Microbial Degradation of Pyridine: a Complete Pathway in Arthrobacter sp. Strain 68b Deciphered

ABSTRACT Pyridine and its derivatives constitute the majority of heterocyclic aromatic compounds that occur largely as a result of human activities and contribute to environmental pollution. It is known that they can be degraded by various bacteria in the environment; however, the degradation of unsubstituted pyridine has not yet been completely resolved. In this study, we present data on the pyridine catabolic pathway in Arthrobacter sp. strain 68b at the level of genes, enzymes, and metabolites. The pyr gene cluster, responsible for the degradation of pyridine, was identified in a catabolic plasmid, p2MP. The pathway of pyridine metabolism consisted of four enzymatic steps and ended by the formation of succinic acid. The first step in the degradation of pyridine proceeds through a direct ring cleavage catalyzed by a two-component flavin-dependent monooxygenase system, encoded by pyrA (pyridine monooxygenase) and pyrE genes. The genes pyrB, pyrC, and pyrD were found to encode (Z)-N-(4-oxobut-1-enyl)formamide dehydrogenase, amidohydrolase, and succinate semialdehyde dehydrogenase, respectively. These enzymes participate in the subsequent steps of pyridine degradation. The metabolites of these enzymatic reactions were identified, and this allowed us to reconstruct the entire pyridine catabolism pathway in Arthrobacter sp. 68b. IMPORTANCE The biodegradation pathway of pyridine, a notorious toxicant, is relatively unexplored, as no genetic data related to this process have ever been presented. In this paper, we describe the plasmid-borne pyr gene cluster, which includes the complete set of genes responsible for the degradation of pyridine. A key enzyme, the monooxygenase PyrA, which is responsible for the first step of the catabolic pathway, performs an oxidative cleavage of the pyridine ring without typical activation steps such as reduction or hydroxylation of the heterocycle. This work provides new insights into the metabolism of N-heterocyclic compounds in nature.

Dual control of NAD+ synthesis by purine metabolites in yeast

Metabolism is a highly integrated process resulting in energy and biomass production. While individual metabolic routes are well characterized, the mechanisms ensuring crosstalk between pathways are poorly described, although they are crucial for homeostasis. Here, we establish a co-regulation of purine and pyridine metabolism in response to external adenine through two separable mechanisms. First, adenine depletion promotes transcriptional upregulation of the de novo NAD+ biosynthesis genes by a mechanism requiring the key-purine intermediates ZMP/SZMP and the Bas1/Pho2 transcription factors. Second, adenine supplementation favors the pyridine salvage route resulting in an ATP-dependent increase of intracellular NAD+. This control operates at the level of the nicotinic acid mononucleotide adenylyl-transferase Nma1 and can be bypassed by overexpressing this enzyme. Therefore, in yeast, pyridine metabolism is under the dual control of ZMP/SZMP and ATP, revealing a much wider regulatory role for these intermediate metabolites in an integrated biosynthesis network.

Short Term Effect of Caffeine on Purine, Pyrimidine and Pyridine Metabolism in Rice (Oryza sativa) Seedlings

As part of our studies on the physiological and ecological function of caffeine, we investigated the effect of exogenously supplied caffeine on purine, pyrimidine and pyridine metabolism in rice seedlings. We examined the effect of 1 mM caffeine on the in situ metabolism of 14C-labelled adenine, guanine, inosine, uridine, uracil, nicotinamide and nicotinic acid. The segments of 4-day-old dark-grown seedlings were incubated with these labelled compounds for 6 h. For purines, the incorporation of radioactivity from [8-14C]adenine and [8-14C]guanine into nucleotides was enhanced by caffeine; in contrast, incorporation into CO2 were reduced. The radioactivity in ureides (allantoin and allantoic acid) from [8-14C]guanine and [8-14C]inosine was increased by caffeine. For pyrimidines, caffeine enhanced the incorporation of radioactivity from [2-14C]uridine into nucleotides, which was accompanied by a decrease in pyrimidine catabolism. Such difference was not found in the metabolism of [2-14C]uracil. Caffeine did not influence the pyridine metabolism of [carbonyl-14C]-nicotinamide and [2-14C]nicotinic acid. The possible control steps of caffeine on nucleotide metabolism in rice are discussed.

Pyridine Metabolism and Trigonelline Synthesis in Leaves of the Mangrove Legume trees Derris indica (Millettia pinnata) and Caesalpinia crista

The aim of this study was to reveal the pyridine metabolism in leaves of two mangrove legumes, Derris indica (= Millettia pinnata or Pongamia pinnata) and Caesalpinia crista. Radioactivity from [carbonyl-14C]nicotinamide supplied exogenously to young leaf disks was recovered in nicotinic acid, nicotinic acid mononucleotide, NAD, NADP, nicotinamide mononucleotide and trigonelline. These mangrove species, especially D. indica, have strong ability to convert nicotinamide to trigonelline, but not to nicotinic acid glucoside. The endogenous trigonelline content in leaves of D. indica was more than 830 μg/g dry weight. This value is 5-12 times greater than that in leaves of Glycine max. There was little short-term effect of 250 and 500 mM NaCl (equivalent to ca. 50% and 100% sea water) on nicotinamide metabolism.

Pyridine metabolism by a denitrifying bacterium

A denitrifying bacterium capable of pyridine degradation was isolated from contaminated soil. The Gram-negative bacterium, which was identified as an Alcaligenes sp., rapidly metabolized pyridine under anaerobic conditions with nitrate as electron acceptor. [14C]Pyridine was converted to 14CO2, unidentified polar metabolic products, and labeled biomass. During pyridine metabolism, nitrate was reduced to nitrogen gas via nitrite and nitrous oxide. The molar ratio of pyridine to nitrate strongly affected pyridine metabolism. Maximum pyridine degradation occurred at a nitrate concentration above 5 mM, a temperature of 22–36 °C, and a pH of 6.8–8.0. Key words: pyridine, anaerobic metabolism, denitrifying bacteria, Alcaligenes sp.