and [14C]monensin (P > .05). Based on the binding capacity of mixed ruminal bacteria, ruminal protozoa, and feed particles, there would be little free ionophore in ruminal fluid.

The effects of lipid composition on the binding of lasalocid A to small unilamellar vesicles.

The binding of the carboxylic ionophore lasalocid A (X537A) to small unilamellar phospholipid vesicles of varying composition was examined in an effort to determine what structural features of the phospholipid membrane influence the ionophore-membrane interaction. Apparent dissociation constants (Kapp) were calculated for both the acidic and anionic forms of the ionophore using the change in fluorescence intensity observed for lasalocid A upon addition of phospholipid vesicles. The Kapp for binding to fluid phase dimyristoylphosphatidylcholine (DMPC) vesicles is 46 microM for the anion and 14 microM for the acid. While the phase transition of DMPC had no effect on the Kapp of the anion, an increase was observed in the Kapp of the acid below the phase transition temperature. The Kapp of the anion was not affected by the incorporation of 10% dimyristoylphosphatidylethanolamine (DMPE), but increased slightly upon incorporation of cholesterol. The pKa values of the ionophore were the same in DMPC and DMPC/DMPE membranes. Incorporation of the negative lipids phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine (at pH 9.4 where PE carries a negative charge) decreases binding of the anion in accord with the increase in surface potential estimated from Gouy-Chapman theory. The CD spectrum of membrane-bound lasalocid A anion indicated the ionophore to be in an extended acyclic conformation on the membrane surface with the C-1 carboxylate rotated out of the plane of the salicylate ring. The out-of-plane rotation of the carboxylate may be the result of facial binding by the amphiphilic ionophore on the membrane surface or of weak ion pairing to the polar lipid head groups. These results suggest that the primary determinants of binding of the anionic ionophore on the membrane surface are packing density of the polar head groups and membrane surface potential. There is no evidence of strong hydrogen bond formation between the lipid polar head groups and the ionophore as has previously been suggested.

The effect of auranofin on polymorphonuclear granulocytes.

The effects of auranofin on the function of neutrophil polymorphonuclear granulocytes (PMN) have been studied in vitro and in vivo. Preincubation of human PMN with auranofin (1-4 micrograms/ml) increased their adherence to nylon fibres and f-met-leu-phe-(fMLP) induced aggregation. PMN migration, phagocytosis, bactericidal capacity and phagocytosis-associated enzyme release were all significantly inhibited by auranofin in a dose-dependent way. Enzyme release stimulated by f-MLP, chemoluminescence and the release of superoxide anions all showed a biphasic response to preincubation with auranofin. They showed an increase at low concentrations and inhibition at high concentrations. In studies of 3H-fMLP binding auranofin did not affect receptor numbers but increased binding affinity. Auranofin at higher concentrations decreased phorbolmyristate acetate and fMLP induced changes in surface charge and membrane potential. In vivo, auranofin administered to rats, did not prevent either the neutropenia induced by zymosan-activated serum or the corresponding rise in plasma lactoferrin levels. PMNs from six rheumatoid arthritis patients treated with auranofin (6 mg/day) for 23 weeks showed changes in bactericidal activity, chemotaxis and chemiluminescence independent of the clinical response. Enzyme release, however, was reduced in PMNs from clinical responders and showed no change in non-responders.

Conformational dynamics of the carboxylic ionophore lasalocid A underlying cation complexation-decomplexation and membrane transport.

The conformational dynamics of lasalocid A have been studied in a series of solvents of graded polarity by means of circular dichroism (CD) and computer-generated molecular models. In high polarity solvents, the uncomplexed anionic ionophore assumes as acyclic conformation minimizing intrinsic molecular strain energy. In this state, the dipoles of the liganding oxygens in the carbon backbone and the terminal carboxylate are stabilized by a high degree of solvent association. As the solvent polarity decreases, the dynamic conformational equilibrium progressively shifts toward a cyclic conformation which predominates at low polarity. Cyclization proceeds by rotation about three carbon-carbon hinge bonds. The resulting twist of the backbone introduces torsional strain which is offset at low polarity by electrostatic stabilization gained through intramolecular hydrogen bonding. Formation of a cation inclusion complex also stabilizes the cyclic conformer, even in relatively polar solvents. These observations suggest a scenario for carboxylic ionophore mediated transmembrane monovalent cation transport at the molecular level. The cation encounters an acyclic ionophore at the membrane interface where it ion pairs to the terminal carboxylate moiety, initiating formation of a lipophilic, cyclic cation inclusion complex. The complex, no longer constrained to the polar interface, diffuses across the membrane interior to the opposite face. There it reequilibrates with the polar environment, the ionophore reassuming the low energy, acyclic conformation and concomitantly releasing the enclosed cation. The free, acyclic ionophore is now confined to the opposite polar interface where it awaits the capture of a new cation to complete its catalytic transport cycle.

Lasalocid-sulfur amino acid interrelationship in the chick.

Several experiments were conducted with young healthy chicks to investigate the relationship between lasalocid and sulfur-containing amino acids (SAA). Chicks were housed in heated starter batteries in all experiments and were fed, in all but one trial, a completely purified crystalline amino acid diet. As previously determined in our laboratory with monensin, the quantitative requirement for SAA, expressed as a percent of the diet, was neither increased not decreased by 125 mg/kg dietary lasalocid. Unlike that observed with monensin, a consistent and striking SAA X lasalocid interaction was apparent. Hence, lasalocid elicited a marked gain and gain/feed response when added to diets severely deficient in SAA, but it resulted in a marked depression in chick performance when added to diets adequate in SAA. At moderate levels of SAA deficiency, such as those existing in practical corn-soybean meal broiler diets, 125 mg/kg lasalocid neither increased nor decreased rate and efficiency of weight gain. Indeed, no evidence of the SAA X lasalocid interaction was seen when lasalocid was provided in a typical corn-soybean meal chick starter diet.