Substrate-mediated purification and characterization of a 3-hydroxybenzoic acid-6-hydroxylase from Micrococcus

3-Hydroxybenzoic acid-6-hydroxylase from Micrococcus sp. was purified to homogeneity in a single step using the substrate-mediated interaction of the enzyme with blue-Sepharose. The enzyme was bound to the affinity matrix in the presence of 3-hydroxybenzoic acid and was eluted in its absence. The molecular weight of the purified enzyme is 70,000 with no subunit structure. The flavoenzyme required the exogenous addition of FAD for its complete activity and had a strict preference for NADH over NADPH. The activity of the enzyme was drastically inhibited by Cu2+ and Hg2+ and the inhibition was reversed by thiol reagents.

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.

Demonstration of IgE Antibodies to Nucleic Acid Antigens in Patients with Sle

Using a combination of avidin-biotin microELISA and solid phase radioimmunoassay, we examined sera from 23 patients with systemic lupus erythematosus (SLE), two patients with established sensitivity to ingested shrimp, and 15 healthy normal subjects. In addition to IgG antibodies, varying amounts of IgE antibodies specific for native DNA (nDNA), denatured or single-stranded DNA (dnDNA), RNA, and tRNA were demonstrable in the sera of SLE patients, but not in the sera of normal subjects. A comparison of the specificity of nucleic acid-specific IgE antibodies present in the sera of shrimp-sensitive patients with those present in the sera of seven SLE patients revealed that the IgE antibodies in the sera of shrimp-sensitive patients specifically recognized shrimp tRNA but not yeast tRNA, calf thymus RNA, or calf thymus DNA, while those present in the sera of patients with SLE recognized all these nucleic acid antigens. The IgE antibodies directed against nDNA, dnDNA, RNA, and tRNA may mediate mast cell and basophil degranulation and thus contribute both to immediate-type hypersensitivity phenomena including hives seen in patients with SLE and to the localization of IgE-nucleic acid complexes in target

Acid-Catalyzed Thermal Rearrangement of Gamm,Delta-Unsaturated Ketones

Thermal activation of gamma,delta-unsaturated ketones (1, 9 and 12) in the presence of a catalytic amount of propionic acid causes a rearrangement to give new gamma,delta-unsaturated ketones (2, 10 and 14) via an intramolecular ene reaction followed by a retro-ene reaction.

Definitions and nomenclature of nucleic acid structure parameters

At an EMBO Workshop on DNA Curvature and Bending, held at Churchill College, Cambridge, on 10-15 September 1988, two sessions were scheduled on definitions of parameters used to describe the geometry of nucleic acid chains and helices, and a common nomenclature for these parameters. The most widely used library of helix analysis programs, HELIB (Fratini et al., 1982; Dickerson, 1985) suffers from the fact that the translations and rotations as defined are not fully independent and depend to a certain extent upon the choice of overall helix axis. Several research groups have been engaged independently in developing alternative programs for the geometrical analysis of polynucleotide chains, but with different definitions of quantities calculated and with widely different nomenclature even when the same parameter was involved.

Bile-Acids in Asymmetric-Synthesis.2. Cholic-Acid Derived Novel Chiral Auxiliaries For Asymmetric Diels-Alder Reactions

Cholic acid-based chiral acrylate 5 yields a Diels-Alder adduct with cyclopent

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.

Insights into conformational and packing features in a series of aryl substituted ethyl-6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate

The supramolecular structures of eight aryl protected ethyl-6-methyl-4-phenyl-2-oxo-1,2,3,4-tetrahydropyrimidine- 5-carboxylates have been analyzed to determine the role of different functional groups on the molecular geometry, conformational characteristics and the packing of these molecules in the crystal lattice. Out of these the para fluoro substituted compound on the aryl ring exhibits conformational polymorphism, due to the different conformation of the ester moiety. This behaviour has been characterized using both powder and single-crystal X-ray diffraction, optical microscopy and differential scanning calorimetry performed on both these polymorphs. The compounds pack via the cooperative interplay of strong N-H center dot center dot center dot O=C intermolecular dimers and chains forming a sheet like structure. In addition, weak C-H center dot center dot center dot O=C and C-H center dot center dot center dot pi interactions impart additional stability to the crystal packing.

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.

Solubility of sulfur dioxide at low partial pressures in dilute sulfuric acid solutions

Equilibrium of dissolution of sulfur dioxide at ppm levels in aqueous solutions of dilute sulfuric acid is analyzed, and a general expression is derived relating the total concentration of sulfur dioxide in the liquid phase to the partial pressure of SO2 in the gas and to the concentration of sulfuric acid in the solution. The equation is simplified for zero and high concentrations of the acid. Experiments at high concentrations of sulfuric acid have enabled the direct determination of Henry’s constant and its dependency on temperature. Heat of dissolution is -31.47 kJ/mol. Experiments in the absence of sulfuric acid and the related simplified expression have led to the determination of the equilibrium constant of the hydrolysis of aqueous sulfur dioxide and its temperature dependency.The heat of hydrolysis is 15.69 kJ/mol. The model equation with these parameters predicts the experimental data of the present work as well as the reported data very well.

Functional Analysis of an Acid Adaptive DNA Adenine Methyltransferase from Helicobacter pylori 26695

HP0593 DNA-(N-6-adenine)-methyltransferase (HP0593 MTase) is a member of a Type III restriction-modification system in Helicobacter pylori strain 26695. HP0593 MTase has been cloned, overexpressed and purified heterologously in Escherichia coli. The recognition sequence of the purified MTase was determined as 5'-GCAG-3' and the site of methylation was found to be adenine. The activity of HP0593 MTase was found to be optimal at pH 5.5. This is a unique property in context of natural adaptation of H. pylori in its acidic niche. Dot-blot assay using antibodies that react specifically with DNA containing m6A modification confirmed that HP0593 MTase is an adenine-specific MTase. HP0593 MTase occurred as both monomer and dimer in solution as determined by gel-filtration chromatography and chemical-crosslinking studies. The nonlinear dependence of methylation activity on enzyme concentration indicated that more than one molecule of enzyme was required for its activity. Analysis of initial velocity with AdoMet as a substrate showed that two molecules of AdoMet bind to HP0593 MTase, which is the first example in case of Type III MTases. Interestingly, metal ion cofactors such as Co2+, Mn2+, and also Mg2+ stimulated the HP0593 MTase activity. Preincubation and isotope partitioning analyses clearly indicated that HP0593 MTase-DNA complex is catalytically competent, and suggested that DNA binds to the MTase first followed by AdoMet. HP0593 MTase shows a distributive mechanism of methylation on DNA having more than one recognition site. Considering the occurrence of GCAG sequence in the potential promoter regions of physiologically important genes in H. pylori, our results provide impetus for exploring the role of this DNA MTase in the cellular processes of H. pylori.

Hybrid Polypeptides: Gabapentin as a Stereochemically Constrained gamma-Amino Acid Residue

The design of folded structures in peptides containing the higher homologues of alpha-amino acid residues requires the restriction of the range of local conformational choices In alpha-amino acids stereochemically constrained residues like alpha,alpha-dialkylated residue, aminoisobutyric acid (Aib), and D-Proline ((D)Pro) have proved extremely useful in the design of helices and hairpins in short peptides Extending this approach, backbone substitution and cyclization are anticipated to bc useful in generating conformationally constrained beta- and gamma-residues This brief review provides a survey of work on hybrid peptide sequences concerning the conformationally constrained gamma-amino acid residue 1-aminomethyl cyclohexane acetic acid, gabapentin (Gpn) This achiral, beta,beta-disubstituted, gamma-residue strongly favors gauche-gauche conformations about the C-alpha-C-beta (0(2)) and C-alpha-C-gamma (0(1)) bonds, facilitating local folding The Gpn residue can adopt both C-7 (NH1 -> CO1) and C-9 (CO1 (I)<- NH1+I) hydrogen bonds which are analogous to the C-5 and C7 (gamma-turn) conformations at alpha-residues In conjunction with adjacent residues, Gpn may be used in ay and gamma alpha segments to generate C-12 hydrogen bonded conformations which may be considered as expanded analogs of conventional beta-turns The structural characterization of C-12 helices, C-12/C-10 helices with mixed hydrogen bond directionalities and beta-hairpins incorporating Gpn residues at the turn segment is illustrated (C) 2010 Wiley Periodicals, Inc Biopolymers (Pept Sci) 94 733-741 2010