Microbial metabolism of the pyridine ring. Metabolic pathways of pyridine biodegradation by soil bacteria.

1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.     

The formation of pyridine haemochromogen.

1. Titration of haem with pyridine in alkaline media of low ionic strength yields a true pyridine haemochromogen, compound III, at very low concentrations of pyridine. 2. Graphical analysis of this titration gives the first spectrophotometric evidence for a dimeric haem. 3. Compound III is unstable and tends to aggregate to a second compound, compound II, whose formation is enhanced under those conditions favourable to hydrophobic bonding. 4. At higher concentrations of pyridine, compound II is dispersed to yield the classical pyridine haemochromogen, compound I, whose spectral properties are essentially those of pyridine haemochromogen in a non-aqueous medium.     

Molecular properties and odour. Effects of substituents on pyridine and pyridine odour

It is investigated how the odour of pyridine changes when substituents of different kinds are inserted on the pyridine ring. The relation between the space filling and electronic properties of molecules and odour is studied. Far infrared spectra have been recorded for pyridines in order to test a hypothetical correlation between odour and vibrational energies.     

Sesquiterpene pyridine alkaloids from Peritassa campestris.

An investigation of the methanol and ethyl acetate extracts from the roots of Peritassa campestris (Hippocrateaceae) afforded the sesquiterpene pyridine alkaloid, 4-hydroxy-7-epi-chuchuhuanine E-V, and nine known alkaloids, forrestine, euonimine, ebenifoline E-I, wilforine, euojaponine F, euonine, wilforjine, neowilforine, and wilforzine. The structures of the isolates were elucidated on the basis of spectral data, particularly HMQC and HMBC experiments.     

Pre-translational induction of pentoxyresorufin O-depentylase by pyridine.

Pentoxyresorufin O-depentylase activity, mainly associated with phenobarbital-inducible cytochrome P450IIB1 (designated CYP2B1), was increased after a single treatment of pyridine (250 mg/kg, i.p.), and further increased by repeated treatments for 5 days. The catalytic activity and immunoreactive protein of CYP2B recognized by polyclonal antibodies were significantly induced by a relatively high dose of pyridine (250 mg/kg, i.p.) while ethanol-inducible cytochrome P450IIE1 (CYP2E1) could be induced by a low dosage (25 mg/kg, i.p.). Unlike CYP2E1 induction without changing its mRNA level, the induction of CYP2B by pyridine was accompanied by an elevation of its mRNA, indicating a pre-translational activation of this enzyme. These results indicate that pyridine induces various isozymes of cytochromes P450 by different induction mechanisms.     

The stereospecificity of nitrate reductase for hydrogen removal from reduced pyridine nucleotides.

The stereospecificity of the hydrogen removal from reduced pyridine nucleotides catalyzed by nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1, and NAD(P)H : nitrate oxidoreductase, EC 1.6.6.2) was investigated. A high degree of enzyme purification was required to obtain conclusive results. Improvements are described for the purification of nitrate reductase from Chlorella fusca and from spinach (Spinacea oleracea, L.) leaves. The latter enzyme is shown to contain a cytochrome. With highly purified nitrate reductase preparations from Cl. fusca, Neurospora crassa, Rhodotorula glutinis and spinach leaves the stereospecificity of the reaction was determined to be predominantly of the A-type in all cases.     

A large-scale preparative electrophoretic method for the purification of pyridine nucleotide-linked dehydrogenases.

A large-scale preparative polyacrylamide gel electrophoresis (PAGE) method that uses a 1.5- or a 2.0-cm-thick slab gel has been developed for the purification of NAD-dependent dehydrogenases. With the 2.0-cm-thick gel, a maximum volume (up to about 160 ml) of enzyme sample was applied to a gel plate, resulting in the application of a large amount of protein and enzyme. After the electrophoretic run, the enzyme band on the gel was detected by activity staining and recovered from the gel by extraction with a fairly loose-fitting glass-Teflon homogenizer. NAD-dependent alanine dehydrogenase, leucine dehydrogenase, and glycerol dehydrogenase were purified in high yields (more than 80%) by the preparative PAGE method. The method can be carried out using a simple slab gel apparatus, which is modified from the conventional analytical apparatus for the purpose of preparative PAGE under conditions used for routine analytical runs. Thus, the method may be suitable for use in purifying NAD(P)-dependent dehydrogenases and many other enzymes after conventional chromatography such as dye-ligand affinity chromatography or ion-exchange chromatography.     

Mechanism of aromatic hydroxylation. Properties of a model for pyridine nucleotide-dependent flavoprotein hydroxylases

A model (NADH-phenazine methosulfate-O₂) formally similar to pyridine nucleotide-dependent flavoprotein hydroxylases catalyzed the hydroxylation of several aromatic compounds. The hydroxylation was maximal at acid pH and was inhibited by ovine Superoxide dismutase, suggesting that perhydroxyl radicals might be intermediates in this process. The stoichiometry of the reaction indicated that a univalent reduction of oxygen was occurring. The correlation between the concentration of semiquinone and hydroxylation, and the inhibition of hydroxylation by ethanol which inhibited semiquinone oxidation, suggested the involvement of phenazine methosulfate-semiquinone. Activation of hydroxylation by Fe³⁺ and Cu²⁺ supported the contention that univalently reduced species of oxygen was involved in hydroxylation. Catalase was without effect on the hydroxylation by the model, ruling out H₂O₂ as an intermediate. A reaction sequence, involving a two-electron reduction of phenazine methosulfate to reduced phenazine methosulfate followed by disproportionation with phenazine methosulfate to generate the semiquinone, was proposed. The semiquinone could donate an electron to O₂ to generate O₂ which could be subsequently protonated to form the perhydroxyl radical.     

Reduced pyridine nucleotide oxidases of Eugena gracilis var. bacillaris.

Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 muM; NADPH oxidase 150 muM ; NADH lipoyl dehydrogenase, 0.35 muM. Enzyme activities after storage at -5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.     

Structure of pyridine nucleotide transhydrogenase from Azotobacter vinelandii.

1. Pyridine nucleotide transhydrogenase of Azotobacter vinelandii purified by affinity chromatography consists of a mixture of polydisperse rods at neutral pH. No other structures are seen by electron microscopy. 2. At high pH (8.5--9.0) the rods depolymerize. Complete depolymerization can be achieved in 0.1 M Tris-Cl pH 9.0. The depolymerized enzyme has a molecular weight of 421000 (sedimentation equilibrium), its sedimentation coefficient s20, w = 15 S and its Stokes' radius Rs = 7 nm. Since gel electrophoresis in the presence of sodium dodecyl sulphate shows that transhydrogenase consists of a single polypeptide chain of molecular weight (54 +/- 2) X 10(3) it follows that the depolymerized enzyme has an octameric quaternary structure. We propose that this octamer serves as the functional monomeric unit ('unimer') from which the polymeric form of transhydrogenase is constructed. 3. Gel filtration and sucrose gradient centrifugation studies of cell-free extracts from A. vinelandii show the unimer to be the predominant active species.     

Hysteretic behavior of nitrate reductase. Evidence of an allosteric binding site for reduced pyridine nucleotides.

In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.     

Studies on the cyclophorase system. XIX. Interconversion of pyridine nucleotides

The enzymatic synthesis of triphosphopyridine nucleotide (TPN) from diphosphopyridine nucleotide (DPN) and adenosine triphosphate (ATP), and the conversion of TPN to DPN has been shown in fractions derived from the cyclophorase-type particulate preparation from rabbit liver and kidney. The formation of DPN from TPN occurs in the soluble fraction also.