Design,synthesis and antifungal activity of novel fenfuram-diarylamine hybrids

Ten novel fenfuram-diarylamine hybrids were designed and synthesized. And their antifungal activities against four phytopathogenic fungi have been evaluated in vitro and most of the compounds demonstrated a significant antifungal activities against Rhizoctonia solani and Sclerotinia sclerotiorum. Compound 5e exhibited the most potent antifungal activity against R. solani with an EC₅₀ value of 0.037 mg/L, far superior to the commercially available fungicide boscalid (EC₅₀ = 1.71 mg/L) and lead fungicide fenfuram (EC₅₀ = 6.18 mg/L). Furthermore, scanning electron microscopy images showed that the mycelia on treated media grew abnormally with tenuous, wizened and overlapping colonies compared to the negative control. Molecular docking studies revealed that compound 5e featured a higher affinity for succinate dehydrogenase (SDH) than fenfuram. Furthermore, it was shown that the 3-chlorophenyl group in compound 5e formed a CH-π interaction with B/Trp-206 and a Cl-π interaction with D/Tyr-128, rendering compound 5e more active than fenfuram against SDH.     

Investigation of various N-heterocyclic substituted piperazine versions of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol: Effect on affinity and selectivity for dopamine D3 receptor

Here we report on the design and synthesis of several heterocyclic analogues belonging to the 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol series of molecules. Compounds were subjected to [³H]spiperone binding assays, carried out with HEK-293 cells expressing either D2 or D3 dopamine receptors, in order to evaluate their inhibition constant (K_i) at these receptors. Results indicate that N-substitution on the piperazine ring can accommodate various substituted indole rings. The results also show that in order to maintain high affinity and selectivity for the D3 receptor the heterocyclic ring does not need to be connected directly to the piperazine ring as the majority of compounds included here are linked either via an amide or a methylene linker to the heterocyclic moiety. The enantiomers of the most potent racemic compound 10e exhibited differential activity with (?)-10e (K_i; D2 = 47.5 nM, D3 = 0.57 nM) displaying higher affinity at both D2 and D3 receptors compared to its enantiomer (+)-10e (K_i; D2 = 113 nM, D3 = 3.73 nM). Additionally, compound (?)-10e was more potent and selective for the D3 receptor compared to either 7-OH-DPAT or 5-OH-DPAT. Among the bioisosteric derivatives, the indazole derivative 10g and benzo[b]thiophene derivative 10i exhibited the highest affinity for D2 and D3 receptors. In the functional GTPγS binding study, one of the lead molecules, (?)-15, exhibited potent agonist activity at both D2 and D3 receptors with preferential affinity at D3.     

Design,synthesis, biological evaluation and molecular modelling studies of novel diaryl substituted pyrazolyl thiazolidinediones as potent pancreatic lipase inhibitors

A series of novel diaryl substituted pyrazolyl 2,4-thiazolidinediones were synthesized via reaction of appropriate pyrazolecarboxaldehydes with 2,4-thiazolidinedione (TZD) and nitrobenzyl substituted 2,4-thiazolidinedione. The resulting compounds were screened in vitro for pancreatic lipase (PL) inhibitory activity. Two assay protocols were performed viz., methods A and B using p-nitrophenyl butyrate and tributyrin as substrates, respectively. Compound 11e exhibited potent PL inhibitory activity (IC₅₀ = 4.81 µM and X_i50 = 10.01, respectively in method A and B), comparable to that of the standard drug, orlistat (IC₅₀ = 0.99 µM and X_i50 = 3.72). Presence of nitrobenzyl group at N-3 position of TZD and nature of substituent at para position of phenyl ring at C-3 position of pyrazole ring notably affected the PL inhibitory activity of the tested compounds. Enzyme inhibition kinetics of 11e revealed its reversible competitive inhibition, similar to that of orlistat. Molecular docking studies validated the rationale of pharmacophoric design and are in accordance to the in vitro results. Compound 11e exhibited a potential MolDock score of ?153.349 kcal/mol. Further, the diaryl pyrazolyl wing exhibited hydrophobic interactions with the amino acids of the hydrophobic lid domain. Moreover, the carbonyl group at 2^nd position of the TZD ring existed adjacent to Ser 152 (≈3 Å) similar to that of orlistat. A 10 ns molecular dynamics simulation of 11e–PL complex revealed a stable binding conformation of 11e in the active site of PL (Maximum RMSD ≈ 3 Å). The present study identified novel thiazolidinedione based leads with promising PL inhibitory activity. Further development of the leads might result in potent PL inhibitors.     

Alpha and quantum yield of aquatic plants derived from PAM fluorometry: Uses and misuses

The slope of the initial linear range of a photosynthesis–irradiance (P–I) curve, alpha (α), is frequently, but often incorrectly, used to denote the maximal quantum yield (or the “efficiency” of photosynthesis) of higher plants and macroalgae under the conditions for which the P–I curve was measured. When using the increasingly popular method of pulse amplitude modulated (PAM) fluorometry, the determination of α from so-called rapid light curves (RLC) may lead to misinterpretations when comparing photosynthetic efficiencies under different environmental conditions. Furthermore, since PAM fluorometry measures the quantum yield (Y) directly, there may be no need to estimate it from the initial slopes of RLCs.We compared photosynthetic parameters derived from RLCs of Ulva sp. measured during winter and summer, and show large differences in α when electron transport rates (ETR) were plotted against incident irradiance (I_i) [α = 0.26 ± 0.00 versus 0.08 ± 0.01 during the winter (November–December) and summer (July–August), respectively], as is usually done. On the other hand, no differences in the initial slopes of the RLCs were apparent when plotting ETR versus the absorbed irradiance (I_a) (initial slope = 0.75 ± 0.01 versus 0.62 ± 0.12 during the winter and summer, respectively); this is called for since also ETR is calculated using I_a. Using the I_a based RLCs, it was also found that the values of the initial slopes equalled those of the first Y-value measurements of the RLCs (Y₀) (t-test, p > 0.05, r² = 0.85). Therefore, when using PAM fluorometry, we suggest (a) to present the x-axis of RLCs as I_a (I_i × AF × 0.5), and ETR on the y-axis as Y × I_a, and (b) that Y₀ can be taken as a correct measure of the maximal quantum yield instead of estimating it from an RLC.     

Light thresholds derived from seagrass loss in the coastal zone of the northern Great Barrier Reef,Australia

There is a world-wide trend for deteriorating water quality and light levels in the coastal zone, and this has been linked to declines in seagrass abundance. Localized management of seagrass meadow health requires that water quality guidelines for meeting seagrass growth requirements are available. Tropical seagrass meadows are diverse and can be highly dynamic and we have used this dynamism to identify light thresholds in multi-specific meadows dominated by Halodule uninervis in the northern Great Barrier Reef, Australia. Seagrass cover was measured at ∼3 month intervals from 2008 to 2011 at three sites: Magnetic Island (MI) Dunk Island (DI) and Green Island (GI). Photosynthetically active radiation was continuously measured within the seagrass canopy, and three light metrics were derived. Complete seagrass loss occurred at MI and DI and at these sites changes in seagrass cover were correlated with the three light metrics. Mean daily irradiance (I_d) above 5 and 8.4 mol m⁻² d⁻¹ was associated with gains in seagrass at MI and DI, however a significant correlation (R = 0.649, p < 0.05) only occurred at MI. The second metric, percent of days below 3 mol m⁻² d⁻¹, correlated the most strongly (MI, R = −0.714, p < 0.01 and DI, R = −0.859, p = <0.001) with change in seagrass cover with 16–18% of days below 3 mol m⁻² d⁻¹ being associated with more than 50% seagrass loss. The third metric, the number of hours of light saturated irradiance (H_sat) was calculated using literature-derived data on saturating irradiance (E_k). H_sat correlated well (R = 0.686, p < 0.01; and DI, R = 0.704, p < 0.05) with change in seagrass abundance, and was very consistent between the two sites as 4 H_sat was associated with increases in seagrass abundance at both sites, and less than 4 H_sat with more than 50% loss. At the third site (GI), small seasonal losses of seagrass quickly recovered during the growth season and the light metrics did not correlate (p > 0.05) with change in percent cover, except for I_d which was always high, but correlated with change in seagrass cover. Although distinct light thresholds were observed, the departure from threshold values was also important. For example, light levels that are well below the thresholds resulted in more severe loss of seagrass than those just below the threshold. Environmental managers aiming to achieve optimal seagrass growth conditions can use these threshold light metrics as guidelines; however, other environmental conditions, including seasonally varying temperature and nutrient availability, will influence seagrass responses above and below these thresholds.     

The reliability and criterion validity of 2D video assessment of single leg squat and hop landing

The objective was to assess the intra-tester, within and between day reliability of measurement of hip adduction (HADD) and frontal plane projection angles (FPPA) during single leg squat (SLS) and single leg landing (SLL) using 2D video and the validity of these measurements against those found during 3D motion capture. 15 healthy subjects had their SLS and SLL assessed using 3D motion capture and video analysis. Inter-tester reliability for both SLS and SLL when measuring FPPA and HADD show excellent correlations (ICC_2,1 0.97–0.99). Within and between day assessment of SLS and SLL showed good to excellent correlations for both variables (ICC_3,1 0.72–91). 2D FPPA measures were found to have good correlation with knee abduction angle in 3-D (r = 0.79, p = 0.008) during SLS, and also to knee abduction moment (r = 0.65, p = 0.009). 2D HADD showed very good correlation with 3D HADD during SLS (r = 0.81, p = 0.001), and a good correlation during SLL (r = 0.62, p = 0.013). All other associations were weak (r < 0.4). This study suggests that 2D video kinematics have a reasonable association to what is being measured with 3D motion capture.     

The effects of feeding on the swimming performance and metabolic response of juvenile southern catfish,Silurus meridionalis,acclimated at different temperatures

To test whether the effects of feeding on swimming performance vary with acclimation temperature in juvenile southern catfish (Silurus meridionalis), we investigated the specific dynamic action (SDA) and swimming performance of fasting and feeding fish at acclimation temperatures of 15, 21, 27, and 33 °C. Feeding had no effect on the critical swimming speeding (U_crit) of fish acclimated at 15 °C (p = 0.66), whereas it elicited a 12.04, 18.70, and 20.98% decrease in U_crit for fish acclimated at 21, 27 and 33 °C, respectively (p < 0.05). Both the maximal postprandial oxygen consumption rate (VO_2peak) and the active metabolic rate (VO_2active, maximal aerobic sustainable metabolic rate of fasting fish) increased significantly with temperature (p < 0.05). The postprandial maximum oxygen consumption rates during swimming (VO_2max) were higher than the VO_2active of fasting fish at all temperature groups (p < 0.05). The VO_2max increased with increasing temperature, but the relative residual metabolic scope (VO_2max? VO_2peak) during swimming decreased with increasing in temperature. The present study showed that the impairment of postprandial swimming performance increased with increasing temperature due to the unparalleled changes in the catfish's central cardio-respiratory, peripheral digestive and locomotory capacities. The different metabolic strategies of juvenile southern catfish at different temperatures may relate to changes in oxygen demand, imbalances in ion fluxes and dissolved oxygen levels with changes in temperature.     

Elements of margin of safety,toxicity and action of sodium selenite in a lipopolysaccharide rat model

ProjectBoth septic shock and sodium selenite (Na₂SeO₃) lead to multiple organ failure through oxidation. Na₂SeO₃ has direct oxidant effects above the nutritional level and indirect anti-oxidant properties.In a lipopolysaccharide (LPS) rat model we assessed margin of safety, toxicity and beneficial effect of pentahydrate Na₂SeO₃ (5H₂O·Na₂SeO₃) at oxidant doses.ProcedureIn a three-step study on 204 rats we: (i) observed toxic effects of Na₂SeO₃ injected intraperitoneously (IP) and determined its Minimum Dose Without Toxic effect (MDWT) 0.25–0.35 mg/kg selenium (Se) content; (ii) injected IP LPS at 70% lethal dose (LD) followed, or not, one hour later by IP Na₂SeO₃ at MDWT and (iii) by doses > MDWT. At 48 h, in survivors, we measured plasma creatinine, lactate, aspartate and alanine aminotransferase (AST, ALT), nitric oxide (NO) and Se concentrations.Results(i) Na₂SeO₃ alone did not increase NO and lactate. Encephalopathy appeared at 1 mg Se/kg. Creatinine increased at 1–1.75 mg Se/kg, AST, ALT at 3–4.5 mg Se/kg, and the minimum LD was 3 mg Se/kg. (ii) Mortality after LPS was 37/50 (74%, [62–86%]) vs. 20/30 (67%, [50–84%]) when followed by Na₂SeO₃ at MDWT (p = 0.483) with a decreased in NO (−31%, p = 0.038) a trend for lactate decrease (−19%, p = 0.068) and an increased Se in plasma of survivals. (iii) All rats died at doses ≥0.6 mg/kg (p < 0.001).ConclusionMechanisms of LPS and Na₂SeO₃ toxicity differ (i.e. NO, lactate). In septic shock 5H₂O·Na₂SeO₃ toxicity increased, margin of safety decrease, but IP administration of dose considered as oxidant of 5H₂O·Na₂SeO₃ showed beneficial effects.     

Natural product hybrid and its superacid synthesized analogues: Dodoneine and its derivatives show selective inhibition of carbonic anhydrase isoforms I,III, XIII and XIV

The natural product dodoneine (a dihydropyranone phenolic compound), extracted from African mistletoe Agelanthus dodoneifolius, has been investigated as inhibitor of several human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms. By using superacid chemistry, analogues of the lactone phenolic hybrid lead compound have been synthesized and tested as CA inhibitors. Small chemical modifications of the basic scaffold revealed strong changes in the selectivity profile against different CA isoforms. These new compounds selectively inhibited isoforms CA I (K_Is in the range of 0.13–0.76 μM), III (K_Is in the range of 5.13–10.80 μM), XIII (K_Is in the range of 0.34–0.96 μM) and XIV (K_Is in the range of 2.44–7.24 μM), and can be considered as new leads, probably acting as non-zinc-binders, similar to other phenols/lactones investigated earlier.     

1,2,4-Triazolyl octahydropyrrolo[2,3-b]pyrroles: A new series of potent and selective dopamine D3 receptor antagonists

A novel series of 1,2,4-triazolyl octahydropyrrolo[2,3-b]pyrroles showing high affinity and selectivity at the DA D3 receptor is reported here. Compounds endowed with high selectivity over the hERG channel were identified and their pharmacokinetic properties thoroughly analyzed. A few derivatives with appropriate developability characteristics were selected for further studies and progression along the screening cascade. In particular, derivative 60a, (DA D3 pK_i = 8.4, DA D2 pK_i = 6.0 and hERG fpK_i = 5.2) showed a balanced profile and further refinements are in progress around this molecule.     

New material of Diacodexis (Mammalia,Artiodactyla) from the early Eocene of Southern Europe

Diacodexeidae are the first representatives of Artiodactyla in the fossil record. Their first occurrence is at the very base of the Ypresian (earliest Eocene, 56.0 Ma) with Diacodexis, a genus well diversified during the early Eocene in Europe, especially during the MP7–MP8 + 9 interval. However, most of European species are documented by scarce material, retrieved from single localities. In this work, we describe new Diacodexis material from ～MP7 and ～MP8 + 9 localities of Southern Europe, including material of D. antunesi from Silveirinha, considered as the most primitive European Diacodexis species, and material from three localities from Southern France (Fordones, Palette, and La Borie). The new material documents Diacodexis premolar morphology and deciduous dentition which bear potentially important phylogenetic information, as well as astragali, including a specimen from Silveirinha that constitutes the earliest occurrence of an astragalus of the genus Diacodexis in the European fossil record. Investigation of the enamel microstructure reveals that early European species had a simple enamel pattern with one-layered Schmelzmuster composed of ‘basic’ radial enamel only, instead of the two-layered Schmelzmuster (thin radial enamel + thick layer of Hunter-Schreger bands) observed on North American species and so far considered to represent the primitive condition within Artiodactyla. In accordance with previous studies, our observations highlight that Diacodexis gigasei from Belgium is morphologically closer to the North American species D. ilicis than to D. antunesi from Portugal. The latter species, together with D. aff. antunesi from Fordones, appears to be morphologically closer to the Asiatic taxa D. indicus and D. pakistanensis. Finally, we found numerous similarities between D. cf. gigasei from Palette and D. gigasei, a result that challenges the intra-European provincialism that characterizes the earliest Ypresian. Diacodexis gigasei could be one of the rare species shared by the northwestern and southwestern European bioprovinces.     

Plants as bioindicator for temperature interpolation purposes: Analyzing spatial correlation between botany based index of thermophily and integrated temperature characteristics

The success of interpolation techniques relies heavily on the density and regularity of field reference data points. For instance temperature interpolations in the Arctic are hampered by few and scattered meteorological stations. The major objective of this study is to analyze the spatial relationship between plants, defined in terms of an index of thermophily (I_t) and temperature distribution. The study area is located in Kongsfjorden, northwest Spitsbergen (Svalbard). A systematic recording of floristic data covering the study area was made within quadrates of 1 km × 1 km (93 units). For each of them, the I_t was calculated. I_t provides a synthetic measure by which plants are taken as temperature indicators at a long time scale. Temperature values were recorded by means of 39 temperature loggers during the summer 2000. The model for spatial interpolation of temperature was developed using multiple regression of remote sensed data (Landsat TM) and topographical features derived from a digital elevation model (DEM). Continuous temperature layers were calculated at a spatial resolution of 50 m × 50 m, and aggregated to a resolution of 1 km × 1 km in order to correspond with the observed botanical units. Different maps were produced showing spatial distribution of the modelled temperature and I_t. Correlations between the I_t and temperature values derived from the modelled temperature layers were systematically explored. Correlation between the I_t and temperatures works well as standard deviation of residues is 0.7 °C only. Highest correlations (r) of I_t and the spatial distribution of temperature were obtained for: (a) maximum average temperature for August, excluding all areas higher than 100 m above sea level (0.75), (b) average daily maximum temperature for July–October (0.67), (c) average temperature for July and August (0.64, 0.65), and (d) when temperature range is >8 °C (0.55). Areas with low correlations between I_t and temperature were mainly attributed to the fact that these measurements represent (a) different time scales and (b) different spatial scales. However, results from this study have shown that calculating I_t provide a mean for restoring selected temperature parameters and thus can contribute to fill in and extend the network of field data points for temperature interpolation purposes.     

Synthesis,in vitro evaluation and molecular docking studies of novel amide linked triazolyl glycoconjugates as new inhibitors of α-glucosidase

A series of N-substituted amide linked triazolyl β-d-glucopyranoside derivatives (4a-l) were synthesized and their in vitro inhibitory activity against yeast α-glucosidase enzyme [EC.3.2.1.20] was assessed. Compounds 4e (IC₅₀ = 156.06 μM), 4f (IC₅₀ = 147.94 μM), 4k (IC₅₀ = 127.71 μM) and 4l (IC₅₀ = 121.33 μM) were identified as the most potent inhibitors for α-glucosidase as compared to acarbose (IC₅₀ = 130.98 μM) under the same in vitro experimental conditions. Kinetic study showed that both 4e and 4f inhibit the enzyme in a competitive manner with p-nitrophenyl α-d-glucopyranoside as substrate. Molecular docking studies of 4e, 4f, 4k and 4l were also carried out using homology model of α-glucosidase to find out the binding modes responsible for the inhibitory activity. This study revealed that the binding affinity of compounds 4e, 4f, 4k and 4l for α-glucosidase were −8.2, −8.6, −8.3 and −8.5 kcal/mol respectively, compared to that of acarbose (−8.9 kcal/mol). The results suggest that the N-substituted amide linked triazole glycoconjugates can reasonably mimic the substrates for the yeast α-glucosidase.     

Synthesis,antichagasic in vitro evaluation,cytotoxicity assays,molecular modeling and SAR/QSAR studies of a 2-phenyl-3-(1-phenyl-1H-pyrazol-4-yl)-acrylic acid benzylidene-carbohydrazide series

Chagas disease (American trypanosomiasis) is one of the most important parasitic diseases with serious social and economic impacts mainly on Latin America. This work reports the synthesis, in vitro trypanocidal evaluation, cytotoxicity assays, and molecular modeling and SAR/QSAR studies of a new series of N-phenylpyrazole benzylidene-carbohydrazides. The results pointed 6k (X = H, Y = p-NO₂, pIC₅₀ = 4.55 M) and 6l (X = F, Y = p-CN, pIC₅₀ = 4.27 M) as the most potent derivatives compared to crystal violet (pIC₅₀ = 3.77 M). The halogen-benzylidene-carbohydrazide presented the lowest potency whereas 6l showed the most promising profile with low toxicity (0% of cell death). The best equation from the 4D-QSAR analysis (Model 1) was able to explain 85% of the activity variability. The QSAR graphical representation revealed that bulky X-substituents decreased the potency whereas hydrophobic and hydrogen bond acceptor Y-substituents increased it.     

Structural changes of the myofibrillar proteins in common carp (Cyprinus carpio) muscle exposed to a hydroxyl radical-generating system

Oxidation is a leading cause for quality deterioration during processing and storage of food. The objective of the present study was to examine the sensitivity of common carp (Cyprinus carpio) myofibrillar protein (MP) to oxidising radicals produced by a hydroxyl radical-generating system. Both structural and functional changes of common carp MP were evaluated. With increasing H₂O₂ concentrations and oxidation time, the protein carbonyl content, surface hydrophobicity and turbidity of MP increased (P < 0.05), while total sulfhydryl groups decreased (P < 0.05). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed protein polymerisation in oxidised MP. The oxidative process destroyed (P < 0.05) the texture (springiness and hardness) of MP gels and decreased their water-binding capacity and whiteness. The thermal gelation profile analysis indicated that oxidation led to a great reduction in the elasticity of samples. Taken together, proteins are susceptible to free radical attack, and oxidative stress had a detrimental effect on protein structure and the general functionality of MP.     

Novel boronated chlorin e6-based photosensitizers: Synthesis,binding to albumin and antitumour efficacy

Chlorins, a class of plant porphyrins, are perspective as photosensitizing agents due to light absorption in the long wavelength spectral region and deeper photodamage of tissues. Aiming at optimization of antitumour properties of chlorins, we synthesized a series of boronated derivatives of chlorin e₆ and their complexes containing Zn(II), Pd(II) or Sn(IV). The compounds were synthesized by alkylation of amino or hydroxy derivatives of chlorin e₆ with 1-trifluoromethanesulfonylmethyl-o-carborane. Chlorin e₆ 13(1)-N-{2-[N-(o-carboran-1-yl)methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ester (compound 5) formed complexes with serum albumin, a major porphyrin carrier. The binding constant of these complexes was ～4 times bigger than the respective value for the complexes of albumin with boron-free aminochlorin e₆. Compound 5 potently sensitized rat fibroblasts to illumination with monochromatic red light: >98% of cells were necrotic by 24 h post-illumination with 1 μM of 5. This compound demonstrated high efficacy in photodynamic therapy of rat M-1 sarcoma. After PDT with 25 mg/kg of 5 the residual tumours were significantly smaller than in animals subjected to PDT with equal concentration of boron-free aminochlorin e₆. No signs of general toxicity were detectable after PDT with 5. Thus, boronation can enhance the potency of chlorins in PDT, in particular, due to an increased binding to albumin. Our data expand the therapeutic applicability of boronated chlorins beyond boron neutron capture therapy; these agents emerge as dual efficacy photoradiosensitizers.     

Cloning and characterization of a NADH-dependent aldo-keto reductase from a newly isolated Kluyveromyces lactis XP1461

An aldo-keto reductase gene (klakr) from Kluyveromyces lactis XP1461 was cloned and heterologously expressed in Escherichia coli. The aldo-keto reductase KlAKR was purified and found to be NADH-dependent with a molecular weight of approximately 36 kDa. It is active and stable at 30 °C and pH 7.0. The maximal reaction rate (v_max), apparent Michaelis–Menten constant (K_m) for NADH and t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (1a) and catalytic number (k_cat) were calculated as 7.63 U mg⁻¹, 0.204 mM, 4.42 mM and 697.4 min⁻¹, respectively. Moreover, the KlAKR has broad substrate specificity to a range of aldehydes, ketones and keto-esters, producing chiral alcohol with e.e. or d.e. >99% for the majority of test substrates.     

Cloning,characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea

We have cloned, purified and characterized the γ-carbonic anhydrase (CA, EC 4.2.1.1) present in the genome of the Antarctic bacterium Colwellia psychrerythraea, which is an obligate psychrophile. The enzyme shows a significant catalytic activity for the physiologic reaction of CO₂ hydration to bicarbonate and protons, with the following kinetic parameters: k_cat of 6.0 × 10⁵ s⁻¹ and a k_cat/K_m of 4.7 × 10⁶ M⁻¹ × s⁻¹. This activity was inhibited by the sulfonamide CA inhibitor (CAI) acetazolamide, with a K_I of 502 nM. A range of anions was also investigated for their inhibitory action against the new enzyme CpsCA. Perchlorate, tetrafluoroborate, fluoride and bromide were not inhibitory, whereas cyanate, thiocyanate, cyanide, hydrogensulfide, carbonate and bicarbonate showed K_Is in the range of 1.4–4.4 mM. Diethyldithiocarbamate was a better inhibitor (K_I of 0.58 mM) whereas sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid were the most effective inhibitors detected, with K_Is ranging between 8 and 38 μM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here.