Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Image by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2×10−4 M and 5.9×10−5 M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.

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.

Liquid-Liquid Interphase (Biphasic) as the Reaction Medium in the Assembly of a Hierarchy of Structures of 4,4 `-Azodibenzoic Acid with Zinc and Cadmium

The compounds Zn(C12H8N2)](2)C12N2H8(COO)(2)](2)center dot(C6H12O)center dot(H2O), I, Zn(C12H8N2)]C12N2H8(COO)(2)], II, Cd(C12H8N2)(H2O)]C12N2H8(COO)(2)]center dot(H2O), III, Zn(C10N2H8)]C12N2H8(COO)(2)]center dot 0.5(C10N2H8), IV, Cd(C12N2H8(COO)(2)center dot H2O], V, and Zn-3(mu(2)-O)(mu(3)-O)(3)]C12N2H8(COO)(2)], VI, have been synthesized by using a biphasic approach (I, III, V, VI) or regular hydrothermal method (II, IV). The compounds exhibit one (I and II), two (In), and three dimensionally (IV, V, VI) extended structures. The flexible azodibenzoate ligand gives rise to a 3-fold interpenetration (IV) when the synthesis was carried out using normal hydrothermal methods. The biphasic approach forms structures without any interpenetrations, especially in the three-dimensional structures of V and VI. Formation of Cd2O2 dimers in V and extended M-O(H)-M two-dimensional layers in VI suggests the subtle structural control achieved by the biphasic method. Transformation studies indicate that it is possible to transform I to II. Lewis acid catalytic studies have been performed to evaluate the role of the coordination environment in such reactions. All the compounds have been characterized by a variety of techniques that includes powder X-ray diffraction, infrared, thermogravitric analysis, UV-vis, photoluminescence studies.

Synthon polymorphism and pseudopolymorphism in co-crystals. The 4,4 `-bipyridine-4-hydroxybenzoic acid structural landscape

Co-crystals of 4,4'-bipyridine and 4-hydroxybenzoic acid (1 : 2) show synthon polymorphism with the former being more stable. A 2 : 1 co-crystal is pseudopolymorphic within the same structural landscape with the structural roles of the two bipyridine N-atoms being distinct, as evidenced by mimicry by 4-phenylpyridine.

Drug-drug co-crystals: Temperature-dependent proton mobility in the molecular complex of isoniazid with 4-aminosalicylic acid

Two drug-drug co-crystals of the anti-tuberculosis drugs isoniazid (INH), pyrazinamide (PYR) and 4-aminosalicylic acid (PAS) are reported. The first is the 1 : 1 molecular complex of INH and PAS. The second is the monohydrate of the 1 : 1 complex of PYR and PAS. The crystal structures of both co-crystals are characterized by a number of hydrogen bonded synthons. Hydrogen bonding of the COOH center dot center dot center dot N-pyridine type is found in both cases. In the INH : PAS co-crystal, there are two symmetry independent COOH center dot center dot center dot center dot N-pyridine hydrogen bonds. In one of these, the H-atom is located on the carboxylic group and is indicative of a co-crystal. In the second case, partial proton transfer occurs across the hydrogen bond, and the extent of proton transfer depends on the temperature. This is more indicative of a salt. Drug-drug co-crystals may have some bearing in the treatment of tuberculosis.

Determination of C-13 Chemical Shift Anisotropy Tensors and Molecular Order of 4-Hexyloxybenzoic Acid

4-Alkoxy benzoic acids belong to an important class of thermotropic liquid crystals that are structurally simple and often used as starting materials for many novel mesogens. 4-Hexyloxybenzoic acid (HBA) is a homologue of the same series and exhibits an enantiotropic nematic phase. As this molecule could serve as an ideal model compound, high resolution C-13 NMR studies of HEA in solution, solid, and liquid crystalline phases have been undertaken. In the solid state, two-dimensional separation of undistorted powder patterns by effortless recoupling (2D SUPER) experiments have been carried out to estimate the magnitude of the components of the chemical shift anisotropy (GSA) tensor of all the aromatic carbons. These values have been used subsequently for calculating the orientational order parameters in the liquid crystalline phase. The GSA values computed by density functional theory (DFT) calculations showed good agreement with the 2D SUPER values. Additionally, C-13-H-1 dipolar couplings in the nematic phase have been determined by separated local field (SLF) spectroscopy at various temperatures and were used for computing the order parameters, which compared well with those calculated by using the chemical shifts. It is anticipated that the CSA values determined for MBA would be useful for the assignment of carbon chemical shifts and for the study of order and dynamics of structurally similar novel mesogens in their nematic phases.

Crystal chemistry and photomechanical behavior of 3,4-dimethoxycinnamic acid: correlation between maximum yield in the solid-state topochemical reaction and cooperative molecular motion

A new monoclinic polymorph, form II (P2(1)/c, Z = 4), has been isolated for 3,4-dimethoxycinnamic acid (DMCA). Its solid-state 2 + 2 photoreaction to the corresponding alpha-truxillic acid is different from that of the first polymorph, the triclinic form I (P (1) over bar, Z = 4) that was reported in 1984. The crystal structures of the two forms are rather different. The two polymorphs also exhibit different photomechanical properties. Form I exhibits photosalient behavior but this effect is absent in form II. These properties can be explained on the basis of the crystal packing in the two forms. The nanoindentation technique is used to shed further insights into these structure-property relationships. A faster photoreaction in form I and a higher yield in form II are rationalized on the basis of the mechanical properties of the individual crystal forms. It is suggested that both Schmidt-type and Kaupp-type topochemistry are applicable for the solid-state trans-cinnamic acid photodimerization reaction. Form I of DMCA is more plastic and seems to react under Kaupp-type conditions with maximum molecular movements. Form II is more brittle, and its interlocked structure seems to favor Schmidt-type topochemistry with minimum molecular movement.

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.

Conformations of Heterochiral and Homochiral Proline-Pseudoproline Segments in Peptides: Context Dependent Cis-Trans Peptide Bond Isomerization

The pseudoproline residue (Psi Pro, L-2,2-dimethyl-1,3-thiazolidine-4-carboxylic acid) has been introduced into heterochiral diproline segments that have been previously shown to facilitate the formation of beta-hairpins, containing central two and three residue turns. NMR studies of the octapeptide Boc-Leu-Phe-Val-(D)Pro-Psi Pro-Leu-Phe-Val-OMe (1), Boc-Leu-Val-Val-(D)Pro-Psi Pro-Leu-Val-Val-OMe (2), and the nonapeptide sequence Boc-Leu-Phe-Val-(D)Pro-Psi Pro-(D)Ala-Leu-Phe-Val-OMe (3) established well-registered beta-hairpin structures in chloroform solution, with the almost exclusive population of the trans conformation for the peptide bond preceding the Psi Pro residue. The beta-hairpin conformation of 1 is confirmed by single crystal X-ray diffraction. Truncation of the strand length in Boc-Val-(D)Pro-Psi Pro-Leu-OMe (4) results in air increase in the population of the cis conformer, with a cis/trans ratio of 3.65. Replacement of Psi Pro in 4 by (L)Pro in 5, results in almost exclusive population of the trans form, resulting in an incipient beta-hairpin conformation, stabilized by two intramolecular hydrogen bonds. Further truncation of the sequence gives an appreciable rise in the population of cis conformers in the tripeptide piv-(D)Pro-Psi Pro-Leu-OMe (6). In the homochiral segment Piv-Pro Psi Pro-Leu-OMe (7) only the cis form is observed with the NMR evidence strongly supporting a type VIa beta-turn conformation, stabilized by a 4 -> 1 hydrogen bond between the Piv (CO) and Leu (3) NH groups. The crystal structure of the analog peptide 7a (Piv-Pro-Psi(H,CH3)Pro-Leu-NHMe) confirms the cis peptide bond geometry for the Pro-Psi(H,CH3)pro peptide bond, resulting in a type VIa beta-turn conformation.

Two Novel Hexadepsipeptides with Several Modified Amino Acid Residues Isolated from the Fungus

Two new cyclohexadepsipeptides have been isolated from the fungus Isaria. Fungal growth in solid media yielded hyphal strands from which peptide fractions were readily isolable by organic-solvent extraction. Two novel cyclodepsipeptides, isaridin A and isaridin B, have been isolated by reverse-phase HPLC, and characterized by ESI-MS and 1H-NMR. Single crystals of both peptides have been obtained, and their 3D structures were elucidated by X-ray diffraction. The isaridins contain several unusual amino acid residues. The sequences are cyclo(β-Gly-HyLeu-Pro-Phe-NMeVal-NMePhe) and cyclo(β-Gly-HyLeu-β-MePro-Phe-NMeVal-NMePhe), where NMeVal is N-methylvaline, NMePhe N-methylphenylalanine, and HyLeu hydroxyleucine (=2-hydroxy-4-methylpentanoic acid). The two peptides differ from one another at residue 3, isaridin A having an (S)-proline at this position, while β-methyl-(S)-proline (=(2S,3S)-2,3,4,5-tetrahydro-3-methyl-1H-pyrrole-2-carboxylic acid) is found in isaridin B. The solid-state conformations of both cyclic depsipeptides are characterized by the presence of two cis peptide bonds at HyLeu(2)-Pro(3)/HyLeu(2)-β-MePro(3) and NMeVal(5)-NMePhe(6), respectively. In isaridin A, a strong intramolecular H-bond is observed between Phe(4)CO⋅⋅⋅HNβ-Gly(1), and a similar, but weaker, interaction is observed between β-Gly(1)CO⋅⋅⋅HNPhe(4). In contrast, in isaridin B, only a single intramolecular H-bond is observed between β-Gly(1)CO⋅⋅⋅HNPhe(4

Synthon Modularity in 4-Hydroxybenzamide-Dicarboxylic Acid Cocrystals

A family of 4-hydroxybenzamide-dicarboxylic acid cocrystals has been designed and subsequently isolated and characterized. The design strategy follows from an understanding of synthon modularity in crystal structures of monocomponent crystals such as gamma-quinol, 4,4'-biphenol and 4-hydroxybenzoic acid. These monocomponent structures contain infinite O-H center dot center dot center dot O-H center dot center dot center dot O-H center dot center dot center dot cooperative synthons linked with molecular connectors such as phenyl and biphenyl, and supramolecular connectors such as the acid dimer in 4-hydroxybenzoic acid. The cocrystal design was influenced by the anticipation that dicarboxylic acids can form a supramolecular connector mediated by acid-amide synthons with 4-hydroxybenzamide, which can then form the phenol O-H center dot center dot center dot O-H center dot center dot center dot O-H center dot center dot center dot infinite synthon. Effectively, the acid-amide and phenol synthons are insulated. The short axis of such a structure will be around 5.12 angstrom and this is borne out in 2:1 cocrystals of 4-hydroxybenzamide with oxalic, succinic, fumaric, glutaric (two forms) and pimelic acids. Hydrated variations of this structure type are seen in the cocrystals obtained with adipic and sebacic acids.

Purification and Properties of l-4-Hydroxymandelate Oxidase from Pseudomonas convexa

An inducible membrane-bound l-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of l-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of O2 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 oC. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 oC which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandelic acid was calculated to be 1.8 × 10−4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents.

Purification and properties of Image -mandelate-4-hydroxylase from Pseudomonas convexa

An inducible Image -mandelate-4-hydroxylase has been partially purified from crude extracts of Pseudomonas convexa. This enzyme catalyzed the hydroxylation of Image -mandelic acid to 4-hydroxymandelic acid. It required tetrahydropteridine, NADPH, Fe2+, and O2 for its activity. The approximate molecular weight of the enzyme was assessed as 91,000 by gel filtration on Sephadex G-150. The enzyme was optimally active at pH 5.4 and 38 °C. A classical Michaelis-Menten kinetic pattern was observed with Image -mandelate, NADPH, and ferrous sulfate and Km values for these substrates were found to be 1 × 10−4, 1.9 × 10−4, and 4.7 × 10−5 Image , respectively. The enzyme is very specific for Image -mandelate as substrate. Thiol inhibitors inhibited the enzyme reaction, indicating that the sulfhydryl groups may be essential for the enzyme action. Treatment of the partially purified enzyme with denaturing agents inactivated the enzyme.

Metabolism of mandelic acid by Neurospora crassa

Preliminary studies on the metabolism of mandelic acid by Neurospora crassa reveal the operation of a pathway for its degradation which involves benzoyl formic acid, benzaldehyde, benzoic acid, 4-hydroxybenzoic acid, and protocatechuic acid as the intermediates. This pathway is different from that followed by bacterial systems and is the same as that observed in Aspergillus niger.

Studies in the polarographic behaviour of zinc-amino acid complexes

1. The polarographic behaviour of amino-acid complexes of zinc has been studied using seven amino acids as complexing agents. 2. The effect of varying the pH of the base solution and the concentration of amino-acid anion on the polarographic behaviour of zinc in these solutions have indi cated the formation of twelve amino-acid complexes. The stability constants could not be calculated due to the irreversible nature of the waves. 3. The effect of sodium hydroxide, sodium carbonate, and ammonia on the polarographic behaviour of zinc has been investigated. The results can be interpreted as due to the formation of mixed complexes in many systems. 4. Amino-acid base solutions have been found to be suitable for the polarographic estimation of zinc.