The major Arabidopsis thaliana apurinic/apyrimidinic endonuclease,ARP is involved in the plant nucleotide incision repair pathway

Apurinic/apyrimidinic (AP) endonucleases are important DNA repair enzymes involved in two overlapping pathways: DNA glycosylase-initiated base excision (BER) and AP endonuclease-initiated nucleotide incision repair (NIR). In the BER pathway, AP endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases, whereas in NIR, the same AP endonucleases incise DNA 5' to a wide variety of oxidized bases. The flowering plant Arabidopsis thaliana contains three genes encoding homologues of major human AP endonuclease 1 (APE1): Arp, Ape1L and Ape2. It has been shown that all three proteins contain AP site cleavage and 3'-repair phosphodiesterase activities; however, it was not known whether the plant AP endonucleases contain the NIR activity. Here, we report that ARP proteins from Arabidopsis and common wheat (Triticum aestivum) contain NIR and 3' → 5' exonuclease activities in addition to their AP endonuclease and 3'-repair phosphodiesterase functions. The steady-state kinetic parameters of reactions indicate that Arabidopsis ARP cleaves oligonucleotide duplexes containing α-anomeric 2'-deoxyadenosine (αdA) and 5,6-dihydrouridine (DHU) with efficiencies (k_cat/K_M = 134 and 7.3 μM⁻¹·min⁻¹, respectively) comparable to those of the human counterpart. However, the ARP-catalyzed 3'-repair phosphodiesterase and 3' → 5' exonuclease activities (k_cat/K_M = 314 and 34 μM⁻¹·min⁻¹, respectively) were about 10-fold less efficient as compared to those of APE1. Interestingly, homozygous A. thaliana arp^–/– mutant exhibited high sensitivity to methyl methanesulfonate and tert-butyl hydroperoxide, but not to H₂O₂, suggesting that ARP is a major plant AP endonuclease that removes abasic sites and specific types of oxidative DNA base damage. Taken together, these data establish the presence of the NIR pathway in plants and suggest its possible role in the repair of DNA damage generated by oxidative stress.     

Processing of the abasic sites clustered with the benzo[a]pyrene adducts by the base excision repair enzymes

The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5′-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5′ direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3′ direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3′ direction (AP₊₃-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP₊₃-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3′ → 5′ exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.     

Design,synthesis, and DNA binding characteristics of a group of orthogonally positioned diamino,N-formamido,pyrrole- and imidazole-containing polyamides

Orthogonally positioned diamino/dicationic polyamides (PAs) have good water solubility and enhanced binding affinity, whilst retaining DNA minor groove and sequence specificity compared to their monoamino/monocationic counterparts. The synthesis and DNA binding properties of the following diamino PAs: f-IPI (3a), f-IPP (4), f-PIP (5), and f-PPP (6) are described. P denotes the site where a 1-propylamino group is attached to the N1-position of the heterocycle. Binding of the diamino PAs to DNA was assessed by DNase I footprinting, thermal denaturation, circular dichroism titration, biosensor surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) studies. According to SPR studies, f-IPI (3a) bound more strongly (K_eq = 2.4 × 10⁸ M^?1) and with comparable sequence selectivity to its cognate sequence 5′-ACGCGT-3′ when compared to its monoamino analog f-IPI (1). The binding of f-IPI (3a) to 5′-ACGCGT-3′ via the stacked dimer motif was balanced between enthalpy and entropy, and that was quite different from the enthalpy-driven binding of its monoamino parent f-IPI (1). f-IPP (4) also bound more strongly to its cognate sequence 5′-ATGCAT-3′ (K_eq = 7.4 × 10⁶ M^?1) via the side-by-side stacked motif than its monoamino analog f-IPP (2a). Although f-PPP (6) bound via a 1:1 motif, it bound strongly to its cognate sequence 5′-AAATTT-3′ (K_eq = 4.8 × 10⁷ M^?1), 15-times higher than the binding of its monoamino analog f-PPP (2c), albeit f-PPP bound via the stacked motif. Finally, f-PIP (5) bound to its target sequence 5′-ATCGAT-3′ as a stacked dimer and it has the lowest affinity among the diamino PAs tested (K_eq <1 × 10⁵ M^?1). This was about two times lower in affinity than the binding of its monoamino analog f-PIP (2b). The results further demonstrated that the ‘core rules’ of DNA recognition by monoamino PAs also apply to their diamino analogs. Specifically, PAs that contain a stacked IP core structure bind most strongly (highest binding constants) to their cognate GC doublet, followed by the binding of PAs with a stacked PP structure to two degenerate AT base pairs, and finally the binding of PAs with a PI core to their cognate CG doublet.     

Metal ion dependence of DNA cleavage by SepMI and EhoI restriction endonucleases

Most of type II restriction endonucleases show an absolute requirement for divalent metal ions as cofactors for DNA cleavage. While Mg²⁺ is the natural cofactor other metal ions can substitute it and mediate the catalysis, however Ca²⁺ (alone) only supports DNA binding. To investigate the role of Mg²⁺ in DNA cleavage by restriction endonucleases, we have studied the Mg²⁺ and Mn²⁺ concentration dependence of DNA cleavage by SepMI and EhoI. Digestion reactions were carried out at different Mg²⁺ and Mn²⁺ concentrations at constant ionic strength. These enzymes showed different behavior regarding the ions requirement, SepMI reached near maximal level of activity between 10 and 20 mM while no activity was detected in the presence of Mn²⁺ and in the presence of Ca²⁺ cleavage activity was significantly decreased. However, EhoI was more highly active in the presence of Mn²⁺ than in the presence of Mg²⁺ and can be activated by Ca²⁺. Our results propose the two-metal ion mechanism for EhoI and the one-metal ion mechanism for SepMI restriction endonuclease. The analysis of the kinetic parameters under steady state conditions showed that SepMI had a K_m value for pTrcHisB DNA of 6.15 nM and a V_max of 1.79 × 10^?2 nM min^?1, while EhoI had a K_m for pUC19 plasmid of 8.66 nM and a V_max of 2 × 10^?2 nM min^?1.     

Anion inhibition studies of two new β-carbonic anhydrases from the bacterial pathogen Legionella pneumophila

We investigated the cloning, catalytic activity and anion inhibition of the β-class carbonic anhydrases (CAs, EC 4.2.1.1) from the bacterial pathogen Legionella pneumophila. Two such enzymes, lpCA1 and lpCA2, were found in the genome of this pathogen. These enzymes were determined to be efficient catalysts for CO₂ hydration, with k_cat values in the range of (3.4–8.3) × 10⁵ s⁻¹ and k_cat/K_M values of (4.7–8.5) × 10⁷ M⁻¹ s⁻¹. A set of inorganic anions and small molecules was investigated to identify inhibitors of these enzymes. Perchlorate and tetrafluoroborate were not acting as inhibitors (K_I >200 mM), whereas sulfate was a very weak inhibitor for both lpCA1 and lpCA2 (K_I values of 77.9–96.5 mM). The most potent lpCA1 inhibitors were cyanide, azide, hydrogen sulfide, diethyldithiocarbamate, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 6 to 94 μM. The most potent lpCA2 inhibitors were diethyldithiocarbamate, sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid, with K_I values ranging from 2 to 13 μM. As these enzymes seem to be involved in regulation of phagosome pH during Legionella infection, inhibition of these targets may lead to antibacterial agents with a novel mechanism of action.     

Triazolopyridyl ketones as a novel class of antileishmanial agents. DNA binding and BSA interaction

A new series of triazolopyridyl pyridyl ketones has been synthetized by regioselective lithiation of the corresponding [1,2,3]triazolo[1,5-a]pyridine at 7 position followed by reaction with different electrophiles. The in vitro antileishmanial activity of these compounds was evaluated against Leishmania infantum, Leishmania braziliensis, Leishmania guyanensis and Leishmania amazonensis. Compounds 6 and 7 were found to be the most active leishmanicidal agents. Both of them showed activities at micromolar concentration against cultured promastigotes of Leishmania spp. (IC₅₀ = 99.8–26.8 μM), without cytotoxicity on J774 macrophage cells. These two compounds were also tested in vivo in a murine model of acute infection by L. infantum. The triazolopyridine derivative 6 was effective against both spleen and liver parasites forms, while 7 was inactive against liver parasites. Mechanistic aspects of the antileishmanial activity were investigated by means of DNA binding studies (UV-titration and viscosimetry). Results have revealed that these active ligands are able to interact strongly with DNA [K_b = 1.14 × 10⁵ M⁻¹ (6) and 3.26 × 10⁵ M⁻¹ (7)]. Moreover, a DNA groove binding has been proposed for both 6 and 7. To provide more insight on the mode of action of compounds 6 and 7 under biological conditions, their interaction with bovine serum albumin (BSA) was monitored by fluorescence titrations and UV–visible spectroscopy. The quenching constants and binding parameters were determined. Triazolopyridine ketones 6 and 7 have exhibited significant affinity towards BSA [K_b = 2.5 × 10⁴ M⁻¹ (6) and 1.9 × 10⁴ M⁻¹ (7)]. Finally, to identify the binding location of compounds 6 and 7 on the BSA, competitive binding experiments were carried out, using warfarin, a characteristic marker for site I, and ibuprofen as one for site II. Results derived from these studies have indicated that both compounds interact at BSA site I and, to a lesser extent, at site II.     

Synthesis,X-ray crystal structure,DNA/protein binding and cytotoxicity studies of five α-aminophosphonate N-derivatives

Five new α-aminophosphonates are synthesized and characterized by EA, FT-IR, ¹H NMR, ¹³C NMR, ³¹P NMR, ESI-MS and X-ray crystallography. The X-ray analyses reveal that the crystal structures of 1–5 are monoclinic or triclinic system with the space group P 21/c, P − 1, P − 1, P2(1)/c and P − 1, respectively. All P atoms of 1–5 have tetrahedral geometries involving two O-ethyl groups, one C_α atom, and a double bond O atom. The binding interaction of five new α-aminophosphonate N-derivatives (1–5) with calf thymus(CT)-DNA have been investigated by UV–visible and fluorescence emission spectrometry. The apparent binding constant (Kapp) values follows the order: 1 (3.38 × 10⁵ M⁻¹) > 2 (3.04 × 10⁵ M⁻¹) > 4 (2.52 × 10⁵ M⁻¹) > 5 (2.32 × 10⁵ M⁻¹) > 3 (2.10 × 10⁵ M⁻¹), suggesting moderate intercalative binding mode between the compounds and DNA. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with the compounds 1–5 showed that the quenching mechanism might be a static quenching procedure. For the compounds 1–5, the number of binding sites were about one for BSA and the binding constants follow the order: 1 (2.72 × 10⁴ M⁻¹) > 2 (2.27 × 10⁴ M⁻¹) > 4 (2.08 × 10⁴ M⁻¹) > 5 (1.79 × 10⁴ M⁻¹) > 3 (1.17 × 10⁴ M⁻¹). Moreover, the DNA cleavage abilities of 1 exhibit remarkable changes and the in vitro cytotoxicity of 1 on tumor cells lines (MCF-7, HepG2 and HT29) have been examined by MTT and shown antitumor effect on the tested cells.     

Synthesis,characterization and biological evaluation of novel chalcone sulfonamide hybrids as potent intestinal alkaline phosphatase inhibitors

Alkaline phosphatase (AP) and ecto-5′-nucleotidase (e5′NT) belong to same family that hydrolyze the extracellular nucleotides and ensure the bioavailability of nucleotides and nucleosides at purinergic receptors. During pathophysiological conditions, the over expression of AP and e5′NT lead to an increased production of adenosine that enhance tumor proliferation, invasiveness, neoangiogenesis and disrupts the body antitumor response. As both enzymes are abundantly expressed in above mentioned conditions, therefore it is of great interest to synthesize and develop potent inhibitors of these enzymes that augment the antitumor therapy. Herein we reported the synthesis and biological activity of a new series of chalcone-sulfonamide hybrids (4a-j). These derivatives were then evaluated for their inhibitory potential against two members of ecto-nucleotidase family, e5′NT (human and rat) and APs isozyme (intestinal and tissue nonspecific). Only six derivatives were found to inhibit both human and rat e5′NT enzymes. Compounds 4e and 4d showed maximum inhibition of human and rat e5′NT with an IC₅₀ ± SEM = 0.26 ± 0.01 and 0.33 ± 0.004 μM, respectively. Moreover, on APs, these derivatives were identified as the selective inhibitors of calf intestinal AP (c-IAP). The derivative 4a exhibited maximum inhibition of c-IAP with an IC₅₀ ± SEM = 0.12 ± 0.02 μM. In conclusion, these chalcone-sulfonamide hybrids exhibited dual inhibition of both family of isozymes but was more selective towards c-IAP enzyme.     

Comparison of two type IV hyperthermophilic adenylyl cyclases characterizations from the archaeon Pyrococcus furiosus

In this paper, two genes that encoded two soluble type IV adenylyl cyclases (AC) from the hyperthermophilic archaeon Pyrococcus furiosus (PFAC I and PFAC II) were cloned and expressed in Escherichia coli (E. coli) BL21 (DE3). Amino acid sequence analysis of the two enzymes showed 29% homology. PFAC I and PFAC II were both Mn²⁺ activated enzyme. They were purified by His-trap chromatography and had a specific activity of 3.1 × 10³ U/mg at pH 10.0, 95 °C (PFAC I) and 2.0 × 10³ U/mg at pH 11.0, 95 °C (PFAC II), respectively. The K_m and k_cat of PFAC I was 1.38 mM and 1.11 s⁻¹. The K_m and k_cat of PFAC II was 1.44 mM and 0.80 s⁻¹. The thermostability of PFAC I and PFAC II were higher than the soluble type IV adenylyl cyclases from Yersinia pestis (YpAC-IV). All of the properties suggested that these two adenylyl cyclases may be useful for the industrial producing of cyclic adenosine 3′,5′-monophosphate (cAMP).     

DNA binding and photocleavage properties and apoptosis-inducing activities of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)2Cl]Cl and its heterometallic derivatives

The interactions of a ruthenium porphyrin complex [(Py-3′)TPP-Ru(phen)₂Cl]Cl (phen = 1,10-phenanthroline, (Py-3′)TPP = 5-(3′-pyridyl-10,15,20-triphenylporphyrin) (1) and its heterometallic derivatives, [Ni(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (2) and [Cu(Py-3′)TPP-Ru(phen)₂Cl][PF₆] (3), with calf thymus DNA have been investigated by spectroscopic and viscosity measurements in this study. The results showed that these synthetic complexes can bind to double strand helix DNA in groove binding mode, and the intrinsic binding constants of complexes 1, 2 and 3, as calculated according to the decay of the Soret absorption, are (1.35 ± 0.5) ×10⁵ M^?1 (s = 4.2), (1.29 ± 0.5) × 10⁵ M^?1 (s = 5.6) and (1.22 ± 0.5) × 10⁵ M^?1 (s = 6.2) (s is the binding-site size), respectively, which are consistent with those obtained from ethidium bromide-quenching experiments. Further investigations on the photocleavage properties of these complexes on plasmid pBR 322 DNA showed that complexes 1, 2 and 3 could cleave single chain DNA and convert DNA molecules from supercoiled form to the nicked form. As determined by MTT assay, the complexes were also identified as potent antiproliferative agents against A375 human melanoma cells, MCF-7 human breast adrenocarcinoma cells, Colo201 human colon adenocarcinoma cells and HepG2 human liver cancer cells. Complex 1 inhibits the growth of A375 cells through induction of apoptotic cell death and G0/G1 cell cycle arrest. Further investigation on intracellular mechanisms indicated that Complex 1 induced depletion of mitochondrial membrane potential (ΔΨ_m) in A375 cells through regulating the expression of pro-survival and pro-apoptotic Bcl-2 family members. Our results suggest that ruthenium porphyrin complexes could be candidates for further evaluation as chemopreventive and chemotherapeutic agents for human cancers.     

Characterization of bi-functional CYP154 from Nocardia farcinica IFM10152 in the O-dealkylation and ortho-hydroxylation of formononetin

Among the 27 cytochrome P450s (CYPs) of Nocardia farcinica IFM10152, three CYPs have been identified as having O-dealkylation catalytic activity. Of the two that encode CYP154 subfamilies, the one encoded by the nfa22930 gene showed distinct O-dealkylation and subsequent hydroxylation of formononetin. Firstly, formononetin was O-dealkylated into daidzein, which was subsequently mono-hydroxylated at the 3′-position of the B-ring into ortho-dihydroxy-isoflavone. Apparent k_cat/K_m values of CYP154 for the O-dealkylation of formononetin and the hydroxylation of daidzein were 3.57 and 1.84 μM⁻¹ min⁻¹, respectively. The dissociation constants of CYP154 based on spectral changes upon binding to each substrate were 5.16 and 3.11 μM, respectively. Homology modeling and docking simulation found that Thr247 is responsible for the 3′-position hydroxylation reaction by forming a hydrogen bond with the 4′-hydroxyl group of daidzein that forces the proton at the 3′-position to face the heme center. Site-directed mutagenesis of Thr247 to alanine drastically decreased the binding affinity for daidzein (9.73 μM) as well as 3′-position hydroxylation catalytic activity by 3 fold (0.48 μM⁻¹ min⁻¹).     

Characterization and directed evolution of BliGO,a novel glycine oxidase from Bacillus licheniformis

Glycine oxidase (GO) has great potential for use in biosensors, industrial catalysis and agricultural biotechnology. In this study, a novel GO (BliGO) from a marine bacteria Bacillus licheniformis was cloned and characterized. BliGO showed 62% similarity to the well-studied GO from Bacillus subtilis. The optimal activity of BliGO was observed at pH 8.5 and 40 °C. Interestingly, BliGO retained 60% of the maximum activity at 0 °C, suggesting it is a cold-adapted enzyme. The kinetic parameters on glyphosate (K_m, k_cat and k_cat/K_m) of BliGO were 11.22 mM, 0.08 s⁻¹, and 0.01 mM⁻¹ s⁻¹, respectively. To improve the catalytic activity to glyphosate, the BliGO was engineered by directed evolution. With error-prone PCR and two rounds of DNA shuffling, the most evolved mutant SCF-4 was obtained from 45,000 colonies, which showed 7.1- and 8-fold increase of affinity (1.58 mM) and catalytic efficiency (0.08 mM⁻¹ s⁻¹) to glyphosate, respectively. In contrast, its activity to glycine (the natural substrate of GO) decreased by 113-fold. Structure modeling and site-directed mutation study indicated that Ser51 in SCF-4 involved in the binding of enzyme with glyphosate and played a crucial role in the improvement of catalytic efficiency.     

Novel 3-arylfuran-2(5H)-one-fluoroquinolone hybrid: Design,synthesis and evaluation as antibacterial agent

3-Arylfuran-2(5H)-one, a novel antibacterial pharmacophore targeting tyrosyl-tRNA synthetase (TyrRS), was hybridized with the clinically used fluoroquinolones to give a series of novel multi-target antimicrobial agents. Thus, twenty seven 3-arylfuran-2(5H)-one-fluoroquinolone hybrids were synthesized and evaluated for their antimicrobial activities. Some of the hybrids exhibited merits from both parents, displaying a broad spectrum of activity against resistant strains including both Gram-negative and Gram-positive bacteria. The most potent compound (11) in antibacterial assay shows MIC₅₀ of 0.11 μg/mL against Multiple drug resistant Escherichia coli, being about 51-fold more potent than ciprofloxacin. The enzyme assays reveal that 11 is a potent multi-target inhibitor with IC₅₀ of 1.15 ± 0.07 μM against DNA gyrase and 0.12 ± 0.04 μM against TyrRS, respectively. Its excellent inhibitory activities against isolated enzymes and intact cells strongly suggest that 11 deserves to further research as a novel antibiotic.     

Syntheses,structures, and magnetic properties of heterobimetallic complexes based on tetracyanometallic building blocks

Two tetracyanometalate building blocks, [Fe(5,5′-dmbipy)(CN)₄]^? (2) and [Fe(4,4′-dmbipy)(CN)₄]^? (3) (5,5′-dmbipy = 5,5′-dimethyl-2,2′-bipyridine; 4,4′-dmbipy = 4,4′-dimethyl-2,2′-bipyridine), and two cyano-bridged heterobimetallic complexes, [Cu₂(bpca)₂(H₂O)₂Fe₂(5,5′-dmbipy)₂(CN)₈] · 2[Cu(bpca)Fe(5,5′-dmbipy)(CN)₄] · 4H₂O (4) and [Cu(bpca)Fe(4,4′-dmbipy)(CN)₄]_n (5) (bpca = bis(2-pyridylcarbonyl)amidate), have been synthesized and structurally characterized. Complex 4 contains two dinuclear and one tetranuclear heterobimetallic clusters in an asymmetric unit whereas the structure of complex 5 features a one-dimensional heterobimetallic zigzag chain. The Cu(II) ion is penta-coordinated in the form of a distorted square-based pyramid. Magnetic studies show ferromagnetic coupling between Cu(II) and Fe(III) ions with g = 2.28, J₁ = 2.64 cm^?1, J₂ = 5.40 cm^?1 and TIP = ?2.36 × 10^?3 for complex 4, and g = 2.17, J = 4.82 cm^?1 and zJ′ = 0.029 cm^?1 for complex 5.     

Semi-interpenetrating polymer networks (semi-IPNs) for entrapment of laccase and their use in Acid Orange 52 decolorization

Laccase enzyme (L) from Trametes versicolor was entrapped in three hydrogel structures namely poly(acrylamide-N-isopropylacrylamide), P(AAm-NIPA), and semi-interpenetrating networks of poly(acrylamide)/alginate, P(AAm)/Alg, and poly(acrylamide-N-isopropylacrylamide)/alginate, P(AAm-NIPA)/Alg. The optimum temperatures for free and all immobilized systems were found to be 40 °C. For free and immobilized laccase systems of P(AAm-NIPA)-L, P(AAm)/Alg-L and P(AAm-NIPA)/Alg-L, K_m values were found to be 6.7 × 10^?3, 8.8 × 10^?2, 5.5 × 10^?2 and 1.8 × 10^?2 mM; V_max values were calculated as 1.8 × 10^?3, 2.5 × 10^?2, 1.5 × 10^?2 and 6.1 × 10^?3 mM min^?1, respectively. For free and the same immobilized systems, the enzymes retained 42%, 91%, 79% and 86% of their initial activities at the end of 56 days of storage. After using the mentioned immobilized systems repeatedly 10 times, they retained 77%, 71% and 84% of their original activities, respectively. For free and the same immobilized systems, decolorization of Acid Orange 52 (AO52) in 6 h were found to be 63%, 50%, 48% and 66%, respectively. Addition of 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, into the assay medium increased these values up to 73%, 73%, 74% and 75%, respectively.     

Cloning,characterization and anion inhibition study of the δ-class carbonic anhydrase (TweCA) from the marine diatom Thalassiosira weissflogii

We investigated the catalytic activity and inhibition of the δ-class carbonic anhydrase (CA, EC 4.2.1.1) from the marine diatom Thalassiosira weissflogii, TweCA. The enzyme, obtained by cloning the synthetic gene, was an efficient catalyst for the CO₂ hydration, its physiological reaction, with a k_cat of 1.3 × 10⁵ s⁻¹ and a k_cat/K_M of 3.3 × 10⁷ M⁻¹ s⁻¹. A range of inorganic anions and small molecules were investigated as inhibitors of TweCA. Chloride and sulfate did not inhibit the enzyme (K_Is >200 mM) whereas other halides and pseudohalides were submillimolar–millimolar inhibitors (K_Is in the range of 0.93–8.3 mM). The best TweCA inhibitors were hydrogen sulfide, sulfamate, sulfamide, phenylboronic acid and phenylarsonic acid, with K_Is in the range of 9–90 μM, whereas acetazolamide inhibited the enzyme with a K_I of 83 nM. This is the first kinetic and inhibition study of a δ-class CA. However, these enzymes are widespread in the marine phytoplankton, being present in haptophytes, dinoflagellates, diatoms, and chlorophytic prasinophytes, contributing to the CO₂ fixation by sea organisms. A phylogenetic analysis with all five genetic families of CAs showed that α- and δ-CAs are evolutionarily more related to each other with respect to the γ-CAs, although these three families clustered all together. On the contrary, the β- and ζ-CAs are also related to each other but phylogenetically much more distant from the α-, γ and δ-CA cluster. Thus, the study of δ-CAs is essential for better understanding this superfamily of metalloenzymes and their potential biotechnological applications in biomimetic CO₂ capture processes, as these enzymes are part of the carbon concentrating mechanism used by many photosynthetic organisms.     

Cloning,overexpression, purification,and site-directed mutagenesis of xylitol-2-dehydrogenase from Candida albicans

Xylitol-2-dehydrogenase from Candida albicans was cloned and overexpressed in Escherichia coli. The purified recombinant XDH has an apparent molecular weight of 40 kDa which belongs to the medium chain alcohol dehydrogenase family and exclusively uses NAD⁺ as a cofactor. The recombinant caXDH has a K_M of 8.8 mM and 37.7 μM using the substrate xylitol and NAD⁺, respectively, and its catalytic efficiency is 53,200 min^?1 mM^?1. Following site-directed mutagenesis, one of the engineered caXDHs with six mutations at Ser95Cys, Ser98Cys, Tyr101Cys, Asp206Ala, Ile207Arg, and Phe208Ser shifted its cofactor dependence from NAD⁺ to NADP⁺ in which the K_M and k_cat/K_M towards NADP⁺ are 119 μM and 26,200 min^?1 mM^?1, respectively.     

Characterization and PCR optimization of the thermostable family B DNA polymerase from Thermococcus guaymasensis

The gene encoding Thermococcus guaymasensis DNA polymerase (Tgu DNA polymerase) was cloned and sequenced. The 2328 bp Tgu DNA polymerase gene encoded a 775 amino acid residue protein. Alignment of the entire amino acid sequence revealed a high degree of sequence homology between Tgu DNA polymerase and other archaeal family B DNA polymerases. The Tgu DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL. The expressed enzyme was then purified by heat treatment followed by two steps of chromatography. The optimum pH and temperature were 7.5 and 80 °C, respectively. The optimal buffer for PCR with Tgu DNA polymerase consisted of 50 mM Tris–HCl (pH 8.2), 4 mM MgCl₂, 50 mM KCl, and 0.02% Triton X-100. Tgu DNA polymerase revealed 4-fold higher fidelity (3.17 × 10^?6) than Taq DNA polymerase (12.13 × 10^?6) and a faster amplification rate than Taq and Pfu DNA polymerases. Tgu DNA polymerase had an extension rate of 30 bases/s and a processivity of 150 nucleotides (nt). Thus, Tgu DNA polymerase has some faster elongation rate and a higher processivity than Pfu DNA polymerase. Use of different ratios of Taq and Tgu DNA polymerases determined that a ratio of 4:1 efficiently facilitated long PCR (approximately 15 kb) and a 3-fold lower error rate (4.44 × 10^?6) than Taq DNA polymerase.     

Sulfation of (1→3)-β-d-glucan from the fruiting bodies of Russula virescens and antitumor activities of the modifiers

A water-insoluble (1→3)-β-d-glucan isolated from the fresh fruiting bodies of Russula virescens was sulfated using sulfur trioxide-pyridine complex as reagent in dimethyl sulfoxide. Depending on the reaction conditions, the products showed different degrees of sulfation (DS) ranging from 0.17 to 1.17 and different weight average molecular weights (M_ws) ranging from 2.5 × 10⁴ to 1.2 × 10⁵ Da. Moreover, the antitumor activities of the five sulfated derivatives against Sarcoma 180 tumor cell were tested both in vitro and in vivo. The results indicated that the native (1→3)-β-d-glucan did not show antitumor activity, while the sulfated derivatives exhibited enhanced antitumor activities. This study demonstrated that DS and M_w could influence the antitumor activities of the sulfated derivatives.     

Purification and biochemical characterization of angiotensin I-converting enzyme (ACE) from ostrich lung: The effect of 2,2,2-trifluoroethanol on ACE conformation and activity

This work reports the purification and biochemical characterization of angiotensin I-converting enzyme (ACE) from ostrich (Struthio camelus) lung. The molecular weight of the purified enzyme was approximately evaluated to be 200 kDa and the maximum enzyme activity was observed at pH 7.5. The enzyme activity was increased by detergents of Triton X-100 (0.01%), cetyltrimethylammonium bromide (CTAB) (0.1 and 1 mM) and sodium dodecyl sulfate (SDS) (0.1 mM), while decreased by Triton X-100 (1% and 10%) and SDS (1 mM and 10 mM). The secondary and tertiary structure and activity of ACE in the absence and presence of trifluoroethanol (TFE) were investigated using circular dichroism, fluorescence quenching and UV–visible spectroscopy, respectively. Our results revealed that TFE stabilizes ACE at low concentrations, while acts as a denaturant at higher concentration (20%). The K_m, K_cat and K_cat/K_m values of ostrich ACE towards FAPGG were 0.8 × 10^?4 M, 59,240 min^?1 and 74 × 10⁷ min^?1 M^?1, respectively. The values of IC₅₀ and K_i for captopril were determined to be 36.5 nM and 16.6 nM, respectively. In conclusion, ostrich lung ACE is a new enzyme which could be employed as a candidate for studying ACE structure and its natural or synthetic inhibitors.