Identification, functional expression and kinetic analysis of two primary amine oxidases from Rhodococcus opacus

Two native copper-containing amine oxidases (EC 1.4.3.21) have been isolated from Rhodococcus opacus and reveal phenotypic plasticity and catalytic activity with respect to structurally diverse natural and synthetic amines. Altering the amine growth substrate has enabled tailored and targeted oxidase upreg-ulation, which with subsequent treatment by precipitation, ion exchange and gel filtration, achieved a 90–150 fold purification. MALDI-TOF mass spectrometric and genomic analysis has indicated multiple gene activation with complex biodegradation pathways and regulatory mechanisms. Additional post-purification characterisation has drawn on the use of carbonyl reagent and chelating agent inhibitors. Michaelis–Menten kinetics for common aliphatic and aromatic amine substrates and several structural analogues demonstrated a broad specificity and high affinity with Michaelis constants (K M) ranging from 0.1 to 0.9 mM for C 1 –C 5 aliphatic mono-amines and <0.2 mM for a range of aromatic amines. Potential exploitation of the enzymatic versatility of the two isolated oxidases in biosensing and bioprocessing is discussed.

Role of amine oxidase expression to maintain putrescine homeostasis in Rhodococcus opacus

While applications of amine oxidases are increasing, few have been characterised and our understanding of their biological role and strategies for bacteria exploitation are limited. By altering the nitrogen source (NH4Cl, putrescine and cadaverine (diamines) and butylamine (monoamine)) and concentration, we have identified a constitutive flavin dependent oxidase (EC 1.4.3.10) within Rhodococcus opacus. The activity of this oxidase can be increased by over two orders of magnitude in the presence of aliphatic diamines. In addition, the expression of a copper dependent diamine oxidase (EC 1.4.3.22) was observed at diamine concentrations>1mM or when cells were grown with butylamine, which acts to inhibit the flavin oxidase. A Michaelis-Menten kinetic treatment of the flavin oxidase delivered a Michaelis constant (KM)=190μM and maximum rate (kcat)=21.8s(-1) for the oxidative deamination of putrescine with a lower KM (=60μM) and comparable kcat (=18.2s(-1)) for the copper oxidase. MALDI-TOF and genomic analyses have indicated a metabolic clustering of functionally related genes. From a consideration of amine oxidase specificity and sequence homology, we propose a putrescine degradation pathway within Rhodococcus that utilises oxidases in tandem with subsequent dehydrogenase and transaminase enzymes. The implications of PUT homeostasis through the action of the two oxidases are discussed with respect to stressors, evolution and application in microbe-assisted phytoremediation or bio-augmentation.

Desorption electrospray ionisation mass spectrometry reveals in situ modification of a hindered amine light stabiliser resulting from direct N–OR bond cleavage

Detection and characterisation of structural modifications of a hindered amine light stabiliser (HALS) directly from a polyester-based coil coating have been achieved by desorption electrospray ionisation mass spectrometry (DESI-MS) for the first time. In situ detection is made possible by exposing the coating to an acetone vapour atmosphere prior to analysis. This is a gentle and non-destructive treatment that allows diffusion of analyte to the surface without promoting lateral migration. Using this approach a major structural modification of the HALS TINUVIN®123 (bis(1-octyloxy-2,2,6,6-tetramethyl-4-piperidyl) sebacate) was discovered where one N-ether piperidine moiety (N-OC8H17) is converted to a secondary piperidine (N–H). With the use of 2-dimensional DESI-MS imaging the modification was observed to arise during high curing temperatures (ca. 260 °C) and under simulated physiological conditions (80 °C, full solar spectrum). It is proposed that the secondary piperidine derivative is a result of a highly reactive aminyl radical intermediate produced by N–O homolytic bond cleavage. The nature of the bond cleavage is also suggested by ESR spin-trapping experiments employing α-phenyl-N-tert-butyl nitrone (PBN) in toluene at 80 °C. The presence of a secondary piperidine derivative in situ and the implication of N–OR competing with NO–R bond cleavage suggest an alternative pathway for generation of the nitroxyl radical—an essential requirement in anti-oxidant activity that has not previously been described for the N-ether sub-class of HALS.

Structural identification of hindered amine light stabilisers in coil coatings using electrospray ionisation tandem mass spectrometry

Hindered amine light stabilisers (HALS) are the most effective antioxidants currently available for polymer systems in post-production, in-service applications, yet the mechanism of their action is still not fully understood. Structural characterisation of HALS in polymer matrices, particularly the identification of structural modifications brought about by oxidative conditions, is critical to aid mechanistic understanding of the prophylactic effects of these molecules. In this work, electrospray ionisation tandem mass spectrometry (ESI-MS/MS) was applied to the analysis of a suite of commercially available 2,2,6,6-tetramethylpiperidine-based HALS. Fragmentation mechanisms for the \[M + H](+) ions are proposed, which provide a rationale for the product ions observed in the MS/MS and MS(3) mass spectra of N-H, N-CH(3), N-C(O)CH(3) and N-OR containing HALS (where R is an alkyl substituent). A common product ion at m/z 123 was identified for the group of antioxidants containing N-H, N-CH3 or N-C(0)CH3 functionality, and this product ion was employed in precursor ion scans on a triple quadrupole mass spectrometer to identify the HALS species present in a crude extract from of a polyester-based coil coating. Using MS/MS, two degradation products were unambiguously identified. This technique provides a simple and selective approach to monitoring HALS structures within complex matrices. Copyright (C) 2010 John Wiley & Sons, Ltd.

Crystal structures of the co-crystalline adduct 5-(4-bromophenyl)-1,3,4-thiadiazol-2-amine--4-nitrobenzoic acid (1/1) and the salt 2-amino-5-(4-bromophenyl)-1,3,4-thiadiazol-3-ium 2-carboxy-4,6-dinitrophenolate

The structures of the 1:1 co-crystalline adduct C8H6BrN3S . C7H5NO4 (I) and the salt C8H7BrN3S+ C7H3N2O7- (II) from the interaction of 5-(4-bromophenyl)-1,3,4-thiadiazol-2-amine with 4-nitrobenzoic acid and 3,5-dinitrosalicylic acid, respectively, have been determined. The primary inter-species association in both (I) and (II) is through duplex R2/2(8) (N-H...O/O-H...O) or (N-H...O/N-H...O) hydrogen bonds, respectively, giving heterodimers. In (II), these are close to planar [dihedral angles between the thiadiazole ring and the two phenyl rings are 2.1(3)deg. (intra) and 9.8(2)deg. (inter)], while in (I) these angles are 22.11(15) and 26.08(18)deg., respectively. In the crystal of (I), the heterodimers are extended into a one-dimensional chain along b through an amine N-...N(thiadiazole) hydrogen bond but in (II), a centrosymmetric cyclic heterotetramer structure is generated through N-H...O hydrogen bonds to phenol and nitro O-atom acceptors and features, together with the primary R2/2(8) interaction, conjoined R4/6(12), R2/1(6) and S(6) ring motifs. Also present in (I) are pi--pi interactions between thiadiazole rings [minimum ring centroid separation, 3.4624(16)deg.] as well as short Br...O(nitro) interactions in both (I) and (II) [3.296(3)A and 3.104(3)A, respectively].

Preparation of lamellar and hexagonal forms of mesoporous silica and zirconiaby the neutral amine route: lamellar-hexagonal transformation in the solid state

The chain length of the surfactant and the solvent composition are two of the factors that determine whether the lamellar or the hexagonal form of mesoporous SiO2 (or ZrO2) is formed by the neutral amine route; a lamellar-hexagonal transformation occurs on removal of the amine from the former.

Hydrogen and fluorine in crystal engineering: systematics from crystallographic studies of hydrogen bonded tartrate-amine complexes and fluoro-substituted coumarins, styrylcoumarins and butadienes

Three aspects of crystal engineering in molecular crystals are presented to emphasize the role of intermolecular interactions and factors influencing crystal packing. Hydrogen bonded tartrate-amine complexes have been analyzed with the propensity for formation of multidirectional hydrogen bonding as a key design element in the generation of materials for second harmonic generation (SHG). The invariance of the framework in DBT and its possible implications on SHG is outlined. The role of Fluorine in orienting molecules of coumarins, styrylcoumarins and butadienes for photodimerization is described with particular emphasis on its steering capability. Usage of coumarin as an design element for the generation of polymorphs of substituted styrylcoumarins is examined with specific examples.

Microbial transformation of a fungicide-derived amine, dimethyl-p-phenylene diamine by Alcaligenes DM4

Abstract Microbial transformation of N, N-dimethyl-p-phenylene diamine (DMPDA), a microbial product formed from the fungicide fenaminosulf (p-dimethylaminobenzenediazo sodium sulfonate) was studied by enriching microbes in soils treated with the amine. Microorganisms isolated from DMPDA-treated soil belonged to the genera of Micrococcus, Alcaligenes, and Corynebacterium. Of the various isolates, Alcaligenes DM4 showed maximal growth on DMPDA utilizing it as sources of carbon and nitrogen. When grown in mineral salts basal medium containing 0.05% DMPDA to serve as carbon and nitrogen sources, Alcaligenes DM4 grew exponentially up to 18 h. Even though the characterization of the complete pathway of microbial degradation of DMPDA could not be carried out due to the auto-oxidation of the compound, the initial transformation product of DMPDA by Alcaligenes DM4 has been identified as a dimer. The dimer is generated into the culture medium presumably by the extra-cellular oxidase of Alcaligenes DM4. It is suggested that the risk-benefit evaluation on the use of fenaminosulf is to be made taking into consideration the microbial transformations of the fungicide.

Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions

A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated.

X-Ray Structure Of A Ternary Metal-Complex - Copper(Ii) Inosine 5'-Monophosphate Imidazole - Metal-Binding To The N(7) Atom Of The Nucleotide In A Mixed-Ligand System Containing An Aromatic Amine

A ternary metal-nucleotide complex, Na2[Cu(5’-IMP)2(im)o,8(H20)l,2(H20)2h]as~ 1be2e.n4 pHr2ep0a,r ed and its structure analyzed by X-ray diffraction (5’-IMP = inosine 5’-monophos hate; im = imidazole). The complex crystallizes in space group C222, with a = 8.733 (4) A, b = 23.213 (5) A, c = 21.489 (6) 1, and Z = 4. The structure was solved by the heavy-atom method and refined by full-matrix least-squares technique on the basis of 2008 observed reflections to a final R value of 0.087. Symmetry-related 5’-IMP anions coordinate in cis geometry through the N(7) atoms of the bases. The other cis positions of the coordination plane are statistically occupied by nitrogen atoms of disordered im groups and water oxygens with occupancies 0.4 and 0.6, respectively. Water oxygens in axial positions complete the octahedral coordination of Cu(I1). The complex is isostructural with C~S-[P~(S’-IMP),(NH~)~a] m”,o del proposed for Pt(I1) binding to DNA. The base binding observed in the present case is different from the typical ”phosphate only” binding shown from earlier studies on metal-nucleotide complexes containing various other ?r-aromatic amines.

Theoretical studies of lithium bonding in lithium chloride/aliphatic amine complexes

In the systematic study of amine … LiCl [amines = NH3, CH3NH2, (CH3)2NH] complexes the possibility of an ion-pair structure and the effect of methylation on the stabilization energy is investigated. ΔEis evaluated by the SCF/4-31G method and augmented by the approximate dispersion energy calculated perturbationally. The interaction energy decreases with the increasing number of methyl groups in the amine. The dispersion energy plays a negligible role in the stabilization of complexes. None of the systems studied are ion pairs; their Li bonds are of a so-called molecular type. Due to the divergence of the multipole expansion, the attempt to correct the 4-31G stabilization energies via the electrostatic energy fails. The relative order of the ΔE in the series of complexes is verified instead in the extended basis set calculation. The lithium bonds are compared with their H-bonded analogues.

Thermal decomposition of ethyl and isopropyl amine perchlorates

Thermal decomposition of ethyl and isopropyl amine perchlorates has been studied by methods such as DTA, TG, isothermal weight loss measurements and the decomposition products have been analyzed in a mass spectrometer. Activation energy values for thermal decomposition have been calculated fromagr-t plots. The proton transfer dissociation mechanism proposed for the thermal decomposition of ammonium perchlorate (AP) has been extended to explain the decomposition products of these twosubstituted amine perchlorates.

Amine biosynthesis in Lathyrus sativus seedlings

The biosynthesis of certain amines in Lathyrus sativus seedlings was studied in isolated shoots and cotyledons. In shoots, arginine was about 14 times more efficient than ornithine for the synthesis of agmatine, putrescine, spermidine and spermine. Isotope dilution experiments, and the changes in specific activities of the 4 amines with time when 14C-arginine served as the precursor, indicated that putrescine and the polyamines were formed mainly from arginine, via agmatine. Similar experiments showed that cadaverine was formed at least in part from homoarginine, though lysine was ca 4 times more effective as a precursor. The pattern of changes in specific activity of homoagmatine and cadaverine with time when 14C-homoarginine served as the precursor support the conclusion that homoarginine and arginine follow analogous metabolic routes in the biosynthesis of putrescine and cadaverine respectively.

Effect of 2-allyl-2-isopropylacetamide on poly(adenylic acid)-containing ribonucleic acid.

A single administration of 2-allyl-2-isopropylacetamide, a porphyrinogenic drug, enhanced the 32P-labelling of nucleoplasmic as well as cytoplasmic poly(A)-containing RNA in rat liver. The synthesis of total microsomal RNA is only marginally increased under these conditions. The drug enhances the labelling of a variety of cytoplasmic poly(A)-containing RNA species, and this effect is counteracted by the simultaneous administration of haemin. 2-Allyl-2-isopropylacetamide also enhanced the release of RNA from the nucleus to the cytoplasm.

Amine levels in Lathyrus sativus seedlings during development

In growing Lathyrus sativus seedlings, the levels of DNA, RNA and protein markedly decreased in the cotyledons and progressively increased in the embryo-axis. In cotyledons, spermidine and spermine contents were substantially reduced while those of agmatine and putrescine were sharply increased. By contrast the embryo-axis progressively accumulated relatively larger amounts of agmatine, homoagmatine. putrescine, cadaverine, spermidine and spermine in parallel with similar changes in its DNA, RNA and protein content. While the cotyledons contained ca 50% of the total agmatine and putrescine present in the plant embryo by day 10, the embryo-axis, though representing less than 20% of the dry wt, contained 90 and 75% of total cadaverine and homoagmatine respectively of the seedlings. Spermidine and spermine levels of this tissue were also comparatively higher, being of the order of 80 and 50% respectively of the total. The root and shoot portions of the embryo-axis also exhibited a similar relationship between changes in DNA, RNA and protein and all the above amines during development. However, the polyamine content of the shoots was relatively higher than those of the roots during the growth period.