Structural requirements for the induction and inhibition of 2,3-dihydroxybenzoic acid decarboxylase ofAspergillus niger

2,3-Dihydroxybenzoic acid decarboxylase inAspergillus niger was induced by many substrate analogs including salicylate and gentisate. Catechol, which is the product, induced the enzyme tenfold. The purified enzyme was competitively inhibited by manyortho substituted benzoic acids. The Ki values for salicylate,o-fluoro ando-chloro benzoic acids were 0.12 mM, 0.12 mM, and 0.13 mM respectively; these values were lower than the Km value for the substrate. As the size of the group in theortho position increased, as in the case of bromo- and iodo-derivatives, there was an increase in their Ki values. The C-2 hydroxyl group was essential both for the induction and for interaction with the enzyme. The C-3 hydroxyl group was not necessary for induction or inhibition, but it might be essential for the catalysis.

Molecular structure of Lantadene-B&C, triterpenoids ofantana camara, red variety: Lantadene-B, 22β-angeloyloxy-3-oxoolean-12-en-28-oic acid; Lantadene-C, 22 β-(S)-2′-methyl butanoyloxy-3-oxoolean-12-en-28-oic acid

(I)Lantadene-B: C35H52O5,M r =552.80, MonoclinicC2,a=25.65(1),b=6.819(9),c=18.75(1) Å,beta=100.61(9),V=3223(5) Å3,Z=4,D x =1.14 g cm–3 CuKagr (lambda=1.5418A),mgr=5.5 cm–1,F(000)=1208,R=0.118,wR=0.132 for 1527 observed reflections withF o ge2sgr(F o ). (II)Lantadene-C: C35H54O5·CH3OH,Mr=586.85, Monoclinic,P21,a=9.822(3),b=10.909(3),c=16.120(8)Å,beta=99.82(4),V=1702(1)Å3,Z=2,D x =1.145 g cm–3, MoKagr (lambda=0.7107Å), mgr=0.708 cm–1 F(000)=644,R=0.098, wR=0.094 for 1073 observed reflections. The rings A, B, C, D, and E aretrans, trans, trans, cis fused and are in chair, chair, sofa, half-chair, chair conformations, respectively, in both the structures. In the unit cell the molecules are stabilized by O-HctdotO hydrogen bonds in both the structures, however an additional C-HctdotO interaction is observed in the case of Lantadene-C.

Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

High-Throughput Study of the Cu(CH3COO)(2)center dot H2O-5-Nitroisophthalic Acid Heterocyclic Ligand System: Synthesis, Structure, Magnetic, and Heterogeneous Catalytic Studies of Three Copper Nitroisophthalates

A high-throughput screening was employed to identify new compounds in Cu(CH3COO)(2)center dot H2O-NIPA-heterocyclic ligand systems. Of the compounds identified, three compounds, Cu-3{(NO2)-C6H3-(COO)(2)}(3)(C3N6H6)] (1), Cu-2(mu(3)-OH)(H2O){(NO2)-C6H3-(COO)(2)}(CN4H)]center dot-(H2O) (II), and Cu-2(mu(3)-OH)(H2O){(NO2)-C6H3-(COO)(2}-)(CN5H2)]center dot 2(H2O) (III), have been isolated as good quality single crystals by employing conventional hydrothermal methods. Three other compounds, Cu-2{(NO2)-C6H3-(COO)(2)}-(CN4H)(H2O) (IIa), Cu-2{(NO2)-C6H3-(COO)(2)}(CN5H2) (IIIa), and Cu-2{(NO2)-C6H3-(COO)(2)}{(CN5H2)(2)}2H(2)O (IIIb), were identified by a combination of elemental analysis, thermogravimetric analysis (TGA), and IR spectroscopic studies, although their structures are yet to be determined. The single crystalline compounds were also characterized by elemental analysis, TGA, IR, UV vis, magnetic, and catalytic studies. The structures of the compounds have paddle wheel (I) and infinite Cu 0(H) Cu chains (II and HI) connected with NLPA and heterocyclic ligands forming two-(II) and three-dimensional (I and III) structures. The bound and lattice water molecules in 11 and 111 could be reversibly removed/inserted without affecting the structure. In the case of II, the removal of water gives rise to a structural transition, but the dehydrated phase reverts back to the original phase on prolonged exposure to atmospheric conditions. Magnetic studies indicate an overall antiferromagnetism in all of the compounds. Lewis acid catalytic studies indicate that compounds II and HI are active for cyanosilylation of imines.

Identification of the active-site peptide of 2,3-dihydroxybenzoic acid decarboxylase from Aspergillus oryzae

The non-oxidative decarboxylation of aromatic acids is a poorly understood reaction. The transformation of 2,3-dihydroxybenzoic acid to catechol in the fungal metabolism of indole is a prototype of such a reaction. 2,3-Dihydroxybenzoic acid decarboxylase (EC 4.1.1.46) which catalyzes this reaction was purified to homogeneity from anthranilate induced cultures of Aspergillus oryzae using affinity chromatography. The enzyme did not require cofactors like NAD(+), PLP, TPP or metal ions for its activity. There was no spectral evidence for the presence of enzyme bound cofactors. The preparation, which was adjudged homogeneous by the criteria of SDS-PAGE, sedimentation analysis and N-terminal analysis, was characterized for its physicochemical and kinetic parameters. The enzyme was inactivated by group-specific modifiers like diethyl pyrocarbonate (DEPC) and N-ethylmaleimide (NEM). The kinetics of inactivation by DEPC suggested the presence of a single class of essential histidine residues, the second order rate constant of inactivation for which was 12.5 M(-1) min(-1). A single class of cysteine residues was modified by NEM with a second order rate constant of 33 M(-1) min(-1). Substrate analogues protected the enzyme against inactivation by both DEPC and NEM, suggesting the Location of the essential histidine and cysteine to be at the active site of the enzyme. The incorporation of radiolabelled NEM in a differential labelling experiment was 0.73 mol per mol subunit confirming the presence of a single essential cysteine per active-site. Differentially labelled enzyme was enzymatically cleaved and the peptide bearing the label was purified and sequenced. The active-site peptide LLGLAETCK and the N-terminal sequence MLGKIALEEAFALPRFEEKT did not bear any similarity to sequences reported in the Swiss-Prot Protein Sequence Databank, a reflection probably of the unique primary structure of this novel enzyme. The sequences reported in this study will appear in the Swiss-Prot Protein Sequence Databank under the accession number P80402.

Crystal structure of nonadentate tricompartmental ligand derived from pyridine-2,6-dicarboxylic acid: Spectroscopic, electrochemical and thermal investigations of its transition metal(II) complexes

The coordinating behavior of a new dihydrazone ligand, 2,6-bis(3-methoxysalicylidene) hydrazinocarbonyl]pyridine towards manganese(II), cobalt(II), nickel(II), copper(II), zinc(II) and cadmium(II) has been described. The metal complexes were characterized by magnetic moments, conductivity measurements, spectral (IR, NMR, UV-Vis, FAB-Mass and EPR) and thermal studies. The ligand crystallizes in triclinic system, space group P-1, with alpha=98.491(10)degrees, beta=110.820(10)degrees and gamma=92.228(10)degrees. The cell dimensions are a=10.196(7)angstrom, b=10.814(7)angstrom, c=10.017(7)angstrom, Z=2 and V=1117.4(12). IR spectral studies reveal the nonadentate behavior of the ligand. All the complexes are neutral in nature and possess six-coordinate geometry around each metal center. The X-band EPR spectra of copper(II) complex at both room temperature and liquid nitrogen temperature showed unresolved broad signals with g(iso) = 2.106. Cyclic voltametric studies of copper(II) complex at different scan rates reveal that all the reaction occurring are irreversible. (C) 2011 Elsevier B.V. All rights reserved.

Synthesis and supramolecular assembly of the bifunctional borinic acid 1,2-fcB(OH)](2)

Treatment of the chloro-substituted diboradiferrocene derivative 1 with Me3SiOMe and subsequent hydrolysis resulted in formation of the novel organometallic bis(borinic acid) derivative 3. The assembly of 3 into supramolecular structures via hydrogen bonding and reversible covalent boron-oxygen bond formation was explored. Upon crystallization from acetone or THF one-dimensional chains form in which molecules of 3 alternately serve as hydrogen bond donors and acceptors. The additional OH hydrogens that are not involved in hydrogen bonding within the polymeric chains undergo hydrogen bonding to the solvent molecules. Removal of the solvent was achieved at moderate temperature under high vacuum. While the polymeric chains remain intact, in the absence of the solvent as a hydrogen bond acceptor, short contacts to the Cp rings of neighboring polymer strands lead to a network-like structure. At higher temperatures, further dehydration occurs with formation of B-O-B linkages as confirmed by MALDI-TOF mass spectrometry. Oligomers with up to 15 repeating units (30 ferrocenes) were detected.

The Structural Characterization of Folded Peptides Containing the Conformationally Constrained beta-Amino Acid Residue beta(2,2)Ac(6)c

Backbone alkylation has been shown to result in a dramatic reduction in the conformational space that is sterically accessible to a-amino acid residues in peptides. By extension, the presence of geminal dialkyl substituents at backbone atoms also restricts available conformational space for beta and ? residues. Five peptides containing the achiral beta 2,2-disubstituted beta-amino acid residue, 1-(aminomethyl)cyclohexanecarboxylic acid (beta 2,2Ac6c), have been structurally characterized in crystals by X-ray diffraction. The tripeptide Boc-Aib-beta 2,2Ac6c-Aib-OMe (1) adopts a novel fold stabilized by two intramolecular H-bonds (C11 and C9) of opposite directionality. The tetrapeptide Boc-Aib-beta 2,2Ac6c]2-OMe (2) and pentapeptide Boc-Aib-beta 2,2Ac6c]2-Aib-OMe (3) form short stretches of a hybrid a beta C11 helix stabilized by two and three intramolecular H-bonds, respectively. The structure of the dipeptide Boc-Aib-beta 2,2Ac6c-OMe (5) does not reveal any intramolecular H-bond. The aggregation pattern in the crystal provides an example of an extended conformation of the beta 2,2Ac6c residue, forming a polar sheet like H-bond. The protected derivative Ac-beta 2,2Ac6c-NHMe (4) adopts a locally folded gauche conformation about the C beta?Ca bonds (?=-55.7 degrees). Of the seven examples of beta 2,2Ac6c residues reported here, six adopt gauche conformations, a feature which promotes local folding when incorporated into peptides. A comparison between the conformational properties of beta 2,2Ac6c and beta 3,3Ac6c residues, in peptides, is presented. Backbone torsional parameters of H-bonded a beta/beta a turns are derived from the structures presented in this study and earlier reports.

Co-evolution of specific amino acid in sigma 1.2 region and nucleotide base in the discriminator to act as sensors of small molecule effectors of transcription initiation in mycobacteria

The transcription from rrn and a number of other promoters is regulated by initiating ribonucleotides (iNTPs) and guanosine tetra/penta phosphate (p)ppGpp], either by strengthening or by weakening of the RNA polymerase (RNAP)-promoter interactions during initiation. Studies in Escherichia coli revealed the importance of a sequence termed discriminator, located between -10 and the transcription start site of the responsive promoters in this mode of regulation. Instability of the open complex at these promoters is attributed to the lack of stabilizing interactions between the suboptimal discriminator and the 1.2 region of sigma 70 (Sig70) in RNAP holoenzyme. We demonstrate a different pattern of interaction between the promoters and sigma A (SigA) of Mycobacterium tuberculosis to execute similar regulation. Instead of cytosine and methionine, thymine at three nucleotides downstream to -10 element and leucine 232 in SigA are found to be essential for iNTPs and pppGpp mediated response at the rrn and gyr promoters of the organism. The specificity of the interaction is substantiated by mutational replacements, either in the discriminator or in SigA, which abolish the nucleotide mediated regulation in vitro or in vivo. Specific yet distinct bases and the amino acids appear to have co-evolved' to retain the discriminator-sigma 1.2 region regulatory switch operated by iNTPs/pppGpp during the transcription initiation in different bacteria.

Stepwise Crystallization: Illustrative Examples of the Use of Metalloligands Cu-6(mna)(6)](6-) and (Ag-6(Hmna)(2)(mna)(4)](4-) (H(2)mna=2-Mercapto Nicotinic Acid) in the Formation of Heterometallic Two- and Three-Dimensional Assemblies with brucite, pcu, and sql Topologies

Three new inorganic coordination polymers, {Mn(H2O)(6)]-Mn-2(H2O)(6)](Cu-6(mna)(6)]center dot 6H(2)O}, 1, {Mn-4(OH)(2)(H2O)(10)] (Cu-6(mna)6]center dot 8H(2)O}, 2, and {Mn-2(H2O)(5)]Ag-6(Hmna)(2)(mna)(4)]center dot 20H(2)O}, 3, have been synthesized at room temperature through a sequential crystallization route. In addition, we have also prepared and characterized the molecular precursor Cu-6(Hmna)(6)]. Compounds 1 and 3 have a two-dimensional structure, whereas 2 has a three-dimensional structure. The formation of 2 has been achieved by minor modification in the synthetic composition, suggesting the subtle relationship between the reactant composition and the structure. The hexanudear copper and silver duster cores have Cu center dot center dot center dot Cu and Ag center dot center dot center dot Ag distances close to the sum of the van der Waals radii of Cu1+ and Ag1+, respectively. The connectivity between Cu-6(mna)(6)](6-) cluster units and Mn2+ ions gives rise to a brucite related layer in 1 and a pcu-net in 2. The Ag-6(Hmna)(2)(mna)(4)](4-) cluster in 3, on the other hand, forms a sql-net with Mn2+. Compound 1 exhibits an interesting and reversible hydrochromic behavior, changing from pale yellow to red, on heating at 70 degrees C or treatment under a vacuum. Electron paramagnetic resonance studies indicate no change in the valence states, suggesting the color change could be due to changes in the coordination environment only. The magnetic studies indicate weak antiferromagnetic behavior. Proton conductivity studies indicate moderate proton migrations in 1 and 3. The present study dearly establishes sequential crystallization as an important pathway for the synthesis of heterometallic coordination polymers.

Exploring the Crystal Structure Landscape with a Heterosynthon Module: Fluorobenzoic Acid:1,2-Bis(4-pyridyl)ethylene 2:1 Cocrystals

The crystal structure landscape of the 2:1 benzoic acid:dipyridylethylene cocrystal (BA:DPE-I) is explored experimentally with fluoro-substituted benzoic acids and extended with studies employing the Cambridge Structural Database (CSD). The interpretation of the cocrystal landscape is facilitated by considering the kinetically favored and robust acidpyridine heterosynthon as a modular unit. Information based on high-throughput crystallography shows that polymorphs and pseudopolymorphs may belong to the same landscape but arise from different crystallization pathways because of complex and different kinetic features, and secondary synthon preferences. Using the CSD as a guide, the coformer was changed from 1,2-bis(4-pyridyl)ethylene (DPE-I) to 1,2-bis(4-pyridyl)ethane (DPE-II) and this provides an extended interpretation of the BA:DPE-I cocrystal landscape, also highlighting the complexity of the kineticthermodynamic dichotomy during the molecule-to-crystal progression.