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.     

Microbial metabolism of the pyridine ring. The metabolism of pyridine-3,4-diol (3,4-dihydroxypyridine) by Agrobacterium sp

1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.     

Microbial metabolism of the pyridine ring. Metabolism of 2- and 3-hydroxypyridines by the maleamate pathway in Achromobacter sp

1. Washed suspensions of two Achromobacter species (G2 and 2L), capable of growth upon 2- and 3-hydroxypyridine respectively as sources of C and N, rapidly oxidized their growth substrate pyridine-2,5-diol (2,5-dihydroxypyridine) and the putative ring-cleavage product maleamate without a lag. Suspensions derived from fumarate plus (NH(4))(2)SO(4) cultures were unable to do so. 2. Extracts of both bacteria oxidized pyridine-2,5-diol with the stoicheiometry of an oxygenase forming 1mol of NH(3)/mol of substrate. 3. Heat-treated extracts, however, formed maleamate and formate with little free NH(3). 4. The conversion of maleamate into maleate plus NH(3) by extracts of strain 2L, fractionated with (NH(4))(2)SO(4), and the metabolism of maleamate and maleate to fumarate by extracts of both strains demonstrated the existence of the enzymes catalysing each reaction of the maleamate pathway in these bacteria. 5. The pyridine-2,5-diol dioxygenase (mol.wt. approx. 340000) in extracts of these Achromobacter species required Fe(2+) (1.7mum) to restore full activity after dialysis or treatment with chelating agents; the enzyme from strain 2L also had a specific requirement for l-cysteine (6.7mm), which could not be replaced by GSH or dithiothreitol. 6. The oxygenase was strongly inhibited in a competitive manner by the isomeric pyridine-2,3- and -3,4-diols.     

Microbial metabolism of dietary phenolic compounds in the colon

Plant foods contain substantial amounts of phenolic compounds. Dietary interventions with phenolic supplementation show that phenolic compounds are transformed into phenolic acids or lactone structures by intestinal microbiota. The colon is the main site of microbial fermentation. The metabolites circulate in plasma and are excreted via urine. The entero-hepatic circulation ensures that their residence time in plasma is extended compared to that of their parent compounds. Thus these metabolites may exert systemic effects, which however have not been studied adequately. In particular the health implications of microbial metabolites of flavonoids, mostly phenolic acids, are unknown. This review aims to elucidate the microbial metabolism of most of the phenolic classes: flavonoids, isoflavonoids, lignans, phenolic acids and tannins. Some examples of biological activity studies of flavonoid and lignan metabolites are given. Biological significance of enterolactone, a mammalian plant lignan metabolite, has been studied quite extensively, but convincing evidence of the health benefits of the diverse pool of microbial metabolites is still scarce. Hopefully, novel tools in systems biology and the constant search for biomarkers will elucidate the role of the phenolic metabolome in health and in the prevention of chronic diseases. In conclusion, the colon is not only an excretion route, but also an active site of metabolism and deserves further attention from the scientific community.     

Construction and functionalization of fused pyridine ring leading to novel compounds as potential antitubercular agents

A series of fused and functionalized pyridine derivatives were designed, synthesized and tested for their potential antitubercular properties. All these novel compounds were prepared by using multistep methods involving the construction of pyridine ring as a key synthetic step. Some of these compounds were found to be interesting when tested for their antitubercular properties in vitro and one of them appeared as an attractive and potential antitubercular agent.     

Microbial metabolism of phenolic amines: degradation of dl-synephrine by an unidentified arthrobacter.

Microorganisms capable of degrading dl-synephrine were isolated from soil of Citrus gardens by enrichment culture, with dl-synephrine as the sole source of carbon and nitrogen. An organism which appears to be an arthrobacter, but which cannot be identified with any of the presently recognized species was predominant in these isolates. It was found to metabolize synephrine by a pathway involving p-hydroxyphenylacetaldehyde, p-hydroxyphenylacetic acid, and 3,4-dihydroxyphenylacetic acid as intermediates. Some of the enzymes of this pathway were demonstrated in cell-free extracts. An aromatic oxygenase, which could also be readily obtained in a cell-free system, was found to degrade 3,4-dihydroxyphenylacetic acid by meta cleavage.     

Thioacylating agents as ultimate intermediates in the beta-lyase catalysed metabolism of S-(pentachloro-butadienyl)-L-cysteine

The transformation of the hexachloro-1,3-butadiene metabolite S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-L-cysteine (PCBC) by bacterial cysteine conjugate beta-lyase (beta-lyase) and by N-dodecylpyridoxal bromide (PLP-Br) was investigated using GC/MS to identify products formed. PCBC was transformed by both bacterial beta-lyase and PLP-Br to the major products 2,3,4,4-tetrachlorobutenoic acid and 2,3,4,4-tetrachlorothiobutenoic acid, and to the minor metabolites trichloroacetic acid and S-(1,2,3,4,4-pentachlorobuta-1,3-dienyl)-mercaptoacetic acid. In the presence of diethylamine as model nucleophile, PLP-Br transformed PCBC to yield 2,3,4,4-tetrachlorothiobutenoic acid diethylamide; attempts to trap 1,2,3,4,4-pentachlorobutadienyl thiol, the initial metabolite formed by beta-elimination from PCBC, were unsuccessful. The results obtained suggest that the formation of a thioacylating intermediate (a thioketene or a thiono acyl chloride) may be the decisive reaction during the beta-lyase dependent activation of PCBC.     

Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.

The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity.     

The metabolism of aromatic acids by micro-organisms. Metabolic pathways in the fungi

1. The metabolic pathways of aromatic-ring fission were examined in a range of fungal genera that utilize several compounds related to lignin. 2. Most of the genera, after growth on p-hydroxybenzoate, protocatechuate or compounds that are degraded to the latter (e.g. caffeate, ferulate or vanillate), rapidly oxidized these compounds, but not catechol. 3. Such genera possessed a protocatechuate 3,4-oxygenase and accumulated beta-carboxymuconate as the product of protocatechuate oxidation. This enzyme had a high pH optimum in most organisms; the Rhodotorula enzyme was competitively inhibited by catechol. 4. beta-Carboxymuconate was converted by all competent fungi into beta-carboxymuconolactone, which was isolated and characterized. None of the fungi produced or utilized at significant rates the corresponding bacterial intermediate gamma-carboxymuconolactone. 5. The lactonizing enzymes of Rhodotorula and Neurospora crassa had a pH optimum near 5.5 and approximate molecular weights of 19000 and 190000 respectively. 6. The fungi did not degrade the isomeric (+)-muconolactone, gamma-carboxymethylenebutanolide or beta-oxoadipate enol lactone at significant rates, and thus differ radically from bacteria, where beta-oxoadipate enol lactone is the precursor of beta-oxoadipate in all strains examined. 7. The end product of beta-carboxymuconolactone metabolism by extracts was beta-oxoadipate. 8. Evidence for a coenzyme A derivative of beta-oxoadipate was found during further metabolism of this keto acid. 9. A few anomalous fungi, after growth on p-hydroxybenzoate, had no protocatechuate 3,4-oxygenase, but possessed all the enzymes of the catechol pathway. Catechol was detected in the growth medium in one instance. 10. A strain of Penicillium sp. formed pyruvate but no beta-oxoadipate from protocatechuate, suggesting the existence also of a ;meta' type of ring cleavage among fungi.