<Emphasis Type="Italic">CrMAPK3</Emphasis> regulates the expression of iron-deficiency-responsive genes in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

Under iron-deficient conditions, Chlamydomonas exhibits high affinity for iron absorption. Nevertheless, the response, transmission, and regulation of downstream gene expression in algae cells have not to be investigated. Considering that the MAPK pathway is essential for abiotic stress responses, we determined whether this pathway is involved in iron deficiency signal transduction in Chlamydomonas.

Results

Arabidopsis MAPK gene sequences were used as entry data to search for homologous genes in Chlamydomonas reinhardtii genome database to investigate the functions of mitogen-activated protein kinase (MAPK) gene family in C. reinhardtii under iron-free conditions. Results revealed 16 C. reinhardtii MAPK genes labeled CrMAPK2–CrMAPK17 with TXY conserved domains and low homology to MAPK in yeast, Arabidopsis, and humans. The expression levels of these genes were then analyzed through qRT-PCR and exposure to high salt (150 mM NaCl), low nitrogen, or iron-free conditions. The expression levels of these genes were also subjected to adverse stress conditions. The mRNA levels of CrMAPK2, CrMAPK3, CrMAPK4, CrMAPK5, CrMAPK6, CrMAPK8, CrMAPK9, and CrMAPK11 were remarkably upregulated under iron-deficient stress. The increase in CrMAPK3 expression was 43-fold greater than that in the control. An RNA interference vector was constructed and transformed into C. reinhardtii 2A38, an algal strain with an exogenous FOX1:ARS chimeric gene, to silence CrMAPK3. After this gene was silenced, the mRNA levels and ARS activities of FOX1:ARS chimeric gene and endogenous CrFOX1 were decreased. The mRNA levels of iron-responsive genes, such as CrNRAMP2, CrATX1, CrFTR1, and CrFEA1, were also remarkably reduced.

Conclusion

CrMAPK3 regulates the expression of iron-deficiency-responsive genes in C. reinhardtii.

     

Synthesis,SAR and in vitro therapeutic potentials of thiazolidine-2,4-diones

Background

Thiazolidinedione is a pentacyclic moiety having five membered unsaturated ring system composed with carbon, oxygen, nitrogen and sulfur molecules at 1 and 3 position of the thiazole ring and widely found throughout nature in various form. They favourably alter concentration of the hormones secreted by adipocytes, particularly adiponectin. They also increase total body fat and have mixed effects on circulating lipids. Thiazolidinedione nucleus is present in numerous biological moieties and has different pharmacological activities likes, e.g. antimalarial, antimicrobial, antimycobacterial, anticonvulsant, antiviral, anticancer, anti-inflammatory, antioxidant, anti-HIV (human immunodeficiency virus) and antituberculosis.

Results and discussion

The synthesized compounds were screened for their in vitro antimicrobial potential against Gram (positive and negative) bacterial and fungal strains by tube dilution technique. In this series, compound 10 exhibited significant antimicrobial activity against B. subtilis and S. aureus with MIC?=?4.2?×?10^?2 µM/ml, compound 15 showed significant activity against K. pneumonia with MIC?=?2.60?×?10^?2 µM/ml and compound 4 displayed potent antibacterial activity against E. coli with MIC?=?4.5?×?10^?2 µM/ml. Compound 10 had most potent antifungal activity against C. albicans and A. niger with MIC?=?4.2?×?10^?2 µM/ml. Compounds 12 and 15 were found as most active antidiabetic agents having IC₅₀?=?27.63 μg/ml and 22.35 μg/ml, respectively, using DPPH assay. Antioxidant activity results indicated that compounds 3 and 9 displayed good antioxidant agent with IC₅₀?=?29.04 μg/ml and 27.66 μg/ml respectively, using α amylase assay.

Conclusion

All the synthesized derivatives exhibited good antimicrobial, antidiabetic and antioxidant activities using specific methods then compared with mentioned standard drugs. Especially, compounds 3, 4, 9, 10, 12 and 15 displayed highest activity. Structure activity relationship demonstrated that presence of electron withdrawing group (o-NO₂, p-Cl, p-Br) enhanced the antibacterial activity against E. coli as well as increased the antioxidant activity while the presence of electron releasing group (o/p-OCH₃, 3,4,5-trimethoxy) enhanced the antibacterial activity against S. aureus, B. subtilis, S. typhi, K. pneumonia, C. albicans and A. niger as well as the antidiabetic activity.

     

Riboflavin synthase of <Emphasis Type="Italic">Schizosaccharomyces pombe.</Emphasis> Protein dynamics revealed by <Superscript>19</Superscript>F NMR protein perturbation experiments

Background

Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.

Results

A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg^-1 min^-1 with an apparent K_M of 5.7 microM. ¹⁹F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. ¹⁹F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.

Conclusion

Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The ¹⁹F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.

     

Linalool isomerase,a membrane-anchored enzyme in the anaerobic monoterpene degradation in <Emphasis Type="Italic">Thauera linaloolentis</Emphasis> 47Lol

Background

Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ¹-Δ²-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool.

Results

The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42?±?0.28 nkat mg * protein^?1 and a k_M value of 455?±?124 μM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C.

Conclusion

The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.

     

Structural insight into the inactivation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> non-classical transpeptidase Ldt<Subscript>Mt2</Subscript> by biapenem and tebipenem

Background

The carbapenem subclass of β-lactams is among the most potent antibiotics available today. Emerging evidence shows that, unlike other subclasses of β-lactams, carbapenems bind to and inhibit non-classical transpeptidases (L,D-transpeptidases) that generate 3 → 3 linkages in bacterial peptidoglycan. The carbapenems biapenem and tebipenem exhibit therapeutically valuable potencies against Mycobacterium tuberculosis (Mtb).

Results

Here, we report the X-ray crystal structures of Mtb L,D-transpeptidase-2 (Ldt_Mt2) complexed with biapenem or tebipenem. Despite significant variations in carbapenem sulfur side chains, biapenem and tebipenem ultimately form an identical adduct that docks to the outer cavity of Ldt_Mt2. We propose that this common adduct is an enzyme catalyzed decomposition of the carbapenem adduct by a mechanism similar to S-conjugate elimination by β-lyases.

Conclusion

The results presented here demonstrate biapenem and tebipenem bind to the outer cavity of Ldt_Mt2, covalently inactivate the enzyme, and subsequently degrade via an S-conjugate elimination mechanism. We discuss structure based drug design based on the findings and propose that the S-conjugate elimination can be leveraged to design novel agents to deliver and locally release antimicrobial factors to act synergistically with the carbapenem carrier.

     

Ca<Superscript>2+</Superscript> binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein

Background

The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca²⁺ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca²⁺ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca²⁺ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca²⁺ cage within CR-domains from this family of receptors that function in endocytosis and signalling.

Results

We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca²⁺ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca²⁺, and show that CR56 binds 2 Ca²⁺ ions with an average affinity of K_D = 10.6 microM, and there is no indication of additional Ca²⁺ binding sites within this receptor fragment.

Conclusions

Both CR-domains of CR56 bind a single Ca²⁺ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.

     

Characterization of <Emphasis Type="Italic">Pf</Emphasis>TrxR inhibitors using antimalarial assays and <Emphasis Type="Italic">in silico</Emphasis>techniques

Background

The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies.

Results

The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC₅₀ values of?<?20 μM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme.

Conclusions

In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.

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Low solubility of unconjugated bilirubin in dimethylsulfoxide – water systems: implications for pK<Subscript><Emphasis Type="Italic">a</Emphasis></Subscript> determinations

Background

Aqueous pK _a values of unconjugated bilirubin are important determinants of its solubility and transport. Published pK _a data on an analog, mesobilirubin-XIIIα, studied by ¹³C-NMR in buffered solutions containing 27 and 64 vol% (C²H₃)₂SO because of low aqueous solubility of mesobilirubin, were extrapolated to obtain pK _a values in water of 4.2 and 4.9. Previous chloroform-water partition data on bilirubin diacid led to higher estimates of its pK _a , 8.12 and 8.44, and its aqueous solubility. A thermodynamic analysis, using this solubility and a published solubility in DMSO, suggested that the systems used to measure ¹³C-NMR shifts were highly supersaturated. This expectation was assessed by measuring the residual concentrations of bilirubin in the supernatants of comparable DMSO-buffer systems, after mild centrifugation to remove microprecipitates.

Results

Extensive sedimentation was observed from numerous systems, many of which appeared optically clear. The very low supernatant concentrations at the lowest pH values (4.1-5.9) were compatible with the above thermodynamic analysis. Extensive sedimentation and low supernatant concentrations occurred also at pH as high as 7.2.

Conclusions

The present study strongly supports the validity of the aqueous solubility of bilirubin diacid derived from partition data, and, therefore, the corresponding high pK _a values. Many of the mesobilirubin systems in the ¹³C-NMR studies were probably supersaturated, contained microsuspensions, and were not true solutions. This, and previously documented errors in pH determinations that caused serious errors in pK _a values of the many soluble reference acids and mesobilirubin, raise doubts regarding the low pK _a estimates for mesobilirubin from the ¹³C-NMR studies.

     

Synthesis,characterization, biological evaluation and molecular docking studies of 2-(1H-benzo[d]imidazol-2-ylthio)-<Emphasis Type="Italic">N</Emphasis>-(substituted 4-oxothiazolidin-3-yl) acetamides

Background

A series of 2-(1H-benzo[d]imidazol-2-ylthio)-N-(substituted 4-oxothiazolidin-3-yl) acetamides was synthesized and characterized by physicochemical and spectral means. The synthesized compounds were evaluated for their in vitro antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Candida albicans and Aspergillus niger by tube dilution method. The in vitro cytotoxicity study of the compounds was carried out against human colorectal (HCT116) cell line. The most promising anticancer derivatives (5l, 5k, 5i and 5p) were further docked to study their binding efficacy to the active site of the cyclin-dependent kinase-8.

Results

All the compounds possessed significant antimicrobial activity with MIC in the range of 0.007 and 0.061 µM/ml. The cytotoxicity study revealed that almost all the derivatives were potent in inhibiting the growth of HCT116 cell line in comparison to the standard drug 5-fluorouracil. Compounds 5l and 5k (IC₅₀ = 0.00005 and 0.00012 µM/ml, respectively) were highly cytotoxic towards HCT116 cell line in comparison to 5-fluorouracil (IC₅₀ = 0.00615 µM/ml) taken as standard drug.

Conclusion

The molecular docking studies of potent anticancer compounds 5l, 5k, 5i and 5p showed their putative binding mode and significant interactions with cyclin-dependent kinase-8 as prospective agents for treating colon cancer.

     

Soluble expression of recombinant midgut zymogen (native propeptide) proteases from the <Emphasis Type="Italic">Aedes aegypti</Emphasis> Mosquito Utilizing <Emphasis Type="Italic">E. coli</Emphasis> as a host

Background

Studying proteins and enzymes involved in important biological processes in the Aedes aegypti mosquito is limited by the quantity that can be directly isolated from the mosquito. Adding to this difficulty, digestive enzymes (midgut proteases) involved in metabolizing blood meal proteins require a more oxidizing environment to allow proper folding of disulfide bonds. Therefore, recombinant techniques to express foreign proteins in Escherichia coli prove to be effective in producing milligram quantities of the expressed product. However, with the most commonly used strains having a reducing cytoplasm, soluble expression of recombinant proteases is hampered. Fortunately, new E. coli strains with a more oxidizing cytoplasm are now available to ensure proper folding of disulfide bonds.

Results

Utilizing an E. coli strain with a more oxidizing cytoplasm (SHuffle® T7, New England Biolabs) and changes in bacterial growth temperature has resulted in the soluble expression of the four most abundantly expressed Ae. aegypti midgut proteases (AaET, AaSPVI, AaSPVII, and AaLT). A previous attempt of solubly expressing the full-length zymogen forms of these proteases with the leader (signal) sequence and a modified pseudo propeptide with a heterologous enterokinase cleavage site led to insoluble recombinant protein expression. In combination with the more oxidizing cytoplasm, and changes in growth temperature, helped improve the solubility of the zymogen (no leader) native propeptide proteases in E. coli. Furthermore, the approach led to autocatalytic activation of the proteases during bacterial expression and observable BApNA activity. Different time-points after bacterial growth induction were tested to determine the time at which the inactive (zymogen) species is observed to transition to the active form. This helped with the purification and isolation of only the inactive zymogen forms using Nickel affinity.

Conclusions

The difficulty in solubly expressing recombinant proteases in E. coli is caused by the native reducing cytoplasm. However, with bacterial strains with a more oxidizing cytoplasm, recombinant soluble expression can be achieved, but only in concert with changes in bacterial growth temperature. The method described herein should provide a facile starting point to recombinantly expressing Ae. aegypti mosquito proteases or proteins dependent on disulfide bonds utilizing E. coli as a host.

     

1-Aryl-3-(1<Emphasis Type="Italic">H</Emphasis>-imidazol-1-yl)propan-1-ol esters: synthesis,anti-<Emphasis Type="Italic">Candida</Emphasis>potential and molecular modeling studies

Background

An increased incidence of fungal infections, both invasive and superficial, has been witnessed over the last two decades. Candida species seem to be the main etiology of nosocomial fungal infections worldwide with Candida albicans, which is commensal in healthy individuals, accounting for the majority of invasive Candida infections with about 30-40% of mortality.

Results

New aromatic and heterocyclic esters 5a-k of 1-aryl-3-(1H-imidazol-1-yl)propan-1-ols 4a-d were successfully synthesized and evaluated for their anti-Candida potential. Compound 5a emerged as the most active congener among the newly synthesized compounds 5a-k with MIC value of 0.0833 μmol/mL as compared with fluconazole (MIC value >1.6325 μmol/mL). Additionally, molecular modeling studies were conducted on a set of anti-Candida albicans compounds.

Conclusion

The newly synthesized esters 5a-k showed more potent anti-Candida activities than fluconazole. Compounds 7 and 8 revealed significant anti-Candida albicans activity and were able to effectively satisfy the proposed pharmacophore geometry, using the energy accessible conformers (E_conf?<?20 kcal/mol).

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The DNA relaxation activity and covalent complex accumulation of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> topoisomerase I can be assayed in <Emphasis Type="Italic">Escherichia coli</Emphasis>: application for identification of potential FRET-dye labeling sites

Background

Mycobacterium tuberculosis topoisomerase I (MtTOP1) and Escherichia coli topoisomerase I have highly homologous transesterification domains, but the two enzymes have distinctly different C-terminal domains. To investigate the structure-function of MtTOP1 and to target its activity for development of new TB therapy, it is desirable to have a rapid genetic assay for its catalytic activity, and potential bactericidal consequence from accumulation of its covalent complex.

Results

We show that plasmid-encoded recombinant MtTOP1 can complement the temperature sensitive topA function of E. coli strain AS17. Moreover, expression of MtTOP1-G116 S enzyme with the TOPRIM mutation that inhibits DNA religation results in SOS induction and loss of viability in E. coli. The absence of cysteine residues in the MtTOP1 enzyme makes it an attractive system for introduction of potentially informative chemical or spectroscopic probes at specific positions via cysteine mutagenesis. Such probes could be useful for development of high throughput screening (HTS) assays. We employed the AS17 complementation system to screen for sites in MtTOP1 that can tolerate cysteine substitution without loss of complementation function. These cysteine substitution mutants were confirmed to have retained the relaxation activity. One such mutant of MtTOP1 was utilized for fluorescence probe incorporation and fluorescence resonance energy transfer measurement with fluorophore-labeled oligonucleotide substrate.

Conclusions

The DNA relaxation and cleavage complex accumulation of M. tuberculosis topoisomerase I can be measured with genetic assays in E. coli, facilitating rapid analysis of its activities, and discovery of new TB therapy targeting this essential enzyme.

     

Synthesis and structure of bis(1,2-diaminoethane) copper(II) bis(<Emphasis Type="Italic">o</Emphasis>-hydroxybenzoate) monohydrate and <Emphasis Type="Italic">trans</Emphasis>-diaquabis(1,2-diaminoethane)copper(II) bis(<Emphasis Type="Italic">o</Emphasis>-aminobenzoate)

X-ray structural analysis has been performed for two complex compounds: Cu(en)₂(o-HB)₂H₂O (I) (a = 16.873(4) Å, b = 8.713(2) Å, c = 14.803(3) Å, β = 91.15(2)°, V = 2175.8(8) ų, C2/c, Z = 4, R(F) = 0.0263, 1516 reflections with I > 3σ (I)) and [Cu(en)₂(OH₂)₂]²⁺(o-AB^?)₂ (II) (a = 7.488(5) Å, b = 22.122(8) Å, c = 7.856(5) Å, β = 118.77(2)°, V = 1140.7(11) ų, P2₁/n, Z = 2, R(F) = 0.0432, 1684 reflections with I > 3σ(I)) synthesized under identical conditions (en is ethylenediamine, o-HB is o-hydroxybenzoate, and o-AB is o-aminobenzoate). Although the compounds were assumed to have similar structures and the Cu-Lig bond lengths and the cis and trans angles are acceptable for an octahedral structure, the geometric parameters of o-HB suggest that the copper atom has a plane square environment.     

Study of variation in thermophysical properties of RE<Subscript>6</Subscript>UO<Subscript>12</Subscript>(s) along the lanthanide series

The novel ligand N,N,N′′′′,N′′′′-tetrabutyl-N′′′,N′′′-(N″,N″-diethyl)-ethidene bisdiglycolamide (TBEE-BisDGA) and other eight analogous extractants have been synthesized and characterized by NMR and HRMS. The solvent extraction of Th⁴⁺, UO₂ ²⁺ and Eu³⁺ from nitric acid solution using the above BisDGA extractants was investigated in 1-dodecanol at 30 ± 1 °C. The extractants exhibited higher affinity toward Th⁴⁺ than UO₂ ²⁺ and Eu³⁺ in the present system. The maximum value of separation factor SF _Th(IV)/U(VI) and SF _{Th(IV)/Eu(III)} is 78.5 and 53.3 respectively for TBEE-BisDGA, 88.1 and 69.5 respectively in the case of TB ^i-PE-BisDGA at 3 M HNO₃ solution.     

The <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background

The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.

Results

In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (Mt CS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant Mt CS. The bifunctionality of Mt CS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN_ox and Mt CS. FMN_ox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.

Conclusion

This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C₄-proS hydrogen is being transferred during the reduction of FMN_ox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMN_ox. The results on enzyme kinetics described here provide evidence for the mode of action of Mt CS and should thus pave the way for the rational design of antitubercular agents.

     

Synthesis,structure, and antibacterial properties of ternary rare-earth complexes with <Emphasis Type="Italic">o</Emphasis>-methylbenzoic Acid and 1,10-phenanthroline

Ternary rare-earth complexes with o-methylbenzoic acid (o-MBA) and 1,10-phenanthroline (Phen) Ln₂(o-MBA)₆(Phen)₂ · nH₂O(n = 0, 1) (Ln = La, Pr, Y, Yb) were synthesized and characterized by elemental analysis, IR, X-ray diffraction, and TG-DTG means. The complex La₂(o-MBA)₆(Phen)₂ · H₂O (I) is composed of two species of binuclear molecules in which the La³⁺ ion is coordinated with two nitrogen atoms of Phen and seven oxygen atoms of carboxylate groups. The carboxylate groups were bonded to La³⁺ in three modes: chelating-bidentate, bridging-bidentate, and chelating-bridging tridentate. The La³⁺ ion adopted a vigorous distorted monocapped square antiprism geometry. Complex I belongs to the triclinic crystal system, P space group, lattice parameters: a = 13.058(3), b = 12.7584(11), c = 20.773(4) Å, α = 101.18(3)°, β = 93.88(3)°, γ = 115.82(3)°, V = 3283.0(11)ų, Z = 2, ρ_calcd = 1.484 mg/m³, M _r = 1467.06, F(000) = 1476, μ = 1.350 mm^-1. The structure was refined to R _l = 0.0631 and wR ₂ = 0.1504. The antibacterial activity test indicates that these complexes exhibit better antibacterial ability against Escherichia coli and Staphylococcus aureus than the corresponding rare-earth chloride or o-MBA.     

Increased enzymatic hydrolysis of sugarcane bagasse by a novel glucose- and xylose-stimulated β-glucosidase from <Emphasis Type="Italic">Anoxybacillus flavithermus</Emphasis> subsp. <Emphasis Type="Italic">yunnanensis</Emphasis> E13<Superscript>T</Superscript>

Background

β-Glucosidase is claimed as a key enzyme in cellulose hydrolysis. The cellulosic fibers are usually entrapped with hemicelluloses containing xylose. So there is ongoing interest in searching for glucose- and xylose-stimulated β-glucosidases to increase the efficiency of hydrolysis of cellulosic biomass.

Results

A thermostable β-glucosidase gene (Bglp) was cloned from Anoxybacillus flavithermus subsp. yunnanensis E13^T and characterized. Optimal enzyme activity was observed at 60 °C and pH 7.0. Bglp was relatively stable at 60 °C with a 10-h half-life. The kinetic parameters V _max and K _m for p-nitrophenyl-β-D-glucopyranoside (pNPG) were 771?±?39 μmol/min/mg and 0.29?±?0.01 mM, respectively. The activity of Bglp is dramatically stimulated by glucose or xylose at concentrations up to 1.4 M. After Bglp was added to Celluclast® 1.5 L, the conversion of sugarcane bagasse was 48.4?±?0.8%, which was much higher than of Celluclast® 1.5 L alone. Furthermore, Bglp showed obvious advantages in the hydrolysis when initial concentrations of glucose and xylose are high.

Conclusions

The supplementation of BglP significantly enhanced the glucose yield from sugarcane bagasse, especially in the presence of high concentrations of glucose or xylose. Bglp should be a promising candidate for industrial applications.

     

Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

Background

The regulation of lipid biosynthesis is essential in photosynthetic eukaryotic cells. This regulation occurs during the direct synthesis of fatty acids and triacylglycerols (TAGs), as well as during other controlling processes in the main carbon metabolic pathway.

Results

In this study, the mRNA levels of Chlamydomonas citrate synthase (CrCIS) were found to decrease under nitrogen-limited conditions, which suggests suppressed gene expression. Gene silencing by RNA interference (RNAi) was conducted to determine whether CrCIS suppression affected the carbon flux in TAG biosynthesis. Results showed that the TAG level increased by 169.5%, whereas the CrCIS activities in the corresponding transgenic algae decreased by 16.7% to 37.7%. Moreover, the decrease in CrCIS expression led to the increased expression of TAG biosynthesis-related genes, such as acyl-CoA:diacylglycerol acyltransferase and phosphatidate phosphatase. Conversely, overexpression of CrCIS gene decreased the TAG level by 45% but increased CrCIS activity by 209% to 266% in transgenic algae.

Conclusions

The regulation of CrCIS gene can indirectly control the lipid content of algal cells. Our findings propose that increasing oil by suppressing CrCIS expression in microalgae is feasible.

     

Preparation,crystal structure and mechanism of thermal decomposition of complex [Dy(<Emphasis Type="Italic">p</Emphasis>-MOBA)<Subscript>3</Subscript>Phen]<Subscript>2</Subscript>

1,10-Phenanthrolinetris(4-methoxybenzoate)dysprosium, Dy(p-MOBA)₃Phen (where p-MOBA = p-methoxybenzoate and Phen = 1,10-phenanthroline), (I) has been synthesized. The complex was characterized by various techniques including elemental analysis, UV, IR, XRD, molar conductance, and TG-DTG. The crystals consist of binuclear molecules and monoclinic, space group P2 ₁/n: a = 14.143(6) Å, b = 17.550(7) Å, c = 14.493(6) Å, β = 117.357(4)°, Z = 2, ρ _c = 1.655 g cm^?3, F(000) = 1588; R ₁ = 0.0176, wR ₂ = 0.0455. In the complex, each Dy³⁺ ion is nine-coordinate to one 1,10-phenanthroline molecule, one bidentate chelating carboxylate group, and four bridging carboxylate groups in which the carboxylate groups are bonded to the Dy³⁺ ions in three modes: bridging bidentate, bridging tridentate, and chelating bidentate. The thermal decomposition mechanism of I has been determined on the basis of thermal analysis. In addition, the lifetime equation at a weight-loss of 10% was deduced as lnτ = ?28.8361 + 19478.37/T by isothermal thermogravimetric analysis.     

Crystal structure of EuLnAgS<Subscript>3</Subscript> (Ln = Gd and Ho) compounds

The crystal structures of the first prepared EuLnAgS₃ (Ln = Gd and Ho) compounds, which have two polymorphs, were determined by X-ray powder diffraction. α-EuLnAgS₃ phases are isostructural to BaErAgS₃ (monoclinic crystal system, space group C2/m): a = 17.3168(10) Å, b = 3.9683(2) Å, c = 8.3174(4) Å, β = 103.94° (EuGdCuS₃); a = 17.1729(12) Å, b = 3.9367(3) Å, c = 8.2905(6) Å, β = 103.9° (EuHoCuS₃). β-EuLnAgS₃ phases belong to the AgBiS₂ structure type (cubic crystal system, space group Fm-3m): a = 5.739(2) Å (EuGdCuS₃) and a = 5.678 Å (EuHoCuS₃). In the α-EuLnAgS₃ crystal structure, LnS₆ octahedra and AgS₅ trigonal bipyramids share edges to form a three-dimensional (3D) structure with channels accommodating Eu²⁺ ions. A decrease in Ln³⁺ ionic radius gives rise to the crystal-chemical contraction of the 3D structure.